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Sample records for malpighian tubule polytene

  1. Malpighian Tubules as Novel Targets for Mosquito Control

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    Peter M. Piermarini

    2017-01-01

    Full Text Available The Malpighian tubules and hindgut are the renal excretory tissues of mosquitoes; they are essential to maintaining hemolymph water and solute homeostasis. Moreover, they make important contributions to detoxifying metabolic wastes and xenobiotics in the hemolymph. We have focused on elucidating the molecular mechanisms of Malpighian tubule function in adult female mosquitoes and developing chemical tools as prototypes for next-generation mosquitocides that would act via a novel mechanism of action (i.e., renal failure. To date, we have targeted inward rectifier potassium (Kir channels expressed in the Malpighian tubules of the yellow fever mosquito Aedes aegypti and malaria mosquito Anopheles gambiae. Inhibition of these channels with small molecules inhibits transepithelial K+ and fluid secretion in Malpighian tubules, leading to a disruption of hemolymph K+ and fluid homeostasis in adult female mosquitoes. In addition, we have used next-generation sequencing to characterize the transcriptome of Malpighian tubules in the Asian tiger mosquito Aedes albopictus, before and after blood meals, to reveal new molecular targets for potentially disrupting Malpighian tubule function. Within 24 h after a blood meal, the Malpighian tubules enhance the mRNA expression of genes encoding mechanisms involved with the detoxification of metabolic wastes produced during blood digestion (e.g., heme, NH3, reactive oxygen species. The development of chemical tools targeting these molecular mechanisms in Malpighian tubules may offer a promising avenue for the development of mosquitocides that are highly-selective against hematophagous females, which are the only life stage that transmits pathogens.

  2. Sodium pumps in the Malpighian tubule of Rhodnius sp.

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    CARUSO-NEVES CELSO

    2000-01-01

    Full Text Available Malpighian tubule of Rhodnius sp. express two sodium pumps: the classical ouabain-sensitive (Na+ + K+ATPase and an ouabain-insensitive, furosemide-sensitive Na+-ATPase. In insects, 5-hydroxitryptamine is a diuretic hormone released during meals. It inhibits the (Na+ + K+ATPase and Na+ -ATPase activities indicating that these enzymes are involved in fluid secretion. Furthermore, in Rhodnius neglectus, proximal cells of Malpighian tubule exposed to hyperosmotic medium, regulate their volume through a mechanism called regulatory volume increase. This regulatory response involves inhibition of the (Na+ + K+ATPase activity that could lead to accumulation of active osmotic solute inside the cell, influx of water and return to the normal cell volume. Adenosine, a compound produced in stress conditions, also inhibits the (Na+ + K+ATPase activity. Taken together these data indicate that (Na+ + K+ATPase is a target of the regulatory mechanisms of water and ions transport responsible for homeostasis in Rhodnius sp.

  3. Structural and functional alterations in Malpighian tubules as biomarkers of environmental pollution: synopsis and prospective.

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    Giglio, Anita; Brandmayr, Pietro

    2017-08-01

    Although a number of biomarkers of pollutant exposure have been identified in invertebrate species, little is known about the effect on Malpighian tubules playing an essential role in excretion and osmoregulation. Analyses of structural and functional alterations on this organ can be useful to predict the effects at the organism and population level in monitoring studies of environmental pollution. The aim of the present review is to provide a synthesis of existing knowledge on cellular damages induced by xenobiotics in Malpighian tubules both under laboratory and field conditions. We compared studies of exposure to pesticides and heavy metals as mainly environmental contaminants from anthropogenic activities. This report provided evidence that the exposure to xenobiotics has an effect on this organ and reinforces the need for further research integrating molecular biomarkers with analysis on Malpighian tubules. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  4. Proteomic changes in response to crystal formation in Drosophila Malpighian tubules.

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    Chung, Vera Y; Konietzny, Rebecca; Charles, Philip; Kessler, Benedikt; Fischer, Roman; Turney, Benjamin W

    2016-04-02

    Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.

  5. The Single Kinin Receptor Signals to Separate and Independent Physiological Pathways in Malpighian Tubules of the Yellow Fever Mosquito

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    2010-06-10

    mortar and pestle . On the day of the experiment a female mosquito, 4-7 days post-eclosion, was cold anesthetized and decapitated. Malpighian tubules...tubules was analyzed with the electron probe as described previously (7, 62), except that we used the JEOL JXA 8900 EPMA Microprobe (Tokyo, Japan

  6. Cadmium transport by the gut and Malpighian tubules of Chironomus riparius

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    Leonard, Erin M.; Pierce, Laura M.; Gillis, Patricia L.; Wood, Chris M.; O'Donnell, Michael J.

    2009-01-01

    Many aquatic insects are very insensitive to cadmium in short-term laboratory studies. LC50 values for larvae of the midge Chironomus riparius are over 25,000 times the Criterion Maximum Concentration in the United States Environmental Protection Agency (U.S. EPA (2000)) species sensitivity distribution (SSD). Excretion or sequestration of cadmium may contribute to insensitivity and we have therefore examined cadmium transport by isolated guts and renal tissues of C. riparius larvae. Regional differences of Cd transport along the gut were identified using a Cd 2+ -selective microelectrode in conjunction with the Scanning Ion-Selective Electrode Technique (SIET). Cd is transported into the anterior midgut (AMG) cells from the lumen and out of the cells into the hemolymph. The transport of Cd from the gut lumen to the hemolymph exposes other tissues such as the nervous system and muscles to Cd. The gut segments which remove Cd from the hemolymph at the highest rate are the posterior midgut (PMG) and the ileum. In addition, assays using an isolated Malpighian (renal) tubule preparation have shown that the Malpighian tubules (MT) both sequester and secrete Cd. For larvae bathed in 10 μmol l -1 Cd, the tubules can secrete the entire hemolymph burden of Cd in ∼15 h.

  7. The single kinin receptor signals to separate and independent physiological pathways in Malpighian tubules of the yellow fever mosquito

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    In the past we have used the leucokinins, the kinins of the cockroach Leucophaea, to evaluate the mechanism of diuretic action of kinin peptides in Malpighian tubules of the yellow fever mosquito Aedes aegypti. Now using aedeskinins, the kinins of Aedes, are available, we find that in isolated Aede...

  8. Proteomic-based insight into Malpighian tubules of silkworm Bombyx mori.

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    Xiao-wu Zhong

    Full Text Available Malpighian tubules (MTs are highly specific organs of arthropods (Insecta, Myriapoda and Arachnida for excretion and osmoregulation. In order to highlight the important genes and pathways involved in multi-functions of MTs, we performed a systematic proteomic analysis of silkworm MTs in the present work. Totally, 1,367 proteins were identified by one-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry, and as well as by Trans Proteomic Pipeline (TPP and Absolute protein expression (APEX analyses. Forty-one proteins were further identified by two-dimensional gel electrophoresis. Some proteins were revealed to be significantly associated with various metabolic processes, organic solute transport, detoxification and innate immunity. Our results might lay a good foundation for future functional studies of MTs in silkworm and other lepidoptera.

  9. Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea).

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    Mampumbu, André Roberto; Mello, Maria Luiza S

    2008-08-01

    Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation

  10. A technique for preparing polytene chromosomes from Aedes aegypti (Diptera, Culicinae

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    Jairo Campos

    2003-04-01

    Full Text Available Polytene chromosome preparations were obtained from larval, pupal and adult female Malpighian tubules of Aedes aegypti. The Malpighian tubules of the pupae (0-4 h old from larvae reared at 20ºC provided the best cytogenetic analysis. The interaction of nucleic acids and proteins that influence the spreading of the chromosomes could be reduced with the preparation technique of the sheets submitted to a stronger treatment starting with the hypotony of tissue and successive bathings with acetic acid. A simple technique should facilitate molecular cytogenetics used in the location of resistance and vector competence genes.

  11. A novel member of the trehalose transporter family functions as an H+-dependent trehalose transporter in the reabsorption of trehalose in Malpighian tubules.

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    Shingo eKikuta

    2012-07-01

    Full Text Available In insects, Malpighian tubules are functionally analogous to mammalian kidneys in that they not only are essential to excrete waste molecules into the lumen but also are responsible for the reabsorption of indispensable molecules, such as sugars, from the lumen to the principal cells. Among sugars, the disaccharide trehalose is highly important to insects because it is the main hemolymph sugar to serve as a source of energy and carbon. The trehalose transporter TRET1 participates in the transfer of newly synthesized trehalose from the fat body across the cellular membrane into the hemolymph. Although transport proteins must play a pivotal role in the reabsorption of trehalose in Malpighian tubules, the molecular context underlying this process remains obscure. Previously, we identified a Tret1 homolog (Nlst8 that is expressed principally in the Malpighian tubules of the brown planthopper (BPH. Here, we used the Xenopus oocyte expression system to show that NlST8 exerts trehalose transport activity that is elevated under low pH conditions. These functional assays indicate that Nlst8 encodes a proton-dependent trehalose transporter (H-TRET1. To examine the involvement of Nlst8 in trehalose reabsorption, we analyzed the sugar composition of honeydew by using BPH with RNAi gene silencing. Trehalose was detected in the honeydew as waste excreted from Nlst8-dsRNA-injected BPH under hyperglycemic conditions. However, trehalose was not expelled from GFP-dsRNA-injected BPH even under hyperglycemic conditions. We conclude that NlST8 could participate in trehalose reabsorption driven by a H+ gradient from the lumen to the principal cells of the Malpighian tubules.

  12. RNA-Seq Comparison of Larval and Adult Malpighian Tubules of the Yellow Fever Mosquito Aedes aegypti Reveals Life Stage-Specific Changes in Renal Function

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    Yiyi Li

    2017-05-01

    Full Text Available Introduction: The life history of Aedes aegypti presents diverse challenges to its diuretic system. During the larval and pupal life stages mosquitoes are aquatic. With the emergence of the adult they become terrestrial. This shifts the organism within minutes from an aquatic environment to a terrestrial environment where dehydration has to be avoided. In addition, female mosquitoes take large blood meals, which present an entirely new set of challenges to salt and water homeostasis.Methods: To determine differences in gene expression associated with these different life stages, we performed an RNA-seq analysis of the main diuretic tissue in A. aegypti, the Malpighian tubules. We compared transcript abundance in 4th instar larvae to that of adult females and analyzed the data with a focus on transcripts that encode proteins potentially involved in diuresis, like water and solute channels as well as ion transporters. We compared our results against the model of potassium- and sodium chloride excretion in the Malpighian tubules proposed by Hine et al. (2014, which involves at least eight ion transporters and a proton-pump.Results: We found 3,421 of a total number of 17,478 (19.6% unique transcripts with a P < 0.05 and at least a 2.5 fold change in expression levels between the two groups. We identified two novel transporter genes that are highly expressed in the adult Malpighian tubules, which have not previously been part of the transport model in this species and may play important roles in diuresis. We also identified candidates for hypothesized sodium and chloride channels. Detoxification genes were generally higher expressed in larvae.Significance: This study represents the first comparison of Malpighian tubule transcriptomes between larval and adult A. aegypti mosquitoes, highlighting key differences in their renal systems that arise as they transform from an aquatic filter-feeding larval stage to a terrestrial, blood-feeding adult stage.

  13. A de novo transcriptome of the Malpighian tubules in non-blood-fed and blood-fed Asian tiger mosquitoes Aedes albopictus: insights into diuresis, detoxification, and blood meal processing

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    Carlos J. Esquivel

    2016-03-01

    Full Text Available Background. In adult female mosquitoes, the renal (Malpighian tubules play an important role in the post-prandial diuresis, which removes excess ions and water from the hemolymph of mosquitoes following a blood meal. After the post-prandial diuresis, the roles that Malpighian tubules play in the processing of blood meals are not well described. Methods. We used a combination of next-generation sequencing (paired-end RNA sequencing and physiological/biochemical assays in adult female Asian tiger mosquitoes (Aedes albopictus to generate molecular and functional insights into the Malpighian tubules and how they may contribute to blood meal processing (3–24 h after blood ingestion. Results/Discussion. Using RNA sequencing, we sequenced and assembled the first de novo transcriptome of Malpighian tubules from non-blood-fed (NBF and blood-fed (BF mosquitoes. We identified a total of 8,232 non-redundant transcripts. The Malpighian tubules of NBF mosquitoes were characterized by the expression of transcripts associated with active transepithelial fluid secretion/diuresis (e.g., ion transporters, water channels, V-type H+-ATPase subunits, xenobiotic detoxification (e.g., cytochrome P450 monoxygenases, glutathione S-transferases, ATP-binding cassette transporters, and purine metabolism (e.g., xanthine dehydrogenase. We also detected the expression of transcripts encoding sodium calcium exchangers, G protein coupled-receptors, and septate junctional proteins not previously described in mosquito Malpighian tubules. Within 24 h after a blood meal, transcripts associated with active transepithelial fluid secretion/diuresis exhibited a general downregulation, whereas those associated with xenobiotic detoxification and purine catabolism exhibited a general upregulation, suggesting a reinvestment of the Malpighian tubules’ molecular resources from diuresis to detoxification. Physiological and biochemical assays were conducted in mosquitoes and isolated

  14. Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster.

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    Verma, Puja; Tapadia, Madhu G

    2015-08-01

    In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. Here we provide evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. We show that early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. We also show that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down regulated and activation and translocation of transcription factor Relish is also affected. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. The adult Drosophila malphigian tubules are maintained by multipotent stem cells | Center for Cancer Research

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    All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue.

  16. Drosophila polytene chromosome bands formed by gene introns.

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    Zhimulev, I F; Boldyreva, L V; Demakova, O V; Poholkova, G V; Khoroshko, V A; Zykova, T Yu; Lavrov, S A; Belyaeva, E S

    2016-01-01

    Genetic organization of bands and interbands in polytene chromosomes has long remained a puzzle for geneticists. It has been recently demonstrated that interbands typically correspond to the 5'-ends of house-keeping genes, whereas adjacent loose bands tend to be composed of coding sequences of the genes. In the present work, we made one important step further and mapped two large introns of ubiquitously active genes on the polytene chromosome map. We show that alternative promoter regions of these genes map to interbands, whereas introns and coding sequences found between those promoters correspond to loose grey bands. Thus, a gene having its long intron "sandwiched" between to alternative promoters and a common coding sequence may occupy two interbands and one band in the context of polytene chromosomes. Loose, partially decompacted bands appear to host large introns.

  17. Polytene chromosome map and inversion polymorphism in Drosophila mediopunctata

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    Galina Ananina

    2002-07-01

    Full Text Available Drosophila mediopunctata belongs to the tripunctata group, and is one of the commonest Drosophila species collected in some places in Brazil, especially in the winter. A standard map of the polytene chromosomes is presented. The breakpoints of the naturally occurring chromosomal rearrangements are marked on the map. The distribution of breaking points through the chromosomes of D. mediopunctata is apparently non-random. Chromosomes X, II and IV show inversion polymorphisms. Chromosome II is the most polymorphic, with 17 inversions, 8 inversions in the distal region and 9 in the proximal region. Chromosome X has four different gene arrangements, while chromosome IV has only two.

  18. Polytene chromosome maps and RAPD polymorphisms in Glossina austeni

    International Nuclear Information System (INIS)

    Gariou-Papalexiou, A.; Yannopoulos, G.; Zacharopoulou, A.; Robinson, A.S.

    2000-01-01

    A combined methodology of cloned RAPD (random amplification of polymorphic DNA) polymorphic bands and in situ hybridisation to polytene chromosomes is an efficient way to initiate construction of a physical and genetic map of insect disease vectors (Dimopoulos et al. 1996, Mutebi et al. 1997). The studies presented here are the first step in developing this approach in tsetse flies. This technology will be used to support tsetse sterile insect technique (SIT) programmes by providing tools with which population structure and isolation can be assessed and genetic markers that can be used to differentiate released flies from wild flies identified. An added benefit is their possible use in unravelling epidemiological complexity and problems regarding speciation (Besansky et al. 1997). Polytene chromosomes of Diptera have been shown to be excellent material for the study of chromosome structure and function as well as for an understanding of the genetics of natural populations (Lefevre 1976). They provide a means for the accurate mapping of chromosome rearrangements and the precise localisation of genes, using both rearrangement analysis and in situ hybridisation. Previous reports on the cytology of the tsetse flies (Riordan 1968, Maudlin 1970, 1979, Southern et al. 1972, Southern and Pell 1973, Davies and Southern 1976, Southern 1980) have described the basic mitotic karyotype in several Glossina species, and demonstrated the presence of well banded polytene chromosomes in pupal trichogen cells (Southern and Pell 1974, 1981, Pell and Southern 1976). Polytene chromosomes were described for G. austeni Newstead, G. morsitans morsitans Westwood, G. pallidipes Austen and G. fuscipes fuscipes Newstead, but these descriptions are difficult to work with as they are drawings of polytene chromosome elements. In this paper, the photographic chromosome maps of pupal scutellar bristles of G. austeni are presented. They show that these chromosomes can be used with much greater ease

  19. Chromatin Structure in Bands and Interbands of Polytene Chromosomes Imaged by Atomic Force Microscopy

    NARCIS (Netherlands)

    de Grauw, C.J.; de Grauw, C.J.; Avogadro, A.; van den Heuvel, D.J.; van den Heuvel, D.J.; van der Werf, Kees; Otto, Cornelis; Kraan, Yvonne M.; van Hulst, N.F.; Greve, Jan

    1998-01-01

    Polytene chromosomes from Drosophila melanogaster, observed from squash preparations, and chromosomes from Chironomus thummi thummi, investigated under physiological conditions, are imaged using an Atomic Force Microscope. Various chromatin fiber structures can be observed with high detail in fixed

  20. Rapid change of chromomeric and pairing patterns of polytene chromosome tips in D. melanogaster: migration of polytene-nonpolytene transition zone?

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    Roberts, P A

    1979-07-01

    The high variability of chromomeric patterns in near-terminal regions of polytene chromosome arms has been explored in a number of races, strains and hybrids of Drosophila melanogaster. Traditional explanations for tip differences between strains (differential compaction of chromatin, somatic or germinal deletion) are examined and, in the light of the reported observations, rejected. The range of polytene tip variability and rates of change in wild races are greater than has been supposed: strains formerly considered to be terminally deleted appear to gain terminal bands; others, formerly considered normal, appear to have lost them. Strains with high cell-to-cell tip variability are also described. Cell-to-cell variations, as well as much of the observed rapid changes in tip appearance, are probably due to heritable differences in the location of an abrupt transition zone between polytene and nonpolytene chromatin. A quantitative relationship between the amount of certain subterminal bands present and the frequency of tip association of nonhomologous chromosomes is shown and its possible significance for chromosome is shown and its possible for chromosome pairing discussed.

  1. Cytological and cytochemical characterization of the polytene chromosomes of Chironomus sancticaroli (Diptera: chironomidae)

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    Freitas, F. de; Leoncini, O.

    1985-01-01

    The chromosome complement of a Brazilian Chironomus Species, C. Sancticaroli, was analyzed cytochemically. The polytene Chromosomes were identified and characterized and the nucleolus organizer regions (NORs) were located by a technique of in situ hybridization and immunofluorescence. Constitutive heterochromatin and its distribution in relation to the NORs were studied. (Author) [pt

  2. Cytological and cytochemical characterization of the polytene chromosomes of Chironomus sancticaroli (Diptera: chironomidae)

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    Freitas, F. de; Leoncini, O.; Floeter-Winter, L.M.

    1985-03-01

    The chromosome complement of a Brazilian Chironomus Species, C. Sancticaroli, was analyzed cytochemically. The polytene Chromosomes were identified and characterized and the nucleolus organizer regions (NORs) were located by a technique of in situ hybridization and immunofluorescence. Constitutive heterochromatin and its distribution in relation to the NORs were studied.

  3. [Late-replicating regions in salivary gland polytene chromosomes of Drosophila melanogaster].

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    Kolesnikov, T D; Andreenkova, N G; Beliaeva, E S; Goncharov, F P; Zykova, T Iu; Boldyreva, L V; Pokholkova, g V; Zhimulev, I F

    2013-01-01

    About 240 specific regions that are replicated at the very end of the S-phase have been identified in D. melanogaster polytene chromosomes. These regions have a repressive chromatine state, low gene density, long intergenic distances and are enriched in tissue specific genes. In polytene chromosomes, about a quarter of these regions have no enough time to complete replication. As a result, underreplication zones represented by fewer DNA copy number, appear. We studied 60 chromosome regions that demonstrated the most pronounced under-replication. By comparing the location of these regions on a molecular map with syntenic blocks found earlier for Drosophila species by von Grotthuss et al., 2010, we have shown that across the genus Drosophila, these regions tend to have conserved gene order. This forces us to assume the existence of evolutionary mechanisms aimed at maintaining the integrity of these regions.

  4. /sup 3/H-UTP incorporation in polytene chromosomes of Chironomus permeabilized cells

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    Diez, J L; de la Torre, C [Consejo Superior de Investigacionis Cientificas, Madrid (Spain). Inst. Biologia Celular

    1979-01-01

    A method is described that allows the cytologic detection of the transcriptional activity of endogenous RNA polymerase in fixed Chironomus polytene chromosomes. /sup 3/H-UTP is preferentially incorporated onto nucleoli and Balbiani rings where transcription is specially high. The former incorporation was sensitive to actinomycin D while incorporation on Balbiani rings was sensitive to ..cap alpha..-amanitin. The label pattern and frequency resemble those detected by /sup 3/H-uridine incorporation in the living state.

  5. Binding of 3H-actinomycin D with polytene chromosomes of Drosophila melanogaster

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    Lakhotia, S.C.

    1976-01-01

    Binding of 3 H-AMD (actinomycin D) in different regions of polytene nuclei of late third instar larvae of Drosophila melanogaster has been examined by EM autoradiography. It is observed that the binding capacity of 3 H-AMD is not related to the transcribing activity of a given region of nuclei, but it may be related to the DNA content. (M.G.B.)

  6. Border Structure of Intercalary Heterochromatin Bands of Drosophila melanogaster Polytene Chromosomes.

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    Khoroshko, V A; Zykova, T Yu; Popova, O O; Zhimulev, I F

    2018-03-01

    The precise genomic localization of the borders of 62 intercalary heterochromatin bands in Drosophila polytene chromosomes was determined. A new type of bands containing chromatin of different states was identified. This type is a combination of the gray band and the intercalary heterochromatin band, creating a genetic structure that with a light microscope is identified as a continuous band. The border structure of such bands includes the coding regions of genes with ubiquitous activity.

  7. Identical functional organization of nonpolytene and polytene chromosomes in Drosophila melanogaster.

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    Tatyana Yu Vatolina

    Full Text Available Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.

  8. Binding of doa kinase to specific loci in polytene chromosomes of Drosophila melanogaster

    Czech Academy of Sciences Publication Activity Database

    Kováčiková, M.; Raška, Ivan; Mateášik, A.; Chase, B. A.; Farkaš, R.

    2006-01-01

    Roč. 40, č. 1 (2006), s. 21-27 ISSN 1210-0668 R&D Projects: GA MŠk(CZ) LC535; GA ČR(CZ) GA304/02/0342 Grant - others:VEGA(SK) 2/3025/23; SAV(SK) APVT-51-027402; NSF(US) IBN-97-24006; US-SK(SK) 13/029/01 Institutional research plan: CEZ:AV0Z50110509 Keywords : specific protein kinases * DOA * polytene chromosomes Subject RIV: EB - Genetics ; Molecular Biology

  9. Transmission electron microscopic method for gene mapping on polytene chromosomes by in situ hybridization

    OpenAIRE

    Wu, Madeline; Davidson, Norman

    1981-01-01

    A transmission electron microscope method for gene mapping by in situ hybridization to Drosophila polytene chromosomes has been developed. As electron-opaque labels, we use colloidal gold spheres having a diameter of 25 nm. The spheres are coated with a layer of protein to which Escherichia coli single-stranded DNA is photochemically crosslinked. Poly(dT) tails are added to the 3' OH ends of these DNA strands, and poly(dA) tails are added to the 3' OH ends of a fragmented cloned Drosophila DN...

  10. Different structure of polytene chromosome of phaseolus coccineus suspensors during early embryogenesis

    International Nuclear Information System (INIS)

    Tagliasacchi, A.M.; Forino, L.M.C.; Cionini, P.G.; Cavallini, A.; Durante, M.; Cremonini, R.; Avanzi, S.

    1984-01-01

    Different regions of polytene chromosomes pair VI have been characterized by: 1. morphological observations, 2. incorporation of 3 H-thymidine and 3 H-uridine, 3. cytophotometry of DNA and associated proteins, 4. hybridization with satellite DNA and highly repeated DNA sequences. The collected data indicate that DNA and RNA puffs are organized by heterochromatic segments. DNA puffs show often a clustered pattern of labeling by 3 H-thymidine and RNA puffs are always labeled by 3 H-urindine. Each heterochromatic segment is characterized by a definite ratio between DNA and associated fastgreen stainable proteins. Satellite DNA binds mostly to heterochromatic blocks at centromers, highly repeated DNA sequences bind, with approximately the same frequency, to centromeric heterochromatin and to the main intercalary heterochromatic band. The telomeric portions of euchromatin seem to be also enriched in highly repeated DNA sequences. The results indicate that heterochromatic chromosome segments might be sites of intense localized DNA replication. The same chromosome regions are also engaged in an active transcription process. The response to hybridization suggests that heterochromatic blocks of chromosome pair VI are heterogeneous in nucleotide sequences. The present studies also indicate that DNA and RNA puffs organized by different chromosome sites are specific of particular steps of embryo differentiation. The observed metabolic aspects of the suspensior's polytene chromosomes are discussed in relation to the synthesis of growth regulators which is known to occur in the suspensor. (Author)

  11. Neurohormones implicated in the control of Malpighian tubule secretion in plant sucking Heteropterans: The stink bugs Acrosternum hilare and Nezara viridula

    Science.gov (United States)

    Plant sucking heteropteran bugs feed regularly on small amounts of K+rich plant material, in contrast to their hematophagous relatives which imbibe large volumes of Na+-rich blood. It was anticipated that this would be reflected in the endocrine control of MT secretion. To explore this, neuroendocri...

  12. A Blood Meal Enhances Innexin mRNA Expression in the Midgut, Malpighian Tubules, and Ovaries of the Yellow Fever Mosquito Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Travis L. Calkins

    2017-11-01

    Full Text Available Mosquitoes are vectors of pathogens that cause diseases of medical and veterinary importance. Female mosquitoes transmit these pathogens while taking a blood meal, which most species require to produce eggs. The period after a blood meal is a time of extreme physiological change that requires rapid coordination of specific tissues. Gap junctions (GJ are intercellular channels that aid in the coordination of cells within tissues via the direct transfer of certain small molecules and ions between cells. Evolutionarily distinct groups of proteins form the gap junctions of vertebrate and invertebrate animals (connexins and innexins, respectively. Aedes aegypti mosquitoes possess six genes encoding innexins: inx1, inx2, inx3, inx4, inx7, and inx8. The goal of this study was to identify potential roles of innexins in the physiology of mosquitoes after a blood meal by using qPCR to quantify their mRNA expression in adult females at 3 h and 24 h post-blood meal (PBM relative to non-blood-fed controls. We found that at 24 h PBM, expression levels of inx2, inx3, and inx4 mRNAs increased; inx2 was the most highly upregulated innexin in key tissues associated with blood-meal digestion and egg production (i.e., the midgut and ovaries, respectively. However, knocking down inx2 mRNA levels by over 75% via RNA interference had no significant effect on fecundity. Altogether, our results suggest that a blood meal influences the molecular expression of innexins in mosquitoes, but their specific physiological roles remain to be elucidated.

  13. [Advances in understanding Drosophila salivary gland polytene chromosome and its applications in genetics teaching].

    Science.gov (United States)

    Li, Gang; Chen, Fan-guo

    2015-06-01

    Drosophila salivary gland polytene chromosome, one of the three classical chromosomes with remarkable characteristics, has been used as an outstanding model for a variety of genetic studies since 1934. The greatest contribution of this model to genetics has been providing extraordinary angle of view in studying interphase chromosome structure and gene expression regulation. Additionally, it has been extensively used to understand some special genetic phenomena, such as dosage compensation and position-effect variegation. In this paper, we briefly review the advances in the study of Drosophila salivary gland chromosome, and try to systematically and effectively introduce this model system into genetics teaching practice in order to steer and inspire students' interest in genetics.

  14. Polytene chromosome analysis in relation to genetic sex separation in the Mediterranean fruit fly, Ceratitis capitata (Wied.)

    International Nuclear Information System (INIS)

    Kerremans, P.; Busch-Petersen, E.

    1990-01-01

    The development of stable genetic sexing strains in the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), is hampered by the presence of low levels of male recombination. Such recombination may be reduced by minimizing the distance between the translocation breakpoint and the translocated 'sexing' allele. Cytogenetic analysis of mitotic/meiotic and polytene chromosomes could provide information on the selection of such potentially stable genetic sexing strains. Translocation breakpoints in two genetic sexing strains in the medfly, based on a white female/brown male pupal colour dimorphism, have been determined. Preliminary results are described and the advantages and limitations of polytene chromosome analysis for the isolation of stable genetic sexing strains of the medfly are discussed. (author). 31 refs

  15. Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps.

    Science.gov (United States)

    Schaeffer, Stephen W; Bhutkar, Arjun; McAllister, Bryant F; Matsuda, Muneo; Matzkin, Luciano M; O'Grady, Patrick M; Rohde, Claudia; Valente, Vera L S; Aguadé, Montserrat; Anderson, Wyatt W; Edwards, Kevin; Garcia, Ana C L; Goodman, Josh; Hartigan, James; Kataoka, Eiko; Lapoint, Richard T; Lozovsky, Elena R; Machado, Carlos A; Noor, Mohamed A F; Papaceit, Montserrat; Reed, Laura K; Richards, Stephen; Rieger, Tania T; Russo, Susan M; Sato, Hajime; Segarra, Carmen; Smith, Douglas R; Smith, Temple F; Strelets, Victor; Tobari, Yoshiko N; Tomimura, Yoshihiko; Wasserman, Marvin; Watts, Thomas; Wilson, Robert; Yoshida, Kiyohito; Markow, Therese A; Gelbart, William M; Kaufman, Thomas C

    2008-07-01

    The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.

  16. Homology of polytene elements between Drosophila and Zaprionus determined by in situ hybridization in Zaprionus indianus.

    Science.gov (United States)

    Campos, S R C; Rieger, T T; Santos, J F

    2007-05-09

    The drosophilid Zaprionus indianus due to its economical importance as an insect pest in Brazil deserves more investigation into its genetics. Its mitotic karyotype and a line-drawing map of its polytene chromosomes are already available. This paper presents a photomap of Z. indianus polytene chromosomes, which was used as the reference map for identification of sections marked by in situ hybridization with gene probes. Hybridization signals for Hsp70 and Hsr-omega were detected, respectively, in sections 34B and 32C of chromosome V of Z. indianus, which indicates its homology to the chromosomal arm 3R of Drosophila melanogaster and, therefore, to Muller's element E. The main signal for Hsp83 gene probe hybridization was in section 17C of Z. indianus chromosome III, suggesting its homology to arm 3L of D. melanogaster and to element D of Muller. The Ubi probe hybridized in sections 10C of chromosome II and 17A of chromosome III. Probably the 17A is the polyubiquitin locus, with homology to arm 3L of D. melanogaster and to the mullerian D element, as suggested also by Hsp83 gene location. The Br-C gene was mapped in section 1D, near the tip of the X chromosome, indicating its homology to the X chromosome of D. melanogaster and to mullerian element A. The Dpp gene probe hybridized mainly in the section 32A of chromosome V and, at lower frequencies to other sections, although no signal was observed as expected in the correspondent mullerian B element. This result led to the suggestion of a rearrangement including the Dpp locus in Z. indianus, the secondary signals possibly pointing to related genes of the TGF-beta family. In conclusion, the results indicate that chromosomes X, III, V of Z. indianus are respectively correspondents to elements A, D, and E of Muller. At least chromosome V of Z. indianus seems to share synteny with the 3R arm of D. melanogaster, as indicated by the relative positions of Hsp70 and Hsr-omega, although the Dpp gene indicates a disruption of

  17. Genotoxic effect of copper on salivary gland polytene chromosomes of Chironomus riparius Meigen 1804 (Diptera, Chironomidae)

    Energy Technology Data Exchange (ETDEWEB)

    Michailova, P. [Institute of Zoology, Bulgarian Academy of Sciences, 1 Tzar Osvoboditel boul., Sofia 1000 (Bulgaria)]. E-mail: michailova@zoology.bas.bg; Petrova, N. [Institute of Zoology, Russian Academy of Sciences, St. Petersburg, 199034, Universit. nab. 1, Russia (Russian Federation); Ilkova, J. [Institute of Zoology, Bulgarian Academy of Sciences, 1 Tzar Osvoboditel boul., Sofia 1000 (Bulgaria); Bovero, S. [Department of Animal Biology, University of Turin, via Albertina 13, Turin (Italy); Brunetti, S. [Department of Animal Biology, University of Turin, via Albertina 13, Turin (Italy); White, K. [School of Biological Sciences, University of Manchester, Manchester M13 9PT (United Kingdom); Sella, G. [Department of Animal Biology, University of Turin, via Albertina 13, Turin (Italy)

    2006-11-15

    The genotoxic action of copper (Cu) on the polytene chromosomes of Chironomus riparius was investigated by analysing structural and functional chromosome aberrations of fourth instars larvae hatched from eggs subject to acute (48 h) exposure with three environmentally relevant concentrations of aqueous Cu (0.005, 0.01, 0.05 mg/l). A dose dependent relationship was observed between Cu concentration and frequency of chromosomal aberrations. A significantly higher frequency of functional alterations, specifically decondensed centromeres and telomeres, and reduction in activity of Balbiani rings, was observed in treated material compared to control. A comparison of breakpoints resulting from treatment with chromium and lead from earlier studies with those Cu-induced identified a series of chromosomal weak points particularly vulnerable to trace metals. We also show that the appearance of structural and functional chromosome aberrations are more sensitive indicators of acute Cu toxicity in chironomid larvae than changes in external morphology. - Acute exposure of Chironomus eggs to copper resulted in changes in chromosome structure and function.

  18. Genotoxic effect of copper on salivary gland polytene chromosomes of Chironomus riparius Meigen 1804 (Diptera, Chironomidae)

    International Nuclear Information System (INIS)

    Michailova, P.; Petrova, N.; Ilkova, J.; Bovero, S.; Brunetti, S.; White, K.; Sella, G.

    2006-01-01

    The genotoxic action of copper (Cu) on the polytene chromosomes of Chironomus riparius was investigated by analysing structural and functional chromosome aberrations of fourth instars larvae hatched from eggs subject to acute (48 h) exposure with three environmentally relevant concentrations of aqueous Cu (0.005, 0.01, 0.05 mg/l). A dose dependent relationship was observed between Cu concentration and frequency of chromosomal aberrations. A significantly higher frequency of functional alterations, specifically decondensed centromeres and telomeres, and reduction in activity of Balbiani rings, was observed in treated material compared to control. A comparison of breakpoints resulting from treatment with chromium and lead from earlier studies with those Cu-induced identified a series of chromosomal weak points particularly vulnerable to trace metals. We also show that the appearance of structural and functional chromosome aberrations are more sensitive indicators of acute Cu toxicity in chironomid larvae than changes in external morphology. - Acute exposure of Chironomus eggs to copper resulted in changes in chromosome structure and function

  19. Distribution of X-ray induced chromosome rearrangement breaks along the polytene chromosomes of Anopheles messeae

    International Nuclear Information System (INIS)

    Pleshkova, G.N.

    1983-01-01

    Distribution of chromosomal aberrations localization along polytene chromosomes (aoutosomes) of salivary glands of malarial mosquito. Anopheles messeae is presented. Induced aberrations in F 1 posterity from X-ray irradiated fecundated females are studied. Poipts of breaks of inversions and trapslocations are localized separately. There are no considerable dif-- ferences in the distribution character of two types of aberrations. Over the length of autosomes the breaks are more frequent in distal halves, their frequency in proximal parts anally in near centromeric regions of chromosomes is reduced. Concentration of breaks in certain ''hot points'' of the chromosomes is pointed out. Comparison of distribution of actual and expected frequencies of break points according to chi 2 criterion revealed highly fiducial discrepancies, testifying to uneven participation of different regions of chromosomes in aberration formation. Similarities and differences of the data obtained from analogous ones, demonstrated in Drosophila, as well as possible reasons for the distribution unevennes are discussed. On the basis of analysis of intrinsic and literature data a supposition is made that the ''hot points'' (break concentrations) can be considered as localizaion markers of intercalary heterochromatin

  20. Polytene Chromosomes - A Portrait of Functional Organization of the Drosophila Genome.

    Science.gov (United States)

    Zykova, Tatyana Yu; Levitsky, Victor G; Belyaeva, Elena S; Zhimulev, Igor F

    2018-04-01

    This mini-review is devoted to the problem genetic meaning of main polytene chromosome structures - bands and interbands. Generally, densely packed chromatin forms black bands, moderately condensed regions form grey loose bands, whereas decondensed regions of the genome appear as interbands. Recent progress in the annotation of the Drosophila genome and epigenome has made it possible to compare the banding pattern and the structural organization of genes, as well as their activity. This was greatly aided by our ability to establish the borders of bands and interbands on the physical map, which allowed to perform comprehensive side-by-side comparisons of cytology, genetic and epigenetic maps and to uncover the association between the morphological structures and the functional domains of the genome. These studies largely conclude that interbands 5'-ends of housekeeping genes that are active across all cell types. Interbands are enriched with proteins involved in transcription and nucleosome remodeling, as well as with active histone modifications. Notably, most of the replication origins map to interband regions. As for grey loose bands adjacent to interbands, they typically host the bodies of house-keeping genes. Thus, the bipartite structure composed of an interband and an adjacent grey band functions as a standalone genetic unit. Finally, black bands harbor tissue-specific genes with narrow temporal and tissue expression profiles. Thus, the uniform and permanent activity of interbands combined with the inactivity of genes in bands forms the basis of the universal banding pattern observed in various Drosophila tissues.

  1. Osmosis in Cortical Collecting Tubules

    Science.gov (United States)

    Schafer, James A.; Troutman, Susan L.; Andreoli, Thomas E.

    1974-01-01

    The present experiments were designed to evaluate the effects of varying the osmolality of luminal solutions on the antidiuretic hormone (ADH)-independent water and solute permeability properties of isolated rabbit cortical collecting tubules. In the absence of ADH, the osmotic water permeability coefficient (cm s–1) Pfl→b, computed from volume flows from hypotonic lumen to isotonic bath, was 20 ± 4 x 10–4 (SEM); the value of Pfb→l in the absence of ADH, computed from volume flows from isotonic bath to hypertonic lumen, was 88 ± 15 x 10–4 cm s–1. We also measured apparent urea permeability coefficients (cm s–1) from 14C-urea fluxes from lumen to bath (P DDurea l→b) and from bath to lumen (P DDurea b→l). For hypotonic luminal solutions and isotonic bathing solutions, P DDurea l→b was 0.045 ± 0.004 x 10–4 and was unaffected by ADH. The ADH-independent values of P DDurea l→b and P urea b→l were, respectively, 0.216 ± 0.022 x 10–4 cm s–1 and 0.033 ± 0.002 x 10–4 cm s–1 for isotonic bathing solutions and luminal solutions made hypertonic with urea, i.e., there was an absolute increase in urea permeability and asymmetry of urea fluxes. Significantly, P DDurea l→b did not rise when luminal hypertonicity was produced by sucrose; and, bathing fluid hypertonicity did not alter tubular permeability to water or to urea. We interpret these data to indicate that luminal hypertonicity increased the leakiness of tight junctions to water and urea but not sucrose. Since the value of Pfb→l in the absence of ADH, when tight junctions were open to urea, was approximately half of the value of Pfl→b in the presence of ADH, when tight junctions were closed to urea, we conclude that tight junctions are negligible paracellular shunts for lumen to bath osmosis with ADH. These findings, together with those in the preceding paper, are discussed in terms of a solubility-diffusion model for water permeation in which ADH increases water solubility in

  2. Critical electrolyte concentration of chromatin in polytene chromosomes of Trichosia pubescens (Diptera, Sciaridae

    Directory of Open Access Journals (Sweden)

    Ana Lúcia P. Monteiro

    1998-06-01

    Full Text Available The binding of toluidine blue molecules to nucleic acid phosphates under conditions of competition with Mg2+ ions was studied in Trichosia pubescens polytene chromosome regions differing in composition and organization (RNA-rich areas and puffs, DNA puffs, heterochromatin. The aim was to find variability in the Mg2+ concentration at which metachromasy was completely prevented (= critical electrolyte concentration; CEC and that could reflect differences at the level of nucleoprotein complexes in particular chromosome regions and developmental phases. Although high CEC values have been found in tightly packed chromatin such as that of heterochromatin zones, the CEC values for the other regions have proven to be affected not only by availability and proximity of DNA phosphates but also by RNA richness and other factors. Among these, changes in DNA geometry and packing state have been assumed for explaining increased values after RNA removal in DNA puffs and RNA-rich zones. Based on CEC values it has been suggested that alterations at the level of nucleoprotein complexes may occur in puffs before they are morphologically detectable. CEC results for polytene chromosomes were thus revealed to vary considerably with slight variations in nucleoprotein composition and organization. However, since a complex and apparently contradictory rationale has to be used for explaining part of the results, CEC is not recommended as a useful tool for extensive and comparative studies of this particular model.A ligação de moléculas de azul de toluidina a grupamentos fosfatos de ácido nucléico em condições de competição com íons Mg2+ foi estudada em regiões de cromossomos politênicos de Trichosia pubescens que diferem em composição e organização (áreas ricas em RNA, pufes de RNA e DNA, heterocromatina. O objetivo foi encontrar variabilidade na concentração de Mg2+ na qual a metacromasia fosse completamente abolida (= concentração crítica de eletr

  3. Dentine tubule infection and endodontic therapy implications.

    Science.gov (United States)

    Oguntebi, B R

    1994-07-01

    A critical review of the literature suggests that the microenvironment of dentinal tubules appears to favour the selection of relatively few bacterial types irrespective of the aetiology of the infection process; coronal dental caries or pulpar necrosis. These bacteria may constitute an important reservoir from which root canal infection and reinfection may occur following pulp necrosis or during and after endodontic treatment. Previous studies of this microflora have utilized microbiological culture techniques which need to be supplemented by those that allow in situ demonstration as well as identification of the bacteria. Newer treatment strategies that are designed to eliminate this microflora must include agents that can penetrate the dentinal tubules and destroy these microorganisms, since they are located in an area beyond the host defence mechanisms where they cannot be reached by systemically administered antimicrobial agents.

  4. A Simple Key for Identifying the Sibling Species of the Malaria Vector Anopheles gambiae (Giles Complex by Polytene Chromosome Cytogenetics

    Directory of Open Access Journals (Sweden)

    Music Temitope OBEMBE

    2018-03-01

    Full Text Available It has been established that Anopheles gambiae complex sibling species are the major Plasmodium malaria vectors in Africa; however, not all the sibling species transmit the infection. Easier molecular methods, PCR-based assays, have been developed to distinguish the several members of the A. gambiae complex. However, malaria vector research in less developed countries, particularly sub-Saharan Africa, is being hampered by the lack of PCR facilities in laboratories and the cost of carrying out the assay within lack of funding. Hence, the present study was designed to develop a simple identification key, based on an affordable method of polytene chromosome cytotaxonomy, for identifying the major P. falciparum vectors. The Identification Key was successfully used to identify two members of the A. gambiae complex, A. gambiae sensu stricto and A. arabiensis, which are the most potent malaria vectors in Africa; even so, it could not be used to establish the infective and the refractory strains.

  5. Polytene chromosomes of monogenic and amphogenic Chrysomya species (Calliphoridae, Diptera): analysis of banding patterns and in situ hybridization with Drosophila sex determining gene sequences.

    Science.gov (United States)

    Puchalla, S

    1994-03-01

    Standard maps for the five banded polytene chromosomes found in trichogen cell nuclei of the monogenic blowfly Chrysomya rufifacies and the amphogenic Chrysomya pinguis are presented. The chromosomes are highly homologous in the two species; differences in banding patterns are predominantly caused by one pericentric and ten paracentric inversions. In chromosome 5 of the amphogenic Chrysomya phaonis, also analysed in this paper, an additional paracentric inversion was observed. The distribution of species specific inversions indicates that the monogenic C. rufifacies is phylogenetically older than the amphogenic species. The maternal sex realizer locus F'/f on polytene chromosome 5 of C. rufifacies is not associated with a structural heterozygosity. Chromosome pair 6 of C. rufifacies and the sex chromosome pair of C. pinguis are under-replicated in polytene nuclei; they consist of irregular chromatin granules, frequently associated with nucleolus material. Evolution of heteromorphic sex chromosomes in Chrysomya is probably correlated with heterochromatin accumulation. A search for sex determining genes in Chrysomya was initiated using sex determining sequences from Drosophila melanogaster for in situ hybridization. The polytene band 41A1 on chromosome 5 of monogenic and amphogenic Chrysomya species contains sequences homologous to the maternal sex determining gene daughterless (da). Homology to the zygotic gene Sex-lethal (Sxl) of Drosophila is detected in band 39A1 on chromosome 5 of C. rufifacies. The findings reported here are the first evidence for a possible homology between the da gene of Drosophila and the maternal sex realizer F' of C. rufifacies. An hypothesis for the evolution of the maternal effect sex determination of C. rufifacies is proposed.

  6. Functional and structural rearrangements of salivary gland polytene chromosomes of Chironomus riparius Mg. (Diptera,Chironomidae) in response to freshly neutralized aluminium

    Energy Technology Data Exchange (ETDEWEB)

    Michailova, P.; Ilkova, J.; White, K.N

    2003-05-01

    Aluminium impacts on aquatic biota can be detected using polytene chromosomes as bio-markers - Although recent work has shown that environmentally relevant concentrations of freshly neutralized aluminium (AI) are bioavailable and toxic to freshwater invertebrates, the genotoxicity of Al has not been examined. Here we show that freshly neutralized Al affects structure and function of the salivary gland polytene chromosomes of the ubiquitous chironomid larva Chironomus riparius over three generations. Exposure to 500 {mu}g l{sup -1} added Al for 24-25 days resulted in a significantly higher frequency of numerous somatic aberrations, while no structural aberrations were found in F1 controls and few in the second and third generation. Aberrations also included deletions of sections of chromosome G of C. riparius larvae as well as deletions of one or more Balbiani rings. Changes in functional activity included decreased activity of the Balbiani rings (BR), and an increase in the number of decondensed centromeres. The activity of the nucleolar organizer (NOR) significantly decreased in F1 chironomids exposed to Al, while in the F2 and F3 generations the NOR showed normal (high) activity. First generation chironomids were generally more susceptible to Al although no clear evidence of tolerance was apparent over three generations. The possible use of alterations in chironomid polytene chromosomes as biomarkers of trace metal pollution is discussed.

  7. Functional and structural rearrangements of salivary gland polytene chromosomes of Chironomus riparius Mg. (Diptera,Chironomidae) in response to freshly neutralized aluminium

    International Nuclear Information System (INIS)

    Michailova, P.; Ilkova, J.; White, K.N.

    2003-01-01

    Aluminium impacts on aquatic biota can be detected using polytene chromosomes as bio-markers - Although recent work has shown that environmentally relevant concentrations of freshly neutralized aluminium (AI) are bioavailable and toxic to freshwater invertebrates, the genotoxicity of Al has not been examined. Here we show that freshly neutralized Al affects structure and function of the salivary gland polytene chromosomes of the ubiquitous chironomid larva Chironomus riparius over three generations. Exposure to 500 μg l -1 added Al for 24-25 days resulted in a significantly higher frequency of numerous somatic aberrations, while no structural aberrations were found in F1 controls and few in the second and third generation. Aberrations also included deletions of sections of chromosome G of C. riparius larvae as well as deletions of one or more Balbiani rings. Changes in functional activity included decreased activity of the Balbiani rings (BR), and an increase in the number of decondensed centromeres. The activity of the nucleolar organizer (NOR) significantly decreased in F1 chironomids exposed to Al, while in the F2 and F3 generations the NOR showed normal (high) activity. First generation chironomids were generally more susceptible to Al although no clear evidence of tolerance was apparent over three generations. The possible use of alterations in chironomid polytene chromosomes as biomarkers of trace metal pollution is discussed

  8. Electron microscope mapping of the pericentric and intercalary heterochromatic regions of the polytene chromosomes of the mutant Suppressor of underreplication in Drosophila melanogaster.

    Science.gov (United States)

    Semeshin, F; Belyaeva, S; Zhimulev, F

    2001-12-01

    Breaks and ectopic contacts in the heterochromatic regions of Drosophila melanogaster polytene chromosomes are the manifestations of the cytological effects of DNA underreplication. Their appearance makes these regions difficult to map. The Su(UR)ES gene, which controls the phenomenon, has been described recently. Mutation of this locus gives rise to new blocks of material in the pericentric heterochromatic regions and causes the disappearance of breaks and ectopic contacts in the intercalary heterochromatic regions, thereby making the banding pattern distinct and providing better opportunities for mapping of the heterochromatic regions in polytene chromosomes. Here, we present the results of an electron microscope study of the heterochromatic regions. In the wild-type salivary glands, the pericentric regions correspond to the beta-heterochromatin and do not show the banding pattern. The most conspicuous cytological effect of the Su(UR)ES mutation is the formation of a large banded chromosome fragment comprising at least 25 bands at the site where the 3L and 3R proximal arms connect. In the other pericentric regions, 20CF, 40BF and 41BC, 15, 12 and 9 new bands were revealed, respectively. A large block of densely packed material appears in the most proximal part of the fourth chromosome. An electron microscope analysis of 26 polytene chromosome regions showing the characteristic features of intercalary heterochromatin was also performed. Suppression of DNA underreplication in the mutant transforms the bands with weak spots into large single bands.

  9. Effect of dental materials on gluconeogenesis in rat kidney tubules

    NARCIS (Netherlands)

    Reichl, F.X.; Durner, J.; Mückter, H.; Elsenhans, B.; Forth, W.; Kunzelmann, K.H.; Hickel, R.; Spahl, W.; Hume, W.R.; Moes, G.W.

    1999-01-01

    The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) as well as mercuric chloride (HgCl2) and methylmercury chloride (MeHgCl) on gluconeogenesis was investigated in isolated rat kidney tubules. From starved rats kidney tubules were

  10. Magnetic properties of permalloy-coated organic tubules

    Science.gov (United States)

    Krebs, J. J.; Rubinstein, M.; Lubitz, P.; Harford, M. Z.; Baral, S.; Shashidar, R.; Ho, Y. S.; Chow, G. M.; Qadri, S.

    1991-11-01

    An initial investigation is presented of the ferromagnetic properties of a novel type of magnetic composite, viz., permalloy-coated submicron diameter hollow cylinders or tubules. The tubules form spontaneously from an organic material, a diacetylenic phosopholipid, and were used as templates on which the ferromagnetic material was deposited by electroless deposition. The permalloy-coated tubules were dispersed in an epoxy matrix to measure the magnetization and ferromagnetic resonance (FMR) properties of individual tubules. The nature of the magnetic anisotropy and the FMR spectra observed confirmed that the tubules are well aligned by a magnetic field during the epoxy curing. The FMR spectra are interpreted in terms of a powder pattern distribution of thin-film spectra consistent with the large diameter-to-thickness ratio.

  11. Shape transitions in anisotropic multicomponent lipid tubules

    Directory of Open Access Journals (Sweden)

    Tim eAtherton

    2016-05-01

    Full Text Available Abstract Ternary mixtures of saturated and unsaturated lipids together with cholesterol can be induced to phase separate by photo-peroxidation into lipid-ordered Lo and lipid-disordered Ld domains. Because these have different mechanical properties, the phase separation is accompanied by dramatic changes in morphology. This work considers a tubule composed of Ld phase with Lo phase inclusions that possess greater rigidity; this system has been shown experimentally by Yuan and coworkers to spontaneously adopt either banded or disc configurations following phase separation. The static behaviour of inter-domain interactions is analyzed in each of these geometries by solving the linearized shape equations. These calculations suggest a possible mechanism by which the two structures form.

  12. Dentinal tubules revealed with X-ray tensor tomography.

    Science.gov (United States)

    Jud, Christoph; Schaff, Florian; Zanette, Irene; Wolf, Johannes; Fehringer, Andreas; Pfeiffer, Franz

    2016-09-01

    Dentin is a mineralized material making up most of the tooth bulk. A system of microtubules, so called dentinal tubules, transverses it radially from the pulp chamber to the outside. This highly oriented structure leads to anisotropic mechanical properties directly connected to the tubules orientation and density: the ultimate tensile strength as well as the fracture toughness and the shear strength are largest perpendicular to dentinal tubules. Consequently, the fatigue strength depends on the direction of dentinal tubules, too. However, none of the existing techniques used to investigate teeth provide access to orientation and density of dentinal tubules for an entire specimen in a non-destructive way. In this paper, we measure a third molar human tooth both with conventional micro-CT and X-ray tensor tomography (XTT). While the achievable resolution in micro-CT is too low to directly resolve the dentinal tubules, we provide strong evidence that the direction and density of dentinal tubules can be indirectly measured by XTT, which exploits small-angle X-ray scattering to retrieve a 3D map of scattering tensors. We show that the mean directions of scattering structures correlate to the orientation of dentinal tubules and that the mean effective scattering strength provides an estimation of the relative density of dentinal tubules. Thus, this method could be applied to investigate the connection between tubule orientation and fatigue or tensile properties of teeth for a full sample without cutting one, non-representative peace of tooth out of the full sample. Copyright © 2016 The Academy of Dental Materials. All rights reserved.

  13. Solo and keratin filaments regulate epithelial tubule morphology.

    Science.gov (United States)

    Nishimura, Ryosuke; Kato, Kagayaki; Fujiwara, Sachiko; Ohashi, Kazumasa; Mizuno, Kensaku

    2018-04-28

    Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.

  14. Effects of vasopressin on the isolated perfused human collecting tubule.

    Science.gov (United States)

    Yanagawa, N; Trizna, W; Bar-Khayim, Y; Fine, L G

    1981-05-01

    Cortical collecting tubules (CCT) were dissected from the surviving normal tissue of human kidneys removed at operation for either carcinoma or calculus. These CCT's were perfused in vitro shortly after the nephrectomy was performed. Transtubular potential differences in different tubules varied from +3.2 to -2.0 mV and were reduced towards zero by lowering the temperature or by adding ouabain to the bath. In the absence of vasopressin, tubules were essentially impermeable to water with extremely low net water fluxes even in the presence of a transtubular osmotic gradient. Addition of vasopressin to the bath caused the transtubular osmotic water permeability coefficient to increase to values of 125, 175, and 155 X 10(-4) cm/sec in three tubules thus studied. These results demonstrate close similarities between the human CCT and the more extensively studied rabbit CCT.

  15. Natural history of seminiferous tubule degeneration in Klinefelter syndrome

    DEFF Research Database (Denmark)

    Aksglaede, Lise; Wikström, Anne M; Rajpert-De Meyts, Ewa

    2006-01-01

    Klinefelter syndrome (47,XXY) is characterized by small, firm testis, gynaecomastia, azoospermia and hypergonadotropic hypogonadism. Degeneration of the seminiferous tubules in 47,XXY males is a well-described phenomenon. It begins in the fetus, progresses through infancy and accelerates dramatic......Klinefelter syndrome (47,XXY) is characterized by small, firm testis, gynaecomastia, azoospermia and hypergonadotropic hypogonadism. Degeneration of the seminiferous tubules in 47,XXY males is a well-described phenomenon. It begins in the fetus, progresses through infancy and accelerates...

  16. Sorting Tubules Regulate Blood-Brain Barrier Transcytosis

    Directory of Open Access Journals (Sweden)

    Roberto Villaseñor

    2017-12-01

    Full Text Available Transcytosis across the blood-brain barrier (BBB regulates key processes of the brain, but the intracellular sorting mechanisms that determine successful receptor-mediated transcytosis in brain endothelial cells (BECs remain unidentified. Here, we used Transferrin receptor-based Brain Shuttle constructs to investigate intracellular transport in BECs, and we uncovered a pathway for the regulation of receptor-mediated transcytosis. By combining live-cell imaging and mathematical modeling in vitro with super-resolution microscopy of the BBB, we show that intracellular tubules promote transcytosis across the BBB. A monovalent construct (sFab sorted for transcytosis was localized to intracellular tubules, whereas a bivalent construct (dFab sorted for degradation formed clusters with impaired transport along tubules. Manipulating tubule biogenesis by overexpressing the small GTPase Rab17 increased dFab transport into tubules and induced its transcytosis in BECs. We propose that sorting tubules regulate transcytosis in BECs and may be a general mechanism for receptor-mediated transport across the BBB.

  17. Self-(Un)rolling Biopolymer Microstructures: Rings, Tubules, and Helical Tubules from the Same Material.

    Science.gov (United States)

    Ye, Chunhong; Nikolov, Svetoslav V; Calabrese, Rossella; Dindar, Amir; Alexeev, Alexander; Kippelen, Bernard; Kaplan, David L; Tsukruk, Vladimir V

    2015-07-13

    We have demonstrated the facile formation of reversible and fast self-rolling biopolymer microstructures from sandwiched active-passive, silk-on-silk materials. Both experimental and modeling results confirmed that the shape of individual sheets effectively controls biaxial stresses within these sheets, which can self-roll into distinct 3D structures including microscopic rings, tubules, and helical tubules. This is a unique example of tailoring self-rolled 3D geometries through shape design without changing the inner morphology of active bimorph biomaterials. In contrast to traditional organic-soluble synthetic materials, we utilized a biocompatible and biodegradable biopolymer that underwent a facile aqueous layer-by-layer (LbL) assembly process for the fabrication of 2D films. The resulting films can undergo reversible pH-triggered rolling/unrolling, with a variety of 3D structures forming from biopolymer structures that have identical morphology and composition. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Secretory NaCl and volume flow in renal tubules.

    Science.gov (United States)

    Beyenbach, K W

    1986-05-01

    This review attempts to give a retrospective survey of the available evidence concerning the secretion of NaCl and fluid in renal tubules of the vertebrate kidney. In the absence of glomerular filtration, epithelial secretory mechanisms, which to this date have not been elucidated, are responsible for the renal excretion of NaCl and water in aglomerular fish. However, proximal tubules isolated from glomerular fish kidneys of the flounder, killifish, and the shark also have the capacity to secrete NaCl and fluid. In shark proximal tubules, fluid secretion appears to be driven via secondary active transport of Cl. In another marine vertebrate, the sea snake, secretion of Na (presumably NaCl) and fluid is observed in freshwater-adapted and water-loaded animals. Proximal tubules of mammals can be made to secrete NaCl in vitro together with secretion of aryl acids. An epithelial cell line derived from dog kidney exhibits secondary active secretion of Cl when stimulated with catecholamines. Tubular secretion of NaCl and fluid may serve a variety of renal functions, all of which are considered here. The occurrence of NaCl and fluid secretion in glomerular proximal tubules of teleosts, elasmobranchs, and reptiles and in mammalian renal tissue cultures suggests that the genetic potential for NaCl secretion is present in every vertebrate kidney.

  19. Natural history of seminiferous tubule degeneration in Klinefelter syndrome

    DEFF Research Database (Denmark)

    Aksglaede, Lise; Wikström, Anne M; Rajpert-De Meyts, Ewa

    2006-01-01

    Klinefelter syndrome (47,XXY) is characterized by small, firm testis, gynaecomastia, azoospermia and hypergonadotropic hypogonadism. Degeneration of the seminiferous tubules in 47,XXY males is a well-described phenomenon. It begins in the fetus, progresses through infancy and accelerates dramatic......Klinefelter syndrome (47,XXY) is characterized by small, firm testis, gynaecomastia, azoospermia and hypergonadotropic hypogonadism. Degeneration of the seminiferous tubules in 47,XXY males is a well-described phenomenon. It begins in the fetus, progresses through infancy and accelerates...... summarize current knowledge on the development of the classical endocrinological and histological features of 47,XXY males from fetus to adulthood and review the literature concerning the degeneration of the seminiferous tubules in this syndrome....

  20. Chemically triggered ejection of membrane tubules controlled by intermonolayer friction.

    Science.gov (United States)

    Fournier, J-B; Khalifat, N; Puff, N; Angelova, M I

    2009-01-09

    We report a chemically driven membrane shape instability that triggers the ejection of a tubule growing exponentially toward a chemical source. The instability is initiated by a dilation of the exposed monolayer, which is coupled to the membrane spontaneous curvature and slowed down by intermonolayer friction. Our experiments are performed by local delivery of a basic pH solution to a giant vesicle. Quantitative fits of the data give an intermonolayer friction coefficient b approximately 2x10;{9} J s/m;{4}. The exponential growth of the tubule may be explained by a Marangoni stress yielding a pulling force proportional to its length.

  1. Hierarchically assembled DNA origami tubules with reconfigurable chirality

    International Nuclear Information System (INIS)

    Chen, Haorong; Cha, Tae-Gon; Pan, Jing; Choi, Jong Hyun

    2013-01-01

    The dynamic reconfiguration of a hierarchically assembled tubular structure is demonstrated using the DNA origami technique. Short cylindrical DNA origami monomers are synthesized and linked into elongated tubules, which can then be disassembled via toehold-mediated strand displacement. The disassembled subunits are subsequently linked into tubules of a different chirality. The reconfiguration is performed with the subunits carrying dumbbell hairpin DNA oligonucleotides or gold nanoparticles (AuNPs). The reconfiguration of higher order origami structures presented here is useful for constructing dynamic nanostructures that exceed the size limit of single DNA origami and may facilitate the study of molecular or particle interactions by tuning their relative distance and organization. (paper)

  2. Masonry structures built with fictile tubules: Experimental and numerical analyses

    Science.gov (United States)

    Tiberti, Simone; Scuro, Carmelo; Codispoti, Rosamaria; Olivito, Renato S.; Milani, Gabriele

    2017-11-01

    Masonry structures with fictile tubules were a distinctive building technique of the Mediterranean area. This technique dates back to Roman and early Christian times, used to build vaulted constructions and domes with various geometrical forms by virtue of their modular structure. In the present work, experimental tests were carried out to identify the mechanical properties of hollow clay fictile tubules and a possible reinforcing technique for existing buildings employing such elements. The experimental results were then validated by devising and analyzing numerical models with the FE software Abaqus, also aimed at investigating the structural behavior of an arch via linear and nonlinear static analyses.

  3. Innervation of the renal proximal convoluted tubule of the rat

    International Nuclear Information System (INIS)

    Barajas, L.; Powers, K.

    1989-01-01

    Experimental data suggest the proximal tubule as a major site of neurogenic influence on tubular function. The functional and anatomical axial heterogeneity of the proximal tubule prompted this study of the distribution of innervation sites along the early, mid, and late proximal convoluted tubule (PCT) of the rat. Serial section autoradiograms, with tritiated norepinephrine serving as a marker for monoaminergic nerves, were used in this study. Freehand clay models and graphic reconstructions of proximal tubules permitted a rough estimation of the location of the innervation sites along the PCT. In the subcapsular nephrons, the early PCT (first third) was devoid of innervation sites with most of the innervation occurring in the mid (middle third) and in the late (last third) PCT. Innervation sites were found in the early PCT in nephrons located deeper in the cortex. In juxtamedullary nephrons, innervation sites could be observed on the PCT as it left the glomerulus. This gradient of PCT innervation can be explained by the different tubulovascular relationships of nephrons at different levels of the cortex. The absence of innervation sites in the early PCT of subcapsular nephrons suggests that any influence of the renal nerves on the early PCT might be due to an effect of neurotransmitter released from renal nerves reaching the early PCT via the interstitium and/or capillaries

  4. Active ion transport in the renal proximal tubule. II. Ionic dependence of the Na pump

    OpenAIRE

    1984-01-01

    The dependence of Na pump activity on intracellular and extracellular Na+ and K+ was investigated using a suspension of rabbit cortical tubules that contained mostly (86%) proximal tubules. The ouabain- sensitive rate of respiration (QO2) was used to measure the Na pump activity of intact tubules, and the Na,K-ATPase hydrolytic activity was measured using lysed proximal tubule membranes. The dependence (K0.5) of the Na pump on intracellular Na+ was affected by the relative intracellular conce...

  5. RNA sequencing of kidney distal tubule cells reveals multiple mediators of chronic aldosterone action

    DEFF Research Database (Denmark)

    Poulsen, Søren Brandt; Limbutara, Kavee; Fenton, Robert Andrew

    2018-01-01

    The renal aldosterone-sensitive distal tubule (ASDT) is crucial for sodium reabsorption and blood pressure regulation. The ASDT consists of the late distal convoluted tubule (DCT2), connecting tubule (CNT) and collecting duct. Due to difficulties in isolating epithelial cells from the ASDT in lar...

  6. Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation.

    Science.gov (United States)

    Siddiqa, Abida; Massimi, Paola; Pim, David; Broniarczyk, Justyna; Banks, Lawrence

    2018-03-15

    Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans -Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans -Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex. IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans -Golgi network. Indeed, the loss of

  7. Paracellular transport and energy utilization in the renal tubule.

    Science.gov (United States)

    Yu, Alan S L

    2017-09-01

    Paracellular transport across the tight junction is a general mechanism for transepithelial transport of solutes in epithelia, including the renal tubule. However, why paracellular transport evolved, given the existence of a highly versatile system for transcellular transport, is unknown. Recent studies have identified the paracellular channel, claudin-2, that is responsible for paracellular reabsorption of sodium in the proximal renal tubule. Knockout of claudin-2 in mice impairs proximal sodium and fluid reabsorption but is compensated by upregulation of sodium reabsorption in the loop of Henle. This occurs at the expense of increased renal oxygen consumption, hypoxia of the outer medulla and increased susceptibility to ischemic kidney injury. Paracellular transport can be viewed as a mechanism to exploit the potential energy in existing electrochemical gradients to drive passive transepithelial transport without consuming additional energy. In this way, it enhances the efficiency of energy utilization by transporting epithelia.

  8. Strontium effects on root dentin tubule occlusion and nanomechanical properties.

    Science.gov (United States)

    Saeki, Kuniko; Marshall, Grayson W; Gansky, Stuart A; Parkinson, Charles R; Marshall, Sally J

    2016-02-01

    Dentin hypersensitivity often is treated by promotion of dentin tubule occlusion. In this in vitro study we evaluated nanomechanical properties and degree of tubule occlusion conferred to sound and demineralized human root dentin following treatment with a 10% (w/w) strontium acetate solution and its relation to the treatment duration and delivery method. 24 human cervical root dentin disks (8 groups of 3) were polished through 0.25 μm. 12 disks were subjected to an acid challenge (1% citric acid, pH 3.8) for 2 min. The specimens were incubated in artificial saliva, treated by soaking or brushing with deionized (DI) water or a solution of 10% strontium acetate for 2 min twice a day for 28 days. The occlusion percent and nanomechanical properties were determined at the baseline, 5, 14 and 28 days. Cross-sectioned specimens were prepared to evaluate the depth affected by strontium acetate / dentin interaction by SEM. Statistical analysis was performed using linear mixed effects models. A 10% strontium acetate treatment over 5-28 days significantly increased tubule occlusion for normal root dentin and to a lesser extent for demineralized dentin and increased the AFM based nanomechanical properties of demineralized dentin. Brushing was more effective than soaking in recovery of properties of demineralized dentin when treated with strontium. No difference in tubuleocclusion was found between the two delivery methods. Strontium acetate itself proved to have the ability to occlude dentin tubules and result in small changes in the mechanical properties of dentin. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  9. Cyclophilin B expression in renal proximal tubules of hypertensive rats.

    Science.gov (United States)

    Kainer, D B; Doris, P A

    2000-04-01

    Rat cyclophilin-like protein (Cy-LP) is a candidate hypertension gene initially identified by differential hybridization and implicated in renal mechanisms of salt retention and high blood pressure. We report the molecular characterization of rat cyclophilin B (CypB) and demonstrate, through sequence analysis and an allele-specific polymerase chain reaction primer assay, that CypB but not Cy-LP is expressed in rat kidney. CypB is an endoplasmic reticulum-localized prolyl-isomerase that interacts with elongation initiation factor 2-beta, an important regulator of protein translation and a central component of the endoplasmic reticulum stress response to hypoxia or ATP depletion. Active renal transport of sodium is increased in the spontaneously hypertensive rat (SHR), and there is evidence that this coincides with hypoxia and ATP depletion in the renal cortex. In the present studies we have examined expression of CypB in rat proximal tubules, which contributes to the increased renal sodium reabsorption in this model of hypertension. We report that CypB transcript abundance is significantly elevated in proximal convoluted tubules from SHR compared with the control Wistar-Kyoto strain. This upregulation occurs in weanling animals and precedes the development of hypertension, indicating that it is not a simple response to hypertension in SHR. Further, CypB expression is also higher in a proximal tubule cell line derived from SHR compared with a similar line derived from Wistar-Kyoto rats, indicating that this difference is genetically determined. No sequence differences were observed in the CypB cDNA from these 2 strains. These observations suggest that a genetically determined alteration in proximal tubules from SHR occurs that leads to increased expression of CypB. In view of evidence linking CypB to the regulation of elongation initiation factor-2, the upregulation of CypB may result from metabolic stress.

  10. Reduced Insulin Receptor Expression Enhances Proximal Tubule Gluconeogenesis.

    Science.gov (United States)

    Pandey, Gaurav; Shankar, Kripa; Makhija, Ekta; Gaikwad, Anil; Ecelbarger, Carolyn; Mandhani, Anil; Srivastava, Aneesh; Tiwari, Swasti

    2017-02-01

    Reduced insulin receptor protein levels have been reported in the kidney cortex from diabetic humans and animals. We recently reported that, targeted deletion of insulin receptor (IR) from proximal tubules (PT) resulted in hyperglycemia in non-obese mice. To elucidate the mechanism, we examined human proximal tubule cells (hPTC) and C57BL/6 mice fed with high-fat diet (HFD, 60% fat for 20 weeks). Immunoblotting revealed a significantly lower protein level of IR in HFD compare to normal chow diet (NCD). Furthermore, a blunted rise in p-AKT 308 levels in the kidney cortex of HFD mice was observed in response to acute insulin (0.75 IU/kg body weight, i.p) relative to NCD n = 8/group, P gluconeogenesis. Transcript levels of the gluconeogenic enzyme PEPCK were significantly increased in cAMP/DEXA-stimulated hPTC cells (n = 3, P gluconeogenesis and PEPCK induction was significantly attenuated in IR (siRNA) silenced hPTC (n = 3, P gluconeogenesis. Thus reduced insulin signaling of the proximal tubule may contribute to hyperglycemia in the metabolic syndrome via elevated gluconeogenesis. J. Cell. Biochem. 118: 276-285, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Genetic and cytogenetic analysis of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae).

    Science.gov (United States)

    Mavragani-Tsipidou, P

    2002-09-01

    The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactmcera oleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomes including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubules of this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b) the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter as an accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence of toroid tips of the chromosome arms. The in situ hybridization of genes or DNA sequences to the salivary gland polytene chromosomes of B. oleae provided molecular markers for all five autosomes and permitted the establishment of chromosomal homologies among B. olea, B. tryoni and Ceratitis capitata. The heat shock response of B. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophila melanogaster.

  12. Metabolism of cysteine and cysteinesulfinate in rat kidney tubules

    International Nuclear Information System (INIS)

    De La Rosa, J.; Stipanuk, M.H.

    1986-01-01

    In studies with rat hepatocytes, hypotaurine plus taurine production accounted for less than 5% of the total amount of cysteine (CYS) catabolized, whereas more than 90% of the metabolized cysteinesulfinate (CSA) was converted to taurine plus hypotaurine. Similar studies have been carried out with kidney tubules isolated from fed rats and incubated with 2 mM [1- 14 C]CYS or 25 mM [1- 14 C]CSA at 37 0 C for up to 40 min. The production of 14 CO 2 from CSA (3.1 +/- 1.3 nmol/sup ./ min -1 /sup ./ mg dry wt -1 ) was equivalent to the accumulation of N in NH 4 + plus glutamate. Substantial oxidation of CYS was observed (16 +/- 11 nmol CO 2 x min -1 x mg dry wt -1 ), but only 12% of the expected amount of N was recovered as NH 4 + plus glutamate. Accumulation of hypotaurine plus taurine was equivalent to 20% of the observed rate of 14 CO 2 production from CSA but accounted for only 2% of the observed rate of 14 CO 2 production from CYS. Addition of unlabeled CSA to incubations with varying levels of CYS had no effect on production of 14 CO 2 . Addition of 2 mM α-ketoglutarate to the incubation mixtures resulted in an increased in 14 CO 2 production from CSA to 290% of the control level but had no effect on CYS oxidation. In agreement with the authors findings for rat hepatocytes, these data suggest that most metabolism of CYS by the rat kidney tubule occurs by a CSA-independent pathway. However, in contrast to the metabolism of CSA almost entirely to taurine in the hepatocyte, kidney tubules appeared to metabolize CSA primarily by the transamination pathway

  13. Mechanisms of adaptation to chronic respiratory acidosis in the rabbit proximal tubule.

    OpenAIRE

    Krapf, R

    1989-01-01

    The hyperbicarbonatemia of chronic respiratory acidosis is maintained by enhanced bicarbonate reabsorption in the proximal tubule. To investigate the cellular mechanisms involved in this adaptation, cell and luminal pH were measured microfluorometrically using (2",7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein in isolated, microperfused S2 proximal convoluted tubules from control and acidotic rabbits. Chronic respiratory acidosis was induced by exposure to 10% CO2 for 52-56 h. Tubules from ac...

  14. Digital three-dimensional reconstruction and ultrastructure of the mouse proximal tubule

    DEFF Research Database (Denmark)

    Zhai, X.Y.; Birn, H.; Jensen, K.B.

    2003-01-01

    . In the medullary rays, these are arranged in layers outside the clusters of more superficial tubules. In contrast to rat and human kidney, no major segmental variation in the ultrastructure of the proximal tubule was identified, and no parameters enabled definition of distinct segments in this strain of mice......, detailed analyses of normal mouse kidney structure and organization are lacking. This study describes the 3D organization and ultrastructural, segmental variation of the mouse kidney proximal tubule. A total of 160 proximal tubules in three C57/BL/6J mouse kidneys were analyzed on 800 serial sections from...

  15. Use of poly (amidoamine dendrimer for dentinal tubule occlusion: a preliminary study.

    Directory of Open Access Journals (Sweden)

    Tianda Wang

    Full Text Available The occlusion of dentinal tubules is an effective method to alleviate the symptoms caused by dentin hypersensitivity, a significant health problem in dentistry and daily life. The in situ mineralization within dentinal tubules is a promising treatment for dentin hypersensitivity as it induces the formation of mineral on the sensitive regions and occludes the dentinal tubules. This study was carried out to evaluate the in vitro effect of a whole generation poly(amidoamine (PAMAM dendrimer (G3.0 on dentinal tubule occlusion by inducing mineralization within dentinal tubules. Dentin discs were treated with PAMAM dendrimers using two methods, followed by the in vitro characterization using Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR, X-ray diffraction (XRD, Field emission scanning electron microscopy (FE-SEM and Energy-Dispersive X-ray Spectroscopy (EDS. These results showed that G3.0 PAMAM dendrimers coated on dentin surface and infiltrated in dentinal tubules could induce hydroxyapatite formation and resulted in effective dentinal tubule occlusion. Moreover, crosslinked PAMAM dendrimers could induce the remineralization of demineralized dentin and thus had the potential in dentinal tubule occlusion. In this in vitro study, dentinal tubules occlusion could be achieved by using PAMAM dendrimers. This could lead to the development of a new therapeutic technique for the treatment of dentin hypersensitivity.

  16. Histological and three dimensional organizations of lymphoid tubules in normal lymphoid organ of Penaeus monodon.

    Science.gov (United States)

    Duangsuwan, Pornsawan; Phoungpetchara, Ittipon; Tinikul, Yotsawan; Poljaroen, Jaruwan; Wanichanon, Chaitip; Sobhon, Prasert

    2008-04-01

    The normal lymphoid organ of Penaeus monodon (which tested negative for WSSV and YHV) was composed of two parts: lymphoid tubules and interstitial spaces, which were permeated with haemal sinuses filled with large numbers of haemocytes. There were three permanent types of cells present in the wall of lymphoid tubules: endothelial, stromal and capsular cells. Haemocytes penetrated the endothelium of the lymphoid tubule's wall to reside among the fixed cells. The outermost layer of the lymphoid tubule was covered by a network of fibers embedded in a PAS-positive extracellular matrix, which corresponded to a basket-like network that covered all the lymphoid tubules as visualized by a scanning electron microscope (SEM). Argyrophilic reticular fibers surrounded haemal sinuses and lymphoid tubules. Together they formed the scaffold that supported the lymphoid tubule. Using vascular cast and SEM, the three dimensional structure of the subgastric artery that supplies each lobe of the lymphoid organ was reconstructed. This artery branched into highly convoluted and blind-ending terminal capillaries, each forming the lumen of a lymphoid tubule around which haemocytes and other cells aggregated to form a cuff-like wall. Stromal cells which form part of the tubular scaffold were immunostained for vimentin. Examination of the whole-mounted lymphoid organ, immunostained for vimentin, by confocal microscopy exhibited the highly branching and convoluted lymphoid tubules matching the pattern of the vascular cast observed in SEM.

  17. Clinical Importance of Morphological Appearance of Seminiferous Tubules During MicroTESE in NOA Cases

    Directory of Open Access Journals (Sweden)

    A. H. Haliloglu

    2005-12-01

    Full Text Available Design: Clinical study. Setting: Research Center on Infertility, Ankara University; and Urology Department. Patients: 65 men with nonobstructive azoospermia (NOA.\tInterventions: Microscopical appearance of seminiferous tubules was recorded during TESE surgery. Differing from others, the largest opaque-white in color tubules were cut and removed. When all the tubules have no discriminating appearance, randomized biopsies were obtained. Removed tissue pieces were subjected to mechanical mincing under the stereomicroscope and then enzymatic digestion processes. Using inversion microscope (x32 magnification spermatozoa were searched. Main Outcome Measures: Morphological appearance of seminiferous tubules under optical magnification, spermatozoa recovery rates and histopathological findings were compared.\tRESULTS: In cases of Sertoli cell-only syndrome (SCOS, maturation arrest, hypospermatogenesis and focal spermatogenesis TESE yielded at least one spermatozoon in 37%, 52%, 100% and 63% of the cases, respectively. When all the seminiferous tubules were homogenously swollen, histopathological diagnosis was hypospermatogenesis in 100% of the cases. Homogenously thin and transparent tubules corresponded to SCOS or maturation arrest in 90% and 10% of the cases, respectively. Mature spermatozoa recovery rates were 100% and zero in homogenously-swollen observed and homogenously-thin observed tubules, respectively. CONCLUSIONS: Present data indicate that in cases of all tubules are homogenous in appearance and none of them can be discriminated from others, using microscope has no advantage in selection of the tubuli to be removed, but randomizely selection would also be sufficient. MicroTESE significantly increases the success in NOA cases with seminiferous tubules dispersed heterogeneously.

  18. [Anatomy, physiology and clinical relevance of the connecting tubule].

    Science.gov (United States)

    Miranda, N; Simeoni, M A; Ciriana, E; Panico, C; Cappello, E; Capasso, G B

    2009-01-01

    The cortical distal nephron is the site of fine regulation of salt and water excretion by peptide and mineralocorticoid hormones and the site for specific actions of diuretics. Some data suggest that sodium reabsorption and potassium secretion in the distal convoluted tubule and the connecting tubule (CNT) are sufficient to maintain the sodium and potassium balance, with little or no contribution of the collecting duct. The homeostatic role of the sodium and potassium transport systems in the collecting duct can be questioned, especially in conditions where dietary sodium intake is high and potassium intake is low compared with the physiological needs of the organism. The functional expression of epithelial sodium channels (ENaC) in the CNT is sufficient for furosemide-stimulated urinary acidification and identifies the CNT as a major segment in electrogenic urinary acidification. In the outer renal cortex, the CNT returns to the glomerular hilus and contacts the renal afferent arterioles (Af-Art). This morphology is compatible with a cross-talk between the CNT and Af-Art. This novel regulatory mechanism of the renal microcirculation may participate in the vasodilatation observed during high salt intake, perhaps by antagonizing tubuloglomerular feedback. In conclusion, the cortical distal nephron appears to be a complex site for several physiological mechanisms; it is mainly involved in salt and fluid homeostasis and in acid-base balance maintenance. Furthermore, the CNT segment appears to promote a CNT-Af-Art feedback loop.

  19. Acid-base transport by the renal proximal tubule.

    Science.gov (United States)

    Skelton, Lara A; Boron, Walter F; Zhou, Yuehan

    2010-01-01

    Each day, the kidneys filter 180 L of blood plasma, equating to some 4,300 mmol of the major blood buffer, bicarbonate (HCO3-). The glomerular filtrate enters the lumen of the proximal tubule (PT), and the majority of filtered HCO3- is reclaimed along the early (S1) and convoluted (S2) portions of the PT in a manner coupled to the secretion of H+ into the lumen. The PT also uses the secreted H+ to titrate non-HCO3- buffers in the lumen, in the process creating "new HCO3-" for transport into the blood. Thus, the PT - along with more distal renal segments - is largely responsible for regulating plasma [HCO3-]. In this review we first focus on the milestone discoveries over the past 50+ years that define the mechanism and regulation of acid-base transport by the proximal tubule. Further on in the review, we will summarize research still in progress from our laboratory, work that addresses the problem of how the PT is able to finely adapt to acid-base disturbances by rapidly sensing changes in basolateral levels of HCO3- and CO2 (but not pH), and thereby to exert tight control over the acid-base composition of the blood plasma.

  20. Wild boars (Sus scrofa scrofa seminiferous tubules morphometry

    Directory of Open Access Journals (Sweden)

    Deiler Sampaio Costa

    2006-09-01

    Full Text Available The aim of this data was to analyze morphology and function of the seminiferous tubule in adult wild boars. Testes removed by unilateral castration of five animals were used. The testicular parenchyma was composed by 82.1±2.2% of seminiferous tubule and 17.9±2.2% of intertubular tissue. The tubular diameter was 249.2±33.0 µm and the seminiferous tubule lenght per gram of testis was 19.3±4.9m. The spermatogonial mitoses efficiency coefficient, meiotic index and spermatogenesis efficiency were 10.34, 2.71 and 30.5 respectively. Each Sertoli cell supported about 13 germinatives cells. The hystometric parameters studied were very similar to those related for domestic boars, however, the wild boars intrinsic efficiency of spermatogenesis and Sertoli cells indexes were smaller than in domestic boars.Objetivou-se com esta pesquisa estudar as características morfométricas e funcionais dos túbulos seminíferos de javalis adultos. Utilizaram-se testículos de cinco animais submetidos a orquiectomia unilateral. O parênquima testicular foi composto por 82,1 ± 2,2% de túbulos seminíferos e 17,9 ± 2,2% de tecido intertubular. O diâmetro tubular foi de 249,2 ± 33,0µm e o comprimento dos túbulos seminíferos por grama de testículo foi de 19,3 ± 4,9m. O coeficiente de eficiência das mitoses espermatogônias, o rendimento meiótico e o rendimento geral da espermatogênese foram, respectivamente, 10,34, 2,71 e 30,50. Cada célula de Sértoli suportou cerca de 13 células germinativas. Conclui-se que os parâmetros histométricos estudados nesta pesquisa foram muito semelhantes aos valores relatados para suínos domésticos, entretanto, o rendimento intrínseco da espermatogênese e os índices de células de Sértoli de javalis foram relativamente baixos quando comparados com aqueles animais.

  1. Proximal Tubule Cell Hypothesis for Cardiorenal Syndrome in Diabetes

    Directory of Open Access Journals (Sweden)

    Akihiko Saito

    2011-01-01

    Full Text Available Incidence of cardiovascular disease (CVD is remarkably high among patients with chronic kidney disease (CKD, even in the early microalbuminuric stages with normal glomerular filtration rates. Proximal tubule cells (PTCs mediate metabolism and urinary excretion of vasculotoxic substances via apical and basolateral receptors and transporters. These cells also retrieve vasculoprotective substances from circulation or synthesize them for release into the circulation. PTCs are also involved in the uptake of sodium and phosphate, which are critical for hemodynamic regulation and maintaining the mineral balance, respectively. Dysregulation of PTC functions in CKD is likely to be associated with the development of CVD and is linked to the progression to end-stage renal disease. In particular, PTC dysfunction occurs early in diabetic nephropathy, a leading cause of CKD. It is therefore important to elucidate the mechanisms of PTC dysfunction to develop therapeutic strategies for treating cardiorenal syndrome in diabetes.

  2. Reversible effects of acute hypertension on proximal tubule sodium transporters

    DEFF Research Database (Denmark)

    Zhang, Y; Magyar, C E; Norian, J M

    1998-01-01

    Acute hypertension provokes a rapid decrease in proximal tubule sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of membranes containing apical membrane sodium/hydrogen exchangers (NHE3) [Y. Zhang, A. K. Mircheff, C. B....... Renal cortex lysate was fractionated on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase activity (but not subunit immunoreactivity) decreased one-third to one-half after BP was elevated and recovered after BP was normalized. After BP was elevated, 55% of the apical NHE3 immunoreactivity......, smaller fractions of sodium-phosphate cotransporter immunoreactivity, and apical alkaline phosphatase and dipeptidyl-peptidase redistributed to membranes of higher density enriched in markers of the intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5), whereas density distributions...

  3. A microfluidic renal proximal tubule with active reabsorptive function.

    Directory of Open Access Journals (Sweden)

    Else M Vedula

    Full Text Available In the kidney, the renal proximal tubule (PT reabsorbs solutes into the peritubular capillaries through active transport. Here, we replicate this reabsorptive function in vitro by engineering a microfluidic PT. The microfluidic PT architecture comprises a porous membrane with user-defined submicron surface topography separating two microchannels representing a PT filtrate lumen and a peritubular capillary lumen. Human PT epithelial cells and microvascular endothelial cells in respective microchannels created a PT-like reabsorptive barrier. Co-culturing epithelial and endothelial cells in the microfluidic architecture enhanced viability, metabolic activity, and compactness of the epithelial layer. The resulting tissue expressed tight junctions, kidney-specific morphology, and polarized expression of kidney markers. The microfluidic PT actively performed sodium-coupled glucose transport, which could be modulated by administration of a sodium-transport inhibiting drug. The microfluidic PT reproduces human physiology at the cellular and tissue levels, and measurable tissue function which can quantify kidney pharmaceutical efficacy and toxicity.

  4. Photoelectrochemical reactivity of polyoxophosphotungstates embedded in titania tubules

    International Nuclear Information System (INIS)

    Xie Yibing

    2006-01-01

    A highly ordered and crystallized titania (TiO 2 ) nanotube array is fabricated by a low-voltage anodization plus a post-embedding calcination process. Polyoxophosphotungstate-titania (POPTA-TiO 2 ) composite catalyst is synthesized by embedding POPTA in TiO 2 tubule channels to improve the photoelectrochemical properties. The morphological characteristics and crystal behaviour of POPTA-TiO 2 are examined by field-emission scanning electron microscopy and x-ray diffraction. The stability of the chemical structure has been analysed by Fourier transformed infrared spectroscopy measurements. The photoelectrochemical properties are investigated by means of the polarization current response. Photocatalytic and photoelectrocatalytic reactivities for the degradation of an endocrine disrupting chemical have also been investigated to examine the photoelectrochemical reaction efficiency of POPTA-TiO 2 composite catalyst

  5. Effect of dental materials on gluconeogenesis in rat kidney tubules

    Energy Technology Data Exchange (ETDEWEB)

    Reichl, F.X.; Durner, J.; Mueckter, H.; Elsenhans, B.; Forth, W. [Muenchen Univ. (Germany). Walter-Straub-Institut fuer Pharmakologie und Toxikologie; Kunzelmann, K.H.; Hickel, R. [Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig-Maximilians-University of Munich (Germany); Spahl, W. [Institute of Organic Chemistry, Ludwig-Maximilians-University of Munich (Germany); Hume, W.R. [Dental Research Institute, Univ. of California, Los Angeles, CA (United States); Moes, G.W. [TNO Prins-Maurits-Laboratorium, Rijswijk (Netherlands)

    1999-09-01

    The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) as well as mercuric chloride (HgCl{sub 2}) and methylmercury chloride (MeHgCl) on gluconeogenesis was investigated in isolated rat kidney tubules. From starved rats kidney tubules were prepared and isolated by digestion with collagenase. Every 10 min up to 60 min 1-ml samples were drawn from the cell suspension for quantitating the glucose content. Glucose formation in controls was 3.3 {+-} 0.2 nmol/mg . per min (mean {+-} SEM, n=21). Relative rates of glucose formation were obtained by expressing individual rates as a percentage of the corresponding control. X-Y concentration curves (effective concentration, EC) of the substances were calculated by fitting a four-parametric sigmoid function to the relative rates of glucose formation at various test concentrations. At the end of the incubation period cell viability was assessed by trypan blue exclusion. Cell viability decreased within the 60 min interval from 90 to approx. 80% (controls), <25 (HEMA), <20 (TEGDMA), <10 (MeHgCl), and <10% (HgCl{sub 2}). Values of 50% effective concentration (EC{sub 50}) were calculated from fitted curves. EC{sub 50} values were (mmol; mean {+-} SEM; n=4): HEMA, 17.7 {+-} 2.9; TEGDMA, 1.8 {+-} 0.2; MeHgCl, 0.018 {+-} 0.0005; and HgCl{sub 2}, 0.0016 {+-} 0.0005. The toxic effect of HgCl{sub 2} was {proportional{underscore}to}1000 or 10 000 higher than that of the dental composite components TEGDMA or HEMA, respectively. (orig.)

  6. 3D variations in human crown dentin tubule orientation: a phase-contrast microtomography study.

    Science.gov (United States)

    Zaslansky, Paul; Zabler, Simon; Fratzl, Peter

    2010-01-01

    Tubules dominate the microstructure of dentin, and in crowns of human teeth they are surrounded by thick mineralized peritubular cuffs of high stiffness. Here we examine the three-dimensional (3D) arrangement of tubules in relation to enamel on the buccal and lingual aspects of intact premolars and molars. Specifically we investigate the angular orientation of tubules relative to the plane of the junction of dentin with enamel (DEJ) by means of wet, non-destructive and high-resolution phase-contrast (coherent) tomography. Enamel capped dentin samples (n=16), cut from the buccal and lingual surfaces of upper and lower premolar and molar teeth, were imaged in water by high-resolution synchrotron-based phase-contrast X-ray radiography. Reconstructed 3D virtual images were co-aligned with respect to the DEJ plane. The average tubule orientation was determined at increasing distances from the DEJ, based on integrated projections onto orthogonal virtual planes. The angle and curl of the tubules were determined every 100 microm to a depth of 1.4mm beneath the DEJ. Most tubules do not extend at right angles from the DEJ. Even when they do, tubules always change their orientations substantially within the first half-millimeter zone beneath the DEJ, both on the buccal and lingual aspects of premolar and molar teeth. Tubules also tend to curl and twist within this zone. Student t-tests indicate that lower teeth seem to have greater tilts in the tubule orientations relative to the DEJ normal with an average angle of 42 degrees (+/-2.0 degrees), whereas upper teeth exhibit a smaller change of orientation, with an average of 32 degrees (+/-2.1 degrees). Tubules are a central characteristic of dentin, with important implications on how it is arranged and what the properties are. Knowing about the path that tubules follow is important for various reasons, ranging form improving control over restorative procedures to understanding or simulating the mechanical properties of teeth

  7. Luminal uptake and intracellular transport of insulin in renal proximal tubules

    International Nuclear Information System (INIS)

    Hellfritzsch, M.; Christensen, E.I.; Sonne, O.

    1986-01-01

    It is generally accepted that proteins taken up from the renal tubular fluid are transported into lysosomes in proximal tubule cells. Recently, however, it has been postulated that insulin in isolated perfused rat kidneys did not accumulate in lysosomes but to a certain degree in the Golgi region. The present study was undertaken to investigate the intracellular handling of biologically unaltered insulin in rat renal proximal tubule cells. Rats were prepared for in vivo micropuncture and either a colloidal gold insulin complex or insulin monoiodinated in the A-14 position ( 125 I-insulin) was microinfused into proximal tubules. After 5, 10, 25 or 60 min the tubules were fixed by microinfusion of glutaraldehyde and processed for electron microscopy or electron microscope autoradiography. A qualitative analysis of tubules infused with colloidal gold insulin or 125 I-insulin showed that insulin was taken up by endocytosis and transported to lysosomes, and a quantitative autoradiographic analysis of the 125 I-insulin microinfused tubules showed that the grain density after five min was significantly increased for endocytic vacuoles and for lysosomes. After 60 min the grain density was still significant over lysosomes. The accumulation of grains was non-significant over all other areas analyzed at any time. This study shows that insulin is taken up from the luminal side of the proximal tubule by endocytosis and transported to the lysosomes. There was no significant transport to the Golgi region

  8. Puerarin Facilitates T-Tubule Development of Murine Embryonic Stem Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Lu Wang

    2014-07-01

    Full Text Available Aims: The embryonic stem cell-derived cardiomyocytes (ES-CM is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs. Methods: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development. Results: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3. Conclusion: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts.

  9. In vivo model for microbial invasion of tooth root dentinal tubules

    Directory of Open Access Journals (Sweden)

    Jane L. BRITTAN

    2016-04-01

    Full Text Available ABSTRACT Objective Bacterial penetration of dentinal tubules via exposed dentine can lead to root caries and promote infections of the pulp and root canal system. The aim of this work was to develop a new experimental model for studying bacterial invasion of dentinal tubules within the human oral cavity. Material and Methods Sections of human root dentine were mounted into lower oral appliances that were worn by four human subjects for 15 d. Roots were then fixed, sectioned, stained and examined microscopically for evidence of bacterial invasion. Levels of invasion were expressed as Tubule Invasion Factor (TIF. DNA was extracted from root samples, subjected to polymerase chain reaction amplification of 16S rRNA genes, and invading bacteria were identified by comparison of sequences with GenBank database. Results All root dentine samples with patent tubules showed evidence of bacterial cell invasion (TIF value range from 5.7 to 9.0 to depths of 200 mm or more. A spectrum of Gram-positive and Gram-negative cell morphotypes were visualized, and molecular typing identified species of Granulicatella, Streptococcus, Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas as dentinal tubule residents. Conclusion A novel in vivo model is described, which provides for human root dentine to be efficiently infected by oral microorganisms. A range of bacteria were able to initially invade dentinal tubules within exposed dentine. The model will be useful for testing the effectiveness of antiseptics, irrigants, and potential tubule occluding agents in preventing bacterial invasion of dentine.

  10. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    Science.gov (United States)

    Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

    2014-01-01

    The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

  11. Annexin IV (Xanx-4) has a functional role in the formation of pronephric tubules.

    Science.gov (United States)

    Seville, Rachel A; Nijjar, Sarbjit; Barnett, Mark W; Massé, Karine; Jones, Elizabeth A

    2002-04-01

    Vertebrate kidney organogenesis is characterised by the successive formation of the pronephros, the mesonephros and the metanephros. The pronephros is the first to form and is the functional embryonic kidney of lower vertebrates; although it is vestigial in higher vertebrates, it is a necessary precursor for the other kidney types. The Xenopus pronephros is a simple paired organ; each nephron consists of a single large glomus, one set of tubules and a single duct. The simple organisation of the pronephros and the amenability of Xenopus laevis embryos to manipulation make the Xenopus pronephros an attractive system in which to study organogenesis. It has been shown that pronephric tubules can be induced to form in presumptive ectodermal tissue by treatment with RA and activin. We have used this system in a subtractive hybridisation screen that resulted in the cloning of Xenopus laevis annexin IV (Xanx-4). Xanx-4 transcripts are specifically located to the developing pronephric tubules, and the protein to the luminal surface of these tubules. Temporal expression shows zygotic transcription is upregulated at the time of pronephric tubule specification and persists throughout pronephric development. The temporal and spatial expression pattern of Xanx-4 suggests it may have a role in pronephric tubule development. Overexpression of Xanx-4 yields no apparent phenotype, but Xanx-4 depletion, using morpholinos, produces a shortened, enlarged tubule phenotype. The phenotype observed can be rescued by co-injection of Xanx-4 mRNA. Although the function of annexins is not yet clear, studies have suggested a role for annexins in a number of cellular processes. Annexin IV has been shown to have an inhibitory role in the regulation of epithelial calcium-activated chloride ion conductance. The enlarged pronephric tubule phenotype observed may be attributed to incorrect modulation of exocytosis, membrane plasticity or ion channels and/or water homeostasis. In this study, we

  12. Functionally induced changes in water transport in the proximal tubule segment of rat kidneys

    DEFF Research Database (Denmark)

    Faarup, Poul; von Holstein-Rathlou, Niels-Henrik; Nørgaard, Tove

    2011-01-01

    To eliminate freezing artifacts in the proximal tubule cells, two cryotechniques were applied to normal rat kidneys, ie, freeze substitution and special freeze drying. In addition, salt depletion and salt loading were applied to groups of rats to evaluate whether the segmental structure of the pr......To eliminate freezing artifacts in the proximal tubule cells, two cryotechniques were applied to normal rat kidneys, ie, freeze substitution and special freeze drying. In addition, salt depletion and salt loading were applied to groups of rats to evaluate whether the segmental structure...... segment, representing a structural background for the essential transport of water from the proximal tubules to the peritubular capillaries....

  13. Proximal Tubules Have the Capacity to Regulate Uptake of Albumin.

    Science.gov (United States)

    Wagner, Mark C; Campos-Bilderback, Silvia B; Chowdhury, Mahboob; Flores, Brittany; Lai, Xianyin; Myslinski, Jered; Pandit, Sweekar; Sandoval, Ruben M; Wean, Sarah E; Wei, Yuan; Satlin, Lisa M; Wiggins, Roger C; Witzmann, Frank A; Molitoris, Bruce A

    2016-02-01

    Evidence from multiple studies supports the concept that both glomerular filtration and proximal tubule (PT) reclamation affect urinary albumin excretion rate. To better understand these roles of glomerular filtration and PT uptake, we investigated these processes in two distinct animal models. In a rat model of acute exogenous albumin overload, we quantified glomerular sieving coefficients (GSC) and PT uptake of Texas Red-labeled rat serum albumin using two-photon intravital microscopy. No change in GSC was observed, but a significant decrease in PT albumin uptake was quantified. In a second model, loss of endogenous albumin was induced in rats by podocyte-specific transgenic expression of diphtheria toxin receptor. In these albumin-deficient rats, exposure to diphtheria toxin induced an increase in albumin GSC and albumin filtration, resulting in increased exposure of the PTs to endogenous albumin. In this case, PT albumin reabsorption was markedly increased. Analysis of known albumin receptors and assessment of cortical protein expression in the albumin overload model, conducted to identify potential proteins and pathways affected by acute protein overload, revealed changes in the expression levels of calreticulin, disabled homolog 2, NRF2, angiopoietin-2, and proteins involved in ATP synthesis. Taken together, these results suggest that a regulated PT cell albumin uptake system can respond rapidly to different physiologic conditions to minimize alterations in serum albumin level. Copyright © 2016 by the American Society of Nephrology.

  14. Scattering of neutrons by catalase: a study of molecules, subunits, and tubules

    International Nuclear Information System (INIS)

    Randall, J.; Starling, D.; Baldwin, J.P.; Ibel, K.

    1976-01-01

    The paper deals with small-angle scattering of neutrons by catalase tetramers, dimers, and monomers and with neutron diffraction by helical assemblies of tetramers in the form of tubules. A preliminary study of catalase is described

  15. The development of enamel tubules during the formation of enamel in the marsupial Monodelphis domestica.

    OpenAIRE

    Sasagawa, I; Ferguson, M W

    1991-01-01

    In Monodelphis domestica, although both processes from odontoblasts and projections from ameloblasts were found in developing enamel, the majority of the contents of enamel tubules were probably processes that originated from odontoblasts. Processes from odontoblasts penetrating into enamel touched part of the ameloblasts in the stage of enamel formation. No specialised cell junctions were seen at the adherence between the two. There were no enamel tubules in the aprismatic and pseudoprismati...

  16. Effect of theobromine-containing toothpaste on dentin tubule occlusion in situ.

    Science.gov (United States)

    Amaechi, Bennett T; Mathews, Sapna M; Mensinkai, Poornima K

    2015-01-01

    Dentin hypersensitivity (DH) is treated by either occlusion of dentin tubules or nerve desensitization. This in situ study compared dentin tubules occlusion by theobromine-containing dentifrices with (Theodent-classic-F®, TCF) and without (Theodent-classic®, TC) fluoride with 1,500 ppm fluoride toothpaste, Colgate®-Regular (Fluoride) and Novamin®-containing toothpaste, Sensodyne®-5000-Nupro (Novamin®). Each subject wore four intraoral appliances bearing dentin blocks while using one of four test dentifrices (n = 20/dentifrice) twice daily for 7 days. The four appliances were removed successively after 1, 2, 3, and 7 days. Treated blocks and their control (untreated) blocks were examined with scanning electron microscopy (SEM). Effects were compared statistically (ANOVA/Tukey's) based on percentage of surface area covered by deposited precipitate layer (%DPL) and percentage of fully open (%FOT), partially occluded (%POT), and completely occluded (%COT) tubules in each block calculated relative to the number of tubules in their control blocks. SEM observation indicated an increased %COT and %DPL over time. After 1 and 2 days, %COT was comparable with TC and TCF, and significantly (p Theobromine-containing toothpastes with and without fluoride have equal potential in occluding dentin tubules within a shorter time period than Novamin®-containing toothpaste; however, the three demonstrated equal potential after 1 week, but not the fluoride toothpaste. Theobromine-containing toothpaste promoted dentin tubule occlusion thus shows potential to relief DH.

  17. Prenatal programming of rat cortical collecting tubule sodium transport.

    Science.gov (United States)

    Cheng, Chih-Jen; Lozano, German; Baum, Michel

    2012-03-15

    Prenatal insults have been shown to lead to elevated blood pressure in offspring when they are studied as adults. Prenatal administration of dexamethasone and dietary protein deprivation have demonstrated that there is an increase in transporter abundance for a number of nephron segments but not the subunits of the epithelial sodium channel (ENaC) in the cortical collecting duct. Recent studies have shown that aldosterone is elevated in offspring of protein-deprived mothers when studied as adults, but the physiological importance of the increase in serum aldosterone is unknown. As an indirect measure of ENaC activity, we compared the natriuretic response to benzamil in offspring of mothers who ate a low-protein diet (6%) with those who ate a normal diet (20%) for the last half of pregnancy. The natriuretic response to benzamil was greater in the 6% group (821.1 ± 161.0 μmol/24 h) compared with the 20% group (279.1 ± 137.0 μmol/24 h), consistent with greater ENaC activity in vivo (P sodium transport (-1.9 ± 3.1 pmol·mm(-1)·min(-1)), the offspring of rats that ate a 6% protein diet during the last half of pregnancy had a net sodium flux of 10.7 ± 2.6 pmol·mm(-1)·min(-1) (P = 0.01) in tubules perfused in vitro. Sodium transport was measured using ion-selective electrodes, a novel technique allowing measurement of sodium in nanoliter quantities of fluid. Thus we directly demonstrate that there is prenatal programming of cortical collecting duct sodium transport.

  18. Palladium nanotubes formed by lipid tubule templating and their application in ethanol electrocatalysis.

    Science.gov (United States)

    Wang, Yinan; Ma, Shenghua; Su, Yingchun; Han, Xiaojun

    2015-04-13

    Palladium nanotubes were fabricated by using lipid tubules as templates for the first time in a controlled manner. The positively charged lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) was doped into lipid tubules to adsorb PdCl4 (2-) on the tubule surfaces for further reduction. The lipid tubule formation was optimized by studying the growing dynamics and ethanol/water ratio. The DOTAP-doped tubules showed pH stability from 0 to 14, which makes them ideal templates for metal plating. The Pd nanotubes are open-ended with a tunable wall thickness. They exhibited good electrocatalytic performance in ethanol. Their electrochemically active surface areas were 6.5, 10.6, and 83.2 m(2)  g(-1) for Pd nanotubes with 77, 101, and 150 nm wall thickness, respectively. These Pd nanotubes have great potential in fuel cells. The method demonstrated also opens up a way to synthesize hollow metal nanotubes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Numerical Analysis of the Effect of T-tubule Location on Calcium Transient in Ventricular Myocytes

    Science.gov (United States)

    George, Uduak Z.; Wang, Jun; Yu, Zeyun

    2013-01-01

    Intracellular calcium (Ca2+) signaling in cardiac myocytes is vital for proper functioning of the heart. Understanding the intracellular Ca2+ dynamics would give an insight into the functions of normal and diseased hearts. In the current study, spatiotemporal Ca2+ dynamics is investigated in ventricular myocytes by considering Ca2+ release and re-uptake via sarcolemma and transverse tubules (T-tubules), Ca2+ diffusion and buffering in the cytosol, and the blockade of Ca2+ activities associated with the sarcoplasmic reticulum. This study is carried out using a three dimensional (3D) geometric model of a branch of T-tubule extracted from the electron microscopy (EM) images of a partial ventricular myocyte. Mathematical modeling is done by using a system of partial differential equations involving Ca2+ , buffers, and membrane channels. Numerical simulation results suggest that a lack of T-tubule structure at the vicinity of the cell surface could increase the peak time of Ca2+ concentration in myocytes. The results also show that T-tubules and mobile buffers play an important role in the regulation of Ca2+ transient in ventricular myocytes. PMID:24212025

  20. Jellyfish stinging is driven by the moving front of the nematocyst's tubule

    Science.gov (United States)

    Shavit, Uri; Park, Sinwook; Piriatinskiy, Gadi; Yossifon, Gilad; Lotan, Tamar

    2017-11-01

    Nematocysts are ultra-fast stinging organelles that are utilized by the Cnidaria phylum for prey capture, defense and locomotion. They consist of a capsule and a tubule and exert high pressure and acceleration to penetrate the target organism. Previous studies report that the ejection and elongation of the tubule are driven by a buildup of osmotic potential in the capsule. We question this explanation using a microfluidic system that controls the osmotic potential by directing the tubule through oil, where no osmotic potential can develop, while keeping the capsule in water. It was found that the time needed for elongation through oil is orders of magnitude larger than through water. Our mathematical model shows that the p γGlu concentration in the tubule is higher than in the capsule and the internal pressure that develops there serves as the elongation driving force. These findings imply that modifications of the environment along the tubule route have the potential to slow down the process and reduce its impact. This may shed light on prey defense strategies, human protection against jellyfish stinging, the use of nematocysts for drug delivery and exploration of osmotic based methods for nanotubes production and elongation.

  1. Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips

    Science.gov (United States)

    Homan, Kimberly A.; Kolesky, David B.; Skylar-Scott, Mark A.; Herrmann, Jessica; Obuobi, Humphrey; Moisan, Annie; Lewis, Jennifer A.

    2016-10-01

    Three-dimensional models of kidney tissue that recapitulate human responses are needed for drug screening, disease modeling, and, ultimately, kidney organ engineering. Here, we report a bioprinting method for creating 3D human renal proximal tubules in vitro that are fully embedded within an extracellular matrix and housed in perfusable tissue chips, allowing them to be maintained for greater than two months. Their convoluted tubular architecture is circumscribed by proximal tubule epithelial cells and actively perfused through the open lumen. These engineered 3D proximal tubules on chip exhibit significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls with or without perfusion. Upon introducing the nephrotoxin, Cyclosporine A, the epithelial barrier is disrupted in a dose-dependent manner. Our bioprinting method provides a new route for programmably fabricating advanced human kidney tissue models on demand.

  2. Fluid transport and ion fluxes in mammalian kidney proximal tubule: a model analysis of isotonic transport

    DEFF Research Database (Denmark)

    Larsen, E.H.; Møbjerg, N.; Sørensen, Jens Nørkær

    2006-01-01

    transport similar to rat proximal tubule. Na+ recirculation is required for truly isotonic transport. The tonicity of the absorbate and the recirculation flux depend critically on ion permeabilities of interspace basement membrane. Conclusion: Our model based on solute-solvent coupling in lateral space......Aim: By mathematical modelling, we analyse conditions for near-isotonic and isotonic transport by mammalian kidney proximal tubule. Methods: The model comprises compliant lateral intercellular space (lis) and cells, and infinitely large luminal and peritubular compartments with diffusible species......: Na+, K+, Cl and an intracellular non-diffusible anion. Unknown model variables are solute concentrations, electrical potentials, volumes and hydrostatic pressures in cell and lis, and transepithelial potential. We used data mainly from rat proximal tubule to model epithelial cells and interspace...

  3. Cellular localization of uranium in the renal proximal tubules during acute renal uranium toxicity.

    Science.gov (United States)

    Homma-Takeda, Shino; Kitahara, Keisuke; Suzuki, Kyoko; Blyth, Benjamin J; Suya, Noriyoshi; Konishi, Teruaki; Terada, Yasuko; Shimada, Yoshiya

    2015-12-01

    Renal toxicity is a hallmark of uranium exposure, with uranium accumulating specifically in the S3 segment of the proximal tubules causing tubular damage. As the distribution, concentration and dynamics of accumulated uranium at the cellular level is not well understood, here, we report on high-resolution quantitative in situ measurements by high-energy synchrotron radiation X-ray fluorescence analysis in renal sections from a rat model of uranium-induced acute renal toxicity. One day after subcutaneous administration of uranium acetate to male Wistar rats at a dose of 0.5 mg uranium kg(-1) body weight, uranium concentration in the S3 segment of the proximal tubules was 64.9 ± 18.2 µg g(-1) , sevenfold higher than the mean renal uranium concentration (9.7 ± 2.4 µg g(-1) ). Uranium distributed into the epithelium of the S3 segment of the proximal tubules and highly concentrated uranium (50-fold above mean renal concentration) in micro-regions was found near the nuclei. These uranium levels were maintained up to 8 days post-administration, despite more rapid reductions in mean renal concentration. Two weeks after uranium administration, damaged areas were filled with regenerating tubules and morphological signs of tissue recovery, but areas of high uranium concentration (100-fold above mean renal concentration) were still found in the epithelium of regenerating tubules. These data indicate that site-specific accumulation of uranium in micro-regions of the S3 segment of the proximal tubules and retention of uranium in concentrated areas during recovery are characteristics of uranium behavior in the kidney. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus

    NARCIS (Netherlands)

    Pouwels, J.; Velden, van der T.; Willemse, J.; Borst, J.W.; Lent, van J.W.M.; Bisseling, T.; Wellink, J.E.

    2004-01-01

    Cowpea mosaic virus (CPMV) moves from cell to cell by transporting virus particles via tubules formed through plasmodesmata by the movement protein (MP). On the surface of protoplasts, a fusion between the MP and the green fluorescent protein forms similar tubules and peripheral punctate spots. Here

  5. Tubulation of Class II MHC Compartments Is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells1

    Science.gov (United States)

    Vyas, Jatin M.; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J. Christopher; Van der Veen, Annemarthe G.; Ploegh, Hidde L.

    2009-01-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP. PMID:17513769

  6. Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells.

    Science.gov (United States)

    Vyas, Jatin M; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J Christopher; Van der Veen, Annemarthe G; Ploegh, Hidde L

    2007-06-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.

  7. Developmentally regulated GTP-binding protein 2 is required for stabilization of Rac1-positive membrane tubules.

    Science.gov (United States)

    Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Yoon, Eun Hye; Lee, Byung Ju; Cho, Wha Ja; Park, Jeong Woo

    2017-11-04

    Previously we have reported that developmentally regulated GTP-binding protein 2 (DRG2) localizes on Rab5 endosomes and plays an important role in transferrin (Tfn) recycling. We here identified DRG2 as a key regulator of membrane tubule stability. At 30 min after Tfn treatment, DRG2 localized to membrane tubules which were enriched with phosphatidylinositol 4-monophosphate [PI(4)P] and did not contain Rab5. DRG2 interacted with Rac1 more strongly with GTP-bound Rac1 and tubular localization of DRG2 depended on Rac1 activity. DRG2 depletion led to destabilization of membrane tubules, while ectopic expression of DRG2 rescued the stability of the membrane tubules in DRG2-depleted cells. Our results reveal a novel mechanism for regulation of membrane tubule stability mediated by DRG2. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Renal compensation to chronic hypoxic hypercapnia: downregulation of pendrin and adaptation of the proximal tubule.

    NARCIS (Netherlands)

    Seigneux, S. de; Malte, H.; Dimke, H.; Frokiaer, J.; Nielsen, S.; Frische, S.

    2007-01-01

    The molecular basis for the renal compensation to respiratory acidosis and specifically the role of pendrin in this condition are unclear. Therefore, we studied the adaptation of the proximal tubule and the collecting duct to respiratory acidosis. Male Wistar-Hannover rats were exposed to either

  9. Role of CAP350 in centriolar tubule stability and centriole assembly.

    Directory of Open Access Journals (Sweden)

    Mikael Le Clech

    Full Text Available BACKGROUND: Centrioles are microtubule-based cylindrical structures composed of nine triplet tubules and are required for the formation of the centrosome, flagella and cilia. Despite theirs importance, centriole biogenesis is poorly understood. Centrosome duplication is initiated at the G1/S transition by the sequential recruitment of a set of conserved proteins under the control of the kinase Plk4. Subsequently, the procentriole is assembled by the polymerization of centriolar tubules via an unknown mechanism involving several tubulin paralogs. METHODOLOGY/PRINCIPAL FINDINGS: Here, we developed a cellular assay to study centrosome duplication and procentriole stability based on its sensitivity to the microtubule-depolymerizing drug nocodazole. By using RNA interference experiments, we show that the stability of growing procentrioles is regulated by the microtubule-stabilizing protein CAP350, independently of hSAS-6 and CPAP which initiate procentriole growth. Furthermore, our analysis reveals the critical role of centriolar tubule stability for an efficient procentriole growth. CONCLUSIONS/SIGNIFICANCE: CAP350 belongs to a new class of proteins which associate and stabilize centriolar tubules to control centriole duplication.

  10. SGLT2 Inhibitors: Glucotoxicity and Tumorigenesis Downstream the Renal Proximal Tubule?

    Science.gov (United States)

    Bertinat, Romina; Nualart, Francisco; Yáñez, Alejandro J

    2016-08-01

    At present, diabetes mellitus is the main cause of end-stage renal disease. Effective glycaemic management is the most powerful tool to delay the establishment of diabetic complications, such as diabetic kidney disease. Together with reducing blood glucose levels, new anti-diabetic agents are expected not only to control the progression but also to restore known defects of the diabetic kidney. Sodium-glucose co-transporter 2 (SGLT2) inhibitors are promising anti-diabetic agents that reduce hyperglycaemia by impairing glucose reabsorption in proximal tubule of the kidney and increasing glucosuria. SGLT2 inhibitors have shown to reduce glucotoxicity in isolated proximal tubule cells and also to attenuate expression of markers of overall kidney damage in experimental animal models of diabetes, but the actual renoprotective effect for downstream nephron segments is still unknown and deserves further attention. Here, we briefly discuss possible undesired effects of enhanced glucosuria and albuminuria in nephron segments beyond the proximal tubule after SGLT2 inhibitor treatment, offering new lines of research to further understand the renoprotective action of these anti-diabetic agents. Strategies blocking glucose reabsorption by renal proximal tubule epithelial cells (RPTEC) may be protective for RPTEC, but downstream nephron segments will still be exposed to high glucose and albumin levels through the luminal face. The actual effect of constant enhanced glucosuria over distal nephron segments remains to be established. J. Cell. Physiol. 231: 1635-1637, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  11. P-glycoprotein-deficient mice have proximal tubule dysfunction but are protected against ischemic renal injury

    NARCIS (Netherlands)

    Huls, M.; Kramers, C.; Levtchenko, E.N.; Wilmer, M.J.G.; Dijkman, H.B.P.M.; Kluijtmans, L.A.J.; Hoorn, J.W.A. van der; Russel, F.G.M.; Masereeuw, R.

    2007-01-01

    The multidrug resistance gene 1 product, P-glycoprotein (P-gp), is expressed in several excretory organs, including the apical membrane of proximal tubules. After inducing acute renal failure, P-gp expression is upregulated and this might be a protective function by pumping out toxicants and harmful

  12. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

    NARCIS (Netherlands)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Lamers, Wouter H.; Chaudhry, Farrukh A.; Verlander, Jill W.; Weiner, I. David

    2016-01-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of

  13. X radiation effect in the proximal contorted renal tubules of mice

    International Nuclear Information System (INIS)

    Pacheco, I.P.

    1977-01-01

    Exposing C 57B1, 100 days old, mice to X-ray radiation in doses of 150, 300 and 400 R was observed in the proximal contorted renal tubules. An early appearance of unspecific alterations which are peculiar to ageing [pt

  14. Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques

    Directory of Open Access Journals (Sweden)

    Isuru D. Jayasinghe

    2014-12-01

    Full Text Available The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM, has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

  15. Regulation of MRP2-mediated transport in shark rectal salt gland tubules.

    NARCIS (Netherlands)

    Miller, D.S.; Masereeuw, R.; Karnaky Jr, K.J.

    2002-01-01

    We examined endothelin-1 (ET-1) regulation of the xenobiotic efflux pump, multidrug resistance-associated protein isoform 2 (MRP2), in intact dogfish shark rectal salt gland tubules using a fluorescent substrate sulforhodamine 101 and confocal microscopy. Subnanomolar to nanomolar concentrations of

  16. In Vitro Evaluation of Dentin Tubule Occlusion for Novel Calcium Lactate Phosphate (CLP Paste

    Directory of Open Access Journals (Sweden)

    Jen-Chang Yang

    2017-02-01

    Full Text Available Introduction: The objective of this in vitro study is to evaluate the effective and long-term occlusion of dentinal tubules using a novel calcium lactate phosphate (CLP based desensitizing agent. Methods: Dentin disks (n = 9 were pre-etched using 1 M lactic acid for 30 s and individually treated with Colgate® Pro-Relief™ paste, CLP paste, and double distilled water (ddH2O by a rubber-cupped handpiece. Dentin disks were analyzed under optical micrographs for pre-treatment, directly after treatment, and 14 days post-treatment. One-way ANOVA and post-hoc Tukey’s test were used to determine whether there were any statistically significant differences in dentinal tubule diameter. Results: A significant decrease occurred in the mean tubule diameter for dentin disks treated with CLP paste. A decrease was observed from 3.52 ± 0.83 µm to 2.62 ± 0.42 µm right after treatment, further decreasing to 1.71 ± 0.45 µm after immersion in artificial saliva for 14 days (p < 0.05. Conclusions: The results suggest that the CLP based desensitizing paste has remineralization properties and provides instant and lasting effectiveness in dentinal tubule occlusion.

  17. Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics

    NARCIS (Netherlands)

    Wilmes, Anja; Bielow, Chris; Ranninger, Christina; Bellwon, Patricia; Aschauer, Lydia; Limonciel, Alice; Chassaigne, Hubert; Kristl, Theresa; Aiche, Stephan; Huber, Christian G; Guillou, Claude; Hewitt, Philipp; Leonard, Martin O; Dekant, Wolfgang; Bois, Frederic Y; Jennings, Paul

    2015-01-01

    Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to

  18. Disruption of Core Planar Cell Polarity Signaling Regulates Renal Tubule Morphogenesis but Is Not Cystogenic.

    Science.gov (United States)

    Kunimoto, Koshi; Bayly, Roy D; Vladar, Eszter K; Vonderfecht, Tyson; Gallagher, Anna-Rachel; Axelrod, Jeffrey D

    2017-10-23

    Oriented cell division (OCD) and convergent extension (CE) shape developing renal tubules, and their disruption has been associated with polycystic kidney disease (PKD) genes, the majority of which encode proteins that localize to primary cilia. Core planar cell polarity (PCP) signaling controls OCD and CE in other contexts, leading to the hypothesis that disruption of PCP signaling interferes with CE and/or OCD to produce PKD. Nonetheless, the contribution of PCP to tubulogenesis and cystogenesis is uncertain, and two major questions remain unanswered. Specifically, the inference that mutation of PKD genes interferes with PCP signaling is untested, and the importance of PCP signaling for cystogenic PKD phenotypes has not been examined. We show that, during proliferative stages, PCP signaling polarizes renal tubules to control OCD. However, we find that, contrary to the prevailing model, PKD mutations do not disrupt PCP signaling but instead act independently and in parallel with PCP signaling to affect OCD. Indeed, PCP signaling that is normally downregulated once development is completed is retained in cystic adult kidneys. Disrupting PCP signaling results in inaccurate control of tubule diameter, a tightly regulated parameter with important physiological ramifications. However, we show that disruption of PCP signaling is not cystogenic. Our results suggest that regulating tubule diameter is a key function of PCP signaling but that loss of this control does not induce cysts. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Tubule urate and PAH transport: sensitivity and specificity of serum protein inhibition

    International Nuclear Information System (INIS)

    Grantham, J.J.; Kennedy, J.; Cowley, B.

    1987-01-01

    Macromolecules in rabbit serum inhibit the cellular uptake and transepithelial secretion of [ 14 C]urate and p-[ 3 H]aminohippurate ([ 3 H]PAH) in rabbit S 2 proximal tubule segments. To understand better the potential role these inhibitors may have in the regulation of renal organic anion excretion, the authors examined the specificity and relative inhibitory effects on tubule urate and PAH transport of albumin and γ-globulin, the major inhibitory proteins in rabbit serum. Native rabbit serum markedly inhibited the cellular accumulation or urate and PAH by isolated nonperfused segments. Urate and PAH transport was also inhibited by bovine serum, human serum, Cohn-fractionated rabbit albumin, and rabbit γ-globulin, but not by Cohn-fractionated bovine serum albumin. α-Lactalbumin and β-lactoglobulin, derived from milk, also inhibited urate and PAH transport, but to a lesser extent than albumin and γ-globulin. The transport inhibitory effects of proteins were independent of their binding to urate and PAH. Unidirectional influx and the steady-state intracellular accumulation of urate and PAH in suspensions of proximal tubules were decreased by rabbit serum proteins, suggesting that these inhibitors act on the external face of the cells to diminish the uptake of the organic anions. These studies indicate that the principal plasma proteins (albumin and γ-globulin) significantly inhibit urate and PAH transporters in the basolateral membranes of S 2 proximal tubules. They suggest that circulating plasma proteins that can penetrate the basement membrane of proximal tubules may directly modulate the renal excretion of urate and PAH

  20. Study of the permeability of the various parts of the tubules to sodium and potassium ions

    International Nuclear Information System (INIS)

    Morel, F.; Falbriard, A.

    1959-01-01

    The method of stop flow analysis has been used in rabbits together with radioactive sodium and potassium injected in the middle of a six minutes period of arrest of urine flow during an osmotic diuresis. Urine was subsequently collected in 60 ta 80 mg samples. The specific activities of sodium and potassium suggest that both ions pass directly from the renal interstitial tissue into the urine at different and distinct areas in the tubules. The whole distal segment, including the area of active reabsorption of this ion, is impermeable to sodium in the direction interstitial tissue to lumen. The adjacent, more proximal tubule is, however, extremely permeable. The distal tubular impermeability to potassium is more limited. The specific activity already having reached a maximum at the level of active sodium reabsorption. Reprint of a paper published in 'Revue Francaise d'Etudes Cliniques et Biologiques', n. 5, vol IV, p. 471-474 [fr

  1. Immortalization of canine adipose-derived mesenchymal stem cells and their seminiferous tubule transplantation.

    Science.gov (United States)

    Fang, Jia; Wei, Yudong; Teng, Xin; Zhao, Shanting; Hua, Jinlian

    2018-04-01

    Adipose-derived mesenchymal stem cells (ADSCs) are proven to provide good effects in numerous tissue engineering application and other cell-based therapies. However, the difficulty in the proliferation of ADSCs, known as the "Hayflick limit" in vitro, limits their clinical application. Here, we immortalized canine ADSCs (cADSCs) with SV40 gene and transplanted them into busulfan-induced seminiferous tubules of infertile mice. The proliferation of these immortalized cells was improved significantly. Then, cellular differentiation assays showed that the immortalized cADSCs could differentiate into three-germ-layer cells, osteogenesis, chondrogenesis, adipogenesis phenotypes, and primordial germ cell-like cells (PGCLCs). In addition, the immortalized cADSCs can proliferate in the busulfan-induced seminiferous tubules of infertile mice. These findings confirmed that the immortalized cADSCs maintain the criteria of cADSCs. © 2017 Wiley Periodicals, Inc.

  2. Local pH domains regulate NHE3-mediated Na+ reabsorption in the renal proximal tubule

    Science.gov (United States)

    Burford, James L.; McDonough, Alicia A.; Holstein-Rathlou, Niels-Henrik; Peti-Peterdi, Janos

    2014-01-01

    The proximal tubule Na+/H+ exchanger 3 (NHE3), located in the apical dense microvilli (brush border), plays a major role in the reabsorption of NaCl and water in the renal proximal tubule. In response to a rise in blood pressure NHE3 redistributes in the plane of the plasma membrane to the base of the brush border, where NHE3 activity is reduced. This NHE3 redistribution is assumed to provoke pressure natriuresis; however, it is unclear how NHE3 redistribution per se reduces NHE3 activity. To investigate if the distribution of NHE3 in the brush border can change the reabsorption rate, we constructed a spatiotemporal mathematical model of NHE3-mediated Na+ reabsorption across a proximal tubule cell and compared the model results with in vivo experiments in rats. The model predicts that when NHE3 is localized exclusively at the base of the brush border, it creates local pH microdomains that reduce NHE3 activity by >30%. We tested the model's prediction experimentally: the rat kidney cortex was loaded with the pH-sensitive fluorescent dye BCECF, and cells of the proximal tubule were imaged in vivo using confocal fluorescence microscopy before and after an increase of blood pressure by ∼50 mmHg. The experimental results supported the model by demonstrating that a rise of blood pressure induces the development of pH microdomains near the bottom of the brush border. These local changes in pH reduce NHE3 activity, which may explain the pressure natriuresis response to NHE3 redistribution. PMID:25298526

  3. Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies

    Directory of Open Access Journals (Sweden)

    Beisson Janine

    2001-03-01

    Full Text Available Abstract Background A breakthrough in the understanding of centriole assembly was provided by the characterization of the UNI3 gene in Chlamydomonas. Deletion of this gene, found to encode a novel member of the tubulin superfamily, delta-tubulin, results in the loss of the C-tubule, in the nine microtubule triplets which are the hallmark of centrioles and basal bodies. Delta-tubulin homologs have been identified in the genomes of mammals and protozoa, but their phylogenetic relationships are unclear and their function is not yet known. Results Using the method of gene-specific silencing, we have inactivated the Paramecium delta-tubulin gene, which was recently identified. This inactivation leads to loss of the C-tubule in all basal bodies, without any effect on ciliogenesis. This deficiency does not directly affect basal body duplication, but perturbs the cortical cytoskeleton, progressively leading to mislocalization and loss of basal bodies and to altered cell size and shape. Furthermore, additional loss of B- and even A-tubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry. Conclusions The fact that in two distinct phyla, delta-tubulin plays a similar role provides a new basis for interpreting phylogenetic relationships among delta-tubulins. The role of delta-tubulin in C-tubule assembly reveals that tubulins contribute subtle specificities at microtubule nucleation sites. Our observations also demonstrate the existence of a prepattern for the ninefold symmetry of the organelle which is maintained even if less than 9 triplets develop.

  4. Vitality of Enterococcus faecalis inside dentinal tubules after five root canal disinfection methods

    OpenAIRE

    Vatkar, Niranjan Ashok; Hegde, Vivek; Sathe, Sucheta

    2016-01-01

    Aim: To compare the vitality of Enterococcus faecalis within dentinal tubules after subjected to five root canal disinfection methods. Materials and Methods: Dentin blocks (n = 60) were colonized with E. faecalis. After 4 weeks of incubation, the dentin blocks were divided into one control and five test groups (n = 10 each). The root canals of test groups were subjected to one of the disinfection methods, namely, normal saline (NS), sodium hypochlorite (NaOCl), chlorhexidine digluconate (C...

  5. Ribavirin exposure induces histopathological changes in the seminiferous tubules of testes in albino rats

    International Nuclear Information System (INIS)

    Batool, A.

    2013-01-01

    Study objectives: The objectives of the study are to describe and compare histopathological changes in the seminiferous tubules of testes of rat, with different doses of Ribavirin at different time intervals. Introduction: The chemical disturbances may affect a vast number of potential sites in male reproductive system as well as its complex hormonal regulation. Testicular toxicity may reduce the fertility of the male. The current study was conducted to evaluate the effects of Ribavirin on the histological structure of seminiferous tubules in the testes of albino rats. Materials and Methods: Seventy two sexually mature adult male albino rats weighing 180-200gms were divided into four groups: A, B, C and D; each group having 18 rats. Ribavirin was administered intraperitoneally in different doses to these groups that were 20mg, 100mg and 200mg/kg body weight, while group A was control. Each group was further divided into three subgroups according to three time points which were selected for sacrifice that were 20th, 40th and 60th day from the last exposure to drug. Six randomly selected rats from each group were sacrificed on every sacrifice time. Results and Conclusion: The seminiferous tubules with degenerative changes like appearance of vacuole and necrotic material were observed in comparison to control groups, on 20th day of sacrifice in all groups. In rats sacrificed on day 40th and 60th, the sign of recovery in the form of regeneration of seminiferous epithelium was observed that was more marked in low dose groups than high dose groups which showed late recovery. We conclude that ribavirin being used as antiviral drug induces reversible degenerative changes in the seminiferous tubules of testes of albino rats. (author)

  6. Passive permeability of salicylic acid in renal proximal S2 and S3 tubules

    International Nuclear Information System (INIS)

    Chatton, J.Y.; Roch-Ramel, F.

    1991-01-01

    The role of nonionic diffusion in the transport of salicylic acid across rabbit proximal S2 and S3 segments was investigated using the in vitro isolated perfused tubule technique. The [ 14 C] salicylic acid apparent reabsorptive permeability (P'I-b, 10(-5) cm/s) was measured at 19 degrees C with luminal solutions kept at different pH and bath maintained at pH 7.4. In S2 tubules, P'I-b was 25.0 +/- 3.5 when luminal pH was 6.0; P'I-b decreased to 8.1 +/- 1.4 and to 4.4 +/- 1.2 at a luminal pH of 6.5 and 7.0, respectively. In S3 tubules, P'I-b was 17.6 +/- 2.4, 5.3 +/- 1.1 and 3.4 +/- 1.1 at a luminal pH of 6.0, 6.5 and 7.0, respectively. There was a close correlation between P'I-b and the calculated proportion of nonionized salicylic acid present at each pH, indicating that only the nonionized molecule could diffuse in our conditions. We calculated the apparent permeability of nonionic salicylic acid and found 0.248 +/- 0.032 cm/s for S2 and 0.176 +/- 0.022 cm/s for S3 tubules. These calculated permeabilities were independent of pH

  7. Antioxidant Effects of Selenium on Seminiferous Tubules of Immature Mice Testis

    Directory of Open Access Journals (Sweden)

    Davoud Kushki

    2015-12-01

    Full Text Available Background: The freezing of immature testis tissue and then the transplant of it can be considered as a major step in fertility preservation for young boys with cancer, the survival of animal generation exposed to extinction and cloning animalistic desirable species. One of the most prevalent of damages in during the freezing-thawing process is oxidative stress. Objectives: The objective of this study was to investigate the antioxidant effects of selenium compound (Na2SeO3 on rate of seminiferous tubules injury in during of cryopreservation. Materials and Methods: In this experimental study, 8 BALB/c immature male mice (6 - 8 days old were randomly selected, and the testes removed surgically (n = 16. The testes divided into 2 groups: experimental group, control group (opposite testes. For each of the two experimental and control groups, two types of soluble (freezing solution and thawing solution were prepared. These solutions, which contain 2 mg/mL solution of Na2SeO3 and control solution, were prepared in the DMEM (Dulbecco’s modified eagle medium base medium. Of each group were 4 testes into fast freezing-thawing procedure and 4 testes were into slow freezing-thawing procedure. Then this testis for analyzing injury, after preparatory process, was stained with hematoxylin-eosin. Results: At the slow freezing-thawing procedure, seminiferous tubules injury significantly reduced in experimental group compared to control group. At the fast freezing-thawing procedure, seminiferous tubules injury significantly reduced in experimental group compared with of control group. Conclusions: It seems that Se due to its antioxidant properties, the harmful effects of freezing-thawing process reduces and protects seminiferous tubules from oxidative injury.

  8. Minicollagen-15, a novel minicollagen isolated from Hydra, forms tubule structures in nematocysts.

    Science.gov (United States)

    Adamczyk, Patrizia; Meier, Sebastian; Gross, Thomas; Hobmayer, Bert; Grzesiek, Stephan; Bächinger, Hans Peter; Holstein, Thomas W; Ozbek, Suat

    2008-02-29

    Minicollagens constitute a family of unusually short collagen molecules isolated from cnidarians. They are restricted to the nematocyst, a cylindrical explosive organelle serving in defense and capture of prey. The nematocyst capsule contains a long tubule inside of its matrix, which is expelled and everted during an ultrafast discharge process. Here, we report the cloning and characterization of a novel minicollagen in Hydra, designated minicollagen-15 (NCol-15). NCol-15, like NCol-3 and NCol-4, shows deviations from the canonical cysteine pattern in its terminal cysteine-rich domains (CRDs). Minicollagens share common domain architectures with a central collagen sequence flanked by polyproline stretches and short N- and C-terminal CRDs. The CRDs are involved in the formation of a highly resistant cysteine network, which constitutes the basic structure of the nematocyst capsule. Unlike NCol-1, which is part of the capsule wall, NCol-15 is localized to tubules, arguing for a functional differentiation of minicollagens within the nematocyst architecture. NMR analysis of the altered C-terminal CRD of NCol-15 showed a novel disulfide-linked structure within the cysteine-containing region exhibiting similar folding kinetics and stability as the canonical CRDs. Our data provide evidence for evolutionary diversification among minicollagens, which probably facilitated alterations in the morphology of the nematocyst wall and tubule.

  9. Effects of bioactive glass with and without mesoporous structures on desensitization in dentinal tubule occlusion

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Wen-Cheng [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Kung, Jung-Chang [Department of Family Dentistry, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Chen, Cheng-Hwei [School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Hsiao, Yu-Cheng [Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Shih, Chi-Jen, E-mail: cjshih@kmu.edu.tw [Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chien, Chi-Sheng, E-mail: jannie.gissing@msa.hinet.net [Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan (China); Department of Orthopaedics, Chi Mei Foundation Hospital, Tainan, Taiwan (China); Department of Electrical Engineering, Southern Taiwan University of Science and Technology, Tainan, Taiwan (China)

    2013-10-15

    Bioactive glass (BG) is a potential material for treating dentin hypersensitivity due to its high ability of dissolution. In this study, conventional BG and BG with well-ordered mesopore structures (MBG) were applied for dentinal tubule occlusion. We used X-ray diffractometer (XRD), scanning electronic microscope (SEM), and Fourier transform infrared (FTIR) to investigate the physiochemical properties and the dentinal tubule occlusion ability of BG and MBG groups. The results showed that the major crystallite phase of MBG and BG agents was monocalcium phosphate monohydrate. MBG pastes, mixed with 30 and 40 wt% phosphoric acid hardening solutions, had the ability to create a penetration depth greater than 50 μm. These results showed that BG with mesoporous structures turned the pastes mixed with suitable phosphoric acid solution into a material with great ability for occluding dentinal tubules; it has a short reaction time and good operability, and these agents have better potential for the treatment of dentin hypersensitivity than BG without mesoporous structures.

  10. The Lowe syndrome protein OCRL1 is required for endocytosis in the zebrafish pronephric tubule.

    Directory of Open Access Journals (Sweden)

    Francesca Oltrabella

    2015-04-01

    Full Text Available Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease.

  11. Piecewise-Constant-Model-Based Interior Tomography Applied to Dentin Tubules

    Directory of Open Access Journals (Sweden)

    Peng He

    2013-01-01

    Full Text Available Dentin is a hierarchically structured biomineralized composite material, and dentin’s tubules are difficult to study in situ. Nano-CT provides the requisite resolution, but the field of view typically contains only a few tubules. Using a plate-like specimen allows reconstruction of a volume containing specific tubules from a number of truncated projections typically collected over an angular range of about 140°, which is practically accessible. Classical computed tomography (CT theory cannot exactly reconstruct an object only from truncated projections, needless to say a limited angular range. Recently, interior tomography was developed to reconstruct a region-of-interest (ROI from truncated data in a theoretically exact fashion via the total variation (TV minimization under the condition that the ROI is piecewise constant. In this paper, we employ a TV minimization interior tomography algorithm to reconstruct interior microstructures in dentin from truncated projections over a limited angular range. Compared to the filtered backprojection (FBP reconstruction, our reconstruction method reduces noise and suppresses artifacts. Volume rendering confirms the merits of our method in terms of preserving the interior microstructure of the dentin specimen.

  12. 50 Years of renal physiology from one man and the perfused tubule: Maurice B. Burg.

    Science.gov (United States)

    Hamilton, Kirk L; Moore, Antoni B

    2016-08-01

    Technical advancements in research techniques in science are made in slow increments. Even so, large advances from insight and hard work of an individual with a single technique can have astonishing ramifications. Here, we examine the impact of Dr. Maurice B. Burg and the isolated perfused renal tubule technique and celebrate the 50th anniversary of the publication by Dr. Burg and his colleagues of their landmark paper in the American Journal of Physiology in 1966. In this study, we have taken a scientific visualization approach to study the scientific contributions of Dr. Burg and the isolated perfused tubule preparation as determining research impact by the number of research students, postdoctoral fellows, visiting scientists, and national and international collaborators. Additionally, we have examined the research collaborations (first and second generation scientists), established the migrational visualization of the first generation scientists who worked directly with Dr. Burg, quantified the metrics indices, identified and quantified the network of coauthorship of the first generation scientists with their second generation links, and determined the citations analyses of outputs of Dr. Burg and/or his first generation collaborators as coauthors. We also review the major advances in kidney physiology that have been made with the isolated perfused tubule technique. Finally, we are all waiting for the discoveries that the isolated perfused preparation technique will bring during the next 50 years. Copyright © 2016 the American Physiological Society.

  13. Vitality of Enterococcus faecalis inside dentinal tubules after five root canal disinfection methods.

    Science.gov (United States)

    Vatkar, Niranjan Ashok; Hegde, Vivek; Sathe, Sucheta

    2016-01-01

    To compare the vitality of Enterococcus faecalis within dentinal tubules after subjected to five root canal disinfection methods. Dentin blocks (n = 60) were colonized with E. faecalis. After 4 weeks of incubation, the dentin blocks were divided into one control and five test groups (n = 10 each). The root canals of test groups were subjected to one of the disinfection methods, namely, normal saline (NS), sodium hypochlorite (NaOCl), chlorhexidine digluconate (CHX), neodymium-doped yttrium aluminum garnet (Nd: YAG) laser, and diode laser. The effect of disinfection methods was assessed by LIVE/DEAD BacLight stain under the confocal laser scanning microscopy to determine the "zone of dead bacteria" (ZDB). Mean values were calculated for ZDB and the difference between groups was established. Penetration of E. faecalis was seen to a depth of >1000 μm. Viable bacteria were detected with NS irrigation. NaOCl and CHX showed partial ZDB. When the root canals were disinfected with Nd: YAG and diode lasers, no viable bacteria were found. E. faecalis has the ability to colonize inside dentinal tubules to a depth of >1000 μm. In contrast to conventional irrigants, both Nd: YAG and diode lasers were effective in eliminating the vitality of E. faecalis. NS, NaOCl, and CHX showed viable bacteria remaining in dentinal tubules.

  14. Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes.

    Science.gov (United States)

    Maher, Geoffrey J; McGowan, Simon J; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O M

    2016-03-01

    De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

  15. Subcellular distribution of folate and folate binding protein in renal proximal tubules

    International Nuclear Information System (INIS)

    Sharkey, C.; Hjelle, J.T.; Selhub, J.

    1986-01-01

    High affinity folate binding protein (FBP) found in brush border membranes derived from renal cortices is thought to be involved in the renal conservation of folate. To examine the mechanisms of folate recovery, the subcellular distribution of FBP and 3 H-folate in rabbit renal proximal tubules (PT) was examined using analytical cell fractionation techniques. Tubules contain 3.41 +/- 0.32 picomoles FBP/mg protein (X +/- S.D.; n = 5). Postnuclear supernates (PNS) of PT were layered atop Percoll-sucrose gradients, centrifuged, fractions collected and assayed for various marker enzymes and FBP. Pooled fractions from such gradients were subsequently treated with digitonin and centrifuged in a stoichiometric manner with the activity of the microvillar enzyme, alanylaminopeptidase (AAP); excess FBP distributed with more buoyant particles. Infusion of 3 H-folate into rabbit kidneys followed by tubule isolation and fractionation revealed a time dependent shift in distribution of radiolabel from the AAP-rich gradient fractions to a region containing more buoyant particles; radiolevel was not associated with lysosomal markers. EM-radioautography revealed grains over intracellular vesicles. These results are consistent with the hypothesis that folate is recovered by a process involving receptor-mediated endocytosis or transcytosis

  16. Effects of bioactive glass with and without mesoporous structures on desensitization in dentinal tubule occlusion

    International Nuclear Information System (INIS)

    Chen, Wen-Cheng; Kung, Jung-Chang; Chen, Cheng-Hwei; Hsiao, Yu-Cheng; Shih, Chi-Jen; Chien, Chi-Sheng

    2013-01-01

    Bioactive glass (BG) is a potential material for treating dentin hypersensitivity due to its high ability of dissolution. In this study, conventional BG and BG with well-ordered mesopore structures (MBG) were applied for dentinal tubule occlusion. We used X-ray diffractometer (XRD), scanning electronic microscope (SEM), and Fourier transform infrared (FTIR) to investigate the physiochemical properties and the dentinal tubule occlusion ability of BG and MBG groups. The results showed that the major crystallite phase of MBG and BG agents was monocalcium phosphate monohydrate. MBG pastes, mixed with 30 and 40 wt% phosphoric acid hardening solutions, had the ability to create a penetration depth greater than 50 μm. These results showed that BG with mesoporous structures turned the pastes mixed with suitable phosphoric acid solution into a material with great ability for occluding dentinal tubules; it has a short reaction time and good operability, and these agents have better potential for the treatment of dentin hypersensitivity than BG without mesoporous structures.

  17. Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis.

    Science.gov (United States)

    Zhou, Dong; Fu, Haiyan; Zhang, Lu; Zhang, Ke; Min, Yali; Xiao, Liangxiang; Lin, Lin; Bastacky, Sheldon I; Liu, Youhua

    2017-08-01

    Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of β -catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro , incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication. Copyright © 2017 by the American Society of Nephrology.

  18. Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes

    DEFF Research Database (Denmark)

    Hansen, Gert H; Pedersen, Jens; Niels-Christiansen, Lise-Lotte

    2003-01-01

    microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident...... lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized...... brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion...

  19. Auto-regeneration of mice testicle seminiferous tubules due to malnutrition based on stem cells mobilization using honey

    Directory of Open Access Journals (Sweden)

    Erma Safitri

    2016-03-01

    Conclusions: Results of this study revealed a significantly different of C34 and CD45 expressions between groups, also an increase SSCs expression and testicle seminiferous tubules cells regeneration as well.

  20. Destruction and regeneration of seminiferous tubules after local x-irradiation of testes of the adult rats

    International Nuclear Information System (INIS)

    Kurnosova, T.R.; Rajtsina, S.S.

    1987-01-01

    It was established that the local X-irradiation (1000 R) of testes of the adult rats results in a total destruction of seminiferous tubules. The restitution of the organ structure proceeds via formation of new seminiferous tubules in which spermatogenic epithelium later develops. Rete testis and germ cells preserved in its epithelium from embryogenesis are a source of regeneration material. The results obtained favour the suggestion about the dynamic structure of mammalian testis

  1. Far infrared radiation promotes rabbit renal proximal tubule cell proliferation and functional characteristics, and protects against cisplatin-induced nephrotoxicity.

    Science.gov (United States)

    Chiang, I-Ni; Pu, Yeong-Shiau; Huang, Chao-Yuan; Young, Tai-Horng

    2017-01-01

    Far infrared radiation, a subdivision of the electromagnetic spectrum, is beneficial for long-term tissue healing, anti-inflammatory effects, growth promotion, sleep modulation, acceleration of microcirculation, and pain relief. We investigated if far infrared radiation is beneficial for renal proximal tubule cell cultivation and renal tissue engineering. We observed the effects of far infrared radiation on renal proximal tubules cells, including its effects on cell proliferation, gene and protein expression, and viability. We also examined the protective effects of far infrared radiation against cisplatin, a nephrotoxic agent, using the human proximal tubule cell line HK-2. We found that daily exposure to far infrared radiation for 30 min significantly increased rabbit renal proximal tubule cell proliferation in vitro, as assessed by MTT assay. Far infrared radiation was not only beneficial to renal proximal tubule cell proliferation, it also increased the expression of ATPase Na+/K+ subunit alpha 1 and glucose transporter 1, as determined by western blotting. Using quantitative polymerase chain reaction, we found that far infrared radiation enhanced CDK5R1, GNAS, NPPB, and TEK expression. In the proximal tubule cell line HK-2, far infrared radiation protected against cisplatin-mediated nephrotoxicity by reducing apoptosis. Renal proximal tubule cell cultivation with far infrared radiation exposure resulted in better cell proliferation, significantly higher ATPase Na+/K+ subunit alpha 1 and glucose transporter 1 expression, and significantly enhanced expression of CDK5R1, GNAS, NPPB, and TEK. These results suggest that far infrared radiation improves cell proliferation and differentiation. In HK-2 cells, far infrared radiation mediated protective effects against cisplatin-induced nephrotoxicity by reducing apoptosis, as indicated by flow cytometry and caspase-3 assay.

  2. The proximal straight tubule (PST) basolateral cell membrane water channel: selectivity characteristics.

    Science.gov (United States)

    Gutiérrez, A M; González, E; Echevarría, M; Hernández, C S; Whittembury, G

    1995-02-01

    Proximal straight tubules (PST) were dissected from rabbit kidneys, held by crimping pipettes in a chamber and bathed in a buffered isosmotic (295 mOsm/kg) solution containing 200 mM mannitol (MBS). Changes in tubule diameter were monitored on line with an inverted microscope, TV camera and image processor. The PST were then challenged for 20 sec with MBS made 35 mOsm/kg hyperosmotic by addition of either NaCl, KCl, mannitol (M), glycerol (G), ethylene glycol (E), glycine (g), urea (U), acetamide (A) or formamide (F). With NaCl, KCl, M, G, E, g, U, and A, tubules shrunk osmometrically within 0.5 sec and remained shrunk for as long as 20 sec without recovering their original volume (sometimes A showed some recovery). PST barely shrunk with F and quickly recovered their original volume. The permeability coefficients were 0 microns/sec (NaCl, M, g, E and U), 1 micron/sec (A), 84 microns/sec (F) and 0.02 micron/sec (G). The reflection coefficients sigma = 1.0 (NaCl, KCl, M, G, E, g and U), 0.95 (A) and 0.62 (F). Similar sigma values were obtained by substituting 200 mOsm/kg M in MBS by either NaCl, KCl, G, E, g, U, a or F. The olive oil/water partition coefficients are 5 (M), 15 (U), 85 (A) and 75 (F) (all x 10(-5)). Thus, part of F permeates the cell membrane through the lipid bilayer. The probing molecules van der Waals diameters are 7.4 x 8.2 x 12.0 (M), 3.6 x 5.2 x 5.4 (U), 3.8 x 5.2 x 5.4 (A) and (3.4 x 4.5 x 5.4 (F) A.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Locally formed dopamine inhibits Na+-K+-ATPase activity in rat renal cortical tubule cells

    International Nuclear Information System (INIS)

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J.

    1988-01-01

    Dopamine, generated locally from L-dopa, inhibits Na + -K + -ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na + -K + -ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate (Qo 2 ) and 86 Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive Qo 2 or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive Qo 2 in a concentration-dependent manner, with half-maximal inhibition (K 0.5 ) of 5 x 10 -7 M and a maximal inhibition of 14.1 ± 1.5% at 10 -4 M. L-Dopa also blunted the nystatin-stimulated Qo 2 in a concentration-dependent manner, indicating the L-dopa directly inhibits Na + -K + -ATPase activity and not sodium entry. Ouabain-sensitive 86 Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive Qo 2 and 86 Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive Qo 2 at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na + -K + -ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner

  4. Reconstruction of mouse testicular cellular microenvironments in long-term seminiferous tubule culture.

    Directory of Open Access Journals (Sweden)

    Juho-Antti Mäkelä

    Full Text Available Research on spermatogonia is hampered by complex architecture of the seminiferous tubule, poor viability of testicular tissue ex vivo and lack of physiologically relevant long-term culture systems. Therefore there is a need for an in vitro model that would enable long term survival and propagation of spermatogonia. We aimed at the most simplified approach to enable all different cell types within the seminiferous tubules to contribute to the creation of a niche for spermatogonia. In the present study we describe the establishment of a co-culture of mouse testicular cells that is based on proliferative and migratory activity of seminiferous tubule cells and does not involve separation, purification or differential plating of individual cell populations. The co-culture is composed of the constituents of testicular stem cell niche: Sertoli cells [identified by expression of Wilm's tumour antigen 1 (WT1 and secretion of glial cell line-derived neurotrophic factor, GDNF], peritubular myoid cells (expressing alpha smooth muscle actin, αSMA and spermatogonia [expressing MAGE-B4, PLZF (promyelocytic leukaemia zinc finger, LIN28, Gpr125 (G protein-coupled receptor 125, CD9, c-Kit and Nanog], and can be maintained for at least five weeks. GDNF was found in the medium at a sufficient concentration to support proliferating spermatogonial stem cells (SSCs that were able to start spermatogenic differentiation after transplantation to an experimentally sterile recipient testis. Gdnf mRNA levels were elevated by follicle-stimulating hormone (FSH which shows that the Sertoli cells in the co-culture respond to physiological stimuli. After approximately 2-4 weeks of culture a spontaneous formation of cord-like structures was monitored. These structures can be more than 10 mm in length and branch. They are formed by peritubular myoid cells, Sertoli cells, fibroblasts and spermatogonia as assessed by gene expression profiling. In conclusion, we have managed to

  5. NMR-based urine analysis in rats: prediction of proximal tubule kidney toxicity and phospholipidosis.

    Science.gov (United States)

    Lienemann, Kai; Plötz, Thomas; Pestel, Sabine

    2008-01-01

    The aim of safety pharmacology is early detection of compound-induced side-effects. NMR-based urine analysis followed by multivariate data analysis (metabonomics) identifies efficiently differences between toxic and non-toxic compounds; but in most cases multiple administrations of the test compound are necessary. We tested the feasibility of detecting proximal tubule kidney toxicity and phospholipidosis with metabonomics techniques after single compound administration as an early safety pharmacology approach. Rats were treated orally, intravenously, inhalatively or intraperitoneally with different test compounds. Urine was collected at 0-8 h and 8-24 h after compound administration, and (1)H NMR-patterns were recorded from the samples. Variation of post-processing and feature extraction methods led to different views on the data. Support Vector Machines were trained on these different data sets and then aggregated as experts in an Ensemble. Finally, validity was monitored with a cross-validation study using a training, validation, and test data set. Proximal tubule kidney toxicity could be predicted with reasonable total classification accuracy (85%), specificity (88%) and sensitivity (78%). In comparison to alternative histological studies, results were obtained quicker, compound need was reduced, and very importantly fewer animals were needed. In contrast, the induction of phospholipidosis by the test compounds could not be predicted using NMR-based urine analysis or the previously published biomarker PAG. NMR-based urine analysis was shown to effectively predict proximal tubule kidney toxicity after single compound administration in rats. Thus, this experimental design allows early detection of toxicity risks with relatively low amounts of compound in a reasonably short period of time.

  6. Caveolin-3 is associated with the T-tubules of mature skeletal muscle fibers

    DEFF Research Database (Denmark)

    Ralston, E; Ploug, Thorkil

    1999-01-01

    Caveolae are abundant in skeletal muscle and their coat contains a specific isoform of caveolin, caveolin-3. It has been suggested that during muscle development, caveolin-3 is associated with the T-tubules, but that in adult muscle it is found on the plasma membrane only. We have studied...... the distribution of caveolin-3 in single skeletal muscle fibers from adult rat soleus by confocal immunofluorescence and by immunogold electron microscopy. We found that caveolin-3 occurs at the highest density on the plasma membrane but is also present in the core of the fibers, at the I-band/A-band interface...

  7. Acute hypotension induced by aortic clamp vs. PTH provokes distinct proximal tubule Na+ transporter redistribution patterns

    DEFF Research Database (Denmark)

    Leong, Patrick K K; Yang, Li E; Lin, Harrison W

    2004-01-01

    . This study aimed to determine the effects of acute hypotension, induced by aortic clamp or by high-dose PTH (100 microg PTH/kg), on renal hemodynamics and proximal tubule Na/H exchanger isoform 3 (NHE3) and type IIa Na-P(i) cotransporter protein (NaPi2) distribution. Subcellular distribution was analyzed...... clearance. There was, however, no significant change in glomerular filtration rate (GFR) or subcellular distribution of NHE3 and NaPi2. In contrast, high-dose PTH rapidly (

  8. Automated tubule nuclei quantification and correlation with oncotype DX risk categories in ER+ breast cancer whole slide images

    Science.gov (United States)

    Romo-Bucheli, David; Janowczyk, Andrew; Romero, Eduardo; Gilmore, Hannah; Madabhushi, Anant

    2016-03-01

    Early stage estrogen receptor positive (ER+) breast cancer (BCa) treatment is based on the presumed aggressiveness and likelihood of cancer recurrence. The primary conundrum in treatment and management of early stage ER+ BCa is identifying which of these cancers are candidates for adjuvant chemotherapy and which patients will respond to hormonal therapy alone. This decision could spare some patients the inherent toxicity associated with adjuvant chemotherapy. Oncotype DX (ODX) and other gene expression tests have allowed for distinguishing the more aggressive ER+ BCa requiring adjuvant chemotherapy from the less aggressive cancers benefiting from hormonal therapy alone. However these gene expression tests tend to be expensive, tissue destructive and require physical shipping of tissue blocks for the test to be done. Interestingly breast cancer grade in these tumors has been shown to be highly correlated with the ODX risk score. Unfortunately studies have shown that Bloom-Richardson (BR) grade determined by pathologists can be highly variable. One of the constituent categories in BR grading is the quantification of tubules. The goal of this study was to develop a deep learning neural network classifier to automatically identify tubule nuclei from whole slide images (WSI) of ER+ BCa, the hypothesis being that the ratio of tubule nuclei to overall number of nuclei would correlate with the corresponding ODX risk categories. The performance of the tubule nuclei deep learning strategy was evaluated with a set of 61 high power fields. Under a 5-fold cross-validation, the average precision and recall measures were 0:72 and 0:56 respectively. In addition, the correlation with the ODX risk score was assessed in a set of 7513 high power fields extracted from 174 WSI, each from a different patient (At most 50 high power fields per patient study were used). The ratio between the number of tubule and non-tubule nuclei was computed for each WSI. The results suggests that for BCa

  9. Isolation and Characterization of Adhesive Secretion from Cuvierian Tubules of Sea Cucumber Holothuria forskåli (Echinodermata: Holothuroidea

    Directory of Open Access Journals (Sweden)

    Malgorzata Baranowska

    2011-01-01

    Full Text Available The sea cucumber Holothuria forskåli possesses a specialized system called Cuvierian tubules. During mechanical stimulation white filaments (tubules are expelled and become sticky upon contact with any object. We isolated a protein with adhesive properties from protein extracts of Cuvierian tubules from H. forskåli. This protein was identified by antibodies against recombinant precollagen D which is located in the byssal threads of the mussel Mytilus galloprovincialis. To find out the optimal procedure for extraction and purification, the identified protein was isolated by several methods, including electroelution, binding to glass beads, immunoprecipitation, and gel filtration. Antibodies raised against the isolated protein were used for localization of the adhesive protein in Cuvierian tubules. Immunostaining and immunogold electron microscopical studies revealed the strongest immunoreactivity in the mesothelium; this tissue layer is involved in adhesion. Adhesion of Cuvierian tubule extracts was measured on the surface of various materials. The extracted protein showed the strongest adhesion to Teflon surface. Increased adhesion was observed in the presence of potassium and EDTA, while cadmium caused a decrease in adhesion. Addition of antibodies and trypsin abolished the adhesive properties of the extract.

  10. Diglycolic acid inhibits succinate dehydrogenase activity in human proximal tubule cells leading to mitochondrial dysfunction and cell death.

    Science.gov (United States)

    Landry, Greg M; Dunning, Cody L; Conrad, Taylor; Hitt, Mallory J; McMartin, Kenneth E

    2013-08-29

    Diethylene glycol (DEG) is a solvent used in consumer products allowing the increased risk for consumer exposure. DEG metabolism produces two primary metabolites, 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA). DGA has been shown to be the toxic metabolite responsible for the proximal tubule cell necrosis seen in DEG poisoning. The mechanism of DGA toxicity in the proximal tubule cell is not yet known. The chemical structure of DGA is very similar to citric acid cycle intermediates. Studies were designed to assess whether its mechanism of toxicity involves disruption of cellular metabolic pathways resulting in mitochondrial dysfunction. First, DGA preferentially inhibited succinate dehydrogenase, including human kidney cell enzyme, but had no effect on other citric acid cycle enzyme activities. DGA produces a cellular ATP depletion that precedes cell death. Human proximal tubule (HPT) cells, pre-treated with increasing DGA concentrations, showed significantly decreased oxygen consumption. DGA did not increase lactate levels, indicating no effect on glycolytic activity. DGA increased reactive oxygen species (ROS) production in HPT cells in a concentration and time dependent manner. These results indicate that DGA produced proximal tubule cell dysfunction by specific inhibition of succinate dehydrogenase and oxygen consumption. Disruption of these processes results in decreased energy production and proximal tubule cell death. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  11. Transport of water in proximal kidney tubules from whole tubules to single channels: length and section of the selectivity filter of aquaporin-1.

    Science.gov (United States)

    Whittembury, G; González, E; Hernández, C S; Gutiérrez, A M; Echevarría, M

    1997-06-27

    Proximal straight tubule (PST) were dissected from rabbit kidneys, held with crimping pipettes in a chamber bathed in a buffered mannitol isosmotic solution (MBS, 295 mOsm/kg). Tubule cell volume changes with time (dV/Adt) after steps in MBS osmolality (delta Cs) were monitored on line with an inverted microscope, a TV camera and an image processor. Reflection coefficients sigma and osmotic permeability coefficients, Pos, for several solutes were measured using two methods. Method 1: sigma was calculated from the delta Csiso of impermeant and permeant solutes at which (dV/Adt)t-->0 = 0 (i.e., by a null point method). It is denoted as sigma 1. sigma 1 = 1.00 for mannitol (M), raffinose (R), sucrose (S), glycerol (G), acetamide (A) and urea (U). With formamide (F), sigma 1, Formamide = 0.62 +/- 0.05. These findings confirm our previous value of dp = 4.5 A for the diameter of the selectivity filter of the basolateral PST cell membrane water channel AQP1. Method 2: PST were exposed for 20 s to MBS made hyperosmotic by addition of a delta Cs of 35 mOsm/kg of R, S, M, G, A and U. Cells shrunk within 500 ms of t = 0 to their osmometric volume and remained shrunk for the 20 s of the osmotic challenge. Pos was measured from the shrinking curves. P(os) = 3000 +/- 25 microns/s with R, S, M, G, A and U. Method 2 also allowed to calculate sigma, denoted as sigma 2. sigma 2 = 1.00 for R, S, M, G, A and U. By contrast, the shrinking curve produced by a delta Cs of 35 mOsm/kg F was 1/5th to 1/6th slower and smaller (i.e., subosmometric) than that produced by a delta Cs of 35 mOsm/kg R, S, M, G, A and U. Furthermore, with F cells did not remain shrunk but recovered their original volume within 3 s. P(os) (measured with F) is denoted as P(os)*, P(os)* = 480 +/- 30 microns/s. sigma 2, Formamide = 0.16 +/- 0.01. Use of sigma 1, sigma 2 and P(os)* values in Hill's equations for the bimodal theory of osmosis leads to n = 2-9. Where n is the number of water molecules single filling

  12. Effect of Tamoxifen on Seminiferous Tubules Structure during Pregnancy in Adult Mice

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    J Soleimani Rad

    2016-03-01

    Full Text Available Introduction: Tamoxifen is a nonsteroidal drug which mainly treats breast cancer. It is also applied for stimulation of ovulation and remedy of infertility. Regarding the tamoxifen binding to estrogen receptors and the possible role of estrogens in spermatogenesis, the present study aimed to histologically evaluate spermatogenesis in the seminiferous ducts of mice, whose mothers had received tamoxifen during pregnancy. Methods: In the present study, 30 female and 15 male mice of NMRI race were selected for mating. Since 13th day of pregnancy, the experimental group received tamoxifen with the dosage of 5 mg/kg intra-peritoneally for 7 days, wherease the control group received normal saline. After childbirth of the mated mice, male infants were selected and monitored in the standard laboratory conditions. After reaching the age of puberty (6-8Weeks, adult mice were sacrificed by the cervical dislocation, and the testes were removed for histological evaluation of spermatogenesis. After routine histological processing, the samples were studied by the light microscope. Results: Histological studies showed that spermatogenic and Sertoli cells in the seminiferous tubules in control and experimental groups were significantly different, though no difference was observed in the number of Leydig cells in the both groups. Conclusion: The findings of the present study showed that tamoxifen exposure during development can cause histological changes in the seminiferous tubules, which can lead to infertility in the male rat.

  13. A bioartificial renal tubule device embedding human renal stem/progenitor cells.

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    Anna Giovanna Sciancalepore

    Full Text Available We present a bio-inspired renal microdevice that resembles the in vivo structure of a kidney proximal tubule. For the first time, a population of tubular adult renal stem/progenitor cells (ARPCs was embedded into a microsystem to create a bioengineered renal tubule. These cells have both multipotent differentiation abilities and an extraordinary capacity for injured renal cell regeneration. Therefore, ARPCs may be considered a promising tool for promoting regenerative processes in the kidney to treat acute and chronic renal injury. Here ARPCs were grown to confluence and exposed to a laminar fluid shear stress into the chip, in order to induce a functional cell polarization. Exposing ARPCs to fluid shear stress in the chip led the aquaporin-2 transporter to localize at their apical region and the Na(+K(+ATPase pump at their basolateral portion, in contrast to statically cultured ARPCs. A recovery of urea and creatinine of (20±5% and (13±5%, respectively, was obtained by the device. The microengineered biochip here-proposed might be an innovative "lab-on-a-chip" platform to investigate in vitro ARPCs behaviour or to test drugs for therapeutic and toxicological responses.

  14. Transport of Streptococcus pneumoniae capsular polysaccharide in MHC Class II tubules.

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    Tom Li Stephen

    2007-03-01

    Full Text Available Bacterial capsular polysaccharides are virulence factors and are considered T cell-independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4(+ T cells in a major histocompatibility complex (MHC class II-dependent manner. The mechanism of carbohydrate presentation to CD4(+ T cells is unknown. We show in live murine dendritic cells (DCs that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell-dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide-carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens.

  15. Effects of angiotensin II and ionomycin on fluid and bicarbonate absorption in the rat proximal tubule

    International Nuclear Information System (INIS)

    Chatsudthipong, V.; Chan, Y.L.

    1986-01-01

    Microperfusion of proximal convoluted tubule(PCT) and peritubular capillaries was performed to examine the effects of angiotensin II(Ang II) and ionomycin on fluid and bicarbonate absorption. Bicarbonate was determined by microcalorimetry and C-14 inulin was used as a volume marker. The rates of bicarbonate absorption (JHCO 3 ) was 143 peq/min x mm and fluid absorption(Jv) was 2.70 nl/min x mm, when PCT and capillary perfusate contained normal Ringer solution. Addition of Ang II (10 -6 M) to the capillary perfusate caused reductions of JHCO 3 and Jv by 35%. A similar effect was observed when ionomycin was added to the capillary perfusate. Ang II antagonist, (Sar 1 , Ile 8 )-Angiotensin II(10 -6 M), completely blocked the inhibitory effect of Ang II on Jv and JHCO 3 . Removal of calcium from both luminal and capillary perfusate did not change the effect of Ang II on Jv and JHCO 3 . Our results indicate that Ang II inhibits the sodium-hydrogen exchanger in the proximal tubule via interacting with angiotensin receptor. The mechanism of Ang II action may involve mobilization of intracellular calcium

  16. Role of calcium in the dopaminergic effect on the proximal convoluted tubule of rat kidney

    International Nuclear Information System (INIS)

    Chan, Y.L.; Chatsudthipong, V.; Su-Tsai, S.M.; von Riotte, A.

    1986-01-01

    Microperfusion studies have shown that dopamine inhibits fluid and bicarbonate absorption in the rate proximal tubule. These studies are designed to examine the cellular mechanism underlying the proximal cellular response to dopamine action. In the isolated proximal cells, dopamine, in the concentration of 10 -6 M or less, had no effect on cAMP production. However, dopamine could increase cytosolic calcium concentration (from 90 nM to 210 nM) as measured by fluorospectrometry with fura-2 as a calcium indicator. Ca-45 flux studies have shown that dopamine could increase initial influx but not the steady state uptake of Ca. Dopamine could also increase efflux of Ca. In situ microperfusion of proximal tubule and peritubular capillaries has demonstrated that Ca ionophore, A23187 (10 -6 M), could simulate the inhibitory effects of dopamine on fluid and biocarbonate absorption. There was no additive effect observed when both agents were added together in the capillary perfusate. Removal of calcium from the perfusate could partially blunt the effect of dopamine. These results suggest that intracellular calcium plays a crucial role in the dopaminergic regulation of proximal tubular transport

  17. Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules.

    Science.gov (United States)

    Armour, Eric A; Carson, Robert P; Ess, Kevin C

    2012-08-15

    Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia.

  18. Effect of a novel bioactive glass-ceramic on dentinal tubule occlusion: an in vitro study.

    Science.gov (United States)

    Zhong, Y; Liu, J; Li, X; Yin, W; He, T; Hu, D; Liao, Y; Yao, X; Wang, Y

    2015-03-01

    This in vitro study aimed to assess the ability and efficacy of HX-BGC, a novel bioactive glass-ceramic (SiO2-P2 O5-CaO-Na2 O-SrO), to reduce dentine tubule permeability. Dentine discs from human third molars were etched and randomly allocated into five groups: Group 1--distilled water; Group 2--Sensodyne Repair toothpaste (containing NovaMin®); Group 3--HX-BGC toothpaste (containing 7.5% HX-BGC); Group 4--control toothpaste (without HX-BGC); and Group 5--HX-BGC powder. Specimens were treated daily by brushing with an electric toothbrush for 20 seconds. Between daily treatments (7 days total), specimens were immersed in artificial saliva for 24 hours. Dentine permeability was measured at baseline, after the first treatment, after the first 24-hour immersion in artificial saliva and at the end of day 7. Dentine morphology and surface deposits were observed by scanning electron microscopy after one day and 7 days of treatment, respectively. Sensodyne Repair and bioactive glass-ceramic toothpaste significantly and immediately lowered dentine permeability. The HX-BGC powder group showed the highest reduction in dentine permeability after 7 days of treatment. The novel bioactive glass-ceramic material HX-BGC is effective in reducing dentine permeability by occluding open dentine tubules, indicating that HX-BGC may be a potential treatment for dentine hypersensitivity. © 2015 Australian Dental Association.

  19. Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension

    International Nuclear Information System (INIS)

    Ecay, T.W.; Valentich, J.D.

    1991-01-01

    Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of 3 H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases in inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells

  20. Effects of lead intoxication on intercellular junctions and biochemical alterations of the renal proximal tubule cells.

    Science.gov (United States)

    Navarro-Moreno, L G; Quintanar-Escorza, M A; González, S; Mondragón, R; Cerbón-Solorzáno, J; Valdés, J; Calderón-Salinas, J V

    2009-10-01

    Lead intoxication is a worldwide health problem which frequently affects the kidney. In this work, we studied the effects of chronic lead intoxication (500 ppm of Pb in drinking water during seven months) on the structure, function and biochemical properties of rat proximal tubule cells. Lead-exposed animals showed increased lead concentration in kidney, reduction of calcium and amino acids uptake, oxidative damage and glucosuria, proteinuria, hematuria and reduced urinary pH. These biochemical and physiological alterations were related to striking morphological modifications in the structure of tubule epithelial cells and in the morphology of their mitochondria, nuclei, lysosomes, basal and apical membranes. Interestingly, in addition to the nuclei, inclusion bodies were found in the cytoplasm and in mitochondria. The epithelial cell structure modifications included an early loss of the apical microvillae, followed by a decrement of the luminal space and the respective apposition and proximity of apical membranes, resulting in the formation of atypical intercellular contacts and adhesion structures. Similar but less marked alterations were observed in subacute lead intoxication as well. Our work contributes in the understanding of the physiopathology of lead intoxication on the structure of renal tubular epithelial cell-cell contacts in vivo.

  1. A Molecular Mechanism to Regulate Lysosome Motility for Lysosome Positioning and Tubulation

    Science.gov (United States)

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-01-01

    To mediate the degradation of bio-macromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca2+ channel TRPML1 cause lysosome storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca2+-dependent centripetal movement of lysosomes towards the perinuclear region, where autophagosomes accumulate, upon autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca2+ sensor that associates physically with the minus-end directed dynactin-dynein motor, while PI(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PI(3,5)P2-TRPML1-ALG-2-dynein signaling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Collectively, Ca2+ release from lysosomes provides an on-demand mechanism regulating lysosome motility, positioning, and tubulation. PMID:26950892

  2. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria

    Directory of Open Access Journals (Sweden)

    E.V. Seliverstova

    2015-04-01

    Full Text Available The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes, and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

  3. Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules

    Science.gov (United States)

    Armour, Eric A.; Carson, Robert P.

    2012-01-01

    Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia. PMID:22674026

  4. Early effects of aldosterone on Na-K pump in rat cortical collecting tubules

    International Nuclear Information System (INIS)

    Fujii, Y.; Takemoto, F.; Katz, A.I.

    1990-01-01

    Sustained exposure to aldosterone (Aldo) increases the abundance and activity of the Na-K pump in cortical collecting tubules (CCT). However, the onset and mechanism of the early interaction of Aldo with the CCT pump, especially in adrenal-intact animals, are unclear. We evaluated the short-term effects of the hormone on Na-K-adenosinetriphosphatase (ATPase) activity and on ouabain-sensitive 86Rb uptake, a measure of the transporting rate of the pump, in microdissected CCT from adrenal-intact rats. Incubation with Aldo (10(-8) M, 2 h) had no effect on Na-K-ATPase activity (Vmax), whereas it produced at least a twofold increase in 86Rb uptake. This effect was generated by physiological concentrations of the hormone (threshold 10(-10) M; apparent K1/2 approximately 10(-9) M), after a short lag of less than or equal to 30 min. Incubation with Aldo in the presence of amiloride or nystatin or in a Na-free medium (choline chloride) did not prevent the enhanced 86Rb uptake seen after Aldo alone; possible interpretations of these observations are discussed. We conclude that Aldo produces a rapid stimulation of pump function in CCT that precedes its induction of new pump synthesis; the physiological significance of this effect is suggested by its occurrence in tubules from adrenal-intact animals within the time frame and concentration range of the hormone's effects on electrolyte transport

  5. Cystine uptake by cultured cells originating from dog proximal tubule segments

    International Nuclear Information System (INIS)

    States, B.; Reynolds, R.; Lee, J.; Segal, S.

    1990-01-01

    Large numbers of kidney epithelial cells were cultured successfully from isolated dog proximal tubule segments. Cells in primary culture and in first passage retained the cystine-dibasic amino acid co-transporter system which is found in vivo and in freshly isolated proximal tubule segments. In contrast to other cultured cells, the cystine-glutamate anti-porter was absent in primary cultures. However, this anti-porter system seemed to be developing in cells in first passage. The intracellular ratio of cysteine:reduced glutathione (CSH:GSH) was maintained at 1:36 in both primary cultures and in low passage cells. Incubation of cells in primary culture for 5 min at 37 degrees C with 0.025 mM [ 35 S]L-cystine resulted in incorporation of approximately 36 and 8.5% of the label into intracellular CSH and GSH, respectively. These cultured cells, therefore, seem to be an excellent model system for the eventual elucidation of (a) the inticacies of cystine metabolism and (b) regulation of (1) the cystine-dibasic amino acid co-transporter system and (2) the development of the cysteine-glutamate anti-porter system

  6. A telomerase immortalized human proximal tubule cell line with a truncation mutation (Q4004X in polycystin-1.

    Directory of Open Access Journals (Sweden)

    Brittney-Shea Herbert

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells.

  7. Effects of two desensitizing dentifrices on dentinal tubule occlusion with citric acid challenge: Confocal laser scanning microscopy study

    Directory of Open Access Journals (Sweden)

    Sneha Anil Rajguru

    2017-01-01

    Full Text Available Background: Dentin hypersensitivity results when patent tubules are exposed to pain-inducing external stimuli. Aim: This study aims to compare the effects of two desensitizing dentifrices containing NovaMin and arginine on dentinal tubule occlusion with and without citric acid challenge in vitro using confocal laser scanning microscopy (CLSM. Materials and Methods: Forty dentin discs were randomly divided into Groups I and II containing twenty specimens each, treated with NovaMin and arginine-containing dentifrices, respectively. Groups I and II were divided into subgroups A and B where IA and IIA underwent CLSM analysis to determine the percentage of tubule occlusion while IB and IIB underwent 0.3% citric acid challenge and CLSM analysis. A novel grading system was devised to categorize tubule occlusion. Results: In Group II, the percentage of occluded tubules was highest for IIA (72.25% ± 10.57% and least for IIB (42.55% ± 8.65% having statistical significance (P < 0.0005. In Group I, the difference between IA (49.9% ± 12.96% and IB (43.15% ± 12.43% was statistically insignificant (P = 0.249. On the comparison between IB and IIB statistically indifferent result was obtained (P = 0.901, whereas the difference between IA and IIA was statistically significant (P < 0.001. The results of grading system were for IA 50% of samples belonged to Grade 2, for IIA 60% - Grade 3, and for IB 70% and for IIB 90% - Grade 2. Conclusion: Dentinal tubule occlusion with arginine-containing dentifrice was significantly higher than NovaMin. However, it could not resist citric acid challenge as effectively as NovaMin. The effects of NovaMin were more sustainable as compared to arginine-containing dentifrice, thus proving to be a better desensitizing agent.

  8. Comparative proteomic analysis of kidney distal convoluted tubule and cortical collecting duct cells following long-term hormonal stimulation

    DEFF Research Database (Denmark)

    Wu, Qi; Moller, Hanne; Rosenbaek, Lena Lindtoft

    2017-01-01

    The distal convoluted tubule (DCT) and the cortical collecting ducts (CCD) are portions of renal tubule that are partly responsible for maintaining the systemic concentrations of potassium, sodium, calcium and magnesium. Despite being structurally similar, DCT and CCD cells have different transpo...... FDR threshold in one cell type plus the unique proteins in this cell type. These 1025 mpkDCT specific proteins and 1211 mpkCCD specific proteins under the three conditions were subjected to further bioinformatics analyses including Panther and DAVID gene ontology analyses, E3 ligase...

  9. Locally formed dopamine inhibits Na sup + -K sup + -ATPase activity in rat renal cortical tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J. (Harvard Medical School, Boston, MA (USA) Karolinska Institute, Stockholm (Sweden))

    1988-10-01

    Dopamine, generated locally from L-dopa, inhibits Na{sup +}-K{sup +}-ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na{sup +}-K{sup +}-ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate ({dot Q}o{sub 2}) and {sup 86}Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive {dot Q}o{sub 2} or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive {dot Q}o{sub 2} in a concentration-dependent manner, with half-maximal inhibition (K{sub 0.5}) of 5 {times} 10{sup {minus}7} M and a maximal inhibition of 14.1 {plus minus} 1.5% at 10{sup {minus}4}M. L-Dopa also blunted the nystatin-stimulated {dot Q}o{sub 2} in a concentration-dependent manner, indicating the L-dopa directly inhibits Na{sup +}-K{sup +}-ATPase activity and not sodium entry. Ouabain-sensitive {sup 86}Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} and {sup 86}Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na{sup +}-K{sup +}-ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner.

  10. Self-organization of engineered epithelial tubules by differential cellular motility

    Energy Technology Data Exchange (ETDEWEB)

    Mori, Hidetoshi; Gjorevski, Nikolce; Inman, Jamie L; Bissell, Mina J; Nelson, Celeste M

    2009-02-04

    Patterning of developing tissues arises from a number of mechanisms, including cell shape change, cell proliferation, and cell sorting from differential cohesion or tension. Here, we reveal that differences in cell motility can also lead to cell sorting within tissues. Using mosaic engineered mammary epithelial tubules, we found that cells sorted depending on their expression level of the membrane-anchored collagenase matrix metalloproteinase (MMP)-14. These rearrangements were independent of the catalytic activity of MMP14 but absolutely required the hemopexin domain. We describe a signaling cascade downstream of MMP14 through Rho kinase that allows cells to sort within the model tissues. Cell speed and persistence time were enhanced by MMP14 expression, but only the latter motility parameter was required for sorting. These results indicate that differential directional persistence can give rise to patterns within model developing tissues.

  11. Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

    International Nuclear Information System (INIS)

    Backues, Steven K.; Bednarek, Sebastian Y.

    2010-01-01

    The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.

  12. Human kidney proximal tubule cells are vulnerable to the effects of Rauwolfia serpentina.

    Science.gov (United States)

    Mossoba, Miriam E; Flynn, Thomas J; Vohra, Sanah; Wiesenfeld, Paddy L; Sprando, Robert L

    2015-12-01

    Rauwolfia serpentina (or Snake root plant) is a botanical dietary supplement marketed in the USA for maintaining blood pressure. Very few studies have addressed the safety of this herb, despite its wide availability to consumers. Its reported pleiotropic effects underscore the necessity for evaluating its safety. We used a human kidney cell line to investigate the possible negative effects of R. serpentina on the renal system in vitro, with a specific focus on the renal proximal tubules. We evaluated cellular and mitochondrial toxicity, along with a variety of other kidney-specific toxicology biomarkers. We found that R. serpentina was capable of producing highly detrimental effects in our in vitro renal cell system. These results suggest more studies are needed to investigate the safety of this dietary supplement in both kidney and other target organ systems.

  13. Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties ▿

    Science.gov (United States)

    Sánchez-Navarro, Jesús; Fajardo, Thor; Zicca, Stefania; Pallás, Vicente; Stavolone, Livia

    2010-01-01

    Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway. PMID:20130061

  14. Proximal tubule Na transporter responses are the same during acute and chronic hypertension

    DEFF Research Database (Denmark)

    Magyar, C E; Zhang, Y; Holstein-Rathlou, N H

    2000-01-01

    Acute hypertension in Sprague-Dawley rats (SD) provokes a decrease in renal proximal tubule (PT) salt and fluid reabsorption, redistribution of apical Na/H exchanger isoform 3 (NHE3) and Na-P(i) cotransporter type 2 (NaPi2) out of the brush border into higher density membranes, and inhibition...... to persistent adaptive changes in NHE3 and NaPi2 distribution and/or NKA activity. Renal cortex Na transporter protein density distributions and activities were compared by subcellular fractionation in 1) adult SHR with an acute increase or decrease in arterial pressure and 2) young SD (YSD) and young SHR (YSHR......) vs. adult SD and SHR. In adult hypertensive SHR NHE3 was shifted to membranes of higher densities, analogous to SD with acute hypertension, and there were no further changes with a further increase or decrease in arterial pressure. There was no change in total pool size of NHE3 in cortex in YSHR vs...

  15. Effects of phospho- and calciotropic hormones on electrolyte transport in the proximal tubule

    DEFF Research Database (Denmark)

    Lee, Justin J; Plain, Allein; Beggs, Megan R

    2017-01-01

    ), active vitamin D 3, and fibroblast growth factor 23 (FGF23). The organs central to this are the kidneys, intestine, and bone. In the kidney, the proximal tubule reabsorbs the majority of filtered calcium and phosphate, which amounts to more than 60% and 90%, respectively. The basic molecular mechanisms......Calcium and phosphate are critical for a myriad of physiological and cellular processes within the organism. Consequently, plasma levels of calcium and phosphate are tightly regulated. This occurs through the combined effects of the phospho- and calciotropic hormones, parathyroid hormone (PTH...... as their regulation of active vitamin D 3 synthesis in this nephron segment. The integrative effects of both phospho- and calciotropic hormones on proximal tubular solute transport and subsequently whole body calcium-phosphate balance thus have been further complicated. Here, we first review the molecular mechanisms...

  16. Antioxidant Protection against Pathological Mycotoxins Alterations on Proximal Tubules in Rat Kidney

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    Susan Abdu

    2011-04-01

    Full Text Available Background: Ochratoxin A (OTA was one of the mycotoxins and received attention worldwide because of the hazard it posed to human and animal health, where the kidney was the primary target organ for OTA toxicity. In the other hand, dates served as a good source of natural antioxidants and could potentially be considered as a functional food.Methods: The study was performed in the department of biology in King Abdulaziz University. Animals were gavage administrated and divided into four groups: first group received (sodium bicarbonate, second group received (289 µg OTA /kg B.W. /day, third group received (1mg Ajwa/kg B.W. / day and fourth group received (289 µg OTA /kg B.W./day+ 1mg Ajwa /kg B.W. / day. Serum (creatinine - urea levels were measured in each group at the time of tissue collection , some biopsies were fixed in 10% buffered formalin solution for light microscopy processing stained with Haematoxylin and Eosin (H& E., Periodic Acid-Schiff (PAS and Masson´s Trichrome (M.T..Other biopsies were immediately collected into electron microscopy processing. Results: After 28 days, a significant decrease in body weight, kidney weight and relative weight was detected in OTA treated group. Also, Serum (creatinine - urea level were elevated .The normal cyto-architecture of proximal tubules were lost exhibiting damaged bruch border, degenerated, binucleated and karyomegalic cells. The most destructed ultra-structure was the mitochondria which severely swollen with disintegrated membranes. In Ajwa Date extract-group the proximal tubules were normal, whereas in Ajwa date extract + OTA -group the severity of the lesions was significantly reduced. Conclusion: The present results indicated that, Ajwa date have protective effects and ameliorated the lesions of Ochratoxin nepherotoxicity which might lead to kidney failure.

  17. Dentinal tubule occluding capability of nano-hydroxyapatite; The in-vitro evaluation.

    Science.gov (United States)

    Baglar, Serdar; Erdem, Umit; Dogan, Mustafa; Turkoz, Mustafa

    2018-04-29

    In this in-vitro study, the effectiveness of experimental pure nano-hydroxyapatite (nHAP) and 1%, 2%, and 3% F¯ doped nano-HAp on dentine tubule occlusion was investigated. And also, the cytotoxicity of materials used in the experiment was evaluated. Nano-HAp types were synthesized by the precipitation method. Forty dentin specimens were randomly divided into five groups of; 1-no treatment (control), 2-specimens treated with 10% pure nano-HAp and 3, 4, 5 specimens treated with 1%, 2%, and 3% F - doped 10% nano-HAp, respectively. To evaluate the effectiveness of the materials used; pH, FTIR, and scanning electron microscopy evaluations were performed before and after degredation in simulated body fluid. To determine cytotoxicity of the materials, MTT assay was performed. Statistical evaluations were performed with F and t tests. All of the nano-HAp materials used in this study built up an effective covering layer on the dentin surfaces even with plugs in tubules. It was found that this layer had also a resistance to degradation. None of the evaluated nano-HAp types were have toxicity. Fluoride doping showed a positive effect on physical and chemical stability until a critical value of 1% F - . The all evaluated nano-HAp types may be effectively used in dentin hypersensitivity treatment. The formed nano-HAp layers were seem to resistant to hydrolic deletion. The pure and 1% F - doped nano-HAp showed the highest biocompatibility thus it was assessed that pure and 1% F - doped materials may be used as an active ingredient in dentin hypersensitivity agents. © 2018 Wiley Periodicals, Inc.

  18. Efficacy of Modified Bioactive Glass for Dentin Remineralization and Obstruction of Dentinal Tubules

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    Mahshid Saffarpour

    2017-10-01

    Full Text Available Objectives: This study assessed the efficacy of modified bioactive glass (MBG for dentin remineralization and obstruction of dentinal tubules.Materials and Methods: Thirty-six dentin discs were made from 20 third molars and were stored in 12% lactic acid solution for two weeks to induce demineralization. The samples were divided into three groups (n=12: 1- BG, 2- BG modified with 5% strontium (Sr and 3- BG modified with 10% Sr. After applying the BG, the samples were stored in artificial saliva for 7, 14 and 21 days. Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR, X-ray Diffraction (XRD analysis, Scanning Electron Microscopy (SEM, and Energy-Dispersive X-ray (EDX analysis were used to assess remineralization. Also, 6 dentin discs were divided into three groups of BG, BG modified with 5% Sr and BG modified with 10% Sr, to examine tubular occlusion. The discs were etched using 0.5M of EDTA for two minutes and were stored in artificial saliva for 7 days. Changes in dentin surface morphology were evaluated under SEM.Results: Group 3 showed high rates of remineralization at days 7 and 14, although the rate decreased at day 21. Group 2 exhibited high rates of remineralization at days 7, 14 and 21. Dentinal tubules were partially occluded by BG and BG modified with 5% Sr, while they were almost completely obstructed after the use of BG modified with 10% Sr.Conclusions: Strontium increases remineralization. Addition of 10% Sr to BG enhances apatite formation; however, the apatite dissolves over time. Addition of 5% Sr to BG stabilizes the apatite lattice and increases the remineralization.

  19. Role of diacylglycerol in adrenergic-stimulated sup 86 Rb uptake by proximal tubules

    Energy Technology Data Exchange (ETDEWEB)

    Baines, A.D.; Drangova, R.; Ho, P. (Univ. of Toronto, Ontario (Canada))

    1990-05-01

    We used rat proximal tubule fragments purified by Percoll centrifugation to examine the role of diacylglycerol (DAG) in noradrenergic-stimulated Na+ reabsorption. Tubular DAG concentration and ouabain-inhibitable 86Rb uptake increased within 30 s after adding norepinephrine (NE) and remained elevated for at least 5 min. NE (1 microM) increased DAG content 17% and ouabain-inhibitable 86Rb uptake 23%. Cirazoline-stimulated 86Rb uptake was not inhibited by BaCl, quinidine, or bumetanide (1-10 microM) or by the omission of HCO3- or Cl- from the medium, but it was completely inhibited by ouabain and furosemide. Oleoyl-acetyl glycerol, L-alpha-1,2-dioctanoylglycerol, and L-alpha-1,2-dioleoylglycerol (DOG) increased total 86Rb uptake 8-11%. 12-O-tetradecanoylphorbol-13-acetate (TPA) (5 nM) increased uptake by only 4%. Staurosporine at 5 nM inhibited DOG stimulation completely, whereas 50 nM staurosporine was required to inhibit NE stimulation completely. Sphingosine inhibited DOG stimulation by 66% but did not inhibit NE stimulation. Amiloride (1 mM) completely blocked DOG stimulation. Monensin increased 86Rb uptake 31% and completely blocked the DOG effect but reduced the NE effect by only 26% (P = 0.08). In tubules from salt-loaded rats, NE did not increase DAG concentration, but NE-stimulated 86Rb uptake was reduced by only 23% (P = 0.15). Thus DAG released by NE may stimulate Na+ entry through Na(+)-H+ exchange. NE predominantly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) by activating a protein kinase that is insensitive to DAG and TPA and is inhibited by staurosporine but not by sphingosine. NE may also stimulate K+ efflux through a BaCl-insensitive K+ channel that is inhibited by millimolar furosemide.

  20. Elucidation of density profile of self-assembled sitosterol + oryzanol tubules with small-angle neutron scattering

    NARCIS (Netherlands)

    Bot, A.; Gilbert, E.P.; Bouwman, W.G.; Sawalha, H.I.M.; Adel, den R.; Garamus, V.M.; Venema, P.; Linden, van der E.; Flöter, E.

    2012-01-01

    Small-angle neutron scattering (SANS) experiments have been performed on self-assembled tubules of sitosterol and oryzanol in triglyceride oils to investigate details of their structure. Alternative organic phases (deuterated and non-deuterated decane, limonene, castor oil and eugenol) were used to

  1. Denervation and high-fat diet reduce insulin signaling in T-tubules in skeletal muscle of living mice

    DEFF Research Database (Denmark)

    Lauritzen, Hans P M; Ploug, Thorkil; Ai, Hua

    2008-01-01

    OBJECTIVE: Insulin stimulates muscle glucose transport by translocation of GLUT4 to sarcolemma and T-tubules. Despite muscle glucose uptake playing a major role in insulin resistance and type 2 diabetes, the temporal and spatial changes in insulin signaling and GLUT4 translocation during these co...

  2. Genetic ablation of AQP2 in the mouse connecting tubules results in a mild urinary concentrating defect

    DEFF Research Database (Denmark)

    Kortenoeven, Marleen; Pedersen, Nis Borbye; Miller, Lance

    Body water balance is regulated in the kidney via the vasopressin (AVP) regulated water channel aquaporin-2 (AQP2) expressed in the connecting tubule (CNT) and the collecting duct (CD). Although crucial for the urinary concentrating mechanism, the relative role of AQP2 in the CNT and CD are not f...

  3. Reducing αENaC expression in kidney connecting tubule induces pseudohypoaldosteronism type 1 symptoms during K+ loading

    DEFF Research Database (Denmark)

    Poulsen, Søren Brandt; Praetorius, Jeppe; Damkier, Helle H

    2015-01-01

    Genetic inactivation of the epithelial Na(+) channel α-subunit (αENaC) in the renal collecting duct (CD) does not interfere with Na(+) and K(+) homeostasis in mice. However, inactivation in the CD and a part of the connecting tubule (CNT) induces autosomal recessive pseudohypoaldosteronism type 1...

  4. Numerical variation of cell lysosomes of the proximal convoluted tubules of mice Kidneys submitted to different X-ray doses

    International Nuclear Information System (INIS)

    Silva Lapa, R. de C.R. da; Pacheco, I.P.; Segreto, C.

    1984-01-01

    The number of cell lysosomes of the proximal convoluted tubules of mice Kidneys (Mus musculus) before and after whole-body irradiation with different X-ray doses is confronted. The mice were sacrificed after 72 hours and the cortex fragments were conduct to electron microscopy. A statistically significant numerical reduction of the lysosomas was observed in 72 hours. (M.A.C.) [pt

  5. Adaptive regulation of taurine and beta-alanine uptake in a human kidney cell line from the proximal tubule

    DEFF Research Database (Denmark)

    Jessen, H; Jacobsen, Christian

    1997-01-01

    1. The underlying mechanisms involved in the adaptive regulation of beta-amino acid uptake in the human proximal tubule were examined by use of an immortalized human embryonic kidney epithelial cell line (IHKE). 2. The results indicated that the adaptive response to maintain whole-body taurine...

  6. Morphometry of testis and seminiferous tubules of the adult crab-eating fox (Cerdocyon thous, Linnaeus, 1766

    Directory of Open Access Journals (Sweden)

    Bianca Cabral Caldeira

    2010-10-01

    Full Text Available Body and testicular biometric parameters are very important for establishing reproductive patterns and, consequently, the development of protocols for assisted reproduction in different species. A direct correlation between the testis weight and the sperm population was observed in other studied species, because the testis size primarily reflects the total volume of the seminiferous tubule, its main component. The objective of this study was to determine the testicular volume parameters and correlate data from morphometry of testis and seminiferous tubules with body mass in six adult crab-eating foxes. The mean body weight of the crab-eating foxes in this study was 6.53 kg, with approximately 0.068% allocated to the testicular mass and 0.042% specifically to seminiferous tubules, which represented 87.5% of the testicular parenchyma. The albuginea comprised 12.5% of the testicular mass. The mean diameter of seminiferous tubules was 236 µm, and the mean thickness of the seminiferous epithelium was 62.9 µm. Values of tubular parameters indicate a sperm productivity close to those observed in previously studied carnivores.

  7. Substrate modulation of fatty acid effects on energization and respiration of kidney proximal tubules during hypoxia/reoxygenation.

    Directory of Open Access Journals (Sweden)

    Anja Bienholz

    Full Text Available Kidney proximal tubules subjected to hypoxia/reoxygenation develop a nonesterified fatty acid-induced energetic deficit characterized by persistent partial mitochondrial deenergization that can be prevented and reversed by citric acid cycle substrates. To further assess the role of competition between fatty acids and substrates on inner membrane substrate carriers in the deenergization and the contribution to deenergization of fatty acid effects on respiratory function, digitonin-permeabilized rabbit and mouse tubules were studied using either addition of exogenous oleate after control normoxic incubation or increases of endogenous fatty acids produced by hypoxia/reoxygenation. The results demonstrated major effects of matrix oxaloacetate accumulation on succinate-supported energization and respiration and their modification by fatty acids. Improvements of energization in the presence of fatty acids by glutamate were shown to result predominantly from lowering matrix oxaloacetate rather than from amelioration of transmembrane cycling of fatty acids and uncoupling. Mouse tubules had 2.5 fold higher rates of succinate utilization, which resulted in stronger effects of oxaloacetate accumulation than rabbit tubules. Hypoxia/reoxygenation induced respiratory inhibition that was more severe for complex I-dependent substrates. Fatty acids themselves did not acutely contribute to this respiratory inhibition, but lowering them during 60 min. reoxygenation to allow recovery of ATP during that period alleviated it. These data clarify the basis for the nonesterified fatty acid-induced mitochondrial energetic deficit in kidney proximal tubules that impairs structural and functional recovery and provide insight into interactions that need to be considered in the design of substrate-based interventions to improve mitochondrial function.

  8. A bradykinin-potentiating peptide (BPP-10c) from Bothrops jararaca induces changes in seminiferous tubules.

    Science.gov (United States)

    Gilio, Joyce M; Portaro, Fernanda Cv; Borella, Maria I; Lameu, Claudiana; Camargo, Antonio Cm; Alberto-Silva, Carlos

    2013-11-06

    The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure-activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs - including BPP-10c - are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability

  9. A bradykinin-potentiating peptide (BPP-10c) from Bothrops jararaca induces changes in seminiferous tubules

    Science.gov (United States)

    2013-01-01

    Background The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure–activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs – including BPP-10c – are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. Results The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without

  10. Osteocyte Lacunae are Lenticular/Ellipsoid Spaces or Spiral/Helical Tubules

    Directory of Open Access Journals (Sweden)

    Bijit Kanti Guha

    2017-10-01

    Full Text Available Introduction: According to the current concept of bone structure, the osteocyte lacunae are lenticular or ellipsoid spaces occupied by osteocytes. These osteocytes are thought to communicate with each other through a tubular system made up of Canaliculi. The rest of the structures i.e., Haversian canal, and Volkmann’s canal are also tubular in shape. Considering this existing concept of bone microstructure described by various authors, it is highly unlikely that the lacunae alone would be lenticular/ellipsoid structure. In the present study author wanted to know that amongst the all tubular spaces, what is the reason that only the osteocyte lacunae are lenticular or ellipsoid structure? Also to investigate whether these lenticular spaces are really lenticular/ellipsoid or they are cut sections of tubes, which are lying helically or spirally. It is well known from various previous studies that Haversian canal, Volkmann’s canal and Canaliculi are tubular shaped structures and how it is possible that lenticular/ellipsoid structure can present amongst them. So we thought that, it may be possible that these lenticular spaces are not actually lenticular but this lenticular shape is due to the cut sections of any type of tubule in various possible planes (i.e., transverse, longitudinal and various degrees of oblique plane. Aim: The present study was carried out to reinvestigate the shape of the osteocyte lacunae amongst the tubular system (i.e., Haversian canal, Volkmann’s canal and Canaliculi of compact bone. Materials and Methods: The study is carried out by preparing thin sections of adult bones (ground glass preparation and visualizing them under binocular light microscope and scanelectron microscope after following proper procedure. Results: We observed that the lacunae are actually spirally/helically placed tubules with several branching. These branching are considered as canaliculi. These branching are of various diameters and they

  11. Effects of mercury on lysosomal protein digestion in the kidney proximal tubule

    International Nuclear Information System (INIS)

    Madsen, K.M.; Christensen, E.I.

    1978-01-01

    The effect of mercury on renal lysosomal protein digestion was studied after administration of mercury in vitro and in vivo. Mercuric chloride or methylmercury chloride was added in vitro to lysosomal enzymes isolated from normal rats, and subsequently, digestion experiments were carried out using 125 I-labeled lysozyme or cytochrome c as substrate proteins. Both mercury compounds produced a concentration-dependent inhibition of the degradation of the proteins, mercuric chloride being the strongest inhibitor. Mercuric chloride was also administered to rats in vivo for 5 to 8 months. Renal lysosomal enzymes from these animals also had a decreased ability to digest the two substrate proteins. Furthermore, the digestion of lysozyme intravenously injected into mercury-intoxicated rats was decreased in renal cortical slices incubated in vitro. Electron microscope autoradiography showed that intravenously injected labeled lysozyme was located primarily over lysosomes in proximal tubule cells 1 hour after injection in both control animals and mercury-intoxicated rats. These results suggest a decreased catabolism of low molecular weight proteins in the kidney during chronic mercury intoxication

  12. Effect of alphatocopherol on diameter of proximal convoluted tubules of kidney in diabetic mice

    International Nuclear Information System (INIS)

    Rashid, S.

    2014-01-01

    Objective: To evaluate the effects of alphatocopherol supplement on proximal convoluted tubular diameter of kidney in diabetic mice. Methods: The randomised controlled trials was conducted partly at the National Institute of Health (NIH), Islamabad, and partly in Army Medical College, Rawalpindi, from November 2009 to November 2010. Thirty adult female mice BALB/C were randomly divided into three equal groups. Group A served as the control group. Group B was made diabetic by the intraperitoneal injection of streptozotocin. Group C received injection streptozotocin and was fed with alphatocopherol (vitamin E) supplemented diet. After 12 weeks, the animals were sacrificed and their kidneys were removed for histomorphological study. Results: Diabetes caused significant changes in the diameter of proximal tubule of Experimental Group B (diabetic) compared to the controls in Group A, but these changes were prevented in alphatocopherol treated Group C. Tubular diameter in Group B was significantly reduced compared to the Control Group A (p 0.05). Conclusion: Significant difference in proximal tubular diameter of kidneys between diabetic and alphatocopherol treated diabetic mice confirm that vitamin E does extend a protective role in improving diabetic nephropathy. (author)

  13. Effect of ADH on rubidium transport in isolated perfused rat cortical collecting tubules

    International Nuclear Information System (INIS)

    Schafer, J.A.; Troutman, S.L.

    1986-01-01

    Unidirectional fluxes of 86Rb+ were measured as an indicator of potassium transport in isolated rat cortical collecting tubules perfused and bathed at 38 degrees C with isotonic solutions in which Rb+ replaced K+. Under control conditions the lumen-to-bath flux (Jl----b) was significantly less than the bath-to-lumen flux (Jb----l), indicating net Rb+ secretion. Net secretion increased approximately 180% after addition of 100 microU/ml of arginine vasopressin (ADH) to the bathing solution, due to a rapid and reversible increase in Jb----l from 4.6 +/- 0.8 to 9.0 +/- 1.9 pmol X min-1 X mm-1 with no significant change in Jl----b. The ADH effect was completely inhibited by 2 mM luminal Ba2+. The average transepithelial voltage (Ve) was not significantly different from zero in the control period but became lumen negative (-5 to -10 mV) after ADH. With 10(-5) M amiloride in the lumen Ve was lumen positive (+2 to +4 mV) and was unaltered by ADH or Ba2+, yet ADH produced a significant but attentuated increase in Jb----l with no change in Jl----b. The results indicate that ADH augments net K+ secretion either by an increase in the Ba2+-sensitive conductance of the apical membrane or by an increase in the electrochemical potential driving force for net Rb+ secretion through this pathway

  14. Dual Regulation of Gluconeogenesis by Insulin and Glucose in the Proximal Tubules of the Kidney.

    Science.gov (United States)

    Sasaki, Motohiro; Sasako, Takayoshi; Kubota, Naoto; Sakurai, Yoshitaka; Takamoto, Iseki; Kubota, Tetsuya; Inagi, Reiko; Seki, George; Goto, Moritaka; Ueki, Kohjiro; Nangaku, Masaomi; Jomori, Takahito; Kadowaki, Takashi

    2017-09-01

    Growing attention has been focused on the roles of the proximal tubules (PTs) of the kidney in glucose metabolism, including the mechanism of regulation of gluconeogenesis. In this study, we found that PT-specific insulin receptor substrate 1/2 double-knockout mice, established by using the newly generated sodium-glucose cotransporter 2 (SGLT2)-Cre transgenic mice, exhibited impaired insulin signaling and upregulated gluconeogenic gene expression and renal gluconeogenesis, resulting in systemic insulin resistance. In contrast, in streptozotocin-treated mice, although insulin action was impaired in the PTs, the gluconeogenic gene expression was unexpectedly downregulated in the renal cortex, which was restored by administration of an SGLT1/2 inhibitor. In the HK-2 cells, the gluconeogenic gene expression was suppressed by insulin, accompanied by phosphorylation and inactivation of forkhead box transcription factor 1 (FoxO1). In contrast, glucose deacetylated peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), a coactivator of FoxO1, via sirtuin 1, suppressing the gluconeogenic gene expression, which was reversed by inhibition of glucose reabsorption. These data suggest that both insulin signaling and glucose reabsorption suppress the gluconeogenic gene expression by inactivation of FoxO1 and PGC1α, respectively, providing insight into novel mechanisms underlying the regulation of gluconeogenesis in the PTs. © 2017 by the American Diabetes Association.

  15. Update on Mechanisms of Renal Tubule Injury Caused by Advanced Glycation End Products

    Directory of Open Access Journals (Sweden)

    Hong Sun

    2016-01-01

    Full Text Available Diabetic nephropathy (DN caused by advanced glycation end products (AGEs may be associated with lipid accumulation in the kidneys. This study was designed to investigate whether Nε-(carboxymethyl lysine (CML, a member of the AGEs family increases lipid accumulation in a human renal tubular epithelial cell line (HK-2 via increasing cholesterol synthesis and uptake and reducing cholesterol efflux through endoplasmic reticulum stress (ERS. Our results showed that CML disrupts cholesterol metabolism in HK-2 cells by activating sterol regulatory element-binding protein 2 (SREBP-2 and liver X receptor (LXR, followed by an increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR mediated cholesterol synthesis and low density lipoprotein receptor (LDLr mediated cholesterol uptake and a reduction in ATP-binding cassette transporter A1 (ABCA1 mediated cholesterol efflux, ultimately causing lipid accumulation in HK-2 cells. All of these responses could be suppressed by an ERS inhibitor, which suggests that CML causes lipid accumulation in renal tubule cells through ERS and that the inhibition of ERS is a potential novel approach to treating CML-induced renal tubular foam cell formation.

  16. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

    Science.gov (United States)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Lamers, Wouter H.; Chaudhry, Farrukh A.; Verlander, Jill W.

    2016-01-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4+ with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na+-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression. PMID:27009341

  17. Regulation of transport in the connecting tubule and cortical collecting duct

    Science.gov (United States)

    Staruschenko, Alexander

    2012-01-01

    The central goal of this overview article is to summarize recent findings in renal epithelial transport, focusing chiefly on the connecting tubule (CNT) and the cortical collecting duct (CCD). Mammalian CCD and CNT are involved in fine tuning of electrolyte and fluid balance through reabsorption and secretion. Specific transporters and channels mediate vectorial movements of water and solutes in these segments. Although only a small percent of the glomerular filtrate reaches the CNT and CCD, these segments are critical for water and electrolyte homeostasis since several hormones, e.g. aldosterone and arginine vasopressin, exert their main effects in these nephron sites. Importantly, hormones regulate the function of the entire nephron and kidney by affecting channels and transporters in the CNT and CCD. Knowledge about the physiological and pathophysiological regulation of transport in the CNT and CCD and particular roles of specific channels/transporters has increased tremendously over the last two decades. Recent studies shed new light on several key questions concerning the regulation of renal transport. Precise distribution patterns of transport proteins in the CCD and CNT will be reviewed, and their physiological roles and mechanisms mediating ion transport in these segments will be also covered. Special emphasis will be given to pathophysiological conditions appearing as a result of abnormalities in renal transport in the CNT and CCD. PMID:23227301

  18. Transport of salicylate in proximal tubule (S2 segment) isolated from rabbit kidney

    International Nuclear Information System (INIS)

    Schild, L.; Roch-Ramel, F.

    1988-01-01

    The secretory and the reabsorptive transport of salicylate was studied in the isolated and perfused rabbit proximal tubule (S 2 segment). Salicylate secretion (J sal b→l ) fulfilled the criteria for a carrier-mediated transport system: J sal b→l was saturable, was reversibly inhibited by probenecid, and occurred against a concentration gradient. The K m and V max for this secretory transport were 80 μM and 3,200 fmol·min -1 ·mm -1 , respectively. At luminal pH of 7.4 and 6.6, salicylate reabsorption (J sal l→b ) was low. J sal l→b was stimulated by increasing the bath Pco 2 or by removing basolateral HCO 3 - ; J sal l→b was inhibited by ethoxyzolamide and by SITS in the bath. The results indicate that salicylate reabsorption depends on H + secretion, consistent with reabsorption by simple nonionic diffusion. When salicylate was present in the lumen only, J sal l→b increased after inhibition of the secretory transport by adding ouabain or probenecid in the bath or by lowering the bath temperature. These results are compatible with luminal recycling of salicylate, and suggest the presence of a mediated secretory transporter located at the luminal membrane

  19. Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics.

    Science.gov (United States)

    Wilmes, Anja; Bielow, Chris; Ranninger, Christina; Bellwon, Patricia; Aschauer, Lydia; Limonciel, Alice; Chassaigne, Hubert; Kristl, Theresa; Aiche, Stephan; Huber, Christian G; Guillou, Claude; Hewitt, Philipp; Leonard, Martin O; Dekant, Wolfgang; Bois, Frederic; Jennings, Paul

    2015-12-25

    Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to biokinetics to identify cell stress response pathways induced by cisplatin. The human renal proximal tubular cell line RPTEC/TERT1 was treated with sub-cytotoxic concentrations of cisplatin (0.5 and 2 μM) in a daily repeat dose treating regime for up to 14 days. Biokinetic analysis showed that cisplatin was taken up from the basolateral compartment, transported to the apical compartment, and accumulated in cells over time. This is in line with basolateral uptake of cisplatin via organic cation transporter 2 and bioactivation via gamma-glutamyl transpeptidase located on the apical side of proximal tubular cells. Cisplatin affected several pathways including, p53 signalling, Nrf2 mediated oxidative stress response, mitochondrial processes, mTOR and AMPK signalling. In addition, we identified novel pathways changed by cisplatin, including eIF2 signalling, actin nucleation via the ARP/WASP complex and regulation of cell polarization. In conclusion, using an integrated omic approach together with biokinetics we have identified both novel and established mechanisms of cisplatin toxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Luminal and basolateral uptake of insulin in isolated perfused, proximal tubules

    International Nuclear Information System (INIS)

    Nielsen, S.; Nielsen, J.T.; Christensen, E.I.

    1987-01-01

    The present study was performed to quantitate compare the luminal and the peritubular uptake of 125 I-insulin in isolated, perfused, proximal tubules from rabbit kidneys. 125 I-insulin was added in physiological concentrations to either the perfusate or the bath fluid for 30 min. The luminal uptake in 30 min averaged 0.76 pg/mm at physiological concentrations and 18.0 pg/mm at high insulin concentrations. About 15-41% of the absorbed insulin was digested and 125 I-insulin at physiological and high concentrations in the bath was 0.136 and 0.318 pg, respectively. The data indicates that insulin is bound/absorbed at the basolateral membranes both by a saturable specific mechanism and a nonspecific, nonsaturable mechanism. The basolateral absorption constituted 15.2 and 1.8% of the total tubular extraction of insulin at physiological and high insulin concentrations, respectively. Electron microscope autoradiography showed that, after luminal as well as basolateral endocytosis, insulin was exclusively accumulated in endocytic vacuoles and lysosomes

  1. Premeiotic germ cell defect in seminiferous tubules of Atm-null testis

    International Nuclear Information System (INIS)

    Takubo, Keiyo; Hirao, Atsushi; Ohmura, Masako; Azuma, Masaki; Arai, Fumio; Nagamatsu, Go; Suda, Toshio

    2006-01-01

    Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16 Ink4a activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16 Ink4a -mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16 Ink4a -mediated senescence

  2. The myopathy-causing mutation DNM2-S619L leads to defective tubulation in vitro and in developing zebrafish

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Gibbs

    2014-01-01

    Full Text Available DNM2 is a ubiquitously expressed GTPase that regulates multiple subcellular processes. Mutations in DNM2 are a common cause of centronuclear myopathy, a severe disorder characterized by altered skeletal muscle structure and function. The precise mechanisms underlying disease-associated DNM2 mutations are unresolved. We examined the common DNM2-S619L mutation using both in vitro and in vivo approaches. Expression of DNM2-S619L in zebrafish led to the accumulation of aberrant vesicular structures and to defective excitation-contraction coupling. Expression of DNM2-S619L in COS7 cells resulted in defective BIN1-dependent tubule formation. These data suggest that DNM2-S619L causes disease, in part, by interfering with membrane tubulation.

  3. Protective Effect of Urtica dioica L. (Urticaceae) on Morphometric and Morphologic Alterations of Seminiferous Tubules in STZ Diabetic Rats.

    Science.gov (United States)

    Golalipour, Mohammad Jafar; Kabiri Balajadeh, Babak; Ghafari, Soraya; Azarhosh, Ramin; Khori, Vahid

    2011-09-01

    Urtica dioica L. has been known as a medicinal plant in the world. This study was conducted to determine the effects of the hydroalcoholic extract of Urtica dioica leaves on seminiferous tubules of diabetic rats. Animals were allocated to control, diabetic and protective groups. Treated animals received extract of U. dioica (100 mg/ kg/ day) IP for the first 5 days and STZ injection on the 6th day. After 5 weeks, testes removed and stained with H&E technique. Tubular cell disintegration, sertoli and spermatogonia cell vacuolization, and decrease in sperm concentration observed in diabetic in comparison with control and protective groups. External seminiferous tubular diameter and seminiferous epithelial height significantly reduced (Pdioica, before induction of diabetes; has protective role on seminiferous tubules alterations.

  4. Data on Na,K-ATPase in primary cultures of renal proximal tubule cells treated with catecholamines

    Directory of Open Access Journals (Sweden)

    Mary Taub

    2016-03-01

    Full Text Available This data article is concerned with chronic regulation of Na,K-ATPase by catecholamines. After a chronic treatment, inhibition of Na,K-ATPase activity was observed in cultures with dopamine, while a stimulation was observed in cultures treated with norepinephrine. Following a chronic incubation with guanabenz, an α adrenergic agonist, an increase in Na,K-ATPase α and β subunit mRNAs was observed. This data supports the research article entitled, “Renal proximal tubule Na, K-ATPase is controlled by CREB regulated transcriptional coactivators as well as salt inducible kinase 1” (Taub et al. 2015 [1]. Keywords: Catecholamines, Kidney, Proximal tubule, Na,K-ATPase, Chronic

  5. Direct physical contact between intercalated cells in the distal convoluted tubule and the afferent arteriole in mouse kidneys.

    Directory of Open Access Journals (Sweden)

    Hao Ren

    Full Text Available Recent physiological studies in the kidney proposed the existence of a secondary feedback mechanism termed 'crosstalk' localized after the macula densa. This newly discovered crosstalk contact between the nephron tubule and its own afferent arteriole may potentially revolutionize our understanding of renal vascular resistance and electrolyte regulation. However, the nature of such a crosstalk mechanism is still debated due to a lack of direct and comprehensive morphological evidence. Its exact location along the nephron, its prevalence among the different types of nephrons, and the type of cells involved are yet unknown. To address these issues, computer assisted 3-dimensional nephron tracing was applied in combination with direct immunohistochemistry on plastic sections and electron microscopy. 'Random' contacts in the cortex were identified by the tracing and excluded. We investigated a total of 168 nephrons from all cortical regions. The results demonstrated that the crosstalk contact existed, and that it was only present in certain nephrons (90% of the short-looped and 75% of the long-looped nephrons. The crosstalk contacts always occurred at a specific position--the last 10% of the distal convoluted tubule. Importantly, we demonstrated, for the first time, that the cells found in the tubule wall at the contact site were always type nonA-nonB intercalated cells. In conclusion, the present work confirmed the existence of a post macula densa physical crosstalk contact.

  6. Direct physical contact between intercalated cells in the distal convoluted tubule and the afferent arteriole in mouse kidneys.

    Science.gov (United States)

    Ren, Hao; Liu, Ning-Yu; Andreasen, Arne; Thomsen, Jesper S; Cao, Liu; Christensen, Erik I; Zhai, Xiao-Yue

    2013-01-01

    Recent physiological studies in the kidney proposed the existence of a secondary feedback mechanism termed 'crosstalk' localized after the macula densa. This newly discovered crosstalk contact between the nephron tubule and its own afferent arteriole may potentially revolutionize our understanding of renal vascular resistance and electrolyte regulation. However, the nature of such a crosstalk mechanism is still debated due to a lack of direct and comprehensive morphological evidence. Its exact location along the nephron, its prevalence among the different types of nephrons, and the type of cells involved are yet unknown. To address these issues, computer assisted 3-dimensional nephron tracing was applied in combination with direct immunohistochemistry on plastic sections and electron microscopy. 'Random' contacts in the cortex were identified by the tracing and excluded. We investigated a total of 168 nephrons from all cortical regions. The results demonstrated that the crosstalk contact existed, and that it was only present in certain nephrons (90% of the short-looped and 75% of the long-looped nephrons). The crosstalk contacts always occurred at a specific position--the last 10% of the distal convoluted tubule. Importantly, we demonstrated, for the first time, that the cells found in the tubule wall at the contact site were always type nonA-nonB intercalated cells. In conclusion, the present work confirmed the existence of a post macula densa physical crosstalk contact.

  7. Fabrication of Collagen Gel Hollow Fibers by Covalent Cross-Linking for Construction of Bioengineering Renal Tubules.

    Science.gov (United States)

    Shen, Chong; Zhang, Guoliang; Wang, Qichen; Meng, Qin

    2015-09-09

    Collagen, the most used natural biomacromolecule, has been extensively utilized to make scaffolds for cell cultures in tissue engineering, but has never been fabricated into the configuration of a hollow fiber (HF) for cell culture due to its poor mechanical properties. In this study, renal tubular cell-laden collagen hollow fiber (Col HF) was fabricated by dissolving sacrificial Ca-alginate cores from collagen shells strengthened by carbodiimide cross-linking. The inner/outer diameters of the Col HF were precisely controlled by the flow rates of core alginate/shell collagen solution in the microfluidic device. As found, the renal tubular cells self-assembled into renal tubules with diameters of 50-200 μm post to the culture in Col HF for 10 days. According to the 3D reconstructed confocal images or HE staining, the renal cells appeared as a tight tubular monolayer on the Col HF inner surface, sustaining more 3D cell morphology than the cell layer on the 2D flat collagen gel surface. Moreover, compared with the cultures in either a Transwell or polymer HF membrane, the renal tubules in Col HF exhibited at least 1-fold higher activity on brush border enzymes of alkaline phosphatase and γ-glutamyltransferase, consistent with their gene expressions. The enhancement occurred similarly on multidrug resistance protein 2 and glucose uptake. Such bioengineered renal tubules in Col HF will present great potential as alternatives to synthetic HF in both clinical use and pharmaceutical investigation.

  8. Fabrication and characterization of dendrimer-functionalized nano-hydroxyapatite and its application in dentin tubule occlusion.

    Science.gov (United States)

    Lin, Xuandong; Xie, Fangfang; Ma, Xueling; Hao, Yuhong; Qin, Hejia; Long, Jindong

    2017-06-01

    The occlusion of dentinal tubules is an effective method to alleviate the symptoms of dentin hypersensitivity. In this paper, we successfully modified nano-hydroxyapatite (n-HAP) with carboxyl-terminated polyamidoamine dendrimers by an aqueous-based chemical method and verified by fourier transform infrared spectroscopy (FTIR) and transmission electron microscope (TEM). Then the demineralization dentin discs were randomly divided into 4 groups, corresponding to subsequent brushing experiments: deionized water and kept in artificial saliva (AS), dendrimer-functionalized n-HAP and stored in AS, n-HAP and saved in AS, dendrimer-functionalized n-HAP and stored in deionized water. After 7 days of simulated brushing, dentin discs followed the in vitro characterization using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy and microhardness test. These data suggested that dendrimer-functionalized n-HAP could crosslink with collagen fibers and resulted in effective dentinal tubule occlusion. Moreover, the new material can induce the HAP formation with the help of superficial carboxyl and fill the spaces in dentinal tubules furtherly. The microhardness of dendrimer-functionalized n-HAP-treated specimens was significantly higher than others. In summary, dendrimer-functionalized n-HAP can be a new therapeutic material for the treatment of dentin hypersensitivity.

  9. The transcriptome of the Didelphis virginiana opossum kidney OK proximal tubule cell line.

    Science.gov (United States)

    Eshbach, Megan L; Sethi, Rahil; Avula, Raghunandan; Lamb, Janette; Hollingshead, Deborah J; Finegold, David N; Locker, Joseph D; Chandran, Uma R; Weisz, Ora A

    2017-09-01

    The OK cell line derived from the kidney of a female opossum Didelphis virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, the genomic sequence for D. virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, transcripts sequenced from both species show significant divergence. The M. domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the D. virginiana OK cell line, we characterized its transcriptome via de novo transcriptome assembly and alignment to the M. domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the M. domestica genome, were compared with publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT-specific ion transporters and other key proteins relevant for rodent and human PT function. Additionally, the sequence and expression data reported here provide an important resource for genetic manipulation and other studies on PT cell function using these cells. Copyright © 2017 the American Physiological Society.

  10. Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules

    Science.gov (United States)

    Lawrence, Marlon G.; Altenburg, Michael K.; Sanford, Ryan; Willett, Julian D.; Bleasdale, Benjamin; Ballou, Byron; Wilder, Jennifer; Li, Feng; Miner, Jeffrey H.; Berg, Ulla B.; Smithies, Oliver

    2017-01-01

    How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson’s or Alport’s proteinuric syndromes resulting from defects in GBM structural proteins (laminin β2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm. PMID:28246329

  11. HK2 Proximal Tubule Epithelial Cells Synthesize and Secrete Plasma Proteins Predominantly Through the Apical Surface.

    Science.gov (United States)

    Zhao, Ke-Wei; Murray, Elsa J Brochmann; Murray, Samuel S

    2017-04-01

    Renal proximal tubule epithelial cells (PTECs) are known to reabsorb salts and small plasma proteins filtered through Bowman's capsule. Following acute kidney injury, PTECs assume some characteristics of hepatocytes in producing various plasma proteins. We now demonstrate that even at a resting state, a PTEC cell line, HK2 expresses mRNAs for and synthesizes and secretes plasma proteins in a complex with complement C3, an α 2 -macroglobulin family chaperone, including albumin, transferrin, α 1 -antitrypsin, α 1 -antichymotrypsin, α 2 -HS-glycoprotein, ceruloplasmin, haptoglobin, C1-inhibitor, secreted phosphoprotein-24, and insulin-like growth factor-1. When grown on transwell inserts, HK2 cells predominantly secrete (∼90%) plasma proteins into the apical side and a smaller fraction into the basolateral side as determined by ELISA assays. When cultured in the presence of exogenous cytokines such as IL1β, IL6, TNFα, BMP2, or TGFβ1, HK2 cell mRNA expressions for plasma proteins were variably affected whereas basolateral secretions were elevated to or in excess of those of the apical level. In addition, HK2 cells produce proTGFβ1 with its intact N-terminal latency associated peptide and latent-TGF-β-binding proteins. The complex cannot be dissociated under conditions of SDS, heating, and electrophoresis. Moreover, HK2 cells maintain their ability to quickly uptake exogenously added serum proteins from the culture medium, as if they are recognized differently by the endocytic receptors. These results provide new insight into the hepatization of PTECs. In addition to their unique uptake of plasma proteins and salts from the filtrate, they are a source of urinary proteins under normal conditions as wells as in chronic and acute kidney diseases. J. Cell. Biochem. 118: 924-933, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Fluoride-associated ultrastructural changes and apoptosis in human renal tubule: a pilot study.

    Science.gov (United States)

    Quadri, J A; Sarwar, S; Sinha, A; Kalaivani, M; Dinda, A K; Bagga, A; Roy, T S; Das, T K; Shariff, A

    2018-01-01

    The susceptibility of the kidneys to fluoride toxicity can largely be attributed to its anatomy and function. As the filtrate moves along the complex tubular structure of each nephron, it is concentrated in the proximal and distal tubules and collecting duct. It has been frequently observed that the children suffering from renal impairments also have some symptoms of dental and skeletal fluorosis. The findings suggest that fluoride somehow interferes with renal anatomy and physiology, which may lead to renal pathogenesis. The aim of this study was to evaluate the fluoride-associated nephrotoxicity. A total of 156 patients with childhood nephrotic syndrome were screened and it was observed that 32 of them had significantly high levels ( p ≤ 0.05) of fluoride in urine (4.01 ± 1.83 ppm) and serum (0.1 ± 0.013 ppm). On the basis of urinary fluoride concentration, patients were divided into two groups, namely group 1 (G-1) ( n = 32) containing normal urine fluoride (0.61 ± 0.17 ppm) and group 2 (G-2) ( n = 32) having high urine fluoride concentration (4.01 ± 1.83 ppm). Age-matched healthy subjects ( n = 33) having normal levels of urinary fluoride (0.56 ± 0.15 ppm) were included in the study as control (group 0 (G-0)). Kidney biopsies were taken from G-1 and G-2 only, who were subjected to ultrastructural (transmission electron microscopy) and apoptotic (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling) analysis. Various subcellular ultrastructural changes including nuclear disintegration, chromosome condensation, cytoplasmic ground substance lysis, and endoplasmic reticulum blebbing were observed. Increased levels of apoptosis were observed in high fluoride group (G-2) compared to normal fluoride group (G-1). Various degrees of fluoride-associated damages to the architecture of tubular epithelia, such as cell swelling and lysis, cytoplasmic vacuolation, nuclear condensation, apoptosis, and necrosis, were observed.

  13. Induction of apoptosis in cultured human proximal tubule cells by fumonisins and fumonisin metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Seefelder, W; Humpf, H -U; Schwerdt, G; Freudinger, R; Gekle, M

    2003-10-15

    Fumonisin B{sub 1} (FB{sub 1}) causes apoptosis in a variety of cell types and tissues but the apoptotic potential of other fumonisins and fumonisin metabolites has not been determined and the underlying mechanisms are not completely understood. In our studies we exposed human proximal tubule-derived cells (IHKE cells) to FB{sub 1}, fumonisin B{sub 2} (FB{sub 2}), fumonisin B{sub 3} (FB{sub 3}), hydrolyzed fumonisin B{sub 1} (HFB{sub 1}) and N-palmitoyl-hydrolyzed fumonisin B{sub 1} (N-Pal-HFB{sub 1}) and investigated caspase-3 activation, chromatin condensation and DNA fragmentation. Exposure to 10 {mu}mol/L FB{sub 1} for 24 h led to a significant increase in caspase-3 activity, chromatin condensation and to DNA fragmentation. All other tested compounds did not show any significant activation of caspase-3 activity nor chromatin condensation and DNA-fragmentation. Furthermore, we examined if a sphinganine accumulation is correlated with an induction of apoptosis in IHKE cells. Therefore we used a liquid chromatography/electrospray ionization-mass spectrometry(LC/ESI-MS)-method using phytosphingosine as an internal standard to determine sphinganine and sphingosine concentrations in IHKE cells. Whereas a significant increase of sphinganine (up to 7000% compared to control cells) was observed with all fumonisin-derivates, sphingosine levels nearly remained unchanged indicating that all substrates inhibited ceramide synthase effectively. These results demonstrate that all compounds let to increased sphinganine levels in IHKE cells but only FB{sub 1} was able to induce apoptosis. We conclude that the inhibition of the ceramide synthase is not per se a predictor whether or not fumonisins induce apoptosis.

  14. Induction of apoptosis in cultured human proximal tubule cells by fumonisins and fumonisin metabolites

    International Nuclear Information System (INIS)

    Seefelder, W.; Humpf, H.-U.; Schwerdt, G.; Freudinger, R.; Gekle, M.

    2003-01-01

    Fumonisin B 1 (FB 1 ) causes apoptosis in a variety of cell types and tissues but the apoptotic potential of other fumonisins and fumonisin metabolites has not been determined and the underlying mechanisms are not completely understood. In our studies we exposed human proximal tubule-derived cells (IHKE cells) to FB 1 , fumonisin B 2 (FB 2 ), fumonisin B 3 (FB 3 ), hydrolyzed fumonisin B 1 (HFB 1 ) and N-palmitoyl-hydrolyzed fumonisin B 1 (N-Pal-HFB 1 ) and investigated caspase-3 activation, chromatin condensation and DNA fragmentation. Exposure to 10 μmol/L FB 1 for 24 h led to a significant increase in caspase-3 activity, chromatin condensation and to DNA fragmentation. All other tested compounds did not show any significant activation of caspase-3 activity nor chromatin condensation and DNA-fragmentation. Furthermore, we examined if a sphinganine accumulation is correlated with an induction of apoptosis in IHKE cells. Therefore we used a liquid chromatography/electrospray ionization-mass spectrometry(LC/ESI-MS)-method using phytosphingosine as an internal standard to determine sphinganine and sphingosine concentrations in IHKE cells. Whereas a significant increase of sphinganine (up to 7000% compared to control cells) was observed with all fumonisin-derivates, sphingosine levels nearly remained unchanged indicating that all substrates inhibited ceramide synthase effectively. These results demonstrate that all compounds let to increased sphinganine levels in IHKE cells but only FB 1 was able to induce apoptosis. We conclude that the inhibition of the ceramide synthase is not per se a predictor whether or not fumonisins induce apoptosis

  15. T-tubule disruption promotes calcium alternans in failing ventricular myocytes: mechanistic insights from computational modeling.

    Science.gov (United States)

    Nivala, Michael; Song, Zhen; Weiss, James N; Qu, Zhilin

    2015-02-01

    In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the effects of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in "orphaned" RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Atgl gene deletion predisposes to proximal tubule damage by impairing the fatty acid metabolism

    International Nuclear Information System (INIS)

    Chen, Wen; Zhang, Qiong; Cheng, Shiwu; Huang, Jie; Diao, Ge; Han, Jian

    2017-01-01

    Fibrosis is the final common pathway of chronic kidney disease (CKD). Normal lipid metabolism is integral to renal physiology, and disturbances of renal lipid metabolism are increasingly being linked with CKD, including the fibrosis. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme of lipolysis. In the present study, we used Atgl −/− mice to investigate whether ATGL played a role in the regulation of proximal convoluted tubule (PCT) lipid metabolism and renal fibrosis development. ATGL deficiency led to lipid vacuolation of PCT and tubulointerstitial fibrosis, accompanied by massive albuminuria and decreased creatinine clearance rate (Ccr). In vitro experiments indicated that inhibition of ATGL in proximal tubular cell line HK-2 promoted intracellular lipid deposition, reactive oxygen species (ROS) accumulation and cell apoptosis. Both in vitro and in vivo experiments showed that ATGL inhibition decreased the renal peroxisome proliferator-activated receptorα(PPARα) expression, which implied the suppressed lipid metabolism. The antioxidant N-acetylcysteine (NAC) could partially reverse the effect of ROS accumulation and cell apoptosis, but could not restore the PPARαdecrease. These data raise the possibility that ATGL deficiency could impair the renal fatty acid metabolism though inhibiting PPARαexpression, which may lead to lipid deposition and cell apoptosis of PCT, and finally contribute to the renal fibrosis and dysfunction. - Highlights: • Atgl −/− mice develop tubulointerstitial damage and renal dysfunction. • ATGL deficiency results in lipid accumulation and apoptosis of proximal tubular cells. • ROS scavenger alleviates the ATGL-knockdown mediated lipid accumulation and apoptosis. • PPARαdown-regulation is the reason of ROS elevating in ATGL-knockdown HK-2 cells.

  17. Na sup + -K sup + -ATPase-dependent sodium flux in cortical collecting tubule

    Energy Technology Data Exchange (ETDEWEB)

    Blot-Chabaud, M.; Jaisser, F.; Gingold, M.; Bonvalet, J.P.; Farman, N. (CEntre d' Etudes Nucleaires de Saclay, Gif sur Yvette (France))

    1988-10-01

    The instantaneous rate of efflux of intracellular Na was studied in rabbit isolated cortical collecting tubules (CCT) as a function of temperature and intracellular Na concentration ((Na){sub i}). (Na){sub i} of microdissected CCT was increased by cold and K-free exposure in the presence of {sup 22}Na and the extracellular tracer ({sup 3}H)sorbitol. (Na){sub i} rose rapidly to 40 mM at 30 min, after which it rose more slowly, reaching 120-140 mM at 6 h. Kinetics of Na efflux were studied after rapid rewarming, using a special device allowing measurements at 20-s intervals. Under control conditions, the total Na load was extruded in <8 min, whereas, in the presence of 10{sup {minus}4} M ouabain, only 50% of the load was extruded during this period of time. Ouabain-sensitive Na efflux was first evident at 13{degree}C and gradually increased between 13 and 35{degree}C. At 37{degree}C, Na{sup +}-K{sup +}-ATPase-dependent Na efflux was dependent on (Na){sub i}. This efflux gradually increased, from 0.05 to 0.5 peq{center dot}nl tubular volume{sup {minus}1}{center dot}s{sup {minus}1} as a function of (Na){sub i} and reached a plateau at 70 mM (Na){sub i}. It is concluded that (Na){sub i} is a major modulator of the pump activity in CCT; at normal levels of (Na){sub i}, the pump is operating at only a small fraction of its total capacity.

  18. Regulation of cyclic AMP metabolism by prostaglandins in rabbit cortical collecting tubule cells

    International Nuclear Information System (INIS)

    Sonnenburg, W.K.

    1987-01-01

    In the rabbit cortical collecting tubule (RCCT), prostaglandin E 1 (PGE 1 ) and prostaglandin E 2 (PGE 2 ) at 1 nM inhibit arginine-vasopressin (AVP)-induced water reabsorption, while 100 nM PGE 1 and PGE 2 alone stimulate water reabsorption. Reported here are studies designed to investigate the molecular basis for the biphasic physiological action of PGE 1 and PGE 2 in the collecting duct. In freshly isolated RCCT cells, PGE 1 , PGE 2 , and 16,16-dimethyl-PGE 2 (DM-PGE 2 ) stimulated cAMP synthesis at concentrations ranging from 0.1 to 10 M. Other prostaglandins including the synthetic PGE 2 analogue, sulprostone, failed to stimulate cAMP synthesis. Moreover, sulprostone did not antagonize PGE 2 -stimulated cAMP formation. In contrast, PGE 2 and sulprostone at concentrations ranging from 1 to 100 nM, inhibited AVP-induced cAMP accumulation in freshly isolated RCCT cells. PGE 2 , PGE 1 , DM-PGE 2 and sulprostone at 100 nM were equally effective in inhibiting AVP-induced cAMP formation. Moreover sulprostone inhibited AVP-stimulated adenylate cyclase activity. These results suggest that PGE derivatives mediate either inhibition or activation of adenylate cyclase by stimulating different PGE receptors. To further test this concept, PGE 2 binding to freshly isolated RCCT cell membranes was characterized. Two different classes of PGE 2 binding were detected. / 3 H/PGE 2 binding to the high affinity class of sites was increased by the GTP-analogue, GTP S, while pertussis toxin pretreatment blocked the stimulatory action. In contrast, / 3 H/ PGE 2 binding to the low affinity class of sites was decreased by GTP S; this inhibitory effect was not blocked by pertussis toxin pretreatment

  19. The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster.

    Science.gov (United States)

    Cannell, Elizabeth; Dornan, Anthony J; Halberg, Kenneth A; Terhzaz, Selim; Dow, Julian A T; Davies, Shireen-A

    2016-06-01

    Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH44) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance. Gene expression and labelled DH44 ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH44 in neurons of the pars intercerebralis and DH44 receptor (DH44-R2) in Malpighian tubule principal cells, indicate that suppression of DH44 signalling improves desiccation tolerance of the intact fly. Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH44 neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH44-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH44 and LK signalling pathways. Finally, although a role for DK in desiccation tolerance was not defined, we demonstrate a novel role for Malpighian tubule cell-specific LKR in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Urinary loss of glucose, phosphate, and protein by diffusion into proximal straight tubules injured by D-serine and maleic acid

    International Nuclear Information System (INIS)

    Carone, F.A.; Nakamura, S.; Goldman, B.

    1985-01-01

    In several models of acute renal failure leakage of glomerular filtrate out of the tubule is an important pathogenetic mechanism; however, bidirectional diffusion of solute to account for certain pathophysiologic features of acute renal failure has received meager attention. Using micropuncture and clearance methods, the authors assessed sequentially leakage of solutes and inulin across proximal straight tubules (PST) injured by two nephrotoxins. In d-serine-treated rats with extensive necrosis of PST, the basis for glucosuria and tubular leakage of inulin was studied. Glucose absorption by the proximal convoluted tubule and glucose delivery to the PST were normal, but glucose delivery to the distal tubule was increased nearly 8-fold, indicating diffusion of glucose from interstitial to tubular luminal fluid across the necrotic PST. Total kidney inulin clearance was greatly reduced, but single nephron glomerular filtration rate, based on proximal convoluted tubule samples, was normal, indicating tubular loss of inulin. Urinary recovery of [ 14 C]inulin infused into tubular lumina revealed that proximal convoluted tubule and distal tubule were impermeable to inulin and that inulin diffused out of the necrotic PST. The progressive return over 6 days of tubular impermeability for inulin correlated with relining of PST with new cells. In maleic acid-treated rats the site and extent of tubular necrosis and the nature of urinary loss of solutes were studied. Microdissection revealed that maleic acid caused limited necrosis of PST which averaged 7.4% of total proximal tubular length. Increased urinary excretion of protein, phosphate, and glucose and increased tubular permeability to microinfused [ 14 C]inulin occurred with the onset of PST necrosis, and return of these abnormalities to normal correlated with the degree of cellular repair of the PST

  1. Immunohistochemical profile of some neurotransmitters and neurotrophins in the seminiferous tubules of rats treated by lonidamine.

    Science.gov (United States)

    Artico, M; Bronzetti, E; Saso, L; Felici, L M; D'Ambrosio, A; Forte, F; Grande, C; Ortolani, F

    2007-01-01

    Lonidamine (LND) or [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] is an anticancer and antispermatogenic drug that exerts a large number of effects on tumor cells and germ cells. Sexually mature male Sprague-Dawley rats were housed at 22 degrees C on a 12-h light/12-h dark cycle 1 week before the experiments, with free access to food and water. LND was suspended in 0.5% methylcellulose at a concentration of 10 mg/mL and administered orally at the dose of 10 mL/kg (b.w.) as a single dose. Control rats received an equal amount of vehicle. Testes were removed, fixed for 24 h in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate (pH 7.2 at 22 degrees C), rinsed with the same buffer, and stored at room temperature. From each sample, a block of tissue was removed by sectioning through the organ. After dehydration in ethanol at increasing concentrations (70-100%), each block was embedded in paraffin and serial 5 mm thick sections were cut using a rotatory microtome. The immunoreactivity for NTs has been observed in spermatogonia of untreated rats, while the rats treated with LND showed an immunohistochemical localization in all the stages of germinal cells. The generally well-expressed immunoreactivity for the neurotrophins receptors in treated rats observed in our study is presumably attributable to alterations of the receptors' structure and/or expression leading to changes of the activity, affinity, localization or protein interactions that may depend on sensitization of ion channels (induced by LND). Neurotrophins (NTs) appear to be interesting proteins for the modulation of sperm maturation and motility with a prominent role for the nerve growth factor (NGF), that may exert an autocrine or paracrine role. We therefore investigated the location and distribution of immunoreactivity for some neurotransmitters (SP, VIP, CGRP, nNOS, Chat), neurotrophins (NGF, BDNF, NT-3) and their own receptors (TrKA, TrKB, TrKC, p75) in the seminiferous tubules

  2. Immunohistochemical profile of some neurotransmitters and neurotrophins in the seminiferous tubules of rats treated by lonidamine

    Directory of Open Access Journals (Sweden)

    M Artico

    2009-06-01

    Full Text Available Lonidamine (LND or [1-(2,4-dichlorobenzyl-1H-indazole-3- carboxylic acid] is an anticancer and antispermatogenic drug that exerts a large number of effects on tumor cells and germ cells. Sexually mature male Sprague-Dawley rats were housed at 22°C on a 12-h light/12-h dark cycle 1 week before the experiments, with free access to food and water. LND was suspended in 0.5% methylcellulose at a concentration of 10 mg/mL and administered orally at the dose of 10 mL/kg (b.w. as a single dose. Control rats received an equal amount of vehicle. Testes were removed, fixed for 24 h in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate (pH 7.2 at 22°C, rinsed with the same buffer, and stored at room temperature. From each sample, a block of tissue was removed by sectioning through the organ. After dehydration in ethanol at increasing concentrations (70-100%, each block was embedded in paraffin and serial 5 mm thick sections were cut using a rotatory microtome. The immunoreactivity for NTs has been observed in spermatogonia of untreated rats, while the rats treated with LND showed an immunohistochemical localization in all the stages of germinal cells. The generally well-expressed immunoreactivity for the neurotrophins receptors in treated rats observed in our study is presumably attributable to alterations of the receptors’ structure and/or expression leading to changes of the activity, affinity, localization or protein interactions that may depend on sensitization of ion channels (induced by LND. Neurotrophins (NTs appear to be interesting proteins for the modulation of sperm maturation and motility with a prominent role for the nerve growth factor (NGF, that may exert an autocrine or paracrine role.We therefore investigated the location and distribution of immunoreactivity for some neurotransmitters (SP, VIP, CGRP, nNOS, Chat, neurotrophins (NGF, BDNF, NT-3 and their own receptors (TrKA, TrKB, TrKC, p75 in the seminiferous tubules of

  3. Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Peiying Yu

    2014-01-01

    Full Text Available NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5 gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT and hypertensive subjects (HT. We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and 2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold greater in HT than NT (P=<0.001, n=6–14/group. Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA, effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (−45.1±3.2% vs. mock-siRNA, n=6–8 in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (−32.5±1.8% than HT (−14.8±1.8. In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.

  4. The effect of L-cysteine on the portion-selective uptake of cadmium in the renal proximal tubule

    International Nuclear Information System (INIS)

    Murakami, Masataka; Sano, Kenichi; Webb, M.

    1987-01-01

    Cadmium (Cd), co-administered with an excess of L-cysteine, accumulates rapidly in the kidneys of the rat. After subcutaneous (s.c.) injection of 3 μmol CdCl 2 /kg body wt the concentrations of Cd in the blood and kidneys increase with the dose of cysteine over the range 0.06-5.0 mmol/kg body wt. At cysteine doses of less than 1.5 mmol/kg body wt the ratio of the concentrations of Cd in the outer medulla and cortex of the kidney remains the same as that after the injection of Cd alone. This ratio, however, is more than doubled at dose levels of 5-10 mmol cysteine/kg body wt. Hepatic uptake of Cd is unaffected by doses of cysteine below 1.5 mmol/kg body wt but decreases markedly at higher doses. In animals that are dosed simultaneously with 5 mmol cysteine/kg body wt, renal uptake of 109 Cd is known to occur in the straight segments of the proximal tubules. At a dose level of less than 1.5 mmol cysteine/kg body wt the present autoradiographical studies show that 109 Cd is taken up predominantly by the proximal convoluted tubules of the kidney cortex. At the critical dose level (1.5 mmol/kg body wt), cysteine decreases the retention of Cd at the s.c. injection site, but probably has little effect on the distribution of Cd between protein and other carrier molecules in the blood. This distribution, however, is altered at higher cysteine dose levels. It is suggested that, under the latter conditions, stable Cd-cysteine complexes are formed in the blood and are filtered readily through the glomeruli. These complexes are taken up in the kidney at the sites of cysteine reabsorption which, by studies with L-[ 35 S]-cysteine, are identified as the straight segments of the proximal tubules. (orig.)

  5. N-domain angiotensin-I converting enzyme is expressed in immortalized mesangial, proximal tubule and collecting duct cells.

    Science.gov (United States)

    Mei Wang, Pamella Huey; Andrade, Maria Claudina; Quinto, Beata Marie Redublo; Di Marco, Giovana; Mortara, Renato Arruda; Vio, Carlos P; Casarini, Dulce Elena

    2015-01-01

    Somatic ACE (sACE) is found in glomerulus, proximal tubule and excreted in urine. We hypothesized that N-domain ACE can also be found at these sites. ACE profile was analyzed in mesangial (IMC), proximal (LLC-PK1), distal tubule (MDCK) and collecting duct (IMCD) cells. Cell lysate and culture medium were submitted to gel filtration chromatography, which separated two peaks with ACE activity from cells and medium, except from distal tubule. The first had a high molecular weight and the second, a lower one (65 kDa; N-domain ACE). We focused on N-domain ACE purification and characterization from LLC-PK1. Total LLC-PK1 N-domain ACE purification was achieved by ion-exchange chromatography, which presented only one peak with ACE activity, denominated ACE(int2A). ACE(int2A) activity was influenced by pH, NaCl and temperature. The purified enzyme was inhibited by Captopril and hydrolyzed AngI, Ang1-7 and AcSDKP. Its ability to hydrolyze AcSDKP characterized it as an N-domain ACE. ACE(int2A) also presented high amino acid sequence homology with the N-terminal part of sACE from mouse, rat, human and rabbit. The presence of secreted and intracellular N-domain ACE and sACE in IMC, LLC-PK1 and IMCD cells confirmed our studies along the nephron. We identified, purified and characterized N-domain ACE from LLC-PK1. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Accelerated recovery of renal mitochondrial and tubule homeostasis with SIRT1/PGC-1α activation following ischemia–reperfusion injury

    Energy Technology Data Exchange (ETDEWEB)

    Funk, Jason A., E-mail: funkj@musc.edu [Center for Cell Death, Injury, and Regeneration, Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Schnellmann, Rick G., E-mail: schnell@musc.edu [Center for Cell Death, Injury, and Regeneration, Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Ralph H. Johnson VA Medical Center, Charleston, SC 29401 (United States)

    2013-12-01

    Kidney ischemia–reperfusion (I/R) injury elicits cellular injury in the proximal tubule, and mitochondrial dysfunction is a pathological consequence of I/R. Promoting mitochondrial biogenesis (MB) as a repair mechanism after injury may offer a unique strategy to restore both mitochondrial and organ function. Rats subjected to bilateral renal pedicle ligation for 22 min were treated once daily with the SIRT1 activator SRT1720 (5 mg/kg) starting 24 h after reperfusion until 72 h–144 h. SIRT1 expression was elevated in the renal cortex of rats after I/R + vehicle treatment (IRV), but was associated with less nuclear localization. SIRT1 expression was even further augmented and nuclear localization was restored in the kidneys of rats after I/R + SRT1720 treatment (IRS). PGC-1α was elevated at 72 h–144 h in IRV and IRS kidneys; however, SRT1720 treatment induced deacetylation of PGC-1α, a marker of activation. Mitochondrial proteins ATP synthase β, COX I, and NDUFB8, as well as mitochondrial respiration, were diminished 24 h–144 h in IRV rats, but were partially or fully restored in IRS rats. Urinary kidney injury molecule-1 (KIM-1) was persistently elevated in both IRV and IRS rats; however, KIM-1 tissue expression was attenuated in IRS rats. Additionally, sustained loss of Na{sup +},K{sup +}–ATPase expression and basolateral localization and elevated vimentin in IRV rats was normalized in IRS rats, suggesting restoration of a differentiated, polarized tubule epithelium. The results suggest that SRT1720 treatment expedited recovery of mitochondrial protein expression and function by enhancing MB, which was associated with faster proximal tubule repair. Targeting MB may offer unique therapeutic strategy following ischemic injury. - Highlights: • We examined recovery of mitochondrial and renal function after ischemia–reperfusion. • SRT1720 treatment after I/R induced mitochondrial biogenesis via SIRT1/PGC-1α. • Recovery of mitochondrial function was

  7. Effects of advanced glycation end products on ezrin-dependent functions in LLC-PK1 proximal tubule cells.

    Science.gov (United States)

    Bach, Leon A; Gallicchio, Marisa A; McRobert, E Anne; Tikoo, Anjali; Cooper, Mark E

    2005-06-01

    We have recently shown that advanced glycation products (AGEs) bind to the ERM (ezrin, radixin, moesin) family of proteins. ERM proteins act as cross-linkers between cell membrane proteins and the actin cytoskeleton. They are also involved in signal transduction pathways. They therefore have a critical role in normal cell processes, including modulation of cell shape, adhesion, and motility. We postulate that AGEs may contribute to diabetic complications by disrupting ERM function. In support of this hypothesis, AGEs inhibit ezrin-dependent tubulogenesis of proximal tubule cells. Phosphorylation is an important activating mechanism for ERM proteins, and AGEs inhibit ezrin phosphorylation mediated by the epidermal growth factor receptor.

  8. Antenatal glucocorticoid treatment alters Na+ uptake in renal proximal tubule cells from adult offspring in a sex-specific manner

    OpenAIRE

    Su, Yixin; Bi, Jianli; Pulgar, Victor M.; Figueroa, Jorge; Chappell, Mark; Rose, James C.

    2015-01-01

    We have shown a sex-specific effect of fetal programming on Na+ excretion in adult sheep. The site of this effect in the kidney is unknown. Therefore, we tested the hypothesis that renal proximal tubule cells (RPTCs) from adult male sheep exposed to betamethasone (Beta) before birth have greater Na+ uptake than do RPTCs from vehicle-exposed male sheep and that RPTCs from female sheep similarly exposed are not influenced by antenatal Beta. In isolated RPTCs from 1- to 1.5-yr-old male and femal...

  9. Thiazolidinediones enhance sodium-coupled bicarbonate absorption from renal proximal tubules via PPARγ-dependent nongenomic signaling.

    Science.gov (United States)

    Endo, Yoko; Suzuki, Masashi; Yamada, Hideomi; Horita, Shoko; Kunimi, Motoei; Yamazaki, Osamu; Shirai, Ayumi; Nakamura, Motonobu; Iso-O, Naoyuki; Li, Yuehong; Hara, Masumi; Tsukamoto, Kazuhisa; Moriyama, Nobuo; Kudo, Akihiko; Kawakami, Hayato; Yamauchi, Toshimasa; Kubota, Naoto; Kadowaki, Takashi; Kume, Haruki; Enomoto, Yutaka; Homma, Yukio; Seki, George; Fujita, Toshiro

    2011-05-04

    Thiazolidinediones (TZDs) improve insulin resistance by activating a nuclear hormone receptor, peroxisome proliferator-activated receptor γ (PPARγ). However, the use of TZDs is associated with plasma volume expansion through a mechanism that remains to be clarified. Here we showed that TZDs rapidly stimulate sodium-coupled bicarbonate absorption from the renal proximal tubule in vitro and in vivo. TZD-induced transport stimulation is dependent on PPARγ-Src-EGFR-ERK and observed in rat, rabbit and human, but not in mouse proximal tubules where Src-EGFR is constitutively activated. The existence of PPARγ-Src-dependent nongenomic signaling, which requires the ligand-binding ability, but not the transcriptional activity of PPARγ, is confirmed in mouse embryonic fibroblast cells. The enhancement of the association between PPARγ and Src by TZDs supports an indispensable role of Src in this signaling. These results suggest that the PPARγ-dependent nongenomic stimulation of renal proximal transport is also involved in TZD-induced volume expansion. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Objectives, Outlines, and Preparation for the Resist Tubule Space Experiment to Understand the Mechanism of Gravity Resistance in Plants

    Science.gov (United States)

    Hoson, Takayuki; Akamatsu, Haruhiko; Soga, Kouichi; Wakabayashi, Kazuyuki; Hashimoto, Hirofumi; Yamashita, Masamichi; Hasegawa, Katsuya; Yano, Sachiko; Omori, Katsunori; Ishioka, Noriaki; Matsumoto, Shohei; Kasahara, Haruo; Shimazu, Toru; A. Baba, Shoji; Hashimoto, Takashi

    Gravity resistance is a principal graviresponse in plants. In resistance to hypergravity, the gravity signal may be perceived by the mechanoreceptors located on the plasma membrane, and then transformed and transduced via the structural continuum or physiological continuity of cortical microtubules-plasma membrane-cell wall, leading to an increase in the cell wall rigidity as the final response. The Resist Tubule experiment, which will be conducted in the Kibo Module on the International Space Station, aims to confirm that this hypothesis is applicable to resistance to 1 G gravity. There are two major objectives in the Resist Tubule experiment. One is to quantify the contributions of cortical microtubules to gravity resistance using Arabidopsis tubulin mutants with different degrees of defects. Another objective is to analyze the modifications to dynamics of cortical microtubules and membrane rafts under microgravity conditions on-site by observing green fluorescent protein (GFP)-expressing Arabidopsis lines with the fluorescence microscope in the Kibo. We have selected suitable mutants, developed necessary hardware, and fixed operation procedure for the experiment.

  11. Effects of phospho- and calciotropic hormones on electrolyte transport in the proximal tubule [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Justin J. Lee

    2017-10-01

    Full Text Available Calcium and phosphate are critical for a myriad of physiological and cellular processes within the organism. Consequently, plasma levels of calcium and phosphate are tightly regulated. This occurs through the combined effects of the phospho- and calciotropic hormones, parathyroid hormone (PTH, active vitamin D3, and fibroblast growth factor 23 (FGF23. The organs central to this are the kidneys, intestine, and bone. In the kidney, the proximal tubule reabsorbs the majority of filtered calcium and phosphate, which amounts to more than 60% and 90%, respectively. The basic molecular mechanisms responsible for phosphate reclamation are well described, and emerging work is delineating the molecular identity of the paracellular shunt wherein calcium permeates the proximal tubular epithelium. Significant experimental work has delineated the molecular effects of PTH and FGF23 on these processes as well as their regulation of active vitamin D3 synthesis in this nephron segment. The integrative effects of both phospho- and calciotropic hormones on proximal tubular solute transport and subsequently whole body calcium-phosphate balance thus have been further complicated. Here, we first review the molecular mechanisms of calcium and phosphate reabsorption from the proximal tubule and how they are influenced by the phospho- and calciotropic hormones acting on this segment and then consider the implications on both renal calcium and phosphate handling as well as whole body mineral balance.

  12. The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: Utility of renal specific P450 reductase knockout mouse models

    International Nuclear Information System (INIS)

    Liu, Senyan; Yao, Yunyi; Lu, Shijun; Aldous, Kenneth; Ding, Xinxin; Mei, Changlin; Gu, Jun

    2013-01-01

    The kidney is a primary target for numerous toxic compounds. Cytochrome P450 enzymes (P450) are responsible for the metabolic activation of various chemical compounds, and in the kidney are predominantly expressed in proximal tubules. The aim of this study was to test the hypothesis that renal proximal tubular P450s are critical for nephrotoxicity caused by chemicals such as chloroform. We developed two new mouse models, one having proximal tubule-specific deletion of the cytochrome P450 reductase (Cpr) gene (the enzyme required for all microsomal P450 activities), designated proximal tubule-Cpr-null (PTCN), and the other having proximal tubule-specific rescue of CPR activity with the global suppression of CPR activity in all extra-proximal tubular tissues, designated extra-proximal tubule-Cpr-low (XPT-CL). The PTCN, XPT-CL, Cpr-low (CL), and wild-type (WT) mice were treated with a single oral dose of chloroform at 200 mg/kg. Blood, liver and kidney samples were obtained at 24 h after the treatment. Renal toxicity was assessed by measuring BUN and creatinine levels, and by pathological examination. The blood and tissue levels of chloroform were determined. The severity of toxicity was less in PTCN and CL mice, compared with that of WT and XPT-CL mice. There were no significant differences in chloroform levels in the blood, liver, or kidney, between PTCN and WT mice, or between XPT-CL and CL mice. These findings indicate that local P450-dependent activities play an important role in the nephrotoxicity induced by chloroform. Our results also demonstrate the usefulness of these novel mouse models for studies of chemical-induced kidney toxicity. - Highlights: • New mouse models were developed with varying P450 activities in the proximal tubule. • These mouse models were treated with chloroform, a nephrotoxicant. • Studies showed the importance of local P450s in chloroform-induced nephrotoxicity

  13. Stage-specific and age-dependent profiles of zinc, copper, manganese, and selenium in rat seminiferous tubules

    International Nuclear Information System (INIS)

    Homma-Takeda, S.; Nishimura, Y.; Watanabe, Y.; Imaseki, H.; Yukawa, M.

    2004-01-01

    Stage-specific and age-dependent profiles of zinc, copper, manganese, and selenium in testis were examined in Wistar rats by both inductively coupled argon plasma-mass spectrometry (ICP-MS) with a microdissection technique and in situ elemental imaging of micro-PIXE analysis. The young adult animals (10 weeks old) contained higher levels of zinc and manganese in the seminiferous tubules at stages VII-VIII than stages XI through VI and IX-X and the levels were higher than those of the immature and old animals. Copper and selenium levels at stages VII-VIII of the young adult animals were also higher than those of the immature and old animals. In stages VII and VIII, zinc was higher in the central area of the seminiferous epithelium, where spermatozoa were localized, demonstrating a cell-specific property. (author)

  14. FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.

    Science.gov (United States)

    Begonja, Antonija Jurak; Pluthero, Fred G; Suphamungmee, Worawit; Giannini, Silvia; Christensen, Hilary; Leung, Richard; Lo, Richard W; Nakamura, Fumihiko; Lehman, William; Plomann, Markus; Hoffmeister, Karin M; Kahr, Walter H A; Hartwig, John H; Falet, Hervé

    2015-07-02

    Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation. © 2015 by The American Society of Hematology.

  15. Antimicrobial Effect of Citrus Aurantifolia Extract on Enterococcus Faecalis Within the Dentinal Tubules in The Presence of Smear Layer

    Directory of Open Access Journals (Sweden)

    Sharifian MR

    2011-12-01

    Full Text Available Background and Aims: Instrumentation of the root canals results in formation of smear layer which covers the dentinal tubules. In infected teeth, it is ideal to achieve a material that has the ability to remove the smear layer besides antimicrobial activity. Therefore, this study was designed to evaluate the antimicrobial effect of Citrus aurantifolia extracts (lime juice and rind extract on Enterococcus faecalis within dentinal tubules in the presence of smear layer.Materials and Methods: One-hundred and forty dentin tubes were prepared from bovine incisors. After removal the smear layer, the specimens were infected with Enterococcus faecalis. Then, the smear layer was reformed. Test solutions were used as the irrigants in study roups as follows: group 1: 5.25% NaOCl; group 2: 17% EDTA; group 3: NaOCl+EDTA; group 4: Lime juice; group 5: ethanolic rind extract of C.aurantifolia; group 6: 96% ethanol. Dentin chips were collected from inner and outer layers of dentinal walls and optical density was measured. The data were analyzed using one-way ANOVA and Tamhane tests.Results: In outer layer of dentin, the efficacy of rind extract was less than that of NaOCl+EDTA (P<0.05. Also Lime juice was less effective than EDTA, NaOCl and NaOCl+EDTA (P<0.05. In inner layer of dentin, Lime juice was significantly less effective than NaOCl and NaOCl+EDTA (P<0.05. The efficacy of rind extract was less than that of NaOCl+EDTA (P<0.05.Conclusion: In the presence of smear layer, the antimicrobial activity of Lime juice was less than that of NaOCl but the efficacy of rind extract was similar to that of NaOCl.

  16. Development of a living membrane comprising a functional human renal proximal tubule cell monolayer on polyethersulfone polymeric membrane.

    Science.gov (United States)

    Schophuizen, Carolien M S; De Napoli, Ilaria E; Jansen, Jitske; Teixeira, Sandra; Wilmer, Martijn J; Hoenderop, Joost G J; Van den Heuvel, Lambert P W; Masereeuw, Rosalinde; Stamatialis, Dimitrios

    2015-03-01

    The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a "living membrane", consisting of a tight kidney cell monolayer with preserved functional organic ion transporters on a suitable artificial membrane surface. In this work, we applied a unique conditionally immortalized proximal tubule epithelial cell (ciPTEC) line with an optimized coating strategy on polyethersulfone (PES) membranes to develop a living membrane with a functional proximal tubule epithelial cell layer. PES membranes were coated with combinations of 3,4-dihydroxy-l-phenylalanine and human collagen IV (Coll IV). The optimal coating time and concentrations were determined to achieve retention of vital blood components while preserving high water transport and optimal ciPTEC adhesion. The ciPTEC monolayers obtained were examined through immunocytochemistry to detect zona occludens 1 tight junction proteins. Reproducible monolayers were formed when using a combination of 2 mg ml(-1) 3,4-dihydroxy-l-phenylalanine (4 min coating, 1h dissolution) and 25 μg ml(-1) Coll IV (4 min coating). The successful transport of (14)C-creatinine through the developed living membrane system was used as an indication for organic cation transporter functionality. The addition of metformin or cimetidine significantly reduced the creatinine transepithelial flux, indicating active creatinine uptake in ciPTECs, most likely mediated by the organic cation transporter, OCT2 (SLC22A2). In conclusion, this study shows the successful development of a living membrane consisting of a reproducible ciPTEC monolayer on PES membranes, an important step towards the development of a bioartificial kidney. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  17. Plasticity of the transverse tubules following denervation and subsequent reinnervation in rat slow and fast muscle fibres.

    Science.gov (United States)

    Takekura, Hiroaki; Tamaki, Hiroyuki; Nishizawa, Tomie; Kasuga, Norikatsu

    2003-01-01

    We have studied the effects of short term denervation followed by reinnervation on the ultrastructure of the membrane systems and on the content of and distribution of key proteins involved in calcium regulation of fast-twitch (FT) extensor digitorum longus (EDL) and slow-twitch (ST) soleus (SOL) muscle fibres. Ischiadic nerve freezing resulted in total lack of neuromuscular transmission for 3 days followed by a slow recovery, but no decline in twitch force elicited by direct stimulation. The latter measurements indicate no significant atrophy within this time frame. The membrane systems of skeletal muscle fibres were visualized using Ca92+)-K3Fe(CN)6-OsO4 techniques and observed using a high voltage electron microscope. [3H]nitrendipine binding was used to detect levels of dihydropyridine receptor (DHPR) expression. The Ca2+ pumping free sarcoplasmic reticulum domains were not affected by the denervation, but the Ca2+ release domains were dramatically increased, particularly in the FT-EDL muscle fibres. The increase is evidenced by a doubling up of the areas of contacts between SR and transverse (t-) tubules, so that in place of the normal triadic arrangement, pentadic and heptadic junctions, formed by multiple interacting layers of ST and t-tubules are seen. Frequency of pentads and heptads increases and declines in parallel to the denervation and reinnervation but with a delay. Immunofluorecence and electron microscopy observations show presence of DHPR and ryanodine receptor clusters at pentads and heptads junctions. A significant (P muscle fibres indicating that overexpression of DHPRs accompanies the build up extra junctional contacts. The results indicate that denervation reversibly affects the domains of the membrane systems involved in excitation-contraction coupling.

  18. Antenatal betamethasone attenuates the angiotensin-(1-7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells.

    Science.gov (United States)

    Su, Yixin; Bi, Jianli; Pulgar, Victor M; Chappell, Mark C; Rose, James C

    2017-06-01

    We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na + ) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to angiotensin-(1-7) [Ang-(1-7)]. The present study determined the Na + uptake and nitric oxide (NO) response to low-dose Ang-(1-7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as percentage of basal uptake or area under the curve for Na + or percentage of control for NO. Male Beta RPTC exhibited greater Na + uptake than male vehicle cells (433 ± 28 vs. 330 ± 26%; P 0.05). Ang-(1-7) significantly inhibited Na + uptake in RPTC from vehicle male (214 ± 11%) and from both vehicle (190 ± 14%) and Beta (209 ± 11%) females but failed to attenuate Na + uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1-7) in male but not female RPTC. Both the Na + and NO responses to Ang-(1-7) were blocked by Mas receptor antagonist d-Ala 7 -Ang-(1-7). We conclude that the tubular Ang-(1-7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na + handling by the renal tubules. Copyright © 2017 the American Physiological Society.

  19. Diglycolic acid is the nephrotoxic metabolite in diethylene glycol poisoning inducing necrosis in human proximal tubule cells in vitro.

    Science.gov (United States)

    Landry, Greg M; Martin, Sarah; McMartin, Kenneth E

    2011-11-01

    Diethylene glycol (DEG), a solvent and chemical intermediate, can produce an acute toxic syndrome, the hallmark of which is acute renal failure due to cortical tubular degeneration and proximal tubular necrosis. DEG is metabolized to two primary metabolites, 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA), which are believed to be the proximate toxicants. The precise mechanism of toxicity has yet to be elucidated, so these studies were designed to determine which metabolite was responsible for the proximal tubule cell death. Human proximal tubule (HPT) cells in culture, obtained from normal cortical tissue and passaged 3-6 times, were incubated with increasing concentrations of DEG, 2-HEAA, or DGA separately and in combination for 48 h at pH 6 or 7.4, and various parameters of necrotic and apoptotic cell death were measured. DEG and 2-HEAA did not produce any cell death. DGA produced dose-dependent necrosis at concentrations above 25 mmol/l. DGA did not affect caspase-3 activity and increased annexin V staining only in propidium iodide-stained cells. Hence, DGA induced necrosis, not apoptosis, as corroborated by severe depletion of cellular adenosine triphosphate levels. DGA is structurally similar to citric acid cycle intermediates that are taken up by specific transporters in kidney cells. HPT cells, incubated with N-(p-amylcinnamoyl)anthranilic acid, a sodium dicarboxylate-1 transporter inhibitor showed significantly decreased cell death compared with DGA alone. These studies demonstrate that DGA is the toxic metabolite responsible for DEG-induced proximal tubular necrosis and suggest a possible transporter-mediated uptake of DGA leading to toxic accumulation and cellular dysfunction.

  20. The Influence of Concentration and Temperature on the Formation of ¿-Oryzanol + ß-Sitosterol Tubules in Edible Oil Organogels

    NARCIS (Netherlands)

    Sawalha, H.I.M.; Venema, P.; Bot, A.; Flöter, E.; Linden, van der E.

    2011-01-01

    The gelation process of mixtures of ¿-oryzanol and sitosterol structurants in sunflower oil was studied using light scattering, rheology, and micro-scanning calorimetry (Micro-DSC). The relation between temperature and the critical aggregation concentration (CAC) of tubule formation of ¿-oryzanol

  1. The influence of the type of oil phase on the self-assembly process of ¿-oryzanol + ß-sitosterol tubules in organogel systems

    NARCIS (Netherlands)

    Sawalha, H.I.M.; Margry, G.; Adel, den R.; Venema, P.; Bot, A.; Flöter, E.; Linden, van der E.

    2013-01-01

    Mixtures of ¿-oryzanol and ß-sitosterol were used to structure different oils (decane, limonene, sunflower oil, castor oil and eugenol). The ¿-oryzanol and ß-sitosterol mixtures self-assemble into double-walled hollow tubules (~10 nm in diameter) in the oil phase, which aggregate to form a network

  2. Comparative Evaluation of Efficacy of Iontophoresis with 0.33% Sodium Fluoride Gel and Diode Laser Alone on Occlusion of Dentinal Tubules.

    Science.gov (United States)

    Patil, Anup Raghunath; Varma, Siddhartha; Suragimath, Girish; Abbayya, Keshava; Zope, Sameer Anil; Kale, Vishwajeet

    2017-08-01

    Dentinal Hypersensitivity (DH) is one of the most commonly encountered clinical problems. Literature reveals no specific therapy to satisfactorily eliminate dentinal hypersensitivity. The aim of this study was to assess and compare the efficacy of iontophoresis with 0.33% Sodium Fluoride (NaF) gel and diode laser alone in dentinal tubule occlusion. This in vitro study included 20 teeth with intact root surfaces unaltered by extraction procedure for specimen preparation. Each tooth was cleaned, air dried and cut into three sections. Total 60 sections were prepared (30 longitudinal and 30 transverse sections), which were acid etched. In control group, no treatment was carried. In iontophoresis treatment group, samples were inserted into a foam tray containing 0.33 % NaF Gel and subjected to 1.5 mA output current for three minutes. In laser treatment group, specimens were lased with 980 nm diode laser at 0.5 W/PW (62.2J /cm 2 ) in a noncontact mode for 30 seconds. Specimens were evaluated under Scanning Electron Microscope (SEM) at 10KV to 20KV under x 2000, x5000 magnification for surface characteristics and patency of dentinal tubules. Total number of tubules visible, open, completely and partially occluded were recorded in each microphotograph and compared. On comparison, laser group showed the least number of open tubules i.e., 130 (31.1%) followed by iontophoresis group, 155 (51.32%) and control group 417 (100%). Diode laser application provided better results as compared to iontophoresis on occlusion of dentinal tubules. Hence, it can be used to treat the patients with DH.

  3. Phosphorylation of rat kidney Na-K pump at Ser938 is required for rapid angiotensin II-dependent stimulation of activity and trafficking in proximal tubule cells.

    Science.gov (United States)

    Massey, Katherine J; Li, Quanwen; Rossi, Noreen F; Keezer, Susan M; Mattingly, Raymond R; Yingst, Douglas R

    2016-02-01

    How angiotensin (ANG) II acutely stimulates the Na-K pump in proximal tubules is only partially understood, limiting insight into how ANG II increases blood pressure. First, we tested whether ANG II increases the number of pumps in plasma membranes of native rat proximal tubules under conditions of rapid activation. We found that exposure to 100 pM ANG II for 2 min, which was previously shown to increase affinity of the Na-K pump for Na and stimulate activity threefold, increased the amount of the Na-K pump in plasma membranes of native tubules by 33%. Second, we tested whether previously observed increases in phosphorylation of the Na-K pump at Ser(938) were part of the stimulatory mechanism. These experiments were carried out in opossum kidney cells, cultured proximal tubules stably coexpressing the ANG type 1 (AT1) receptor, and either wild-type or a S938A mutant of rat kidney Na-K pump under conditions found by others to stimulate activity. We found that 10 min of incubation in 10 pM ANG II stimulated activity of wild-type pumps from 2.3 to 3.5 nmol K · mg protein(-1) · min(-1) and increased the amount of the pump in the plasma membrane by 80% but had no effect on cells expressing the S938A mutant. We conclude that acute stimulation of Na-K pump activity in native rat proximal tubules includes increased trafficking to the plasma membrane and that phosphorylation at Ser(938) is part of the mechanism by which ANG II directly stimulates activity and trafficking of the rat kidney Na-K pump in opossum kidney cells.

  4. Efficacy of 4 Irrigation Protocols in Killing Bacteria Colonized in Dentinal Tubules Examined by a Novel Confocal Laser Scanning Microscope Analysis.

    Science.gov (United States)

    Azim, Adham A; Aksel, Hacer; Zhuang, Tingting; Mashtare, Terry; Babu, Jegdish P; Huang, George T-J

    2016-06-01

    The aim of this study was to determine the efficiency of 4 irrigation systems in eliminating bacteria in root canals, particularly in dentinal tubules. Roots of human teeth were prepared to 25/04, autoclaved, and inoculated with Enterococcus faecalis for 3 weeks. Canals were then disinfected by (1) standard needle irrigation, (2) sonically agitating with EndoActivator, (3) XP Endo finisher, or (4) erbium:yttrium aluminum garnet laser (PIPS) (15 roots/group). The bacterial reduction in the canal was determined by MTT assays. For measuring live versus dead bacteria in the dentinal tubules (4 teeth/group), teeth were split open and stained with LIVE/DEAD BackLight. Coronal, middle, and apical thirds of the canal dentin were scanned by using a confocal laser scanning microscope (CLSM) to determine the ratio of dead/total bacteria in the dentinal tubules at various depths. All 4 irrigation protocols significantly eliminated bacteria in the canal, ranging from 89.6% to 98.2% reduction (P bacteria in the coronal, middle, and apical segments at 50-μm depth. On the other hand, PIPS had the greatest bacterial killing efficiency at the 150-μm depth in all 3 root segments. XP Endo appears to be more efficient than other 3 techniques in disinfecting the main canal space and up to 50 μm deep into the dentinal tubules. PIPS appears to be most effective in killing the bacteria deep in the dentinal tubules. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  5. The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

    Science.gov (United States)

    Yang, Chih-Wei; Wu, Mai-Szu; Pan, Ming-Jeng; Hsieh, Wang-Ju; Vandewalle, Alain; Huang, Chiu-Ching

    2002-08-01

    Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

  6. In vitro remineralization of enamel subsurface lesions and assessment of dentine tubule occlusion from NaF dentifrices with and without calcium

    Directory of Open Access Journals (Sweden)

    A R Prabhakar

    2013-01-01

    Full Text Available Currently, fluoride is the most effective preventive treatment for remineralization of incipient carious lesions and dentinal hypersensitivity due to wasting disorders. The products containing fluoride, calcium and phosphate are also claim to remineralize early, non-cavitated enamel demineralization. The aim of this study was to investigate and compare the efficacy of two such products, Tooth Mousse and Clinpro tooth crème on remineralization and tubule occluding ability with 5000ppm fluoride-containing toothpaste. Materials and Methods :Thirty third molar teeth were placed in demineralizing solution for 5 days such that only a window of 1mm x 5mm was exposed to the environment to produce artificial caries-like lesions and randomly assigned to three groups: Group I, 5000ppm sodium fluoride; Group II, GC MI paste plus and Group III, Clinpro tooth crème. Axial longitudinal sections of 140-160 μm of each tooth which included the artificial carious lesion taken and were photographed under polarized light microscope. The demineralized areas were then quantified with a computerized imaging system. The experimental materials were applied onto the tooth sections as a topical coating and subjected to pH-cycling for 28 days. To evaluate tubule occlusion ability, thirty dentin specimens of 2mm thickness were obtained from cervical third of sound third molars. Specimens were ultrasonicated and etched with 6% citric acid for 2 minutes to simulate the hypersensitive dentin. Specimens were randomly divided into above mentioned three groups (n=10. The test agents were brushed over the specimens with an electric toothbrush, prepared and observed under Scanning Electron Microscope for calculation of the percentage of occluded tubules. Results: Group I showed a significantly greater percentage of remineralization than Group III and Group II. Comparison of the remineralization potential between group II and group III were not significant.In case of dentine

  7. Visfatin Reduces Gap Junction Mediated Cell-to-Cell Communication in Proximal Tubule-Derived Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Claire E. Hills

    2013-11-01

    Full Text Available Background/Aims: In the current study we examined if the adipocytokine, visfatin, alters connexin-mediated intercellular communication in proximal tubule-derived epithelial cells. Methods: The effects of visfatin (10-200ng/mL on cell viability and cytotoxicity in HK2-cells were assessed by MTT, crystal violet and lactate dehydrogenase assays. Western blot analysis was used to confirm expression of Cx26, Cx40 and Cx43. The effect of visfatin (10-200ng/mL on TGF-β1 secretion was confirmed by ELISA, and the effects of both TGF-β1 (2-10ng/mL and visfatin (10-200ng/mL on connexin expression were assessed by western blot. Functional intercellular communication was determined using transfer of Lucifer Yellow and paired-whole cell patch clamp electrophysiology. Results: In low glucose (5mM, visfatin (10-200ng/mL did not affect membrane integrity, cytotoxicity or cell viability at 48hrs, but did evoke a concentration-dependent reduction in Cx26 and Cx43 expression. The expression of Cx40 was unaffected. At 48hrs, visfatin (10-200ng/mL increased the secretion of TGF-β1 and the visfatin-evoked changes in connexin expression were mimicked by exogenous application of the pro-fibrotic cytokine (2-10ng/ml. Visfatin reduced dye transfer between coupled cells and decreased functional conductance, with levels falling by 63% as compared to control. Although input resistance was increased following visfatin treatment by 166%, the change was not significant as compared to control. The effects of visfatin on Cx-expression and cell-coupling were blocked in the presence of a TGF-β1 specific neutralizing antibody. Conclusions: The adipocytokine visfatin selectively evoked a non-toxic reduction in connexin expression in HK2-cells. The loss in gap-junction associated proteins was mirrored by a loss in functional conductance between coupled cells. Visfatin increased TGF-β secretion and the pattern of change for connexins expression was mimicked by exogenous

  8. A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells

    International Nuclear Information System (INIS)

    Sarró, Eduard; Jacobs-Cachá, Conxita; Itarte, Emilio; Meseguer, Anna

    2012-01-01

    Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC. Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells. Highlights: ► We used a novel pharmacological approach to elucidate cyclosporine (CsA) toxicity. ► The ability of a broad range of compounds to prevent CsA toxicity was evaluated. ► CsA toxicity was monitored using LDH release assay and PARP cleavage. ► Protein synthesis, PI3K, GSK3, MMP, PKC and caspase inhibitors prevented CsA toxicity. ► We also identified ER

  9. A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Sarró, Eduard, E-mail: eduard.sarro@vhir.org [Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain); Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Jacobs-Cachá, Conxita, E-mail: conxita.jacobs@vhir.org [Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Itarte, Emilio, E-mail: emili.itarte@uab.es [Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain); Meseguer, Anna, E-mail: ana.meseguer@vhir.org [Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Departament de Bioquimica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain)

    2012-01-15

    Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC. Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells. Highlights: ► We used a novel pharmacological approach to elucidate cyclosporine (CsA) toxicity. ► The ability of a broad range of compounds to prevent CsA toxicity was evaluated. ► CsA toxicity was monitored using LDH release assay and PARP cleavage. ► Protein synthesis, PI3K, GSK3, MMP, PKC and caspase inhibitors prevented CsA toxicity. ► We also identified ER

  10. Effect of water on self-assembled tubules in β-sitosterol + γ-oryzanol-based organogels

    Science.gov (United States)

    den Adel, Ruud; Heussen, Patricia C. M.; Bot, Arjen

    2010-10-01

    Mixtures of β-sitosterol and γ-oryzanol form a network in triglyceride oil that may serve as an alternative to the network of small crystallites of triglycerides occurring in regular oil structuring. The present x-ray diffraction study investigates the relation between the crystal forms of the individual compounds and the mixture in oil, water and emulsion. β-Sitosterol and γ-oryzanol form normal crystals in oil, in water, or in emulsions. The crystals are sensitive to the presence of water. The mixture of β-sitosterol + γ-oryzanol forms crystals in water and emulsions that can be traced back to the crystals of the pure compounds. Only in oil, a completely different structure emerges in the mixture of β-sitosterol + γ-oryzanol, which bears no relation to the structures that are formed by both individual compounds, and which can be identified as a self-assembled tubule (diameter 7.2±0.1 nm, wall thickness 0.8±0.2 nm).

  11. Physiological Functions and Regulation of the Na+/H+ Exchanger [NHE1] in Renal Tubule Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Patricia G Vallés

    2015-08-01

    Full Text Available The sodium-hydrogen exchanger isoform-1 [NHE1] is a ubiquitously expressed plasma membrane protein that plays a central role in intracellular pH and cell volume homeostasis by catalyzing an electroneutral exchange of extracellular sodium and intracellular hydrogen. Outside of this important physiological function, the NHE1 cytosolic tail domain acts as a molecular scaffold regulating cell survival and actin cytoskeleton organization through NHE1-dependent signaling proteins. NHE1 plays main roles in response to physiological stress conditions which in addition to cell shrinkage and acidification, include hypoxia and mechanical stimuli, such as cell stretch. NHE1-mediated modulation of programmed cell death results from the exchanger-mediated changes in pHi, cell volume, and/or [Na+]I; and, it has recently become known that regulation of cellular signaling pathways are involved as well. This review focuses on NHE1 functions and regulations. We describe evidence showing how these structural actions integrate with ion translocation in regulating renal tubule epithelial cell survival.

  12. Comprehensive analyses of how tubule occlusion and advanced glycation end-products diminish strength of aged dentin

    Science.gov (United States)

    Shinno, Yuko; Ishimoto, Takuya; Saito, Mitsuru; Uemura, Reo; Arino, Masumi; Marumo, Keishi; Nakano, Takayoshi; Hayashi, Mikako

    2016-01-01

    In clinical dentistry, since fracture is a major cause of tooth loss, better understanding of mechanical properties of teeth structures is important. Dentin, the major hard tissue of teeth, has similar composition to bone. In this study, we investigated the mechanical properties of human dentin not only in terms of mineral density but also using structural and quality parameters as recently accepted in evaluating bone strength. Aged crown and root dentin (age ≥ 40) exhibited significantly lower flexural strength and toughness than young dentin (age mineral density; but showed significantly lower flexural strength than young dentin. Dentin with strong alignment of the c-axis in hydroxyapatite exhibited high fracture strength, possibly because the aligned apatite along the collagen fibrils may reinforce the intertubular dentin. Aged dentin, showing a high advanced glycation end-products (AGEs) level in its collagen, recorded low flexural strength. We first comprehensively identified significant factors, which affected the inferior mechanical properties of aged dentin. The low mechanical strength of aged dentin is caused by the high mineral density resulting from occlusion of dentinal tubules and accumulation of AGEs in dentin collagen.

  13. Urinary Excretion of Tetrodotoxin Modeled in a Porcine Renal Proximal Tubule Epithelial Cell Line, LLC-PK1

    Directory of Open Access Journals (Sweden)

    Takuya Matsumoto

    2017-07-01

    Full Text Available This study examined the urinary excretion of tetrodotoxin (TTX modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK1. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA, l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH, and unaffected by 1-methyl-4-phenylpyridinium (MPP+, oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs and organic cation/carnitine transporters (OCTNs, partially transported by organic anion transporters (OATs and multidrug resistance-associated proteins (MRPs, and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs.

  14. Construction of bioartificial renal tubule assist device in vitro and its function of transporting sodium and glucose.

    Science.gov (United States)

    Dong, Xinggang; Chen, Jianghua; He, Qiang; Yang, Yi; Zhang, Wei

    2009-08-01

    To explore a new way of constructing bioartificial renal tubule assist device (RAD) in vitro and its function of transporting sodium (Na(+)) and glucose and to evaluate the application of atomic force microscope in the RAD construction, rat renal tubular epithelial cell line NRK-52E was cultured in vitro, seeded onto the outer surfaces of hollow fibers in a bioreactor, and then cultured for two weeks to construct RAD. Bioreactor hollow fibers without NRK-52E cells were used as control. The morphologies of attached cells were observed with scanning electron microscope, and the junctions of cells and polysulfone membrane were observed with atomic force microscope. Transportation of Na(+) and glucose was measured. Oubaine and phlorizin were used to inhibit the transporting property. The results showed that NRK-52E cells and polysulfone membrane were closely linked, as observed under atomic force microscope. After exposure to oubaine and phlorizin, transporting rates of Na(+) and glucose were decreased significantly in the RAD group as compared with that in the control group (Pconstructed successfully in vitro, and it is able to selectively transport Na(+) and glucose.

  15. Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

    International Nuclear Information System (INIS)

    Klinefelter, G.R.; Hamilton, D.W.

    1985-01-01

    We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-[35S]methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-[35S]methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB[3H]4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis

  16. Effect of water on self-assembled tubules in {beta}-sitosterol + {gamma}-oryzanol-based organogels

    Energy Technology Data Exchange (ETDEWEB)

    Adel, Ruud den; Heussen, Patricia C M; Bot, Arjen, E-mail: ruud-den.adel@unilever.co [Unilever Research and Development Vlaardingen, Olivier van Noortlaan 120, NL-3133 AT Vlaardingen (Netherlands)

    2010-10-01

    Mixtures of {beta}-sitosterol and {gamma}-oryzanol form a network in triglyceride oil that may serve as an alternative to the network of small crystallites of triglycerides occurring in regular oil structuring. The present x-ray diffraction study investigates the relation between the crystal forms of the individual compounds and the mixture in oil, water and emulsion. {beta}-Sitosterol and {gamma}-oryzanol form normal crystals in oil, in water, or in emulsions. The crystals are sensitive to the presence of water. The mixture of {beta}-sitosterol + {gamma}-oryzanol forms crystals in water and emulsions that can be traced back to the crystals of the pure compounds. Only in oil, a completely different structure emerges in the mixture of {beta}-sitosterol + {gamma}-oryzanol, which bears no relation to the structures that are formed by both individual compounds, and which can be identified as a self-assembled tubule (diameter 7.2{+-}0.1 nm, wall thickness 0.8{+-}0.2 nm).

  17. Effect of water on self-assembled tubules in β-sitosterol + γ-oryzanol-based organogels

    International Nuclear Information System (INIS)

    Adel, Ruud den; Heussen, Patricia C M; Bot, Arjen

    2010-01-01

    Mixtures of β-sitosterol and γ-oryzanol form a network in triglyceride oil that may serve as an alternative to the network of small crystallites of triglycerides occurring in regular oil structuring. The present x-ray diffraction study investigates the relation between the crystal forms of the individual compounds and the mixture in oil, water and emulsion. β-Sitosterol and γ-oryzanol form normal crystals in oil, in water, or in emulsions. The crystals are sensitive to the presence of water. The mixture of β-sitosterol + γ-oryzanol forms crystals in water and emulsions that can be traced back to the crystals of the pure compounds. Only in oil, a completely different structure emerges in the mixture of β-sitosterol + γ-oryzanol, which bears no relation to the structures that are formed by both individual compounds, and which can be identified as a self-assembled tubule (diameter 7.2±0.1 nm, wall thickness 0.8±0.2 nm).

  18. Localization of Secondary Metabolites in Marine Invertebrates: Contribution of MALDI MSI for the Study of Saponins in Cuvierian Tubules of H. forskali

    Science.gov (United States)

    Meriaux, Céline; Bonnel, David; Salzet, Michel; Fournier, Isabelle; Wisztorski, Maxence

    2010-01-01

    Background Several species of sea cucumbers of the family Holothuriidae possess a particular mechanical defense system called the Cuvierian tubules (Ct). It is also a chemical defense system as triterpene glycosides (saponins) appear to be particularly concentrated in Ct. In the present study, the precise localization of saponins in the Ct of Holothuria forskali is investigated. Classical histochemical labeling using lectin was firstly performed but did not generate any conclusive results. Thus, MALDI mass spectrometry Imaging (MALDI-MSI) was directly applied and completed by statistical multivariate tests. A comparison between the tubules of relaxed and stressed animals was realized. Results These analyses allowed the detection of three groups of ions, corresponding to the isomeric saponins of the tubules. Saponins detected at m/z 1287 and 1303 were the most abundant and were apparently localized in the connective tissue of the tubules of both relaxed and stressed individuals. Saponins at m/z 1125 and 1141 were detected in lower amount and were present in tissues of relaxed animals. Finally, saponin ions at 1433, 1449, 1463 and 1479 were observed in some Ct of stressed holothuroids in the outer part of the connective tissue. The saponin group m/z 14xx seems therefore to be stress-specific and could originate from modifications of the saponins with m/z of 11xx. Conclusions All the results taken together indicate a complex chemical defense mechanism with, for a single organ, different sets of saponins originating from different cell populations and presenting different responses to stress. The present study also reflects that MALDI-MSI is a valuable tool for chemical ecology studies in which specific chemical signalling molecules like allelochemicals or pheromones have to be tracked. This report represents one of the very first studies using these tools to provide a functional and ecological understanding of the role of natural products from marine invertebrates

  19. De novo expression of sodium-glucose cotransporter SGLT2 in Bowman's capsule coincides with replacement of parietal epithelial cell layer with proximal tubule-like epithelium.

    Science.gov (United States)

    Tabatabai, Niloofar M; North, Paula E; Regner, Kevin R; Kumar, Suresh N; Duris, Christine B; Blodgett, Amy B

    2014-08-01

    In kidney nephron, parietal epithelial cells line the Bowman's capsule and function as a permeability barrier for the glomerular filtrate. Bowman's capsule cells with proximal tubule epithelial morphology have been found. However, the effects of tubular metaplasia in Bowman's capsule on kidney function remain poorly understood. Sodium-glucose cotransporter 2 (SGLT2) plays a major role in reabsorption of glucose in the kidney and is expressed on brush border membrane (BBM) of epithelial cells in the early segment of the proximal tubule. We hypothesized that SGLT2 is expressed in tubularized Bowman's capsule and used our novel antibody to test this hypothesis. Immunohistochemical analysis was performed with our SGLT2 antibody on C57BL/6 mouse kidney prone to have tubularized Bowman's capsules. Cell membrane was examined with periodic acid-Schiff (PAS) stain. The results showed that SGLT2 was localized on BBM of the proximal tubules in young and adult mice. Bowman's capsules were lined mostly with normal brush border-less parietal epithelial cells in young mice, while they were almost completely covered with proximal tubule-like cells in adult mice. Regardless of age, SGLT2 was expressed on BBM of the tubularized Bowman's capsule but did not co-localize with nephrin in the glomerulus. SGLT2-expressing tubular cells expanded from the urinary pole toward the vascular pole of the Bowman's capsule. This study identified the localization of SGLT2 in the Bowman's capsule. Bowman's capsules with tubular metaplasia may acquire roles in reabsorption of filtered glucose and sodium.

  20. The Influence of Concentration and Temperature on the Formation of γ-Oryzanol + β-Sitosterol Tubules in Edible Oil Organogels.

    Science.gov (United States)

    Sawalha, Hassan; Venema, Paul; Bot, Arjen; Flöter, Eckhard; van der Linden, Erik

    2011-03-01

    The gelation process of mixtures of γ-oryzanol and sitosterol structurants in sunflower oil was studied using light scattering, rheology, and micro-scanning calorimetry (Micro-DSC). The relation between temperature and the critical aggregation concentration (CAC) of tubule formation of γ-oryzanol and sitosterol was determined using these techniques. The temperature dependence of the CAC was used to estimate the binding energy and enthalpic and entropic contribution to the tubular formation process. The binding energy calculated at the corresponding temperatures and CACs were relatively low, in order of 2 RT (4.5 kJ mol(-1)), which is in accord with the reversibility of the tubular formation process. The formation of the tubules was associated with negative (exothermic) enthalpy change (ΔH(0)) compared with positive entropy term (-T ΔS(0) >0), indicating that the aggregation into tubules is an enthalpy-driven process. The oryzanol-sitosterol ratio affected the aggregation process; solutions with ratio of (60 oryzanol-40 sitosterol) started aggregation at higher temperature compared with other ratios.

  1. Dysferlin, annexin A1, and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch.

    Science.gov (United States)

    Waddell, Leigh B; Lemckert, Frances A; Zheng, Xi F; Tran, Jenny; Evesson, Frances J; Hawkes, Joanne M; Lek, Angela; Street, Neil E; Lin, Peihui; Clarke, Nigel F; Landstrom, Andrew P; Ackerman, Michael J; Weisleder, Noah; Ma, Jianjie; North, Kathryn N; Cooper, Sandra T

    2011-04-01

    Mutations in dysferlin cause an inherited muscular dystrophy because of defective membrane repair. Three interacting partners of dysferlin are also implicated in membrane resealing: caveolin-3 (in limb girdle muscular dystrophy type 1C), annexin A1, and the newly identified protein mitsugumin 53 (MG53). Mitsugumin 53 accumulates at sites of membrane damage, and MG53-knockout mice display a progressive muscular dystrophy. This study explored the expression and localization of MG53 in human skeletal muscle, how membrane repair proteins are modulated in various forms of muscular dystrophy, and whether MG53 is a primary cause of human muscle disease. Mitsugumin 53 showed variable sarcolemmal and/or cytoplasmic immunolabeling in control human muscle and elevated levels in dystrophic patients. No pathogenic MG53 mutations were identified in 50 muscular dystrophy patients, suggesting that MG53 is unlikely to be a common cause of muscular dystrophy in Australia. Western blot analysis confirmed upregulation of MG53, as well as of dysferlin, annexin A1, and caveolin-3 to different degrees, in different muscular dystrophies. Importantly, MG53, annexin A1, and dysferlin localize to the t-tubule network and show enriched labeling at longitudinal tubules of the t-system in overstretch. Our results suggest that longitudinal tubules of the t-system may represent sites of physiological membrane damage targeted by this membrane repair complex.

  2. Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Wolff Natascha A

    2010-05-01

    Full Text Available Abstract Background The class 1 carcinogen cadmium (Cd2+ disrupts the E-cadherin/β-catenin complex of epithelial adherens junctions (AJs and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/β-catenin signaling are known to contribute to carcinogenesis. Results We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC. Cd2+ (25 μM, 3-9 h caused nuclear translocation of β-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of β-catenin with AJ components (E-cadherin, α-catenin and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1 were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. Conclusions Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.

  3. Superresolution microscope image reconstruction by spatiotemporal object decomposition and association: application in resolving t-tubule structure in skeletal muscle.

    Science.gov (United States)

    Sun, Mingzhai; Huang, Jiaqing; Bunyak, Filiz; Gumpper, Kristyn; De, Gejing; Sermersheim, Matthew; Liu, George; Lin, Pei-Hui; Palaniappan, Kannappan; Ma, Jianjie

    2014-05-19

    One key factor that limits resolution of single-molecule superresolution microscopy relates to the localization accuracy of the activated emitters, which is usually deteriorated by two factors. One originates from the background noise due to out-of-focus signals, sample auto-fluorescence, and camera acquisition noise; and the other is due to the low photon count of emitters at a single frame. With fast acquisition rate, the activated emitters can last multiple frames before they transiently switch off or permanently bleach. Effectively incorporating the temporal information of these emitters is critical to improve the spatial resolution. However, majority of the existing reconstruction algorithms locate the emitters frame by frame, discarding or underusing the temporal information. Here we present a new image reconstruction algorithm based on tracklets, short trajectories of the same objects. We improve the localization accuracy by associating the same emitters from multiple frames to form tracklets and by aggregating signals to enhance the signal to noise ratio. We also introduce a weighted mean-shift algorithm (WMS) to automatically detect the number of modes (emitters) in overlapping regions of tracklets so that not only well-separated single emitters but also individual emitters within multi-emitter groups can be identified and tracked. In combination with a maximum likelihood estimator method (MLE), we are able to resolve low to medium density of overlapping emitters with improved localization accuracy. We evaluate the performance of our method with both synthetic and experimental data, and show that the tracklet-based reconstruction is superior in localization accuracy, particularly for weak signals embedded in a strong background. Using this method, for the first time, we resolve the transverse tubule structure of the mammalian skeletal muscle.

  4. Interactive toxicity of inorganic mercury and trichloroethylene in rat and human proximal tubules: Effects on apoptosis, necrosis, and glutathione status

    International Nuclear Information System (INIS)

    Lash, Lawrence H.; Putt, David A.; Hueni, Sarah E.; Payton, Scott G.; Zwickl, Joshua

    2007-01-01

    Simultaneous or prior exposure to one chemical may alter the concurrent or subsequent response to another chemical, often in unexpected ways. This is particularly true when the two chemicals share common mechanisms of action. The present study uses the paradigm of prior exposure to study the interactive toxicity between inorganic mercury (Hg 2+ ) and trichloroethylene (TRI) or its metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in rat and human proximal tubule. Pretreatment of rats with a subtoxic dose of Hg 2+ increased expression of glutathione S-transferase-α1 (GSTα1) but decreased expression of GSTα2, increased activities of several GSH-dependent enzymes, and increased GSH conjugation of TRI. Primary cultures of rat proximal tubular (rPT) cells exhibited both necrosis and apoptosis after incubation with Hg 2+ . Pretreatment of human proximal tubular (hPT) cells with Hg 2+ caused little or no changes in GST expression or activities of GSH-dependent enzymes, decreased apoptosis induced by TRI or DCVC, but increased necrosis induced by DCVC. In contrast, pretreatment of hPT cells with TRI or DCVC protected from Hg 2+ by decreasing necrosis and increasing apoptosis. Thus, whereas pretreatment of hPT cells with Hg 2+ exacerbated cellular injury due to TRI or DCVC by shifting the response from apoptosis to necrosis, pretreatment of hPT cells with either TRI or DCVC protected from Hg 2+ -induced cytotoxicity by shifting the response from necrosis to apoptosis. These results demonstrate that by altering processes related to GSH status, susceptibilities of rPT and hPT cells to acute injury from Hg 2+ , TRI, or DCVC are markedly altered by prior exposures

  5. Cationic uremic toxins affect human renal proximal tubule cell functioning through interaction with the organic cation transporter.

    Science.gov (United States)

    Schophuizen, Carolien M S; Wilmer, Martijn J; Jansen, Jitske; Gustavsson, Lena; Hilgendorf, Constanze; Hoenderop, Joost G J; van den Heuvel, Lambert P; Masereeuw, Rosalinde

    2013-12-01

    Several organic cations, such as guanidino compounds and polyamines, have been found to accumulate in plasma of patients with kidney failure due to inadequate renal clearance. Here, we studied the interaction of cationic uremic toxins with renal organic cation transport in a conditionally immortalized human proximal tubule epithelial cell line (ciPTEC). Transporter activity was measured and validated in cell suspensions by studying uptake of the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP(+)). Subsequently, the inhibitory potencies of the cationic uremic toxins, cadaverine, putrescine, spermine and spermidine (polyamines), acrolein (polyamine breakdown product), guanidine, and methylguanidine (guanidino compounds) were determined. Concentration-dependent inhibition of ASP(+) uptake by TPA, cimetidine, quinidine, and metformin confirmed functional endogenous organic cation transporter 2 (OCT2) expression in ciPTEC. All uremic toxins tested inhibited ASP(+) uptake, of which acrolein required the lowest concentration to provoke a half-maximal inhibition (IC50 = 44 ± 2 μM). A Dixon plot was constructed for acrolein using three independent inhibition curves with 10, 20, or 30 μM ASP(+), which demonstrated competitive or mixed type of interaction (K i = 93 ± 16 μM). Exposing the cells to a mixture of cationic uremic toxins resulted in a more potent and biphasic inhibitory response curve, indicating complex interactions between the toxins and ASP(+) uptake. In conclusion, ciPTEC proves a suitable model to study cationic xenobiotic interactions. Inhibition of cellular uptake transport was demonstrated for several uremic toxins, which might indicate a possible role in kidney disease progression during uremia.

  6. Gestational Undernourishment Modifies the Composition of Skeletal Muscle Transverse Tubule Membranes and the Mechanical Properties of Muscles in Newborn Rats

    Directory of Open Access Journals (Sweden)

    Ricardo Tonathiu Ramírez-Oseguera

    2013-10-01

    Full Text Available Backgroud/Aims: Skeletal muscle (SM constitutes more than 40% of the body weight in adulthood. Transports dietary glucose mainly through the insulin-dependent glucose transporter (Glut-4 located in the Transverse tubule membrane system (TT. The TT development ends shortly after birth. The TT membrane hosts the proteins involved in excitation-contraction coupling and glucose uptake. Glycaemic regulation through movement is a key function of fully developed skeletal muscle. In this study, we aimed to characterize the effect of gestational undernourishment (GUN in rats GLUT-4 expression and on the protein/lipid content of the TT membranes. We also examined the effect of GUN on the mechanical properties of muscles as an indication of the metabolic condition of the SM at birth. Methods: Isolated TT membrane from SM of GUN rats were used to study lipid/protein content and protein stability by differential scanning calorimetry. The effect of GUN on the SM mechanical properties was determined in isolated Extensor Digitorum Longus (EDL muscle. Results: We demonstrate that compared to control, GUN in the new-born produces; i decreases body weight; ii diminution in SM mass; iii decreases the formation of TT membranes; iv expresses TT membrane proteins with higher thermal stability. The TT membrane expression of GLUT-4 in GUN offspring was twice that of controls. The isolated EDL of GUN offspring was 20% stronger as measured by contractile force and more resistant to fatigue relative to controls. Conclusion; These results provide the first evidence of adaptive changes of the SM in new-borns exposed to severe gestational food restriction. The effects of GUN on muscle at birth are the first step toward detrimental SM metabolic function, contributing to the physiopathology of metabolic diseases in adulthood.

  7. Gestational undernourishment modifies the composition of skeletal muscle transverse tubule membranes and the mechanical properties of muscles in newborn rats.

    Science.gov (United States)

    Ramírez-Oseguera, Ricardo Tonathiu; Jiménez-Garduño, Aura Matilde; Alvarez, Rocío; Heine, Katharina; Pinzón-Estrada, Enrique; Torres-Saldaña, Ismael; Ortega, Alicia

    2013-01-01

    [corrected] Skeletal muscle (SM) constitutes more than 40% of the body weight in adulthood. Transports dietary glucose mainly through the insulin-dependent glucose transporter (Glut-4) located in the Transverse tubule membrane system (TT). The TT development ends shortly after birth. The TT membrane hosts the proteins involved in excitation-contraction coupling and glucose uptake. Glycaemic regulation through movement is a key function of fully developed skeletal muscle. In this study, we aimed to characterize the effect of gestational undernourishment (GUN) in rats GLUT-4 expression and on the protein/lipid content of the TT membranes. We also examined the effect of GUN on the mechanical properties of muscles as an indication of the metabolic condition of the SM at birth. Isolated TT membrane from SM of GUN rats were used to study lipid/protein content and protein stability by differential scanning calorimetry. The effect of GUN on the SM mechanical properties was determined in isolated Extensor Digitorum Longus (EDL) muscle. We demonstrate that compared to control, GUN in the new-born produces; i) decreases body weight; ii) diminution in SM mass; iii) decreases the formation of TT membranes; iv) expresses TT membrane proteins with higher thermal stability. The TT membrane expression of GLUT-4 in GUN offspring was twice that of controls. The isolated EDL of GUN offspring was 20% stronger as measured by contractile force and more resistant to fatigue relative to controls. These results provide the first evidence of adaptive changes of the SM in new-borns exposed to severe gestational food restriction. The effects of GUN on muscle at birth are the first step toward detrimental SM metabolic function, contributing to the physiopathology of metabolic diseases in adulthood. © 2013 S. Karger AG, Basel

  8. Regulation of the Na+2Cl–K+ cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias.

    Science.gov (United States)

    Warth, R; Bleich, M; Thiele, I; Lang, F; Greger, R

    1998-07-01

    Previously it has been shown that the Na+2Cl–K+ cotransporter accepts NH4 + at its K+ binding site. This property can be used to estimate its transport rates by adding NH4 + to the bath and measuring the initial furosemide-dependent rates of change in BCECF fluorescence. We have utilized this technique to determine the regulation of the furosemide-inhibitable Na+2Cl–K+ cotransporter in in vitroperfused rectal gland tubules (RGT) of Squalus acanthias. Addition of NH4 + to the bath (20 mmol/l) led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4 + uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δfluorescence/time). This acidification could be completely inhibited by furosemide. In the absence of any secretagogue preincubation of RGT in a low Cl– solution (6 mmol/l, low Cl–) for 10 min enhanced the uptake rate significantly from 4.04±0.51 to 12.7±1.30 (n=5). The addition of urea (200 mmol/l) was without effect, but the addition of 300 mmol/l mannitol (+300 mannitol) enhanced the rate significantly from 7.24±1.33 to 14.7±4.6 (n=6). Stimulation of NaCl secretion by a solution maximizing the cytosolic cAMP concentration (Stim) led to a significant increase in NH4 + uptake rate from 5.00±1.33 to 13.3±1.54 (n=6). Similar results were obtained in the additional presence of Ba2+ (1 mmol/l): the uptake rate was increased significantly from 4.23±0.34 to 15.1±1.86 (n=16). In the presence of Stim low Cl– had no additional effect on the uptake rate: 15.1±3.1 versus 15.2±2.8 in high Cl– (n=6). The uptake rate in Stim containing additional +300 mannitol (22.3±4.0, n=5) was not significantly different from that obtained with Stim or +300 mannitol alone. By whatever mechanism the NH4 + uptake rate was increased furosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced furosemide

  9. Does stimulation of NaCl secretion in in vitro perfused rectal gland tubules of Squalus acanthias increase membrane capacitance?

    Science.gov (United States)

    Greger, R; Thiele, I; Warth, R; Bleich, M

    1998-07-01

    NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl– channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl– channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl– channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 µmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G m). The membrane capacitance (C m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C m correlated with the increase in G m with a slope of 51 fF/nS. Next the effect of furosemide (500 µmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na+2Cl–K+ uptake and to reduce cell volume but not membrane trafficking of Cl– channels. Furosemide reduced G m slightly (due to the fall in cytosolic Cl– concentration) and C m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC m/ΔG m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 µmol/l to the pipette solution and dialysed for >10 min) did not alter cell voltage, G m or C m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C m changes observed with Stim reflect a minor cell volume increase and an

  10. Outer retinal tubulation as a predictor of the enlargement amount of geographic atrophy in age-related macular degeneration.

    Science.gov (United States)

    Hariri, Amirhossein; Nittala, Muneeswar G; Sadda, SriniVas R

    2015-02-01

    To determine the prognostic value of outer retinal tubulation (ORT) in the enlargement amount of geographic atrophy (GA) in eyes with age-related macular degeneration (AMD). Cohort study. One hundred eight fellow untreated eyes of 143 patients with GA resulting from AMD enrolled in the MAHALO study (clinicaltrials.gov identifier, NCT01229215) who completely satisfied the study term and had gradable spectral-domain optical coherence tomography (OCT) images obtained at both baseline and month 18 visits. The MAHALO study enrolled 143 subjects into a phase 1b/2 multicenter, randomized, single-masked, sham-injection controlled clinical trial of the safety, tolerability, and evidence of activity of lampalizumab in patients with GA associated with AMD. Spectral-domain optical coherence tomography images were obtained at multiple time points in both eyes, although only the baseline and month 18 data of the fellow (nonstudy) eyes were considered in this exploratory analysis. The Cirrus HD-OCT review software was used for automatic segmentation and measurement of GA areas, with manual correction of segmentation errors by certified OCT graders. Baseline OCT images also were assessed for the presence of ORT. The enlargement amount of GA in eyes with ORT was compared with that of eyes without ORT. Comparison of the enlargement amount of GA in eyes with and without ORT. Twenty-four of these 108 eyes demonstrated evidence of ORT. The amount of enlargement of GA in eyes with ORT was significantly slower than that of eyes without ORT (1.85±0.78 vs. 2.67±1.61; P = 0.001). This difference remained significant when considering subgroups with unifocal or multifocal GA lesions, because eyes with ORT in both subgroups had a slower enlargement amount of GA than eyes without ORT (2.91±1.70 vs. 2.08±0.88 [P = 0.01], in eyes with multifocal GA lesions; and 2.24±1.40 vs. 1.63±0.57 [P = 0.02], in eyes with unifocal GA lesions). In eyes with ORT, GA lesions seem to enlarge at a

  11. Polyploidy and Mitotic Cell Death Are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells.

    Science.gov (United States)

    Payne, Emily H; Ramalingam, Dhivya; Fox, Donald T; Klotman, Mary E

    2018-01-15

    Prior studies have found that HIV, through the Vpr protein, promotes genome reduplication (polyploidy) in infection-surviving epithelial cells within renal tissue. However, the temporal progression and molecular regulation through which Vpr promotes polyploidy have remained unclear. Here we define a sequential progression to Vpr-mediated polyploidy in human renal tubule epithelial cells (RTECs). We found that as in many cell types, Vpr first initiates G 2 cell cycle arrest in RTECs. We then identified a previously unreported cascade of Vpr-dependent events that lead to renal cell survival and polyploidy. Specifically, we found that a fraction of G 2 -arrested RTECs reenter the cell cycle. Following this cell cycle reentry, two distinct outcomes occur. Cells that enter complete mitosis undergo mitotic cell death due to extra centrosomes and aberrant division. Conversely, cells that abort mitosis undergo endoreplication to become polyploid. We further show that multiple small-molecule inhibitors of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including those that target ATR, ATM, and mTOR, indirectly prevent Vpr-mediated polyploidy by preventing G 2 arrest. In contrast, an inhibitor that targets DNA-dependent protein kinase (DNA-PK) specifically blocks the Vpr-mediated transition from G 2 arrest to polyploidy. These findings outline a temporal, molecularly regulated path to polyploidy in HIV-positive renal cells. IMPORTANCE Current cure-focused efforts in HIV research aim to elucidate the mechanisms of long-term persistence of HIV in compartments. The kidney is recognized as one such compartment, since viral DNA and mRNA persist in the renal tissues of HIV-positive patients. Further, renal disease is a long-term comorbidity in the setting of HIV. Thus, understanding the regulation and impact of HIV infection on renal cell biology will provide important insights into this unique HIV compartment. Our work identifies mechanisms that distinguish

  12. 'Special K' and a Loss of Cell-To-Cell Adhesion in Proximal Tubule-Derived Epithelial Cells: Modulation of the Adherens Junction Complex by Ketamine

    Science.gov (United States)

    Hills, Claire E.; Jin, Tianrong; Siamantouras, Eleftherios; Liu, Issac K-K; Jefferson, Kieran P.; Squires, Paul E.

    2013-01-01

    Ketamine, a mild hallucinogenic class C drug, is the fastest growing ‘party drug’ used by 16–24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24–48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1–1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention. PMID:24009666

  13. Organic anion and cation SLC22 "drug" transporter (Oat1, Oat3, and Oct1 regulation during development and maturation of the kidney proximal tubule.

    Directory of Open Access Journals (Sweden)

    Thomas F Gallegos

    Full Text Available Proper physiological function in the pre- and post-natal proximal tubule of the kidney depends upon the acquisition of selective permeability, apical-basolateral epithelial polarity and the expression of key transporters, including those involved in metabolite, toxin and drug handling. Particularly important are the SLC22 family of transporters, including the organic anion transporters Oat1 (originally identified as NKT and Oat3 as well as the organic cation transporter Oct1. In ex vivo cultures of metanephric mesenchyme (MM; the embryonic progenitor tissue of the nephron Oat function was evident before completion of nephron segmentation and corresponded with the maturation of tight junctions as measured biochemically by detergent extractability of the tight junction protein, ZO-1. Examination of available time series microarray data sets in the context of development and differentiation of the proximal tubule (derived from both in vivo and in vitro/ex vivo developing nephrons allowed for correlation of gene expression data to biochemically and functionally defined states of development. This bioinformatic analysis yielded a network of genes with connectivity biased toward Hnf4α (but including Hnf1α, hyaluronic acid-CD44, and notch pathways. Intriguingly, the Oat1 and Oat3 genes were found to have strong temporal co-expression with Hnf4α in the cultured MM supporting the notion of some connection between the transporters and this transcription factor. Taken together with the ChIP-qPCR finding that Hnf4α occupies Oat1, Oat3, and Oct1 proximal promoters in the in vivo differentiating rat kidney, the data suggest a network of genes with Hnf4α at its center plays a role in regulating the terminal differentiation and capacity for drug and toxin handling by the nascent proximal tubule of the kidney.

  14. Influence of extracellular HCO3- and pH on lysine (LYS) and leucine (LEU) uptake and metabolism in swine renal tubules

    International Nuclear Information System (INIS)

    Patience, J.F.; Esteve-Garcia, E.; Austic, R.E.

    1986-01-01

    Fragments of renal tubules prepared by collagenase treatment of renal cortex were suspended to Krebs-Henseleit buffers which were modified to contain 10, 25 and 35 mM HCO 3 - at pH 7.4, or 25 mM HCO 3 - at pH 7.1, 7.4 and 7.7. Buffers were oxygenated with O 2 -CO 2 gas mixtures varying in carbon dioxide concentration prior to incubation. Approximately 100 mg tubules were incubated with shaking at 37 0 C for 30 min in serum-stoppered 25 ml Erlenmeyer flasks in 3.0 ml of buffer containing 0.1% dialyzed bovine serum albumin, 5 mM D-glucose and 0.3 mM L-[U- 14 C]-lysine or L-[1- 14 C]-leucine. The incorporation of carbon-14 into CO 2 and into 10% sulfosalicylic acid (SSA)-soluble and SSA-insoluble fractions of the incubation mixture was determined. Low (10mM) bicarbonate reduced the incorporation of lys and leu into protein but did not substantially affect the recovery of 14 CO 2 from either amino acid. High pH (7.7) resulted in reduced incorporation of lys and leu into protein, and decreased the oxidation of lys but not leu. The specific activity of lys (leu was not determined) in the SSA-soluble fraction was unaffected by bicarbonate or pH. The authors conclude that variations in extracellular pH and HCO 3 - (or pCO 2 ) affect the metabolism of amino acids by renal tubules and that low extracellular HCO 3 - (or pCO 2 ) may depress the incorporation of amino acids into protein

  15. Dancing with the Stars: Using Image Analysis to Study the Choreography of the Endoplasmic Reticulum and Its Partners and of Movement Within Its Tubules.

    Science.gov (United States)

    Griffing, Lawrence R

    2018-01-01

    In this chapter, approaches to the image analysis of the choreography of the plant endoplasmic reticulum (ER) labeled with fluorescent fusion proteins ("stars," if you wish) are presented. The approaches include the analyses of those parts of the ER that are attached through membrane contact sites to moving or nonmoving partners (other "stars"). Image analysis is also used to understand the nature of the tubular polygonal network, the hallmark of this organelle, and how the polygons change over time due to tubule sliding or motion. Furthermore, the remodeling polygons of the ER interact with regions of fundamentally different topology, the ER cisternae, and image analysis can be used to separate the tubules from the cisternae. ER cisternae, like polygons and tubules, can be motile or stationary. To study which parts are attached to nonmoving partners, such as domains of the ER that form membrane contact sites with the plasma membrane/cell wall, an image analysis approach called persistency mapping has been used. To study the domains of the ER that are moving rapidly and streaming through the cell, the image analysis of optic flow has been used. However, optic flow approaches confuse the movement of the ER itself with the movement of proteins within the ER. As an overall measure of ER dynamics, optic flow approaches are of value, but their limitation as to what exactly is "flowing" needs to be specified. Finally, there are important imaging approaches that directly address the movement of fluorescent proteins within the ER lumen or in the membrane of the ER. Of these, fluorescence recovery after photobleaching (FRAP), inverse FRAP (iFRAP), and single particle tracking approaches are described.

  16. Differential patterns of injury to the proximal tubule of renal cortical slices following in vitro exposure to mercuric chloride, potassium dichromate, or hypoxic conditions.

    Science.gov (United States)

    Ruegg, C E; Gandolfi, A J; Nagle, R B; Brendel, K

    1987-09-15

    The innate susceptibility of renal cell types to these agents was investigated using precision-cut rabbit renal cortical slices made perpendicular to the cortical-papillary axis. Slices were incubated in DME/F12 medium containing 10 microM, 100 microM, or 1 mM concentrations of either metal for 12 hr or in Krebs-Hepes buffer gassed with nitrogen (100%) for 0.75 to 5 hr of hypoxic exposure. To simulate postischemic reperfusion, some slices were transferred to vessels gassed with oxygen after an initial hypoxic period. Mercuric chloride (100 microM) exposure resulted in damage to the straight regions of proximal tubules by 12 hr leaving convoluted regions unaffected. Hypoxia (2.25 hr) and potassium dichromate (100 microM for 12 hr) both caused injury to the convoluted proximal tubules without affecting straight proximal tubular regions. Mercury concentrations of 10 microM and 1 mM had no effect or injured all cell types within the slice, respectively. Similar results were observed for hypoxic periods less than 1.5 hr or greater than 3 hr of exposure. Potassium dichromate had no measurable affect at 10 microM, but at 1 mM focal lesions were observed after 4 hr of exposure, and by 12 hr all cell types within the slice were affected. Intracellular potassium content normalized to DNA correlated well, but always preceded the pathological lesions observed. These results demonstrate that injury to specific regions of the proximal tubule by these agents relates to an innate susceptibility of the intoxicated cell type independent of physiologic feedback or blood delivery patterns proposed as mechanisms of selective injury from in vivo studies.

  17. Proximal tubule proteins are significantly elevated in bladder urine of patients with ureteropelvic junction obstruction and may represent novel biomarkers: A pilot study.

    Science.gov (United States)

    Gerber, Claire; Harel, Miriam; Lynch, Miranda L; Herbst, Katherine W; Ferrer, Fernando A; Shapiro, Linda H

    2016-04-01

    Ureteropelvic junction obstruction (UPJO) is the major cause of hydronephrosis in children and may lead to renal injury and early renal dysfunction. However, diagnosis of the degree of obstruction and severity of renal injury relies on invasive and often inconclusive renal scans. Biomarkers from voided urine that detect early renal injury are highly desirable because of their noninvasive collection and their potential to assist in earlier and more reliable diagnosis of the severity of obstruction. Early in response to UPJO, increased intrarenal pressure directly impacts the proximal tubule brush border. We hypothesize that single-pass, apically expressed proximal tubule brush border proteins will be shed into the urine early and rapidly and will be reliable noninvasive urinary biomarkers, providing the tools for a more reliable stratification of UPJO patients. We performed a prospective cohort study at Connecticut Children's Medical Center. Bladder urine samples from 12 UPJO patients were obtained prior to surgical intervention. Control urine samples were collected from healthy pediatric patients presenting with primary nocturnal enuresis. We determined levels of NGAL, KIM-1 (previously identified biomarkers), CD10, CD13, and CD26 (potentially novel biomarkers) by ELISA in control and experimental urine samples. Urinary creatinine levels were used to normalize the urinary protein levels measured by ELISA. Each of the proximal tubule proteins outperformed the previously published biomarkers. No differences in urinary NGAL and KIM-1 levels were observed between control and obstructed patients (p = 0.932 and p = 0.799, respectively). However, levels of CD10, CD13, and CD26 were significantly higher in the voided urine of obstructed individuals when compared with controls (p = 0.002, p = 0.024, and p = 0.007, respectively) (Figure). Targeted identification of reliable, noninvasive biomarkers of renal injury is critical to aid in diagnosing patients at risk, guiding

  18. Equivalent complex conductivities representing the effects of T-tubules and folded surface membranes on the electrical admittance and impedance of skeletal muscles measured by external-electrode method

    Science.gov (United States)

    Sekine, Katsuhisa

    2017-12-01

    In order to represent the effects of T-tubules and folded surface membranes on the electrical admittance and impedance of skeletal muscles measured by the external-electrode method, analytical relations for the equivalent complex conductivities of hypothetical smooth surface membranes were derived. In the relations, the effects of each tubule were represented by the admittance of a straight cable. The effects of the folding of a surface membrane were represented by the increased area of surface membranes. The equivalent complex conductivities were represented as summation of these effects, and the effects of the T-tubules were different between the transversal and longitudinal directions. The validity of the equivalent complex conductivities was supported by the results of finite-difference method (FDM) calculations made using three-dimensional models in which T-tubules and folded surface membranes were represented explicitly. FDM calculations using the equivalent complex conductivities suggested that the electrically inhomogeneous structure due to the existence of muscle cells with T-tubules was sufficient for explaining the experimental results previously obtained using the external-electrode method. Results of FDM calculations in which the structural changes caused by muscle contractions were taken into account were consistent with the reported experimental results.

  19. Hypoxia inducible factor 1-alpha (HIF-1 alpha is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival.

    Directory of Open Access Journals (Sweden)

    Elisa Conde

    Full Text Available Acute tubular necrosis (ATN caused by ischemia/reperfusion (I/R during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α, using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

  20. The in vitro antibacterial effect of iodine-potassium iodide and calcium hydroxide in infected dentinal tubules at different time intervals.

    Science.gov (United States)

    Lin, Shaul; Kfir, Anda; Laviv, Amir; Sela, Galit; Fuss, Zvi

    2009-03-01

    The aim of this study was to evaluate the antibacterial effect of iodine-potassium iodide (IKI) and calcium hydroxide (CH) on dentinal tubules infected with Enterococcus faecalis (E. faecalis) at different time intervals. Hollow cylinders of bovine root dentin (n=45) were infected and divided into three equal groups filled with either IKI or CH and a positive control. After placing each medicament in the infected cylinders for time periods of 10 minutes, 48 hours and 7 days, microbiological samples were analyzed. At the end of each period, four 100 microm thick inner dentin layers (400 microm thick from each specimen) were removed using dental burs of increasing diameters. Dentin powder was cultured on agar plates to quantitatively assess their infection, expressed in colony forming units (cfu). In all layers of the positive control group, heavy bacterial infection was observed. After 10 minutes, IKI reduced the amount of viable bacteria more efficiently than CH, whereas at later time intervals CH showed the best results. For short periods of exposure, IKI has a more efficient antibacterial effect in the dentinal tubules than CH but CH performs better after longer durations of exposure. This research indicates the use of IKI is a better choice for disinfecting the root canal than CH if only a short duration of exposure is used because of its more efficient antibacterial effect. However, if a longer exposure time is used, then CH is a better choice because of its better disinfecting effect over time.

  1. Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor in a cardiac T-tubule/SR microdomain.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms, Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor "dyads." E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B(+/- cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.

  2. Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells.

    Science.gov (United States)

    Gildea, John J; Xu, Peng; Kemp, Brandon A; Carlson, Julia M; Tran, Hanh T; Bigler Wang, Dora; Langouët-Astrié, Christophe J; McGrath, Helen E; Carey, Robert M; Jose, Pedro A; Felder, Robin A

    2018-01-01

    Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte

  3. Experimental type II diabetes and related models of impaired glucose metabolism differentially regulate glucose transporters at the proximal tubule brush border membrane.

    Science.gov (United States)

    Chichger, Havovi; Cleasby, Mark E; Srai, Surjit K; Unwin, Robert J; Debnam, Edward S; Marks, Joanne

    2016-06-01

    What is the central question of this study? Although SGLT2 inhibitors represent a promising treatment for patients suffering from diabetic nephropathy, the influence of metabolic disruption on the expression and function of glucose transporters is largely unknown. What is the main finding and its importance? In vivo models of metabolic disruption (Goto-Kakizaki type II diabetic rat and junk-food diet) demonstrate increased expression of SGLT1, SGLT2 and GLUT2 in the proximal tubule brush border. In the type II diabetic model, this is accompanied by increased SGLT- and GLUT-mediated glucose uptake. A fasted model of metabolic disruption (high-fat diet) demonstrated increased GLUT2 expression only. The differential alterations of glucose transporters in response to varying metabolic stress offer insight into the therapeutic value of inhibitors. SGLT2 inhibitors are now in clinical use to reduce hyperglycaemia in type II diabetes. However, renal glucose reabsorption across the brush border membrane (BBM) is not completely understood in diabetes. Increased consumption of a Western diet is strongly linked to type II diabetes. This study aimed to investigate the adaptations that occur in renal glucose transporters in response to experimental models of diet-induced insulin resistance. The study used Goto-Kakizaki type II diabetic rats and normal rats rendered insulin resistant using junk-food or high-fat diets. Levels of protein kinase C-βI (PKC-βI), GLUT2, SGLT1 and SGLT2 were determined by Western blotting of purified renal BBM. GLUT- and SGLT-mediated d-[(3) H]glucose uptake by BBM vesicles was measured in the presence and absence of the SGLT inhibitor phlorizin. GLUT- and SGLT-mediated glucose transport was elevated in type II diabetic rats, accompanied by increased expression of GLUT2, its upstream regulator PKC-βI and SGLT1 protein. Junk-food and high-fat diet feeding also caused higher membrane expression of GLUT2 and its upstream regulator PKC

  4. Changes in gene expression in human renal proximal tubule cells exposed to low concentrations of S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene

    International Nuclear Information System (INIS)

    Lock, Edward A.; Barth, Jeremy L.; Argraves, Scott W.; Schnellmann, Rick G.

    2006-01-01

    Epidemiology studies suggest that there may be a weak association between high level exposure to trichloroethylene (TCE) and renal tubule cell carcinoma. Laboratory animal studies have shown an increased incidence of renal tubule carcinoma in male rats but not mice. TCE can undergo metabolism via glutathione (GSH) conjugation to form metabolites that are known to be nephrotoxic. The GSH conjugate, S-(1,2-dichlorovinyl)glutathione (DCVG), is processed further to the cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), which is the penultimate nephrotoxic species. We have cultured human renal tubule cells (HRPTC) in serum-free medium under a variety of different culture conditions and observed growth, respiratory control and glucose transport over a 20 day period in medium containing low glucose. Cell death was time- and concentration-dependent, with the EC 5 for DCVG being about 3 μM and for DCVC about 7.5 μM over 10 days. Exposure of HRPTC to sub-cytotoxic doses of DCVC (0.1 μM and 1 μM for 10 days) led to a small number of changes in gene expression, as determined by transcript profiling with Affymetrix human genome chips. Using the criterion of a mean 2-fold change over control for the four samples examined, 3 genes at 0.1 μM DCVC increased, namely, adenosine kinase, zinc finger protein X-linked and an enzyme with lyase activity. At 1 μM DCVC, two genes showed a >2-fold decrease, N-acetyltransferase 8 and complement factor H. At a lower stringency (1.5-fold change), a total of 63 probe sets were altered at 0.1 μM DCVC and 45 at 1 μM DCVC. Genes associated with stress, apoptosis, cell proliferation and repair and DCVC metabolism were altered, as were a small number of genes that did not appear to be associated with the known mode of action of DCVC. Some of these genes may serve as molecular markers of TCE exposure and effects in the human kidney

  5. Epidermal growth factor decreases PEPT2 transport capacity and expression in the rat kidney proximal tubule cell line SKPT0193 cl.2

    DEFF Research Database (Denmark)

    Bravo, Silvina A; Nielsen, Carsten Uhd; Amstrup, Jan

    2004-01-01

    by studies of apical uptake of [14C]glycylsarcosine, rPepT2 mRNA levels, and immunostaining of SKPT cells with a rPEPT2-specific antibody. On the contrary, apical uptake of glucose and lysine was increased in EGF-treated cells, indicating that EGF was not acting generally to decrease apical nutrient uptake...... mechanisms in the proximal tubule cells. Our findings indicate that EGF decreases rPEPT2 expression by lowering transcription of the rat PepT2 gene or by decreasing rat PepT2 mRNA stability. Previous investigators routinely used SKPT cell culture media with a high (10 ng/ml) EGF concentration. Our study...

  6. Distinct mechanisms underlie adaptation of proximal tubule Na+/H+ exchanger isoform 3 in response to chronic metabolic and respiratory acidosis.

    Science.gov (United States)

    Silva, Pedro Henrique Imenez; Girardi, Adriana Castello Costa; Neri, Elida Adalgisa; Rebouças, Nancy Amaral

    2012-04-01

    The Na(+/)H(+) exchanger isoform 3 (NHE3) is essential for HCO(3)(-) reabsorption in renal proximal tubules. The expression and function of NHE3 must adapt to acid-base conditions. The goal of this study was to elucidate the mechanisms responsible for higher proton secretion in proximal tubules during acidosis and to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and, if so, to identify the relevant parameters that may trigger these distinct adaptive responses. We achieved metabolic acidosis by lowering HCO(3)(-) concentration in the cell culture medium and respiratory acidosis by increasing CO(2) tension in the incubator chamber. We found that cell-surface NHE3 expression was increased in response to both forms of acidosis. Mild (pH 7.21 ± 0.02) and severe (6.95 ± 0.07) metabolic acidosis increased mRNA levels, at least in part due to up-regulation of transcription, whilst mild (7.11 ± 0.03) and severe (6.86 ± 0.01) respiratory acidosis did not up-regulate NHE3 expression. Analyses of the Nhe3 promoter region suggested that the regulatory elements sensitive to metabolic acidosis are located between -466 and -153 bp, where two consensus binding sites for SP1, a transcription factor up-regulated in metabolic acidosis, were localised. We conclude that metabolic acidosis induces Nhe3 promoter activation, which results in higher mRNA and total protein level. At the plasma membrane surface, NHE3 expression was increased in metabolic and respiratory acidosis alike, suggesting that low pH is responsible for NHE3 displacement to the cell surface.

  7. Novel Hg2+-Induced Nephropathy in Rats and Mice Lacking Mrp2: Evidence of Axial Heterogeneity in the Handling of Hg2+ Along the Proximal Tubule

    Science.gov (United States)

    Zalups, Rudolfs K.; Joshee, Lucy; Bridges, Christy C.

    2014-01-01

    The role of the multi-resistance protein 2 (Mrp2) in the nephropathy induced by inorganic mercuric mercury (Hg2+) was studied in rats (TR−) and mice (Mrp2−/−), which lack functional Mrp2, and control animals. Animals were exposed to nephrotoxic doses of HgCl2. Forty-eight or 24 hours after exposure, tissues were harvested and analyzed for Hg content and markers of injury. Histological analyses revealed that the proximal tubular segments affected pathologically by Hg2+ were significantly different between Mrp2-deficient animals and controls. In the absence of Mrp2, cellular injury localized almost exclusively in proximal tubular segments in the subcapsular (S1) to midcortical regions (early S2) of the kidney. In control animals, cellular death occurred mainly in the proximal tubular segments in the inner cortex (late S2) and outer stripe of the outer medulla (S3). These differences in renal pathology indicate that axial heterogeneity exists along the proximal tubule with respect to how mercuric ions are handled. Total renal and hepatic accumulation of mercury was also greater in animals lacking Mrp2 than in controls, indicating that Mrp2 normally plays a significant role in eliminating mercuric ions from within proximal tubular cells and hepatocytes. Analyses of plasma creatinine, BUN, and renal expression of Kim-1 and Ngal tend to support the severity of the nephropathies detected histologically. Collectively, our findings indicate that a fraction of mercuric ions is normally secreted by Mrp2 in early portions of proximal tubules into the lumen and then is absorbed downstream in straight portions, where mercuric species typically induce toxic effects. PMID:25145654

  8. Stimulation of Na+-alanine cotransport activates a voltage-dependent conductance in single proximal tubule cells isolated from frog kidney

    Science.gov (United States)

    Robson, L; Hunter, M

    1999-01-01

    The swelling induced by Na+-alanine cotransport in proximal tubule cells of the frog kidney is followed by regulatory volume decrease (RVD). This RVD is inhibited by gadolinium (Gd3+), an inhibitor of stretch-activated channels, but is independent of extracellular Ca2+. In this study, the whole cell patch clamp technique was utilized to examine the effect of Na+-alanine cotransport on two previously identified volume- and Gd3+-sensitive conductances. One conductance is voltage dependent and anion selective (GVD) whilst the other is voltage independent and cation selective (GVI). Addition of 5 mM L-alanine to the bathing solution increased the whole cell conductance and gave a positive (depolarizing) shift in the reversal potential (Vrev, equivalent to the membrane potential in current-clamped cells) consistent with activation of Na+-alanine cotransport. Vrev shifted from -36 ± 4·9 to +12·9 ± 4·2 mV (n= 15). In the presence of alanine, the total whole cell conductance had several components including the cotransporter conductance and GVD and GVI. These conductances were separated using Gd3+, which inhibits both GVD and GVI, and the time dependency of GVD. Of these two volume-sensitive conductances, L-alanine elicited a specific increase in GVD, whereas GVI was unaffected. The L-alanine-induced activation of GVD was significantly reduced when cells were incubated in a hypertonic bathing solution. In summary, in single proximal tubule cells isolated from frog kidney, on stimulation of Na+-alanine cotransport GVD is activated, while GVI is unaffected. Taken with other evidence, this suggests that GVD is activated by cell swelling, consequent upon alanine entry, and may play a role as an anion efflux pathway during alanine-induced volume regulation. PMID:10226159

  9. Arginine vasopressin increases cellular free calcium concentration and adenosine 3',5'-monophosphate production in rat renal papillary collecting tubule cells in culture

    International Nuclear Information System (INIS)

    Ishikawa, S.; Okada, K.; Saito, T.

    1988-01-01

    The role of calcium (Ca) in the cellular action of arginine vasopressin (AVP) was examined in rat renal papillary collecting tubule cells in culture. AVP increased both the cellular free Ca concentration ([Ca2+]i) using fura-2, and cAMP production in a dose-dependent manner. AVP-induced cellular Ca mobilization was totally blocked by the antagonist to the antidiuretic action of AVP, and somewhat weakened by the antagonist to the vascular action of AVP. 1-Deamino-8-D-AVP (dDAVP). an antidiuretic analog of AVP, also increased [Ca2+] significantly. Cellular Ca mobilization was not obtained with cAMP, forskolin (a diterpene activator of adenylate cyclase), or phorbol-12-myristate-13-acetate. The early phase of [Ca2+]i depended on the intracellular Ca pool, since an AVP-induced rise in [Ca2+]i was obtained in cells pretreated with Ca-free medium containing 1 mM EGTA, verapamil, or cobalt, which blocked cellular Ca uptake. Also, AVP increased 45 Ca2+ influx during the initial 10 min, which initiated the sustained phase of cellular Ca mobilization. However, cellular cAMP production induced by AVP during the 10-min observation period was diminished in the cells pretreated with Ca-free medium, verapamil, or cobalt, but was still significantly higher than the basal level. This was also diminished by a high Ca concentration in medium. These results indicate that 1) AVP concomitantly regulates cellular free Ca as well as its second messenger cAMP production; 2) AVP-induced elevation of cellular free Ca is dependent on both the cellular Ca pool and extracellular Ca; and 3) there is an optimal level of extracellular Ca to modulate the AVP action in renal papillary collecting tubule cells

  10. Characterization of rPEPT2-mediated Gly-Sar transport parameters in the rat kidney proximal tubule cell line SKPT-0193 cl.2 cultured in basic growth media

    DEFF Research Database (Denmark)

    Bravo, Silvina A; Nielsen, Carsten Uhd; Frokjaer, Sven

    2005-01-01

    The rat proximal kidney tubule cell line SKPT-0193 cl.2 (SKPT) expresses the di-/tripeptide transporter PEPT2 (rPEPT2) and has been used to study PEPT2-mediated transport. Traditionally, SKPT cells have been cultured in growth media supplemented with epidermal growth factor (EGF), apotransferrin,...

  11. Haematobia irritans dataset of raw sequence reads from Illumina-based transcriptome sequencing of specific tissues and life stages

    Science.gov (United States)

    Illumina HiSeq technology was used to sequence the transcriptome from various dissected tissues and life stages from the horn fly, Haematobia irritans. These samples include eggs (0, 2, 4, and 9 hours post-oviposition), adult fly gut, adult fly legs, adult fly malpighian tubule, adult fly ovary, adu...

  12. The effect of cold acclimation on active ion transport in cricket ionoregulatory tissues

    Czech Academy of Sciences Publication Activity Database

    Des Marteaux, Lauren E.; Khazraeenia, S.; Yerushalmi, G. Y.; Donini, A.; Li, N. G.; Sinclair, B. J.

    2018-01-01

    Roč. 216, FEB 01 (2018), s. 28-33 ISSN 1095-6433 Institutional support: RVO:60077344 Keywords : insect * Gryllus * malpighian tubules Subject RIV: ED - Physiology OBOR OECD: Biology (theoretical, mathematical, thermal, cryobiology, biological rhythm), Evolutionary biology Impact factor: 1.812, year: 2016 https://www.sciencedirect.com/science/article/pii/S1095643317302519?via%3Dihub

  13. The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Cannell, Elizabeth; Dornan, Anthony J.; Halberg, Kenneth Agerlin

    2016-01-01

    Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH44) and Drosophila melanogaster kinin...

  14. Brochosome influence on parasitisation efficiency of Homalodisca coagulata (Say) (Hemiptera: Cicadellidae) egg masses by Gonatocerus ashmeadi Girault (Hymenoptera : Mymaridae)

    NARCIS (Netherlands)

    Velema, H.P.; Hemerik, L.; Hoddle, M.S.; Luck, R.F.

    2005-01-01

    1. Many cicadellid females in the tribe Proconiini (Hemiptera: Cicadellidae) cover their egg masses with specialised, usually rod-shaped, brochosomes as the eggs are being laid. The brochosomes are produced in Golgi complexes in the Malpighian tubules of Cicadellidae. In contrast to the gravid

  15. Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2

    DEFF Research Database (Denmark)

    Iversen, Annette; Cazzamali, Giuseppe; Williamson, Michael

    2002-01-01

    the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification...

  16. A leucokinin mimic elicits aversive behavior in mosquito Aedes aegypti (L.) and inhibits the sugar taste neuron

    Science.gov (United States)

    Insect kinins (leucokinins) are multifunctional peptides acting as neurohormones and neurotransmitters. In females of the mosquito vector Aedes aegypti (L.), aedeskinins are known to stimulate fluid secretion from the renal organs (Malpighian tubules) and hindgut contractions by activating a G prot...

  17. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    2011-07-08

    Jul 8, 2011 ... Non-apoptotic function of apoptotic proteins in the development of Malpighian tubules of Drosophila melanogaster ... Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the ...

  18. Calcineurin inhibitor-induced complement system activation via ERK1/2 signalling is inhibited by SOCS-3 in human renal tubule cells.

    Science.gov (United States)

    Loeschenberger, Beatrix; Niess, Lea; Würzner, Reinhard; Schwelberger, Hubert; Eder, Iris E; Puhr, Martin; Guenther, Julia; Troppmair, Jakob; Rudnicki, Michael; Neuwirt, Hannes

    2018-02-01

    One factor that significantly contributes to renal allograft loss is chronic calcineurin inhibitor (CNI) nephrotoxicity (CIN). Among other factors, the complement (C-) system has been proposed to be involved CIN development. Hence, we investigated the impact of CNIs on intracellular signalling and the effects on the C-system in human renal tubule cells. In a qPCR array, CNI treatment upregulated C-factors and downregulated SOCS-3 and the complement inhibitors CD46 and CD55. Additionally, ERK1/-2 was required for these regulations. Following knock-down and overexpression of SOCS-3, we found that SOCS-3 inhibits ERK1/-2 signalling. Finally, we assessed terminal complement complex formation, cell viability and apoptosis. Terminal complement complex formation was induced by CNIs. Cell viability was significantly decreased, whereas apoptosis was increased. Both effects were reversed under complement component-depleted conditions. In vivo, increased ERK1/-2 phosphorylation and SOCS-3 downregulation were observed at the time of transplantation in renal allograft patients who developed a progressive decline of renal function in the follow-up compared to stable patients. The progressive cohort also had lower total C3 levels, suggesting higher complement activity at baseline. In conclusion, our data suggest that SOCS-3 inhibits CNI-induced ERK1/-2 signalling, thereby blunting the negative control of C-system activation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Antioxidative effects of fermented sesame sauce against hydrogen peroxide-induced oxidative damage in LLC-PK1 porcine renal tubule cells

    Science.gov (United States)

    Song, Jia-Le; Choi, Jung-Ho; Seo, Jae-Hoon; Kil, Jeung-Ha

    2014-01-01

    BACKGROUND/OBJECTIVES This study was performed to investigate the in vitro antioxidant and cytoprotective effects of fermented sesame sauce (FSeS) against hydrogen peroxide (H2O2)-induced oxidative damage in renal proximal tubule LLC-PK1 cells. MATERIALS/METHODS 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (•OH), and H2O2 scavenging assay was used to evaluate the in vitro antioxidant activity of FSeS. To investigate the cytoprotective effect of FSeS against H2O2-induced oxidative damage in LLC-PK1 cells, the cellular levels of reactive oxygen species (ROS), lipid peroxidation, and endogenous antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) were measured. RESULTS The ability of FSeS to scavenge DPPH, •OH and H2O2 was greater than that of FSS and AHSS. FSeS also significantly inhibited H2O2-induced (500 µM) oxidative damage in the LLC-PK1 cells compared to FSS and AHSS (P sauces, FSeS also significantly increased cellular CAT, SOD, and GSH-px activities and mRNA expression (P < 0.05). CONCULUSIONS These results from the present study suggest that FSeS is an effective radical scavenger and protects against H2O2-induced oxidative damage in LLC-PK1 cells by reducing ROS levels, inhibiting lipid peroxidation, and stimulating antioxidant enzyme activity. PMID:24741396

  20. Pathophyisiology analysis overload nephron protein in the proximal tubule damage in the nephrolithiasis size stones of 0.6-1.0 sm

    Directory of Open Access Journals (Sweden)

    Yu. Ye. Rohovyi

    2017-07-01

    Full Text Available Nephrolithiasis as the most common urological disorder characterized by frequent early relapses, acquires a social character, because these patients are 30 to 45% of all urological patients, while in Europe the disease is diagnosed in 2% of the population. The study included 40 patients with nephrolithiasis, 10 patients constituted the control group. Set the “hidden” damage to the proximal nephron in the presence of renal stones with a size of 0.6-1.0 cm in the upper third of the ureter, the upper and middle sections of the calyx, which is confirmed by the inhibition of enzymatic fibrinolytic activity of urine, the presence of tubular proteinuria in the absence of inhibition of proximal reabsorption of sodium ions. The link “hidden” damage to the proximal tubule of the degree of overload of the proximal nephron is a protein graphics multicorrelation dependence between the protein concentration of urine, standardized excretion protein and excretion, which are becoming more significant disharmony form towards control in the following sequence: the upper third of the ureter, medium cup, top cup.

  1. In vitro antimicrobial activity of sodium hypochlorite, chlorhexidine gluconate and octenidine dihydrochloride in elimination of microorganisms within dentinal tubules of primary and permanent teeth.

    Science.gov (United States)

    Tirali, Resmiye-Ebru; Bodur, Haluk; Ece, Gülden

    2012-05-01

    The aim of this study was to evaluate the effectiveness of different irrigation solutions at different time intervals for the elimination of E. faecalis and C. albicans penetrated into the dentine tubules of primary and permanent teeth in vitro. The 4 mm primary and permanent teeth sections were sterilized and contaminated with a mixture of E. faecalis and C. albicans strains. After the application of different irrigation solutions (Sodium hypochlorite, Chlorhexidine gluconate, Octenidine Dihydrochloride, saline) to the contaminated tooth sections according to study groups, neutralizers were applied for inactivation of the solutions after 30 sec, 1 min and 5 min. Dentine shavings were placed into TSB and 10 µL from each tube was inoculated on agar plates, followed by an incubation period of 24 h at 37°C. The colonies were counted macroscopically. The results were compared by using Kruskal-Wallis and Mann Whitney U tests, with a significance level at pteeth, the most effective one was found as 5-minute application of 0.1% Octenidine Dihydrochloride. The antibacterial effects of the tested solutions on the same time periods against C. albicans revealed no significant difference. There were no statistically significant differences between primary and permanent teeth with respect to the antimicrobial activity of the tested solutions. Moreover, Octenidine Dihydrochloride may be used as an alternative endodontic irrigant.

  2. Megalin-mediated specific uptake of chitosan/siRNA nanoparticles in mouse kidney proximal tubule epithelial cells enables AQP1 gene silencing.

    Science.gov (United States)

    Gao, Shan; Hein, San; Dagnæs-Hansen, Frederik; Weyer, Kathrin; Yang, Chuanxu; Nielsen, Rikke; Christensen, Erik I; Fenton, Robert A; Kjems, Jørgen

    2014-01-01

    RNAi-based strategies provide a great therapeutic potential for treatment of various human diseases including kidney disorders, but face the challenge of in vivo delivery and specific targeting. The chitosan delivery system has previously been shown to target siRNA specifically to the kidneys in mice when administered intravenously. Here we confirm by 2D and 3D bioimaging that chitosan formulated siRNA is retained in the kidney for more than 48 hours where it accumulates in proximal tubule epithelial cells (PTECs), a process that was strongly dependent on the molecular weight of chitosan. Chitosan/siRNA nanoparticles, administered to chimeric mice with conditional knockout of the megalin gene, distributed almost exclusively in cells that expressed megalin, implying that the chitosan/siRNA particle uptake was mediated by a megalin-dependent endocytotic pathway. Knockdown of the water channel aquaporin 1 (AQP1) by up to 50% in PTECs was achieved utilizing the systemic i.v. delivery of chitosan/AQP1 siRNA in mice. In conclusion, specific targeting PTECs with the chitosan nanoparticle system may prove to be a useful strategy for knockdown of specific genes in PTECs, and provides a potential therapeutic strategy for treating various kidney diseases.

  3. De novo expression of sodium-glucose cotransporter SGLT2 in Bowman’s capsule coincides with replacement of parietal epithelial cell layer with proximal tubule-like epithelium

    OpenAIRE

    Tabatabai, Niloofar M.; North, Paula E.; Regner, Kevin R.; Kumar, Suresh N.; Duris, Christine B.; Blodgett, Amy B.

    2014-01-01

    In kidney nephron, parietal epithelial cells line the Bowman’s capsule and function as a permeability barrier for the glomerular filtrate. Bowman’s capsule cells with proximal tubule epithelial morphology have been found. However, the effects of tubular metaplasia in Bowman’s capsule on kidney function remain poorly understood. Sodium-glucose cotransporter 2 (SGLT2) plays a major role in reabsorption of glucose in the kidney and is expressed on brush border membrane of epithelial cells in the...

  4. The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions.

    Science.gov (United States)

    Gildea, John J; Xu, Peng; Carlson, Julia M; Gaglione, Robert T; Bigler Wang, Dora; Kemp, Brandon A; Reyes, Camellia M; McGrath, Helen E; Carey, Robert M; Jose, Pedro A; Felder, Robin A

    2015-12-01

    The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake. Copyright © 2015 the American Physiological Society.

  5. Role of l-carnitine in the prevention of seminiferous tubules damage induced by gamma radiation: a light and electron microscopic study

    Energy Technology Data Exchange (ETDEWEB)

    Topcu-Tarladacalisir, Yeter; Kanter, Mehmet [Trakya University, Department of Histology and Embryology, Faculty of Medicine, Edirne (Turkey); Uzal, Mustafa Cem [Trakya University, Department of Radiation Oncology, Faculty of Medicine, Edirne (Turkey)

    2009-08-15

    The present study, we hypothesized that l-carnitine can minimize germ-cell depletion and morphological features of late cell damage in the rat testis following gamma ({gamma})-irradiation. Wistar albino male rats were divided into three groups. Control group received physiological saline 0.2 ml intraperitoneally (i.p.), as placebo. Radiation group received scrotal {gamma}-irradiation of 10 Gy as a single dose plus physiological saline. Radiation + l-carnitine group received scrotal {gamma}-irradiation plus 200 mg/kg i.p. l-carnitine. l-carnitine starting 1 day before irradiation and 21 days (three times per week) after irradiation. Testis samples of the all groups were taken at day 21, 44 and 70 post-irradiation. All samples were processed at the light and electron microscopic levels. Morphologically, examination of {gamma}-irradiated testis revealed presence of marked disorganization and depletion of germ cells, arrest of spermatogenesis, formation of multinucleated giant cells, and vacuolization in the germinal epithelium. The type and extent of these changes varied at different post-treatment intervals. The damage was evident at the 21st day and reached maximum level by the 44th day. By day 44 post-irradiation, the changes were most advanced, and were associated with atrophied seminiferous tubules without germ cells, the increase in the number and size of vacuolizations in germinal epithelium, and the absent multinucleated giant cells due to spermatids had completely disappeared. The increase in nucleus invaginations, the dilatation of smooth endoplasmic reticulum cysternas and the increase in the number and size of lipid droplets in the Sertoli cells were determined at the electron microscopic level. In conclusion, l-carnitine supplementation during the radiotherapy would be effective in protecting against radiation-induced damages in rat testis, and thereby may improve the quality of patient's life after the therapy. (orig.)

  6. Role of l-carnitine in the prevention of seminiferous tubules damage induced by gamma radiation: a light and electron microscopic study

    International Nuclear Information System (INIS)

    Topcu-Tarladacalisir, Yeter; Kanter, Mehmet; Uzal, Mustafa Cem

    2009-01-01

    The present study, we hypothesized that l-carnitine can minimize germ-cell depletion and morphological features of late cell damage in the rat testis following gamma (γ)-irradiation. Wistar albino male rats were divided into three groups. Control group received physiological saline 0.2 ml intraperitoneally (i.p.), as placebo. Radiation group received scrotal γ-irradiation of 10 Gy as a single dose plus physiological saline. Radiation + l-carnitine group received scrotal γ-irradiation plus 200 mg/kg i.p. l-carnitine. l-carnitine starting 1 day before irradiation and 21 days (three times per week) after irradiation. Testis samples of the all groups were taken at day 21, 44 and 70 post-irradiation. All samples were processed at the light and electron microscopic levels. Morphologically, examination of γ-irradiated testis revealed presence of marked disorganization and depletion of germ cells, arrest of spermatogenesis, formation of multinucleated giant cells, and vacuolization in the germinal epithelium. The type and extent of these changes varied at different post-treatment intervals. The damage was evident at the 21st day and reached maximum level by the 44th day. By day 44 post-irradiation, the changes were most advanced, and were associated with atrophied seminiferous tubules without germ cells, the increase in the number and size of vacuolizations in germinal epithelium, and the absent multinucleated giant cells due to spermatids had completely disappeared. The increase in nucleus invaginations, the dilatation of smooth endoplasmic reticulum cysternas and the increase in the number and size of lipid droplets in the Sertoli cells were determined at the electron microscopic level. In conclusion, l-carnitine supplementation during the radiotherapy would be effective in protecting against radiation-induced damages in rat testis, and thereby may improve the quality of patient's life after the therapy. (orig.)

  7. Leucine-rich repeat kinase 2 (LRRK2-deficient rats exhibit renal tubule injury and perturbations in metabolic and immunological homeostasis.

    Directory of Open Access Journals (Sweden)

    Daniel Ness

    Full Text Available Genetic evidence links mutations in the LRRK2 gene with an increased risk of Parkinson's disease, for which no neuroprotective or neurorestorative therapies currently exist. While the role of LRRK2 in normal cellular function has yet to be fully described, evidence suggests involvement with immune and kidney functions. A comparative study of LRRK2-deficient and wild type rats investigated the influence that this gene has on the phenotype of these rats. Significant weight gain in the LRRK2 null rats was observed and was accompanied by significant increases in insulin and insulin-like growth factors. Additionally, LRRK2-deficient rats displayed kidney morphological and histopathological alterations in the renal tubule epithelial cells of all animals assessed. These perturbations in renal morphology were accompanied by significant decreases of lipocalin-2, in both the urine and plasma of knockout animals. Significant alterations in the cellular composition of the spleen between LRRK2 knockout and wild type animals were identified by immunophenotyping and were associated with subtle differences in response to dual infection with rat-adapted influenza virus (RAIV and Streptococcus pneumoniae. Ontological pathway analysis of LRRK2 across metabolic and kidney processes and pathological categories suggested that the thioredoxin network may play a role in perturbing these organ systems. The phenotype of the LRRK2 null rat is suggestive of a complex biology influencing metabolism, immune function and kidney homeostasis. These data need to be extended to better understand the role of the kinase domain or other biological functions of the gene to better inform the development of pharmacological inhibitors.

  8. Smad mediated regulation of inhibitor of DNA binding 2 and its role in phenotypic maintenance of human renal proximal tubule epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mangalakumar Veerasamy

    Full Text Available The basic-Helix-Loop-Helix family (bHLH of transcriptional factors plays a major role in regulating cellular proliferation, differentiation and phenotype maintenance. The downregulation of one of the members of bHLH family protein, inhibitor of DNA binding 2 (Id2 has been shown to induce de-differentiation of epithelial cells. Opposing regulators of epithelial/mesenchymal phenotype in renal proximal tubule epithelial cells (PTEC, TGFβ1 and BMP7 also have counter-regulatory effects in models of renal fibrosis. We investigated the regulation of Id2 by these growth factors in human PTECs and its implication in the expression of markers of epithelial versus myofibroblastic phenotype. Cellular Id2 levels were reduced by TGFβ1 treatment; this was prevented by co-incubation with BMP7. BMP7 alone increased cellular levels of Id2. TGFβ1 and BMP7 regulated Id2 through Smad2/3 and Smad1/5 dependent mechanisms respectively. TGFβ1 mediated Id2 suppression was essential for α-SMA induction in PTECs. Although Id2 over-expression prevented α-SMA induction, it did not prevent E-cadherin loss under the influence of TGFβ1. This suggests that the loss of gate keeper function of E-cadherin alone may not necessarily result in complete EMT and further transcriptional re-programming is essential to attain mesenchymal phenotype. Although BMP7 abolished TGFβ1 mediated α-SMA expression by restoring Id2 levels, the loss of Id2 was not sufficient to induce α-SMA expression even in the context of reduced E-cadherin expression. Hence, a reduction in Id2 is critical for TGFβ1-induced α-SMA expression in this model of human PTECs but is not sufficient in it self to induce α-SMA even in the context of reduced E-cadherin.

  9. Cyclophilin B interacts with sodium-potassium ATPase and is required for pump activity in proximal tubule cells of the kidney.

    Directory of Open Access Journals (Sweden)

    Guillermo Suñé

    Full Text Available Cyclophilins (Cyps, the intracellular receptors for Cyclosporine A (CsA, are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1 that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.

  10. Cyclophilin B interacts with sodium-potassium ATPase and is required for pump activity in proximal tubule cells of the kidney.

    Science.gov (United States)

    Suñé, Guillermo; Sarró, Eduard; Puigmulé, Marta; López-Hellín, Joan; Zufferey, Madeleine; Pertel, Thomas; Luban, Jeremy; Meseguer, Anna

    2010-11-10

    Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.

  11. Effect of isoflavone-enriched soybean flour, zinc (Zn and vitamin E in the ration on testosterone level and total permatogenic cell in seminiferous tubules of rat

    Directory of Open Access Journals (Sweden)

    Astuti

    2008-12-01

    Full Text Available The objective of this experiment are to evaluate the effects of isoflavoneen-enriched soybean flour, zinc (Zn and vitamin E on testosterone level of male rats and total spermatogenic cells in the seminiferous tubules of rat testes as animal model. Diet was given as isonitrogen and isocaloric with 15% of dietary protein. Thirty male Sprague Dawley rats were divided into six groups and treated with isoflavoneen-riched soybean flour, Zn and vitamin E in different combination. Isoflavoneen-riched soybean flour (3mg/day was given by oral administration, whereas Zn and vitamin E were mixed wih the basic diet. The treatment was done for 2 month. Results indicated that complete treatment of isoflavoneen-riched soybean flour, Zn and vitamin E on male rats increased testosteron level and total spermatogenic cells in comparison with single treatment and the other combination. The best results showed in the group that given isoflavoneen-enriched soybean flour with diet containing both Zn and vitamin E i.e; testosteron level 3.49 ± 0.31 ng/ml; while the number of spermatogonia, spermatocytes, early spermatids, late spermatids, and total spermatogenic cells were 37.56 ± 4.48, 67 ± 4.72, 287.11 ± 31.75, 227.22 ± 29.78, and 618.89 ± 47.38, respectively. It was concluded that synergic interaction between isoflavoneen-enriched soybean flour, Zn and vitamin E increased testosteron level and total spermatogenic cells of rat testes.

  12. Effect of acute acid-base disturbances on ErbB1/2 tyrosine phosphorylation in rabbit renal proximal tubules.

    Science.gov (United States)

    Skelton, Lara A; Boron, Walter F

    2013-12-15

    The renal proximal tubule (PT) is a major site for maintaining whole body pH homeostasis and is responsible for reabsorbing ∼80% of filtered HCO3(-), the major plasma buffer, into the blood. The PT adapts its rate of HCO3(-) reabsorption (JHCO3(-)) in response to acute acid-base disturbances. Our laboratory previously showed that single isolated perfused PTs adapt JHCO3(-) in response to isolated changes in basolateral (i.e., blood side) CO2 and HCO3(-) concentrations but, surprisingly, not to pH. The response to CO2 concentration can be blocked by the ErbB family tyrosine kinase inhibitor PD-168393. In the present study, we exposed enriched rabbit PT suspensions to five acute acid-base disturbances for 5 and 20 min using a panel of phosphotyrosine (pY)-specific antibodies to determine the influence of each disturbance on pan-pY, ErbB1-specific pY (four sites), and ErbB2-specific pY (two sites). We found that each acid-base treatment generated a distinct temporal pY pattern. For example, the summated responses of the individual ErbB1/2-pY sites to each disturbance showed that metabolic acidosis (normal CO2 concentration and reduced HCO3(-) concentration) produced a transient summated pY decrease (5 vs. 20 min), whereas metabolic alkalosis produced a transient increase. Respiratory acidosis (normal HCO3(-) concentration and elevated CO2 concentration) had little effect on summated pY at 5 min but produced an elevation at 20 min, whereas respiratory alkalosis produced a reduction at 20 min. Our data show that ErbB1 and ErbB2 in the PT respond to acute acid-base disturbances, consistent with the hypothesis that they are part of the signaling cascade.

  13. Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells

    International Nuclear Information System (INIS)

    Wolff, Natascha A.; Lee, Wing-Kee; Abouhamed, Marouan; Thevenod, Frank

    2008-01-01

    Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membrane (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 μM for 24 h) toxicity was significantly attenuated from 27.3 ± 3.9% in ARF6-WT to 11.1 ± 4.0% in ARF6-DN cells (n = 6, P < 0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0 ± 4.6% versus 3.9 ± 3.9% of ARF6-WT cells, n = 3, P < 0.01) and/or remained near the PM (89.3 ± 5. 6% versus 45.2 ± 14.3% of ARF6-WT cells, n = 3, P < 0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells

  14. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    Energy Technology Data Exchange (ETDEWEB)

    Carette, Diane [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Perrard, Marie-Hélène, E-mail: marie-helene.durand@ens-lyon.fr [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Prisant, Nadia [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Gilleron, Jérome; Pointis, Georges [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); Segretain, Dominique [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Durand, Philippe [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Kallistem SAS Ecole Normale Supérieure de Lyon, Lyon (France)

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  15. Roles of CD34+ cells and ALK5 signaling in the reconstruction of seminiferous tubule-like structures in 3-D re-aggregate culture of dissociated cells from neonatal mouse testes.

    Science.gov (United States)

    Abe, Shin-Ichi; Abe, Kazuko; Zhang, Jidong; Harada, Tomoaki; Mizumoto, Go; Oshikawa, Hiroki; Akiyama, Haruhiko; Shimamura, Kenji

    2017-01-01

    Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFβ signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results

  16. The distribution and function of serotonin in the large milkweed bug, Oncopeltus fasciatus. a comparative study with the blood-feeding bug, Rhodnius prolixus.

    Science.gov (United States)

    Miggiani, L; Orchard, I; TeBrugge, V

    1999-11-01

    The blood-feeding hemipteran, Rhodnius prolixus, ingests a large blood meal at the end of each larval stage. To accommodate and process this meal, its cuticle undergoes plasticisation, and its gut and Malpighian tubules respectively absorb and secrete a large volume of water and salts for rapid diuresis. Serotonin has been found to be integral to the feeding process in this animal, along with a diuretic peptide(s). The large milkweed bug, Oncopeltus fasciatus, tends to feed in a more continuous and abstemious manner, and therefore may have different physiological requirements than the blood feeder. Unlike R. prolixus, O. fasciatus is lacking serotonin-like immunoreactive dorsal unpaired median neurons in the mesothoracic ganglionic mass, and lacks serotonin-like immunoreactive neurohaemal areas and processes on the abdominal nerves, integument, salivary glands, and anterior junction of the foregut and crop. The salivary glands and crop do, however, respond to serotonin with increased levels of cAMP, while the integument and Malpighian tubules do not. In addition, O. fasciatus Malpighian tubules respond to both O. fasciatus and R. prolixus partially purified CNS extracts, which are likely to contain any native diuretic peptides. Thus, while serotonin and diuretic peptides may be involved in tubule control in R. prolixus, the latter may be of greater importance in O. fasciatus.

  17. Transport and activation of S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine in rat kidney proximal tubules

    International Nuclear Information System (INIS)

    Zhang, G.H.; Stevens, J.L.

    1989-01-01

    An important step in understanding the mechanism underlying the tubular specificity of the nephrotoxicity of toxic cysteine conjugates is to identify the rate-limiting steps in their activation. The rate-limiting steps in the activation of toxic cysteine conjugates were characterized using isolated proximal tubules from the rat and 35S-labeled S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAC-DCVC) as model compounds. The accumulation by tubules of 35S radiolabel from both DCVC and NAC-DCVC was time and temperature dependent and was mediated by both Na+-dependent and independent processes. Kinetic studies with DCVC in the presence of sodium revealed the presence of two components with apparent Km and Vmax values of (1) 46 microM and 0.21 nmol/mg min and (2) 2080 microM and 7.3 nmol/mg.min. NAC-DVVC uptake was via a single system with apparent Km and Vmax values of 157 microM and 0.65 nmol/mg.min, respectively. Probenecid, an inhibitor of the renal organic anion transport system, inhibited accumulation of radiolabel from NAC-DCVC, but not from DCVC. The covalent binding of 35S label to cellular macromolecules was much greater from [35S]DCVC than from NAC-[35S]DCVC. Analysis of metabolites showed that a substantial amount of the cellular NAC-[35S]DCVC was unmetabolized while [35S]DCVC was rapidly metabolized to bound 35S-labeled material and unidentified products. The data suggest that DCVC is rapidly metabolized following transport, but that activation of NAC-DCVC depends on a slower rate of deacetylation. The results are discussed with regard to the segment specificity of cysteine conjugate toxicity and the role of disposition in vivo in the nephrotoxicity of glutathione conjugates

  18. Developmental expression of Manduca shade, the P450 mediating the final step in molting hormone synthesis

    DEFF Research Database (Denmark)

    Rewitz, Kim; Rybczynski, Robert; Warren, James T.

    2006-01-01

    body and epidermis with very low expression in the prothoracic gland and nervous system. Developmental variations in E20MO enzymatic activity are almost perfectly correlated with comparable changes in the gene expression of Msshd in the fat body and midgut during the fifth instar and the beginning...... gene shade (shd; CYP314A1) that encodes the E20MO in the tobacco hornworm, Manduca sexta. Manduca Shd (MsShd) mediates the conversion of E to 20E when expressed in Drosophila S2 cells. In accord with the central dogma, the data show that Msshd is expressed mainly in the midgut, Malpighian tubules, fat...... of pupal-adult development. The results indicate three successive and overlapping peaks of expression in the fat body, midgut and Malpighian tubules, respectively, during the fifth larval instar. The data suggest that precise tissue-specific transcriptional regulation controls the levels, and thereby...

  19. The Aquaporin Gene Family of the Yellow Fever Mosquito, Aedes aegypti

    OpenAIRE

    Drake, Lisa L.; Boudko, Dmitri Y.; Marinotti, Osvaldo; Carpenter, Victoria K.; Dawe, Angus L.; Hansen, Immo A.

    2010-01-01

    Background The mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small...

  20. Histologi Tubulus Seminiferus dan Kadar Testosteron Tikus yang Diberi Pakan Imbuhan Tepung Daun Kaliandra dan Kulit Nanas (HISTOLOGY OF SEMINIFEROUS TUBULES AND TESTOSTERONE LEVEL OF RAT GIVEN CALLIANDRA LEAF MEAL AND PINEAPPLE PEELS IN THE DIETS

    Directory of Open Access Journals (Sweden)

    Iriani Setyawati

    2017-09-01

    Full Text Available Calliandra calothyrsus leaf contains 17-28% protein which is hihly potential for use as source of protein supplement for animal feed. However, the plants also contain high level (>10% of condensed tannins as antinutritional effects which may reduce consumtion efficiency of diet. The addition of protease (bromelain into feed containing tannin is expected to decrease the negative effects of tannins. Bromelain can be obtained from the pineapple plant (Ananas comosus including on the peels. This study was conducted to determine the effect of calliandra tannin combined with bromelain protease of pineapple peels in the feed on testicular histology and testosterone level of rats feeded during the growth period. The feeding experiment on post-weaning male rats was conducted using a completely randomized factorial design (4 main factors x 4 subfactors. The main factors were calliandra leaf meal substitution of 0; 10; 17.5 and 25% in the diets and subfactors were addition of pineapple peels, 0; 4.35; 8.70 and 13.05 g/rat/day. Rats were divided into 16 groups and were feeded the diets for two months (during the growth period. The results showed the susbtituion of feed with calliandra leaf had no sigbnificant effecton the thickness of the seminiferous tubules, but it appeared to have significant effect on the histology of seminiferous tubules. Increased pineapple peels level in the diets containing calliandra decreased wall thickness of the seminiferous tubules of the rat testes, indicating that bromalein fastened the maturation of spermatozoa. The addition of pineapple peels into the diet containing calliandra had a significant interaction effect on testoteron levels of male rats, but the testoteron levels among all treated rats were still within the normal range. ABSTRAK Daun kaliandra (Calliandra calothyrsus mengandung protein 17-28% sehingga potensial sebagai sumber protein pakan ternak, namun tanaman ini mengandung condensed tannin cukup tinggi (>10

  1. The Chriz–Z4 complex recruits JIL-1 to polytene chromosomes, a ...

    Indian Academy of Sciences (India)

    Reducing the core component Chriz by RNAi results in destabilization of the complex and a strong reduction of interband-specific ... is a chromodomain protein interacting with Z4 (Eggert et al. 2004 .... Analysis of colocalization was carried out on the digital .... main is localized to bands, indicating that the chromodo- main is ...

  2. Genome-wide mapping of Painting of fourth on Drosophila melanogaster salivary gland polytene chromosomes.

    Science.gov (United States)

    Johansson, Anna-Mia; Larsson, Jan

    2014-12-01

    The protein Painting of fourth (POF) in Drosophila melanogaster specifically targets and stimulates expression output from the heterochromatic 4th chromosome, thereby representing an autosome specific protein [1,2]. Despite the high specificity for chromosome 4 genes, POF is occasionally observed binding to the cytological region 2L:31 in males and females [3] and two loci on the X-chromosome, PoX1 and PoX2 only in females [4]. Here we provide a detailed description of the experimental design and analysis of the tiling array data presented by Lundberg and colleagues in G3: Genes, Genomes, Genetics 2013 [4], where the female specific POF binding to PoX1 and PoX2 loci on the X chromosome was reported. We show the genome-wide high resolution binding profile of the POF protein where these different POF binding sites are detected. The complete data set is available at http://www.ncbi.nlm.nih.gov/geo/ (accession: GSE45402).

  3. Functional correlates of positional and gender-specific renal asymmetry in Drosophila.

    Directory of Open Access Journals (Sweden)

    Venkateswara R Chintapalli

    Full Text Available In humans and other animals, the internal organs are positioned asymmetrically in the body cavity, and disruption of this body plan can be fatal in humans. The mechanisms by which internal asymmetry are established are presently the subject of intense study; however, the functional significance of internal asymmetry (outside the brain is largely unexplored. Is internal asymmetry functionally significant, or merely an expedient way of packing organs into a cavity?Like humans, Drosophila shows internal asymmetry, with the gut thrown into stereotyped folds. There is also renal asymmetry, with the rightmost pair of renal (Malpighian tubules always ramifying anteriorly, and the leftmost pair always sitting posteriorly in the body cavity. Accordingly, transcriptomes of anterior-directed (right-side and posterior-directed (left-side Malpighian (renal tubules were compared in both adult male and female Drosophila. Although genes encoding the basic functions of the tubules (transport, signalling were uniformly expressed, some functions (like innate immunity showed positional or gender differences in emphasis; others, like calcium handling or the generation of potentially toxic ammonia, were reserved for just the right-side or left-side tubules, respectively. These findings correlated with the distinct locations of each tubule pair within the body cavity. Well known developmental genes (like dorsocross, dachshund and doublesex showed continuing, patterned expression in adult tubules, implying that somatic tissues maintain both left-right and gender identities throughout life. Gender asymmetry was also noted, both in defence and in male-specific expression of receptors for neuropeptide F and sex-peptide: NPF elevated calcium only in male tubules.Accordingly, the physical asymmetry of the tubules in the body cavity is directly adaptive. Now that the detailed machinery underlying internal asymmetry is starting to be delineated, our work invites the

  4. Bioengineered kidney tubules efficiently excrete uremic toxins

    NARCIS (Netherlands)

    Jansen, Jitske; Fedecostante, M.; Wilmer, M.; Peters, J.G.; Kreuser, U.M.; Broek, P.H.; Mensink, R.A.; Boltje, T.J.; Stamatialis, Dimitrios; Wetzels, J.F.; van der Heuvel, L.P.; Hoenderop, J.G.; Masereeuw, R.

    2016-01-01

    The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed

  5. A Drosophila genetic model of nephrolithiasis: transcriptional changes in response to diet induced stone formation.

    Science.gov (United States)

    Chung, Vera Y; Turney, Benjamin W

    2017-11-28

    Urolithiasis is a significant healthcare issue but the pathophysiology of stone disease remains poorly understood. Drosophila Malpighian tubules were known to share similar physiological function to human renal tubules. We have used Drosophila as a genetic model to study the transcriptional response to stone formation secondary to dietary manipulation. Wild-type male flies were raised on standard medium supplemented with lithogenic agents: control, sodium oxalate (NaOx) and ethylene glycol (EG). At 2 weeks, Malpighian tubules were dissected under polarized microscope to visualize crystals. The parallel group was dissected for RNA extraction and subsequent next-generation RNA sequencing. Crystal formation was visualized in 20%(±2.2) of flies on control diet, 73%(±3.6) on NaOx diet and 84%(±2.2) on EG diet. Differentially expressed genes were identified in flies fed with NaOx and EG diet comparing with the control group. Fifty-eight genes were differentially expressed (FDR <0.05, p < 0.05) in NaOx diet and 20 genes in EG diet. The molecular function of differentially expressed genes were assessed. Among these, Nervana 3, Eaat1 (Excitatory amino acid transporter 1), CG7912, CG5404, CG3036 worked as ion transmembrane transporters, which were possibly involved in stone pathogenesis. We have shown that by dietary modification, stone formation can be manipulated and visualized in Drosophila Malpighian tubules. This genetic model could be potentially used to identify the candidate genes that influence stone risk hence providing more insight to the pathogenesis of human stone disease.

  6. The influence of vinegars on exposure of dentinal tubules: a SEM evaluation Influência de vinagres na exposição dos túbulos dentinários: avaliação em MEV

    Directory of Open Access Journals (Sweden)

    Daniela Leal Zandim

    2004-03-01

    Full Text Available Dentin hypersensitivity is a common painful condition observed in clinics. Dietary habits have been much associated with its development and persistence during and following periodontal treatment. The aim of this in vitro study was to evaluate the influence of vinegars on the removal of smear layer and exposure of dentinal tubules. Extracted human teeth were submitted to manual scaling with Gracey curettes in order to remove the cementum as well as to form a smear layer. Dentin samples with 3 mm² were obtained and distributed into six experimental groups: one control and five types of vinegars (alcohol, apple, rice, white wine and balsamic. Each group included two methods of vinegar application: topical and friction. After routine preparation for SEM analysis, photomicrographs were assessed by a calibrated and blind examiner using an appropriate index system. Kruskal-Wallis test indicated a significant influence of vinegars on smear layer removal. There was a statistically significant difference between groups treated with apple, white and rice vinegars and the control group (p A hipersensibilidade dentinária cervical é uma condição dolorosa muito comum nos consultórios. A dieta tem sido bastante associada ao seu aparecimento, assim como a sua persistência após o tratamento periodontal. o objetivo deste trabalho foi avaliar in vitro a influência dos vinagres na remoção de "smear layer" e exposição dos túbulos dentinários. Dentes de humanos foram instrumentados com curetas Gracey para a remoção do cemento e formação de "smear layer". Foram obtidas amostras de dentina com 3 mm², divididas entre o grupo controle (água destilada e cinco grupos de vinagre: branco, maçã, arroz, vinho branco e balsâmico. Cada grupo incluiu duas formas de aplicação da substância, tópica ou por fricção. Após o preparo para observação em MEV (microscopia eletrônica de varredura, as fotomicrografias foram avaliadas por um examinador

  7. Diffusion ability of endotoxin through dentinal tubules Avaliação da difusão de endotoxinas através dos túbulos dentinários

    Directory of Open Access Journals (Sweden)

    Luciane Dias de Oliveira

    2005-03-01

    Full Text Available The aim of this study was to evaluate the ability of endotoxin to diffuse through dentinal tubules towards the cement and to observe the period of time needed for it to reach the external root surface. Thirty single-rooted human teeth had their crowns and apices removed in order to standardize the root length to 15 mm. Teeth were instrumented until #30 K-file and made externally impermeable with epoxy adhesive, leaving 10 mm of the exposed root (middle third. The specimens were placed in plastic vials and irradiated (60Co gamma-rays. Then, they were divided into 2 groups (n = 15: G1 Escherichia coli endotoxin was inoculated into the root canal of the specimens and 1 ml of pyrogen-free water was put in the tubes; G2 (control: pyrogen-free water was inoculated into the root canals and 1 ml of pyrogen-free water was put in each tube. After 30 min, 2 h, 6 h, 12 h, 24 h, 48 h, 72 h and 7 days, the water of the tubes was removed and replaced. The removed aliquot was tested for the presence of endotoxin. Considering that the endotoxin is a B-lymphocyte polyclonal activator, at each experimental period, B-lymphocyte culture was stimulated with a sample of water removed from each tube and antibody (IgM production was detected by ELISA technique. The results of IgM production were higher in groups of 24 h, 48 h, 72 h and 7 days in relation to the other studied groups, with statistically significant differences (ANOVA and Tukey's test p O objetivo desta pesquisa foi avaliar in vitro a capacidade e o tempo necessário para a endotoxina se difundir pelos túbulos dentinários em direção ao cemento. Foram utilizados 30 dentes humanos unirradiculados, que tiveram suas coroas e seus ápices seccionados, padronizando-se o tamanho em 15 mm. Os dentes foram instrumentados até a lima K30 e impermeabilizados externamente com adesivo epóxi, deixando-se 10 mm de raiz exposta (terço médio. Os espécimes foram acondicionados em tubos plásticos e submetidos

  8. Análise de prova de carga em tubulão a céu aberto submetido a esforço horizontal em solo não saturado de diabásio da região de Campinas

    OpenAIRE

    Roberto Kassouf

    2012-01-01

    Resumo: O problema de tubulões carregados horizontalmente no topo é freqüente em obras de construção civil e normalmente estes esforços horizontais atuam concomitantemente aos esforços verticais e momentos fletores onde os casos mais freqüentes são as ações dos ventos, torres de transmissão e para energia eólica, obras de arte como pontes e viadutos, muros de arrimo com o empuxo do solo, escavações para subsolo e edificações prediais, havendo uma reduzida bibliografia sobre os parâmetros a s...

  9. Can the exposure of Apis mellifera (Hymenoptera, Apiadae) larvae to a field concentration of thiamethoxam affect newly emerged bees?

    Science.gov (United States)

    Friol, Priscila Sepúlveda; Catae, Aline Fernanda; Tavares, Daiana Antonia; Malaspina, Osmar; Roat, Thaisa Cristina

    2017-10-01

    The use of insecticides on crops can affect non-target insects, such as bees. In addition to the adult bees, larvae can be exposed to the insecticide through contaminated floral resources. Therefore, this study aimed to investigate the possible effects of the exposure of A. mellifera larvae to a field concentration of thiamethoxam (0.001 ng/μL thiamethoxam) on larval and pupal survival and on the percentage of adult emergence. Additionally, its cytotoxic effects on the digestive cells of midgut, Malpighian tubules cells and Kenyon cells of the brain of newly emerged A. mellifera bees were analyzed. The results showed that larval exposure to this concentration of thiamethoxam did not influence larval and pupal survival or the percentage of adult bee emergence. However, this exposure caused ultra-structural alterations in the target and non-target organs of newly emerged bees. The digestive cell of bees that were exposed to the insecticide exhibited a basal labyrinth without long and thin channels and compromised mitochondria. In Malpighian tubules cells, disorganized basal labyrinth, dilated mitochondria with a deformed shape and a loss of cristae, and disorganized microvilli were observed. The results showed that the exposed bees presented Kenyon cells with alterations in the nucleus and mitochondria. These alterations indicate possible tissue degeneration, demonstrating the cytotoxicity of thiamethoxam in the target and non-target organs of newly emerged bees. Such results suggest cellular organelle impairment that can compromise cellular function of the midgut cells, Malpighian tubules cells and Kenyon cells, and, consequently, can compromise the longevity of the bees of the whole colony. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

    Czech Academy of Sciences Publication Activity Database

    Drosopoulou, E.; Nakou, I.; Šíchová, Jindra; Kubíčková, S.; Marec, František; Mavragani-Tsipidou, P.

    2012-01-01

    Roč. 140, 4-6 (2012), s. 169-180 ISSN 0016-6707 R&D Projects: GA AV ČR IAA600960925 Grant - others:Ministry of Agriculture of the Czech Republic(CZ) MZE 0002716202; Grant Agency of the University of South Bohemia(CZ) GAJU 137/2010/P Institutional support: RVO:60077344 Keywords : chromosome painting * FISH * laser microdissection Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.681, year: 2012 http://link.springer.com/article/10.1007/s10709-012-9668-3

  11. The D. melanogaster capa-1 neuropeptide activates renal NF-kB signaling.

    Science.gov (United States)

    Terhzaz, Selim; Overend, Gayle; Sebastian, Sujith; Dow, Julian A T; Davies, Shireen-A

    2014-03-01

    The capa peptide family exists in a very wide range of insects including species of medical, veterinary and agricultural importance. Capa peptides act via a cognate G-protein coupled receptor (capaR) and have a diuretic action on the Malpighian tubules of Dipteran and Lepidopteran species. Capa signaling is critical for fluid homeostasis and has been associated with desiccation tolerance in the fly, Drosophila melanogaster. The mode of capa signaling is highly complex, affecting calcium, nitric oxide and cyclic GMP pathways. Such complex physiological regulation by cell signaling pathways may occur ultimately for optimal organismal stress tolerance to multiple stressors. Here we show that D. melanogaster capa-1 (Drome-capa-1) acts via the Nuclear Factor kappa B (NF-kB) stress signaling network. Human PCR gene arrays of capaR-transfected Human Embryonic Kidney (HEK) 293 cells showed that Drome-capa-1 increases expression of NF-kB, NF-kB regulated genes including IL8, TNF and PTGS2, and NF-kB pathway-associated transcription factors i.e. EGR1, FOS, cJUN. Furthermore, desiccated HEK293 cells show increased EGR1, EGR3 and PTGS2 - but not IL8, expression. CapaR-transfected NF-kB reporter cells showed that Drome-capa-1 increased NF-kB promoter activity via increased calcium. In Malpighian tubules, both Drome-capa-1 stimulation and desiccation result in increased gene expression of the D. melanogaster NF-kB orthologue, Relish; as well as EGR-like stripe and klumpfuss. Drome-capa-1 also induces Relish translocation in tubule principal cells. Targeted knockdown of Relish in only tubule principal cells reduces desiccation stress tolerance of adult flies. Together, these data suggest that Drome-capa-1 acts in desiccation stress tolerance, by activating NF-kB signaling. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Distribution and activity of a Dippu DH31-like peptide in the large milkweed bug Oncopeltus fasciatus.

    Science.gov (United States)

    Te Brugge, V A; Orchard, I

    2008-02-01

    The milkweed bug, Oncopeltus fasciatus, is a plant feeding hemipteran. While there has been much research done on the neurohormonal control of the post-feeding diuresis in the blood-feeding hemipteran, Rhodnius prolixus, little is known about the control of the post-feeding diuresis in O. fasciatus. One of the neurohormones that may play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH31) family of insect peptides. In this study we demonstrate the presence of DH31-like immunoreactivity in the central nervous system (CNS) and gut of O. fasciatus 5th instars. As well, DH31-like material was quantified and partially purified from the CNS of 5th instar O. fasciatus using reversed-phase liquid chromatography (RPLC) and monitored with an enzyme-linked immunosorbent assay (ELISA). When tested on O. fasciatus 5th instar Malpighian tubules, DH31-like peptides significantly increased the rate of secretion over saline controls. The results suggest that there is a DH31-like peptide(s) present in the CNS of O. fasciatus and that this peptide may play a role in the control of Malpighian tubule secretion.

  13. Impact of agrochemicals on non-target species: Calathus fuscipes Goeze 1777 (Coleoptera: Carabidae) as model.

    Science.gov (United States)

    Giglio, Anita; Cavaliere, Francesco; Giulianini, Piero Giulio; Mazzei, Antonio; Talarico, Federica; Vommaro, Maria Luigia; Brandmayr, Pietro

    2017-08-01

    Carabid beetles are important in the biological control of arable crop pests. Agricultural practices can produce over time a delayed toxic effect at the organismal and population levels and can compromise the survival on these species. In this research, we quantified the cumulative sublethal effect on body size, Malpighian tubules and immune responses in Calathus fuscipes adults living in the potato field and exposed to lambda-cyhalothrin and cymoxanil-based commercial formulates. Reductions of morphological parameters such as body, pronotum and elytron in both males and females from the potato field indicated that the pre-imaginal stages (larvae and pupae) suffer the sublethal effects of exposure to the larvicide control action of lambda-cyhalothrin. Ultrastructural alterations recorded in Malpighian tubules at the level of plasma membrane, mitochondria and nucleus indicated the reduction of the detoxification capability. The basal phenoloxidase and lysozyme-like enzyme activities have measured as markers of immune competence. Spectrophometric analyses showed that the chronic exposure in field causes an increase of basal phenoloxidase enzyme activity, while the lytic activity of haemolymph was not affected. As a result, the use of larvicides and fungicides have a harmful effect on beneficial species such C. fuscipes living in the soil of potato fields. These morphological and physiological results recorded at the organismal level can provide useful information of effects at the population and community levels to preserve the biodiversity of agroecosystem. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Effect of Gamma-Irradiation on the Midgut Epithelial Cells of Female Potato Tuber Moth, Phthorimaea operculella, Zeller

    International Nuclear Information System (INIS)

    Haiba, I.M.; Abd El-Meguid, A.A.; Yousef, H.A.A.; Mahmoud, E.A.

    2008-01-01

    The present work deals with the histological studies of the midgut in non- irradiated 4-day-old potato tuber moth, Phthorimaea operculella and after the exposure of 5-day-old pupae to sub sterilizing and sterilizing doses 50 and 150 Gy, respectively. Anatomically, the midgut was a wide straight tube, the proximal part was marked by the connection to the crop and the distal part was determined by the connection of the Malpighian tubules. The arrangement of the Malpighian tubules around the midgut tube took a special pattern symmetrically on each side. Histologically, the midgut was subdivided into three main subdivisions, anterior, middle and posterior regions. The epithelial cells differed in shape and size according to the regions in the midgut. Exposure to gamma-irradiation showed various forms of changes, there was direct relationship between the dose levels and the observed effects. At dose level 150 Gy, the effects were more advanced than those at 50 Gy dose level. The effects of radiation could be summarized as: appearance of vacuoles and vesicles in the cytoplasm, nuclei in some cells were pyknotic or karyolysed, reduction in the cell number, enlargement in nuclei, absence of regenerative cells from some parts and the epithelial cells suffered from hydropic degeneration

  15. Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros.

    Science.gov (United States)

    Yang, J; Yamamoto, M; Ishibashi, J; Taniai, K; Yamakawa, M

    1998-08-01

    An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

  16. Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation

    NARCIS (Netherlands)

    Engelse, M.A.; Laurens, N.; Verloop, R.E.; Koolwijk, P.; Hinsbergh, V.W.M. van

    2008-01-01

    The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA

  17. Utilização de células-tronco autólogas de medula óssea na regeneração do nervo tibial de coelhos mediante técnica de tubulização com prótese de silicone Applyng of autologous stem cells from bone marrow in the regeneration of tibial nerve of rabbits using the tubulization technique with silicone tube

    Directory of Open Access Journals (Sweden)

    Lucas Marques Colomé

    2008-12-01

    Full Text Available Neste estudo é apresentado um modelo experimental de defeito agudo em nervo periférico para avaliação da regeneração nervosa mediante técnica de tubulização associada à inoculação de células-tronco autólogas de medula óssea. Foram utilizados 12 coelhos Nova Zelândia albinos, submetidos à secção bilateral e ao afastamento de 5mm do nervo tibial e posterior reparo mediante utilização de câmara de silicone. Internamente à prótese de tubulização do nervo tibial esquerdo em todos os animais, foram inoculadas células-tronco autólogas de medula óssea, coletadas a partir do úmero. Como grupo controle (nervo tibial direito, mediante aplicação da mesma técnica de reparo, solução de NaCl 0,9% foi administrada internamente à prótese. Após 30 dias de observação, os animais foram eutanasiados e foi realizada a avaliação histológica dos segmentos nervosos por meio das colorações de hematoxilina-eosina, luxol fast blue e azul de toluidina. Com os resultados, foi possível concluir que o transplante de células-tronco autólogas associado à técnica de tubulização apresenta vantagens no processo de regeneração nervosa periférica.This study presents an experimental model of an acute deffect in a peripheral nerve to evaluate neural regeneration using a tubulization technique associated with the inoculation of autologous stem cells from bone marrow. A total of 12 New Zealand white rabbits underwent a bilateral dissection of the tibial nerve followed by repair with silicone tubulization. On the left tibial nerve of all animals, the tube was filled with autologous bone marrow-derived stem cells collected from the humerus. For control, using the same repair technique, the tubes were filled with a NaCl solution in the right tibial nerve. After 30 days of observation, the animals were euthanized and a histological evaluation of the collected nerve segments was performed by staining with hematoxylin-eosin, luxol fast

  18. Mechanistic perspective of mitochondrial fusion: tubulation vs. fragmentation.

    Science.gov (United States)

    Escobar-Henriques, Mafalda; Anton, Fabian

    2013-01-01

    Mitochondrial fusion is a fundamental process driven by dynamin related GTPase proteins (DRPs), in contrast to the general SNARE-dependence of most cellular fusion events. The DRPs Mfn1/Mfn2/Fzo1 and OPA1/Mgm1 are the key effectors for fusion of the mitochondrial outer and inner membranes, respectively. In order to promote fusion, these two DRPs require post-translational modifications and proteolysis. OPA1/Mgm1 undergoes partial proteolytic processing, which results in a combination between short and long isoforms. In turn, ubiquitylation of mitofusins, after oligomerization and GTP hydrolysis, promotes and positively regulates mitochondrial fusion. In contrast, under conditions of mitochondrial dysfunction, negative regulation by proteolysis on these DRPs results in mitochondrial fragmentation. This occurs by complete processing of OPA1 and via ubiquitylation and degradation of mitofusins. Mitochondrial fragmentation contributes to the elimination of damaged mitochondria by mitophagy, and may play a protective role against Parkinson's disease. Moreover, a link of Mfn2 to Alzheimer's disease is emerging and mutations in Mfn2 or OPA1 cause Charcot-Marie-Tooth type 2A neuropathy or autosomal-dominant optic atrophy. Here, we summarize our current understanding on the molecular mechanisms promoting or inhibiting fusion of mitochondrial membranes, which is essential for cellular survival and disease control. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. The application of halloysite tubule nanoclay in drug delivery.

    Science.gov (United States)

    Lvov, Yuri M; DeVilliers, Melgardt M; Fakhrullin, Rawil F

    2016-07-01

    Natural and biocompatible clay nanotubes are among the best inorganic materials for drug nanoformulations. These halloysite tubes with SiO2 on the outermost surface have diameter of ca. 50 nm, length around 1 micrometer and may be loaded with drugs at 10-30 wt. %. Narrow tube openings allow for controllable sustained drug release for hours, days or even weeks. Physical-chemical properties of these nanotubes are described followed by examples of drug-loading capabilities, release characteristics, and control of duration of release through the end tube capping with polymers. Development of halloysite-polymer composites such as tissue scaffolds and bone cement/dentist resin formulations with enhanced mechanical properties and extension of the drug release to 2-3 weeks are described. Examples of the compression properties of halloysite in tablets and capsules are also shown. We expect that clay nanotubes will be used primarily for non-injectable drug formulations, such as topical and oral dosage forms, cosmetics, as well as for composite materials with enhanced therapeutic effects. These include tissue scaffolds, bone cement and dentist resins with sustained release of antimicrobial and cell growth-promoting medicines (including proteins and DNA) as well as other formulations such as compounds for antiseptic treatment of hospitals.

  20. Morphological changes in the sperm storage tubules of the ...

    African Journals Online (AJOL)

    TEM results showed pyknosis, swollen mitochondria, vacuolation and increased number of lysosomes in degenerating SST. The observed morphological changes indicate the ability of carbendazim to disrupt structural integrity of SST as well as its storage capacity. This poses a great threat to the fertility of exposed birds ...

  1. The loop of Henle as the milestone of mammalian kindey concentrating ability: a historical review.

    Science.gov (United States)

    Koulouridis, Efstathios; Koulouridis, Ioannis

    2014-01-01

    The first description of the renal tubules is attributed to Lorenzo Bellini in 1662 and four years later Marcello Malpighi described the glomerulus. In 1842 Sir William Bowman described the capsule that surrounds the Malpighian body and its connection with the renal tubule and introduced the "excretory" hypothesis of urine formation. In the same year, Carl Ludwig introduced the "filtration-reabsorption" hypothesis of urine formation. Bowman's hypothesis was accepted by the so-called "vitalists" and Ludwig's hypothesis by the so-called "mechanists". In the middle of this confliction, Jacob Henle described in 1862 the homonymous "U" shaped loop but his discovery has neglected. In 1942 Werner Kuhn, a physical chemist, proposed that the loop of Henle may be the natural analog of the hairpin countercurrent multiplication system which concentrates urine in mammalian kidneys. In 1951 Kuhn, Hargitay and Wirz showed experimentally that the loop of Henle was the most important part of the countercurrent multiplication system of urine-concentrating mechanism in mammalian kidneys. The new theory was accepted by English-speaking scientists later, in 1958, when Carl Gottschalk and Margaret Mylle published their experimental work and proved that Kuhn's theory was correct. Gottschalk summarized the evidence of the accumulated knowledge in 1962, three centuries after the first description of renal tubules and one century after description of Henle's loop.

  2. Efficient gene transfer into silkworm larval tissues by a combination of sonoporation and lipofection.

    Science.gov (United States)

    Lee, Jae Man; Takahashi, Masateru; Mon, Hiroaki; Koga, Katsumi; Kawaguchi, Yutaka; Kusakabe, Takahiro

    2005-11-01

    Sonoporation (ultrasound treatment) provides a new and attractive nonviral way of in vivo gene transfer. To access the applicability of this method to the silkworm, Bombyx mori, we have compared the efficiencies of gene transfer by means of lipofection (using an appropriate agent, PDD111), sonoporation (ditto, FluoroGene), and lipofection followed by sonoporation. By these methods, a luciferase expression plasmid was found to be markedly transferred into the haemocoel of newly ecdysed fifth instar silkworm larvae, and also into other tissues although with lower rates compared with the haemocoel. In terms of luciferase activity, the efficiencies of transgene by lipofection plus sonoporation were approximately 6 (hemocytes), 20 (silk glands), 8 (mid-gut), 38 (fat body), 10 (Malpighian tubules), 33 (ovaries), and 16 (testes) times as high as those by lipofection or sonoporation alone. These results demonstrated that the present method is useful to introduce the exogenous DNA into insect organs in vivo.

  3. Characterization and functional assay of a fatty acyl-CoA reductase gene in the scale insect, Ericerus pela Chavannes (Hemiptera: Coccoidae).

    Science.gov (United States)

    Hu, Yan-Hong; Chen, Xiao-Ming; Yang, Pu; Ding, Wei-Feng

    2018-04-01

    Ericerus pela Chavannes (Hemiptera: Coccoidae) is an economically important scale insect because the second instar males secrete a harvestable wax-like substance. In this study, we report the molecular cloning of a fatty acyl-CoA reductase gene (EpFAR) of E. pela. We predicted a 520-aa protein with the FAR family features from the deduced amino acid sequence. The EpFAR mRNA was expressed in five tested tissues, testis, alimentary canal, fat body, Malpighian tubules, and mostly in cuticle. The EpFAR protein was localized by immunofluorescence only in the wax glands and testis. EpFAR expression in High Five insect cells documented the recombinant EpFAR reduced 26-0:(S) CoA and to its corresponding alcohol. The data illuminate the molecular mechanism for fatty alcohol biosynthesis in a beneficial insect, E. pela. © 2017 Wiley Periodicals, Inc.

  4. Morphologic and molecular data help adopting the insect-pathogenic nephridiophagids (Nephridiophagidae among the early diverging fungal lineages, close to the Chytridiomycota

    Directory of Open Access Journals (Sweden)

    Renate Radek

    2017-07-01

    Full Text Available Nephridiophagids are poorly known unicellular eukaryotes, previously of uncertain systematic position, that parasitize the Malpighian tubules of insects. Their life cycle includes merogony with multinucleate plasmodia and sporogony leading to small, uninucleate spores. We examined the phylogenetic affiliations of three species of Nephridiophaga, including one new species, Nephridiophaga maderae, from the Madeira cockroach (Leucophaea maderae. In addition to the specific host, the new species differs from those already known by the size of the spores and by the number of spores within the sporogenic plasmodium. The inferred phylogenetic analyses strongly support a placement of the nephridiophagids in the fungal kingdom near its root and with a close, but unresolved, relationship to the chytids (Chytridiomycota. We found evidence for the nephridiophagidean speciation as being strongly coupled to host speciation.

  5. Contamination as the Cause of Erroneous Records of Brochosomes

    Directory of Open Access Journals (Sweden)

    Roman Rakitov

    2011-01-01

    Full Text Available Brochosomes are ultramicroscopic particles produced in large quantities by the Malpighian tubules of leafhoppers (Insecta, Hemiptera, and Cicadellidae and applied by leafhoppers as a coat to their integuments. A recent study has described brochosomes on museum specimens of Heteroptera and Psylloidea, suggesting a wider distribution of brochosomes among Hemiptera. Here, I report that the majority of adult Sthenarus rotermundi (Scholtz (Miridae and Kleidocerys resedae (Panzer (Lygaeidae reared in captivity and handled with clean tools had no brochosomes on them, suggesting that the earlier records of brochosomes in these and perhaps other species outside of the Cicadellidae were due to contamination. Additionally, simple experiments demonstrated that insects can become contaminated with brochosomes via entomological tools that had been in contact with leafhoppers and via preservation in ethanol together with leafhoppers. Contamination of host plants, predators, and parasites of leafhoppers with brochosomes is also expected but remains to be demonstrated.

  6. Birefringent Crystals and Abdominal Discoloration in the Predatory Mite Phytoseiulus persimilis (Acari: Phytoseiidae)

    Science.gov (United States)

    Bjørnson; Steiner; Keddie

    1997-03-01

    In response to grower complaints of poor performance of Phytoseiulus persimilis, mites from 14 commercial insectaries and research colonies were examined for pathogens. Some were found to have abdominal discolorations, manifested initially as two white stripes along the dorsal sides of the body within the Malpighian tubules. Advanced signs appeared as a large, centrally located, white spot or U-shaped discoloration in the distal opisthosoma within the rectum/anal atrium. White material often accumulated and hardened within the anus and formed a rectal plug that inhibited further excretion. Most affected mites were lethargic. Adults and immatures with abdominal discoloration contained numerous densely packed, birefringent, dumbbell-shaped entities. Though occasionally observed in the colon, they occurred most frequently within the Malpighian tubules and/or rectum and anal atrium. Dumbbells measured 2-4 &mgr;m long and contained prominent concentric rings. When observed by transmission electron microscopy, the entities lacked cellular organelles. Asymptomatic mites contained few or no such entities. Dumbbell-shaped inclusions were observed in P. persimilis from all sources examined. High levels of potassium, low levels of phosphorous and sulfur, and traces of chlorine were detected by energy-dispersive X-ray analysis. Guanine and uric acid, known nitrogenous wastes of arachnids, do not contain these elements. The chemical composition and structure indicate that the dumbbells are crystals. Both asymptomatic mites and those specimens exhibiting abdominal discoloration were examined for pathogens using light and transmission electron microscopy. Microsporidia, virus-like particles, and a rickettsia (genus Wolbachia) were observed in some mites but showed no correlation with white abdominal discoloration or associated crystal formation. Neither were pathogens always detected in symptomatic mites. Although birefringent crystals may be naturally occurring excretory

  7. Bombyx mori cecropin A has a high antifungal activity to entomopathogenic fungus Beauveria bassiana.

    Science.gov (United States)

    Lu, Dingding; Geng, Tao; Hou, Chengxiang; Huang, Yuxia; Qin, Guangxing; Guo, Xijie

    2016-05-25

    A cDNA encoding cecropin A (CecA) was cloned from the larvae of silkworm, Bombyx mori, using RT-PCR. It encodes a protein of 63 amino acids, containing a 22 amino acid signal peptide and a 37 amino acid mat peptide of functional domain. The CecA secondary structure contains two typical amphiphilic α-helices. Real-time qPCR analysis revealed that CecA was expressed in all the tissues tested, including cuticle, fat body, hemocytes, Malpighian tubule, midgut and silk gland in the silkworm larvae with the highest expression in the fat body and hemocytes. The gene expression of B. mori CecA was rapidly induced by Beauveria bassiana challenge and reached maximum levels at 36h after inoculation in third instar larvae. In the fifth instar larvae infected with B. bassiana, the relative expression level of CecA was upregulated in fat body and hemocytes, but not in cuticle, Malpighian tubule, midgut and silk gland. The cDNA segment of the CecA was inserted into the expression plasmid pET-30a(+) to construct a recombinant expression plasmid. Western blot results revealed that his-tagged fusion protein was successfully expressed and purified. Then the mat peptide of CecA was chemically synthesized with C-terminus amidation for in vivo antifungal assay and purity achieved 93.7%. Mass spectrometry and SDS-PAGE showed its molecular weight to be 4046.95Da. Antifungal assays indicated that the B. mori CecA had a high antifungal activity to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that the CecA is effective to inhibit B. bassiana inside the body of silkworm. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Colony Collapse Disorder (CCD) and bee age impact honey bee pathophysiology.

    Science.gov (United States)

    vanEngelsdorp, Dennis; Traynor, Kirsten S; Andree, Michael; Lichtenberg, Elinor M; Chen, Yanping; Saegerman, Claude; Cox-Foster, Diana L

    2017-01-01

    Honey bee (Apis mellifera) colonies continue to experience high annual losses that remain poorly explained. Numerous interacting factors have been linked to colony declines. Understanding the pathways linking pathophysiology with symptoms is an important step in understanding the mechanisms of disease. In this study we examined the specific pathologies associated with honey bees collected from colonies suffering from Colony Collapse Disorder (CCD) and compared these with bees collected from apparently healthy colonies. We identified a set of pathological physical characteristics that occurred at different rates in CCD diagnosed colonies prior to their collapse: rectum distension, Malpighian tubule iridescence, fecal matter consistency, rectal enteroliths (hard concretions), and venom sac color. The multiple differences in rectum symptomology in bees from CCD apiaries and colonies suggest effected bees had trouble regulating water. To ensure that pathologies we found associated with CCD were indeed pathologies and not due to normal changes in physical appearances that occur as an adult bee ages (CCD colonies are assumed to be composed mostly of young bees), we documented the changes in bees of different ages taken from healthy colonies. We found that young bees had much greater incidences of white nodules than older cohorts. Prevalent in newly-emerged bees, these white nodules or cellular encapsulations indicate an active immune response. Comparing the two sets of characteristics, we determined a subset of pathologies that reliably predict CCD status rather than bee age (fecal matter consistency, rectal distension size, rectal enteroliths and Malpighian tubule iridescence) and that may serve as biomarkers for colony health. In addition, these pathologies suggest that CCD bees are experiencing disrupted excretory physiology. Our identification of these symptoms is an important first step in understanding the physiological pathways that underlie CCD and factors

  9. El "Status" taxonomico de leishmania garnhami, indicado por su patron de desarrollo en el Vector The taxonomic status of Leishmania garnhami, indicated by its development pattern in the vector

    Directory of Open Access Journals (Sweden)

    N. Añez

    1985-03-01

    Full Text Available Bajo condiciones experimentales, se estabelece el de desarrollo primario de Leishmania garnhami em su vector Lutzomyia townsendi, siguiendo disecciones diarias entre el Iro y 12mo dia post-imnfección. Se demuestra que L. garnhami puede lograr el estabelecimiento inicial de la infección en cualquiera de las regiones del intestino y en los túbulos de Malpighi del insecto vector. Un comportamiento similar fue observado en ejemplares de Lu. longipalpis. Se discute la susceptibilidad de Lu. townsendi a las infecciones por L. mexicana, L. braziliensis y L. garnhami. Se concluye que L. garnhami, a juzgar por su comportamiento en el vector, debe considerarse distinta a las especies del complejo L.mexicana y que su patrón de desarrollo difiere del de las especies de la Sección Suprapylaria, ya que invaden el triângulo pilórico, y del de la Peripylaria por su invasión a los túbulos de Malpighi.Under experimental conditions, the developmental pattern of Leishmania garnhami in its vector Lutzomyia townsendi, was established following daily dissections between the 1st and the 12th day post-infection. L. garnhami succeeded in establishing initial infections in any of the gut regions and in the Malpighian tubules of infected sandflies. Similar behavior was observed in infected Lu. longipalpis. The distribution of L. garnhami in the digestive tract of infected flies, is different to that observed in species of the L. mexicana and L. braziliensis complexes. The susceptibility of Lu. townsendi to infection by L. mexicana, L. braziliensis and L. garnhami is discussed. It is concluded that L. garnhami, according to its behaviour in the vector, must be considered different from the l. mexicana complex, and that its developmental pattern differs from that of the Suprapylaria, because it invades the hind triangle and also from the species of the Peripylaria by its invasion of the Malpighian tubules.

  10. Colony Collapse Disorder (CCD and bee age impact honey bee pathophysiology.

    Directory of Open Access Journals (Sweden)

    Dennis vanEngelsdorp

    Full Text Available Honey bee (Apis mellifera colonies continue to experience high annual losses that remain poorly explained. Numerous interacting factors have been linked to colony declines. Understanding the pathways linking pathophysiology with symptoms is an important step in understanding the mechanisms of disease. In this study we examined the specific pathologies associated with honey bees collected from colonies suffering from Colony Collapse Disorder (CCD and compared these with bees collected from apparently healthy colonies. We identified a set of pathological physical characteristics that occurred at different rates in CCD diagnosed colonies prior to their collapse: rectum distension, Malpighian tubule iridescence, fecal matter consistency, rectal enteroliths (hard concretions, and venom sac color. The multiple differences in rectum symptomology in bees from CCD apiaries and colonies suggest effected bees had trouble regulating water. To ensure that pathologies we found associated with CCD were indeed pathologies and not due to normal changes in physical appearances that occur as an adult bee ages (CCD colonies are assumed to be composed mostly of young bees, we documented the changes in bees of different ages taken from healthy colonies. We found that young bees had much greater incidences of white nodules than older cohorts. Prevalent in newly-emerged bees, these white nodules or cellular encapsulations indicate an active immune response. Comparing the two sets of characteristics, we determined a subset of pathologies that reliably predict CCD status rather than bee age (fecal matter consistency, rectal distension size, rectal enteroliths and Malpighian tubule iridescence and that may serve as biomarkers for colony health. In addition, these pathologies suggest that CCD bees are experiencing disrupted excretory physiology. Our identification of these symptoms is an important first step in understanding the physiological pathways that underlie CCD and

  11. The digestive system of the "stick bug" Cladomorphus phyllinus (Phasmida, Phasmatidae): a morphological, physiological and biochemical analysis.

    Science.gov (United States)

    Monteiro, Emiliano C; Tamaki, Fábio K; Terra, Walter R; Ribeiro, Alberto F

    2014-03-01

    This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatin-mediated acute kidney injury

    NARCIS (Netherlands)

    Li, Shenyang; Nagothu, K.; Ranganathan, G.; Ali, S.M.; Shank, B.; Gokden, N.; Ayyadevara, S.; Megysi, J.; Olivecrona, G.; Chugh, S.S.; Kersten, A.H.; Portilla, D.

    2012-01-01

    Peroxisome proliferator-activated receptor-a (PPARa) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPARa and CP

  13. The Universal Role of Tubulence in the Propagation of Strong Shocks and Detonation Waves

    Science.gov (United States)

    Lee, John H.

    2001-06-01

    The passage of a strong shock wave usually results in irreversible physical and chemical changes in the medium. If the chemical reactions are sufficiently exothermic, the shock wave can be self-propagating, i.e., sustained by the chemical energy release via the expansion work of the reaction products. Although shocks and detonations can be globally stable and propagate at constant velocities (in the direction of motion), their structure may be highly unstable and exhibit large hydrodynamic fluctuations, i.e., turbulence. Recent investigations on plastic deformation of polycrystalline material behind shock waves have revealed particle velocity dispersion at the mesoscopic level, a result of vortical rotational motion similar to that of turbulent fluid flows at high Reynolds number.1 Strong ionizing shocks in noble gases2, as well as dissociating shock waves in carbon dioxide,3 also demonstrate a turbulent density fluctuation in the non-equilibrium shock transition zone. Perhaps the most thoroughly investigated unstable structure is that of detonation waves in gaseous explosives.4 Detonation waves in liquid explosives such as nitromethane also take on similar unstable structure as gaseous detonations.5 There are also indications that detonations in solid explosives have a similar unsteady structure under certain conditions. Thus, it appears that it is more of a rule than an exception that the structure of strong shocks and detonations are unstable and exhibit turbulent-like fluctuations as improved diagnostics now permit us to look more closely at the meso- and micro-levels. Increasing attention is now devoted to the understanding of the shock waves at the micro-scale level in recent years. This is motivated by the need to formulate physical and chemical models that contain the correct physics capable of describing quantitatively the shock transition process. It should be noted that, in spite of its unstable 3-D structure, the steady 1-D conservation laws (in the direction of propagation) apply across the shock transition zone if the downstream equilibrium plane is taken far enough away to ensure the decay of the turbulent fluctuations. Thus, the Hugoniot properties of one-dimensional propagation of shock and detonation waves remain valid. However, the conservation laws do not describe the important propagation mechanisms (i.e., the physical and chemical processes that effect the transition from initial to the final state) in the wave structure. Since gaseous detonations enjoy the advantage of being able to be observed experimentally in great detail, its complex turbulent structure is now quite well established. Furthermore, the equation of state for perfect gases is well known and the chemistry of most gas phase reactions is also sufficiently understood quantitatively to permit detailed numerical simulation of the complex detonation structure. Thus, a good database of information exists for gaseous detonation, and in this paper we shall explore the turbulent structure of gaseous detonation with the aim of answering the question as to "why nature prefers to evoke such a complicated manner to effect its propagation." We will then attempt to generalize the discussion to the "terra incognita" of condensed phase materials where the structure is much less understood. 1. Meshcheryakov, Yu.I., and Atroshenko, S.A., Izv. Vyssh. Uchebn. Zaved. Fiz., 4, 105-123 (1992). 2. Glass, I.I, and Liu, W.S., J. Fluid Mech., 84(1), 55-77 (1978). 3. Griffiths, R.W., Sanderman, R.J., and Hornung, H.G., J. Phys. D., 8, 1681-1691 (1975). 4. Lee, J.H.S., Ann. Rev. Fluid Mech., 16, 311-336 (1984). 5. Mallory, H.D., J. Appl. Physics, 38, 5302-5306 (1967).

  14. Renal Tubule Repair: Is Wnt/β-Catenin a Friend or Foe?

    Science.gov (United States)

    Gewin, Leslie S

    2018-01-24

    Wnt/β-catenin signaling is extremely important for proper kidney development. This pathway is also upregulated in injured renal tubular epithelia, both in acute kidney injury and chronic kidney disease. The renal tubular epithelium is an important target of kidney injury, and its response (repair versus persistent injury) is critical for determining whether tubulointerstitial fibrosis, the hallmark of chronic kidney disease, develops. This review discusses how Wnt/β-catenin signaling in the injured tubular epithelia promotes either repair or fibrosis after kidney injury. There is data suggesting that epithelial Wnt/β-catenin signaling is beneficial in acute kidney injury and important in tubular progenitors responsible for epithelial repair. The role of Wnt/β-catenin signaling in chronically injured epithelia is less clear. There is convincing data that Wnt/β-catenin signaling in interstitial fibroblasts and pericytes contributes to the extracellular matrix accumulation that defines fibrosis. However, some recent studies question whether Wnt/β-catenin signaling in chronically injured epithelia actually promotes fibrosis or repair.

  15. Nanoparticles Formed Onto/Into Halloysite Clay Tubules: Architectural Synthesis and Applications.

    Science.gov (United States)

    Vinokurov, Vladimir A; Stavitskaya, Anna V; Glotov, Aleksandr P; Novikov, Andrei A; Zolotukhina, Anna V; Kotelev, Mikhail S; Gushchin, Pawel A; Ivanov, Evgenii V; Darrat, Yusuf; Lvov, Yuri M

    2018-01-04

    Nanoparticles, being objects with high surface area are prone to agglomeration. Immobilization onto solid supports is a promising method to increase their stability and it allows for scalable industrial applications, such as metal nanoparticles adsorbed to mesoporous ceramic carriers. Tubular nanoclay - halloysite - can be an efficient solid support, enabling the fast and practical architectural (inside / outside) synthesis of stable metal nanoparticles. The obtained halloysite-nanoparticle composites can be employed as advanced catalysts, ion-conducting membrane modifiers, inorganic pigments, and optical markers for biomedical studies. Here, we discuss the possibilities to synthesize halloysite decorated with metal, metal chalcogenide, and carbon nanoparticles, and to use these materials in various fields, especially in catalysis and petroleum refinery. © 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Stem cells from glomerulus to distal tubule: a never-ending story?

    Directory of Open Access Journals (Sweden)

    Melania Puddu

    2016-08-01

    Full Text Available The growing interest of research in the field of renal stem cells and kidney regeneration aims to get results that allow its clinical application, favoring the birth and development of regenerative medicine.Nephrogenesis requires differentiation into epithelial cells of a population of progenitor mesenchymal cells. Since this process ends at 36-38 weeks of gestational age, it is quite likely to imagine that such a population disappears in the human kidney after birth. However, several studies have identified in different parts of the adult kidney cells having the characteristics of stem cells that would be involved in renal regenerative processes. They may be classified as resident mesenchymal/epithelial progenitors and often share the same genetic and epigenetic profile as progenitor stem cells active during embryonic life, thus suggesting a common origin.Current literature includes two lines of thought: one attributes to stem cells a fundamental role in renal regeneration processes while the other sustains the intervention of other mechanisms.The aim of this review is to report on progress made in research in the field of kidney regeneration starting from the past century and arriving at the present, with an analysis of scientific works that have produced the most important results in this field. Proceedings of the 2nd International Course on Perinatal Pathology (part of the 11th International Workshop on Neonatology · October 26th-31st, 2015 · Cagliari (Italy · October 31st, 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano

  17. Gene Expression Analysis in Tubule Interstitial Compartments Reveals Candidate Agents for IgA Nephropathy

    Directory of Open Access Journals (Sweden)

    Jinling Wang

    2014-09-01

    Full Text Available Background/Aims: Our aim was to explore the molecular mechanism underlying development of IgA nephropathy and discover candidate agents for IgA nephropathy. Methods: The differentially expressed genes (DEGs between patients with IgA nephropathy and normal controls were identified by the data of GSE35488 downloaded from GEO (Gene Expression Omnibus database. The co-expressed gene pairs among DEGs were screened to construct the gene-gene interaction network. Gene Ontology (GO enrichment analysis was performed to analyze the functions of DEGs. The biologically active small molecules capable of targeting IgA nephropathy were identified using the Connectivity Map (cMap database. Results: A total of 55 genes involved in response to organic substance, transcription factor activity and response to steroid hormone stimulus were identified to be differentially expressed in IgA nephropathy patients compared to healthy individuals. A network with 45 co-expressed gene pairs was constructed. DEGs in the network were significantly enriched in response to organic substance. Additionally, a group of small molecules were identified, such as doxorubicin and thapsigargin. Conclusion: Our work provided a systematic insight in understanding the mechanism of IgA nephropathy. Small molecules such as thapsigargin might be potential candidate agents for the treatment of IgA nephropathy.

  18. Cdc42 regulates epithelial cell polarity and cytoskeletal function during kidney tubule development

    DEFF Research Database (Denmark)

    Elias, Bertha C; Das, Amrita; Parekh, Diptiben V

    2015-01-01

    The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development and main......The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development...

  19. Balance between Apical Membrane Growth and Luminal Matrix Resistance Determines Epithelial Tubule Shape

    Directory of Open Access Journals (Sweden)

    Bo Dong

    2014-05-01

    Full Text Available The morphological stability of biological tubes is crucial for the efficient circulation of fluids and gases. Failure of this stability causes irregularly shaped tubes found in multiple pathological conditions. Here, we report that Drosophila mutants of the ESCRT III component Shrub/Vps32 exhibit a strikingly elongated sinusoidal tube phenotype. This is caused by excessive apical membrane synthesis accompanied by the ectopic accumulation and overactivation of Crumbs in swollen endosomes. Furthermore, we demonstrate that the apical extracellular matrix (aECM of the tracheal tube is a viscoelastic material coupled with the apical membrane. We present a simple mechanical model in which aECM elasticity, apical membrane growth, and their interaction are three vital parameters determining the stability of biological tubes. Our findings demonstrate a mechanical role for the extracellular matrix and suggest that the interaction of the apical membrane and an elastic aECM determines the final morphology of biological tubes independent of cell shape.

  20. Balance between apical membrane growth and luminal matrix resistance determines epithelial tubule shape.

    Science.gov (United States)

    Dong, Bo; Hannezo, Edouard; Hayashi, Shigeo

    2014-05-22

    The morphological stability of biological tubes is crucial for the efficient circulation of fluids and gases. Failure of this stability causes irregularly shaped tubes found in multiple pathological conditions. Here, we report that Drosophila mutants of the ESCRT III component Shrub/Vps32 exhibit a strikingly elongated sinusoidal tube phenotype. This is caused by excessive apical membrane synthesis accompanied by the ectopic accumulation and overactivation of Crumbs in swollen endosomes. Furthermore, we demonstrate that the apical extracellular matrix (aECM) of the tracheal tube is a viscoelastic material coupled with the apical membrane. We present a simple mechanical model in which aECM elasticity, apical membrane growth, and their interaction are three vital parameters determining the stability of biological tubes. Our findings demonstrate a mechanical role for the extracellular matrix and suggest that the interaction of the apical membrane and an elastic aECM determines the final morphology of biological tubes independent of cell shape. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Metabolic response to low-level toxicant exposure in a novel renal tubule epithelial cell system

    NARCIS (Netherlands)

    Ellis, James Keith; Athersuch, Toby James; Cavill, Rachel; Radford, Robert; Slattery, Craig; Jennings, Paul; McMorrow, Tara; Ryan, Michael P; Ebbels, Timothy Mark David; Keun, Hector Charles

    Toxicity testing is vital to protect human health from exposure to toxic chemicals in the environment. Furthermore, combining novel cellular models with molecular profiling technologies, such as metabolomics can add new insight into the molecular basis of toxicity and provide a rich source of

  2. Pathologic Features of Behçet's Disease in the Tubuler Gut

    Directory of Open Access Journals (Sweden)

    Tuba Kara

    2012-01-01

    Full Text Available Behçet's disease (BD is a vasculitic disorder of relapsing acute inflammation characterized by recurrent oral ulcers, genital ulcers, uveitis, and skin lesions. The disease also affects other organs, including joints, the nervous system, blood vessels, and gastrointestinal (GI system may also be involved and the lower GI tract is the mostly involved part, leading to severe morbidity. The frequency of GI involvement in BD varies among different ethnic groups. Although 50–60% of Japanese patients have GI disease, these manifestations are rare in patients from Mediterranean countries. The gastrointestinal manifestations of BD usually appear 4.5–6 years after the onset of the oral ulcers. The intestinal lesions are usually resistant to medical treatment and recur after surgery. The elementary lesion is apthous ulcer. Deep, round or oval ulcers with a punched-out appearance tend to perforate easily, so that many patients require urgent operation.

  3. Membrane tubulation in lipid vesicles triggered by the local application of calcium ions

    DEFF Research Database (Denmark)

    Ali Doosti, Baharan; Pezeshkian, Weria; Bruhn, Dennis Skjøth

    2017-01-01

    , generates spontaneous curvature and triggers the formation of tubular protrusions that point away from the ion source. This behavior is rationalized by strong binding of the divalent cations to the surface of the charged bilayer which effectively neutralizes the surface charge density of outer leaflet...

  4. Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule

    DEFF Research Database (Denmark)

    Pedersen, Nis Borbye; Hofmeister, Marlene Vind; Rosenbaek, Lena L

    2010-01-01

    The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for renal electrolyte balance and its phosphorylation causes an increase in its transport activity and cellular localization. Here, we generated phospho-specific antibodies against two conserved N-terminal phosphorylation sites...

  5. Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules

    DEFF Research Database (Denmark)

    van Weering, Jan R.T.; Sessions, Richard B.; Traer, Colin J.

    2012-01-01

    that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective 'tip...... and organizes the tubular endosomal network....

  6. Modeling removal of accumulated potassium from T-tubules by inward rectifier potassium channels

    NARCIS (Netherlands)

    Wallinga, W.; Vliek, M.; Wienk, E.D.; Alberink, M.J.; Ypey, D.L.; Ypey, D.L.

    1996-01-01

    The membrane models of Cannon et al. (1993) and Alberink et al. (1995) for mammalian skeletal muscle fibers are based upon Hodgkin-Huxley descriptions of sodium, potassium delayed rectifier and leak conductances and the capacitive current taking into account fast inactivation of sodium channels. Now

  7. Tubule-forming capacity of the movement proteins of alfalfa mosaic virus and brome mosaic virus

    NARCIS (Netherlands)

    Kasteel, D. T.; van der Wel, N. N.; Jansen, K. A.; Goldbach, R. W.; van Lent, J. W.

    1997-01-01

    The structural phenotype of the movement proteins (MPs) of two representatives of the Bromoviridae, alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), was studied in protoplasts. Immunofluorescence microscopy showed that the MPs of these viruses, for which there has been no evidence of a

  8. Observation of endoplasmic reticulum tubules via TOF-SIMS tandem mass spectrometry imaging of transfected cells.

    Science.gov (United States)

    Chini, Corryn E; Fisher, Gregory L; Johnson, Ben; Tamkun, Michael M; Kraft, Mary L

    2018-02-26

    Advances in three-dimensional secondary ion mass spectrometry (SIMS) imaging have enabled visualizing the subcellular distributions of various lipid species within individual cells. However, the difficulty of locating organelles using SIMS limits efforts to study their lipid compositions. Here, the authors have assessed whether endoplasmic reticulum (ER)-Tracker Blue White DPX ® , which is a commercially available stain for visualizing the endoplasmic reticulum using fluorescence microscopy, produces distinctive ions that can be used to locate the endoplasmic reticulum using SIMS. Time-of-flight-SIMS tandem mass spectrometry (MS 2 ) imaging was used to identify positively and negatively charged ions produced by the ER-Tracker stain. Then, these ions were used to localize the stain and thus the endoplasmic reticulum, within individual human embryonic kidney cells that contained higher numbers of endoplasmic reticulum-plasma membrane junctions on their surfaces. By performing MS 2 imaging of selected ions in parallel with the precursor ion (MS 1 ) imaging, the authors detected a chemical interference native to the cell at the same nominal mass as the pentafluorophenyl fragment from the ER-Tracker stain. Nonetheless, the fluorine secondary ions produced by the ER-Tracker stain provided a distinctive signal that enabled locating the endoplasmic reticulum using SIMS. This simple strategy for visualizing the endoplasmic reticulum in individual cells using SIMS could be combined with existing SIMS methodologies for imaging intracellular lipid distribution and to study the lipid composition within the endoplasmic reticulum.

  9. Active lithium transport by rat renal proximal tubule: a micropuncture study

    DEFF Research Database (Denmark)

    Leyssac, P P; Frederiksen, O; Holstein-Rathlou, N H

    1994-01-01

    We tested the hypothesis that proximal tubular Li+ reabsorption is due to passive transport. Clearances of [14C]inulin (CIn) and Li+ (CLi), proximal transepithelial electrical potential difference (PD), and tubular fluid-to-plasma Li+ concentration ratios [(TF/P)Li] were measured in anesthetized ...

  10. Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins

    DEFF Research Database (Denmark)

    Varkey, Jobin; Isas, Jose Mario; Mizuno, Naoko

    2010-01-01

    Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have...... of amphipathic helices alone. Moreover, we frequently observed that a-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that a-synuclein plays a role in vesicle trafficking...

  11. Cisplatin-induced injury of the renal distal convoluted tubule is associated with hypomagnesaemia in mice

    NARCIS (Netherlands)

    Angelen, A.A. van; Glaudemans, B.; Kemp, A. van der; Hoenderop, J.G.J.; Bindels, R.J.M.

    2013-01-01

    Background Cisplatin is an effective anti-neoplastic drug, but its clinical use is limited due to dose-dependent nephrotoxicity. The majority of cisplatin-treated patients develop hypomagnesaemia, often associated with a reduced glomerular filtration rate (GFR), polyuria and other electrolyte

  12. The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules

    DEFF Research Database (Denmark)

    Halberg, Kenneth Agerlin; Rainey, Stephanie M.; Veland, Iben Rønn

    2016-01-01

    Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most...... role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border....

  13. Proximal-tubule-like epithelium in Bowman's capsule in spontaneously hypertensive rats. Changes with age.

    OpenAIRE

    Haensly, W. E.; Granger, H. J.; Morris, A. C.; Cioffe, C.

    1982-01-01

    Kidneys were samples from male spontaneously hypertensive rats (SHR) and normotensive rats (WKY) in four groups. Renal tissues were examined in 64 rats: 6 SHR and 6 WKY rats 8 and 16 weeks of age and 10 SHR and 10 WKY rats 32 and 64 weeks of age. Tissue samples were fixed, processed, and stained by routine histologic procedures. The parietal layer of Bowman's capsule in 100-115 renal corpuscles from right to left kidney sections was classified as squamous or cuboidal epithelium. The cuboidal ...

  14. UV light and urban pollution: Bad cocktail for mosquitoes?

    International Nuclear Information System (INIS)

    Tetreau, Guillaume; Chandor-Proust, Alexia; Faucon, Frédéric; Stalinski, Renaud; Akhouayri, Idir; Prud’homme, Sophie M.; Régent-Kloeckner, Myriam; Raveton, Muriel; Reynaud, Stéphane

    2014-01-01

    Highlights: •Mosquito tolerance to temephos is induced by PAHs and UV exposure. •Toxicity of fluoranthene for mosquito Malpighian tubules cells is induced by UV. •Fluoranthene crystallizes in mosquito Malpighian tubules upon UV exposure. •Mixture of two PAHs is less toxic for mosquitoes than each PAHs separately. •Combination of abiotic parameters (PAHs and UV) affect mosquito physiology. -- Abstract: Mosquito breeding sites consist of water pools, which can either be large open areas or highly covered ponds with vegetation, thus with different light exposures combined with the presence in water of xenobiotics including polycyclic aromatic hydrocarbons (PAHs) generated by urban pollution. UV light and PAHs are abiotic factors known to both affect the mosquito insecticide resistance status. Nonetheless, their potential combined effects on the mosquito physiology have never been investigated. The present article aims at describing the effects of UV exposure alongside water contamination with two major PAH pollutants (fluoranthene and benzo[a]pyrene) on a laboratory population of the yellow fever mosquito Aedes aegypti. To evaluate the effects of PAH exposure and low energetic UV (UV-A) irradiation on mosquitoes, different parameters were measured including: (1) The PAH localization and its impact on cell mortality by fluorescent microscopy; (2) The detoxification capacities (cytochrome P450, glutathione-S-transferase, esterase); (3) The responses to oxidative stress (Reactive Oxygen Species–ROS) and (4) The tolerance of mosquito larvae to a bioinsecticide (Bacillus thuringiensis subsp. israelensis–Bti) and to five chemical insecticides (DDT, imidacloprid, permethrin, propoxur and temephos). Contrasting effects regarding mosquito cell mortality, detoxification and oxidative stress were observed as being dependent on the pollutant considered, despite the fact that the two PAHs belong to the same family. Moreover, UV is able to modify pollutant effects on

  15. UV light and urban pollution: Bad cocktail for mosquitoes?

    Energy Technology Data Exchange (ETDEWEB)

    Tetreau, Guillaume, E-mail: guillaume.tetreau@gmail.com [Laboratoire d’Ecologie Alpine, LECA-UMR 5553, Université de Grenoble 1, BP 53, 38041 Grenoble cedex 09 (France); Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456 (United States); Chandor-Proust, Alexia; Faucon, Frédéric; Stalinski, Renaud; Akhouayri, Idir; Prud’homme, Sophie M.; Régent-Kloeckner, Myriam; Raveton, Muriel; Reynaud, Stéphane [Laboratoire d’Ecologie Alpine, LECA-UMR 5553, Université de Grenoble 1, BP 53, 38041 Grenoble cedex 09 (France)

    2014-01-15

    Highlights: •Mosquito tolerance to temephos is induced by PAHs and UV exposure. •Toxicity of fluoranthene for mosquito Malpighian tubules cells is induced by UV. •Fluoranthene crystallizes in mosquito Malpighian tubules upon UV exposure. •Mixture of two PAHs is less toxic for mosquitoes than each PAHs separately. •Combination of abiotic parameters (PAHs and UV) affect mosquito physiology. -- Abstract: Mosquito breeding sites consist of water pools, which can either be large open areas or highly covered ponds with vegetation, thus with different light exposures combined with the presence in water of xenobiotics including polycyclic aromatic hydrocarbons (PAHs) generated by urban pollution. UV light and PAHs are abiotic factors known to both affect the mosquito insecticide resistance status. Nonetheless, their potential combined effects on the mosquito physiology have never been investigated. The present article aims at describing the effects of UV exposure alongside water contamination with two major PAH pollutants (fluoranthene and benzo[a]pyrene) on a laboratory population of the yellow fever mosquito Aedes aegypti. To evaluate the effects of PAH exposure and low energetic UV (UV-A) irradiation on mosquitoes, different parameters were measured including: (1) The PAH localization and its impact on cell mortality by fluorescent microscopy; (2) The detoxification capacities (cytochrome P450, glutathione-S-transferase, esterase); (3) The responses to oxidative stress (Reactive Oxygen Species–ROS) and (4) The tolerance of mosquito larvae to a bioinsecticide (Bacillus thuringiensis subsp. israelensis–Bti) and to five chemical insecticides (DDT, imidacloprid, permethrin, propoxur and temephos). Contrasting effects regarding mosquito cell mortality, detoxification and oxidative stress were observed as being dependent on the pollutant considered, despite the fact that the two PAHs belong to the same family. Moreover, UV is able to modify pollutant effects on

  16. Functional interpretation of a non-gut hemocoelic tissue aminopeptidase N (APN in a lepidopteran insect pest Achaea janata.

    Directory of Open Access Journals (Sweden)

    Thuirei Jacob Ningshen

    Full Text Available Insect midgut membrane-anchored aminopeptidases N (APNs are Zn(++ dependent metalloproteases. Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific in vitro interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of Achaea janata. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of A. janata showed high structural similarity to the Cry1Aa toxin binding midgut APN of Bombyx mori, especially in the toxin binding region. Structural similarity was further substantiated by in vitro binding of Cry1Aa toxin. RNA interference (RNAi resulted in significant down-regulation of AjAPN1 transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s during post-embryonic development of A. janata. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation.

  17. Role in diuresis of a calcitonin receptor (GPRCAL1 expressed in a distal-proximal gradient in renal organs of the mosquito Aedes aegypti (L..

    Directory of Open Access Journals (Sweden)

    Hyeogsun Kwon

    Full Text Available Evolution of anthropophilic hematophagy in insects resulted in the coordination of various physiological processes for survival. In female mosquitoes, a large blood meal provides proteins for egg production and as a trade-off, rapid elimination of the excess water and solutes (Na(+, Cl(- is critical for maintaining homeostasis and removing excess weight to resume flight and avoid predation. This post-prandial excretion is achieved by the concerted action of multiple hormones. Diuresis and natriuresis elicited by the calcitonin-like diuretic hormone 31 (DH(31 are believed to be mediated by a yet uncharacterized calcitonin receptor (GPRCAL in the mosquito Malpighian tubules (MTs, the renal organs. To contribute knowledge on endocrinology of mosquito diuresis we cloned GPRCAL1 from MT cDNA. This receptor is the ortholog of the DH(31 receptor from Drosophila melanogaster that is expressed in principal cells of the fruit fly MT. Immunofluorescence similarly showed AaegGPRCAL1 is present in MT principal cells in A. aegypti, however, exhibiting an overall gradient-like pattern along the tubule novel for a GPCR in insects. Variegated, cell-specific receptor expression revealed a subpopulation of otherwise phenotypically similar principal cells. To investigate the receptor contribution to fluid elimination, RNAi was followed by urine measurement assays. In vitro, MTs from females that underwent AaegGPRcal1 knock-down exhibited up to 57% decrease in the rate of fluid secretion in response to DH(31. Live females treated with AaegGPRcal1 dsRNA exhibited 30% reduction in fluid excreted after a blood meal. The RNAi-induced phenotype demonstrates the critical contribution of this single secretin-like family B GPCR to fluid excretion in invertebrates and highlights its relevance for the blood feeding adaptation. Our results with the mosquito AaegGPRCAL1 imply that the regulatory function of calcitonin-like receptors for ion and fluid transport in renal organs

  18. Effect of gamma irradiation on sex chromatin body appearance and the sex chromosome aberrations in the potato tuber moth, phthorimaea operculella (Lepidoptera: Gelechiidae)

    International Nuclear Information System (INIS)

    Makee, H.

    2007-01-01

    Genetic sexing technique based on the construction of a Balanced Lethal Strain (BLS) has been proposed for Phthorimaea operculella (Zeller). The isolation of female with T(W. Z) translocation is a fundamental step to develop such strain. Gamma irradiation was used to induce the requested translocations. The availability of sex-linked morphological marker is required to facilitate the detection of such mutations. Since a visible sex-linked marker has not been found in P. operculella, therefore main aim of our study was to determine the possibility of using sex heterochromatin body as a marker to identify the required translocated females. The appearance of sex heterochromatin body and the analysis of sex chromosomes in F1 females of irradiated P. operculella females were investigated. The percentage of abnormality in sex heterochromatin body in highly polyploid Malpighian tubule nuclei was increased by increasing the applied dose. Based on the appearance of this body, 3 mutant lines were isolated: elongated, small, fragmented lines. W chromosome was easily distinguished from Z chromosome when the analysis of pachytene sex chromosome bivalents of P. operculella females was carried out. The aberrations involved W chromosome directly influenced the appearance of sex heterochromatin body in highly polyploid somatic cells of the isolated mutant lines. The results showed that sex heterochromatin could be used as sex determination and cytogenetic marker in P. operculella. (Author)

  19. In vivo 3D PIXE-micron-CT imaging of Drosophila melanogaster using a contrast agent

    Energy Technology Data Exchange (ETDEWEB)

    Matsuyama, Shigeo; Hamada, Naoki; Ishii, Keizo; Nozawa, Yuichiro; Ohkura, Satoru; Terakawa, Atsuki; Hatori, Yoshinobu; Fujiki, Kota; Fujiwara, Mitsuhiro; Toyama, Sho

    2015-04-01

    In this study, we developed a three-dimensional (3D) computed tomography (CT) in vivo imaging system for imaging small insects with micrometer resolution. The 3D CT imaging system, referred to as 3D PIXE-micron-CT (PIXEμCT), uses characteristic X-rays produced by ion microbeam bombardment of a metal target. PIXEμCT was used to observe the body organs and internal structure of a living Drosophila melanogaster. Although the organs of the thorax were clearly imaged, the digestive organs in the abdominal cavity could not be clearly discerned initially, with the exception of the rectum and the Malpighian tubule. To enhance the abdominal images, a barium sulfate powder radiocontrast agent was added. For the first time, 3D images of the ventriculus of a living D. melanogaster were obtained. Our results showed that PIXEμCT can provide in vivo 3D-CT images that reflect correctly the structure of individual living organs, which is expected to be very useful in biological research.

  20. Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect, Panstrongylus megistus (Burmeister (Hemiptera, Reduviidae

    Directory of Open Access Journals (Sweden)

    Garcia Simone L

    2002-01-01

    Full Text Available Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion or death (apoptosis, necrosis responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40°C, 1 h or cold (5 or 0°C, 1 h shock and then subjected to a second shock for 12 h at 40 or 0°C, respectively, after 8, 18, 24 and 72 h at 28°C (control temperature. As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0ºC were more effective than the temperatures of 35 and 5ºC in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.

  1. Effect of gamma irradiation on sex chromatin body appearance and the sex chromosome aberrations in the potato tuber moth, phthorimaea operculella (Lepidoptera: Gelechiidae)

    International Nuclear Information System (INIS)

    Makee, H.

    2006-05-01

    Genetic sexing technique based on the construction of a Balanced Lethal Strain (BLS) has been proposed for Phthorimaea operculella (Zeller). The isolation of female with T(W; Z) translocation is a fundamental step to develop such strain. Gamma irradiation was used to induce the requested translocations. The availability of sex-linked morphological marker is required to facilitate the detection of such mutations. Since a visible sex-linked marker has not been found in P. operculella, therefore main aim of our study was to determine the possibility of using sex heterochromatin body as a marker to identify the required translocated females. The appearance of sex heterochromatin body and the analysis of sex chromosomes in F1 females of irradiated P. operculella females were investigated. The percentage of abnormality in sex heterochromatin body in highly polyploid Malpighian tubule nuclei was increased by increasing the applied dose. Based on the appearance of this body, 3 mutant lines were isolated: elongated, small, fragmented lines. W chromosome was easily distinguished from Z chromosome when the analysis of pachytene sex chromosome bivalents of P. operculella females was carried out. The aberrations involved W chromosome directly influenced the appearance of sex heterochromatin body in highly polyploid somatic cells of the isolated mutant lines. The results showed that sex heterochromatin could be used as sex determination and cytogenetic marker in P. operculella. (Author)

  2. The plasticity of extracellular fluid homeostasis in insects.

    Science.gov (United States)

    Beyenbach, Klaus W

    2016-09-01

    In chemistry, the ratio of all dissolved solutes to the solution's volume yields the osmotic concentration. The present Review uses this chemical perspective to examine how insects deal with challenges to extracellular fluid (ECF) volume, solute content and osmotic concentration (pressure). Solute/volume plots of the ECF (hemolymph) reveal that insects tolerate large changes in all three of these ECF variables. Challenges beyond those tolerances may be 'corrected' or 'compensated'. While a correction simply reverses the challenge, compensation accommodates the challenge with changes in the other two variables. Most insects osmoregulate by keeping ECF volume and osmotic concentration within a wide range of tolerance. Other insects osmoconform, allowing the ECF osmotic concentration to match the ambient osmotic concentration. Aphids are unique in handling solute and volume loads largely outside the ECF, in the lumen of the gut. This strategy may be related to the apparent absence of Malpighian tubules in aphids. Other insects can suspend ECF homeostasis altogether in order to survive extreme temperatures. Thus, ECF homeostasis in insects is highly dynamic and plastic, which may partly explain why insects remain the most successful class of animals in terms of both species number and biomass. © 2016. Published by The Company of Biologists Ltd.

  3. TmCactin plays an important role in Gram-negative and -positive bacterial infection by regulating expression of 7 AMP genes in Tenebrio molitor

    Science.gov (United States)

    Jo, Yong Hun; Jung Kim, Yu; Beom Park, Ki; Hwan Seong, Jeong; Gon Kim, Soo; Park, Soyi; Young Noh, Mi; Seok Lee, Yong; Soo Han, Yeon

    2017-01-01

    Cactin was originally identified as an interactor of the Drosophila IκB factor Cactus and shown to play a role in controlling embryonic polarity and regulating the NF-κB signaling pathway. While subsequent studies have identified the roles for Cactin in the mammalian immune response, the immune function of Cactin in insects has not been described yet. Here, we identified a Cactin gene from the mealworm beetle, Tenebrio molitor (TmCactin) and characterized its functional role in innate immunity. TmCactin was highly expressed in prepupa to last instar stages, and its expression was high in the integument and Malpighian tubules of last instar larvae and adults. TmCactin was induced in larvae after infection with different pathogens and detectable within 3 hours of infection. The highest levels of TmCactin expression were detected at 9 hours post infection. TmCactin RNAi significantly decreased the survival rates of larvae after challenge with Escherichia coli and Staphylococcus aureus, but had no significant effect after challenge with Candida albicans. Furthermore, TmCactin RNAi significantly reduced the expression of seven antimicrobial peptide genes (AMPs) after bacterial challenge. Our results suggest that TmCactin may serve as an important regulator of innate immunity, mediating AMP responses against both Gram-positive and Gram-negative bacteria in T. molitor. PMID:28418029

  4. Post-embrionic development of the digestive tube of Dermatobia hominis (Linnaeus (Diptera, Cuterebridae

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    Ana Maria Vieira

    1996-01-01

    Full Text Available The digestive tube of 2nd and 3rd instar larvae, pupae and newly emerged adults of Dermatobia hominis (Linnaeus, 1781 was studied anatomically. The specimens were dissected in buffer saline under a stereomicroscope, and the digestive tubes were placed on slides and fixed in 10% buffered formalin. Each tube was measured using a micrometric eye piece, and drawings were made with camera lucida. The results showed that the midgut, the hindgut and the Malpighian tubules with their ducts grow gradually during the larval development. The oesophagus and the salivary glands with their ducts grow only during the moult from the 2nd to the 3rd instar. In the pupal period, salivary glands grow gradually but disappeared after the 20th day. After metamorphosis the digestive tube regressed. This is expected since adult D. hominis lives about nine days without feeding. This fly, similar to other calyptratae muscoid flies shows no vestige of a crop during all post-embrionic development, and the adult has no salivary glands.

  5. Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): responses to plant allelochemicals and pesticides.

    Science.gov (United States)

    Giraudo, M; Hilliou, F; Fricaux, T; Audant, P; Feyereisen, R; Le Goff, G

    2015-02-01

    Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment. © 2014 The Royal Entomological Society.

  6. Morphological and molecular description of Blastocrithidia cyrtomeni sp. nov. (Kinetoplastea: Trypanosomatidae associated with Cyrtomenus bergi Froeschner (Hemiptera: Cydnidae from Colombia

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    Ana Milena Caicedo

    2011-05-01

    Full Text Available A new trypanosomatid species, Blastocrithidia cyrtomeni, is herein described using morphological and molecular data. It was found parasitising the alimentary tract of the insect host Cyrtomenus bergi, a polyphagous pest. The morphology of B. cyrtomeni was investigated using light and transmission microscopy and molecular phylogeny was inferred from the sequences of spliced leader RNA (SL rRNA - 5S rRNA gene repeats and the 18S small subunit (SSU rRNA gene. Epimastigotes of variable size with straphanger cysts adhering to the middle of the flagellum were observed in the intestinal tract, hemolymph and Malpighian tubules. Kinetoplasts were always observed anterior to the nucleus. The ultrastructure of longitudinal sections of epimastigotes showed the flagellum arising laterally from a relatively shallow flagellar pocket near the kinetoplast. SL RNA and 5S rRNA gene repeats were positive in all cases, producing a 0.8-kb band. The amplicons were 797-803 bp long with > 98.5% identity, indicating that they originated from the same organism. According to the sequence analysis of the SL-5S rRNA gene repeats and the 18S SSU rRNA gene, B. cyrtomeni is different from all other known species or isolates of Trypanosomatidae. Both analyses indicate that among known species, it is most closely related to Blastocrithidia triatomae.

  7. Molecular Cloning and Characterization of a P-Glycoprotein from the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae)

    Science.gov (United States)

    Tian, Lixia; Yang, Jiaqiang; Hou, Wenjie; Xu, Baoyun; Xie, Wen; Wang, Shaoli; Zhang, Youjun; Zhou, Xuguo; Wu, Qingjun

    2013-01-01

    Macrocyclic lactones such as abamectin and ivermectin constitute an important class of broad-spectrum insecticides. Widespread resistance to synthetic insecticides, including abamectin and ivermectin, poses a serious threat to the management of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), a major pest of cruciferous plants worldwide. P-glycoprotein (Pgp), a member of the ABC transporter superfamily, plays a crucial role in the removal of amphiphilic xenobiotics, suggesting a mechanism for drug resistance in target organisms. In this study, PxPgp1, a putative Pgp gene from P. xylostella, was cloned and characterized. The open reading frame (ORF) of PxPgp1 consists of 3774 nucleotides, which encodes a 1257-amino acid peptide. The deduced PxPgp1 protein possesses structural characteristics of a typical Pgp, and clusters within the insect ABCB1. PxPgp1 was expressed throughout all developmental stages, and showed the highest expression level in adult males. PxPgp1 was highly expressed in midgut, malpighian tubules and testes. Elevated expression of PxPgp1 was observed in P. xylostella strains after they were exposed to the abamectin treatment. In addition, the constitutive expressions of PxPgp1 were significantly higher in laboratory-selected and field-collected resistant strains in comparison to their susceptible counterpart. PMID:24264038

  8. Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin.

    Science.gov (United States)

    Shi, M; Zhao, S; Wang, Z-H; Stanley, D; Chen, X-X

    2016-12-01

    Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species. In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full-length cDNA sequence is composed of 959 bp containing a 321 bp open reading frame that encodes a predicted protein of 106 amino acids, a predicted molecular weight of 11.7 kDa and an isoelectric point of 5.03. PxTrx was mainly expressed in larval Malpighian tubules and the fat body. An enriched recombinant PxTrx had insulin disulphide reductase activity and stimulated Human Embryonic Kidney 293 (HEK293) cell proliferation. It also protected supercoiled DNA and living HEK293 cells from H 2 O 2 -induced damage. Parasitization by Cotesia vestalis and injections of 0.05 and 0.01 equivalents of C. vestalis Bracovirus (CvBv), the symbiotic virus carried by the parasitoid, led to down-regulation of PxTrx expression in host fat body. Taken together, our results indicate that PxTrx contributes to the maintenance of P. xylostella cellular haemostasis. Host fat body expression of PxTrx is strongly attenuated by parasitization and by injections of CvBv. © 2016 The Royal Entomological Society.

  9. Metabolite localization by atmospheric pressure high-resolution scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging in whole-body sections and individual organs of the rove beetle Paederus riparius.

    Science.gov (United States)

    Bhandari, Dhaka Ram; Schott, Matthias; Römpp, Andreas; Vilcinskas, Andreas; Spengler, Bernhard

    2015-03-01

    Mass spectrometry imaging provides for non-targeted, label-free chemical imaging. In this study, atmospheric pressure high-resolution scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) was used for the first time to describe the chemical distribution of the defensive compounds pederin, pseudopederin, and pederon in tissue sections (16 μm thick) of the rove beetle Paederus riparius. The whole-insect tissue section was scanned with a 20-μm step size. Mass resolution of the orbital trapping mass spectrometer was set to 100,000 at m/z 200. Additionally, organ-specific compounds were identified for brain, nerve cord, eggs, gut, ovaries, and malpighian tubules. To confirm the distribution of the specific compounds, individual organs from the insect were dissected, and MSI experiments were performed on the dissected organs. Three ganglia of the nerve cord, with a dimension of 250-500 μm, were measured with 10-μm spatial resolution. High-quality m/z images, based on high spatial resolution and high mass accuracy were generated. These features helped to assign mass spectral peaks with high confidence. Mass accuracy of the imaging experiments was section. Without any labeling, we assigned key lipids for specific organs to describe their location in the body and to identify morphological structures with a specificity higher than with staining or immunohistology methods.

  10. An Emerging Translational Model to Screen Potential Medicinal Plants for Nephrolithiasis, an Independent Risk Factor for Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    San-Yuan Wu

    2014-01-01

    Full Text Available Pharmacological therapy for urolithiasis using medicinal plants has been increasingly adopted for the prevention of its recurrence. A Drosophila melanogaster model developed for translational research of urolithiasis was applied to evaluate agents with potential antilithic effects and calcium oxalate (CaOx formation. Potential antilithic herbs were prepared in a mixture of food in a diluted concentration of 5,000 from the original extract with 0.5% ethylene glycol (EG as the lithogenic agent. The control group was fed with food only. After 3 weeks, flies (n≥150 for each group were killed using CO2 narcotization, and the Malpighian tubules were dissected, removed, and processed for polarized light microscopy examination of the crystals. The crystal formation rate in the EG group was 100.0%. In the study, 16 tested herbal drugs reached the crystal formation rate of 0.0%, including Salviae miltiorrhizae, Paeonia lactiflora, and Carthami flos. Scutellaria baicalensis enhanced CaOx crystal formation. Two herbal drugs Commiphora molmol and Natrii sulfas caused the death of all flies. Our rapid screening methods provided evidence that some medicinal plants have potential antilithic effects. These useful medicinal plants can be further studied using other animal or human models to verify their effects.

  11. Life History of the Emerald Jewel Wasp Ampulex compressa

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    Ryan Arvidson

    2018-04-01

    Full Text Available The Emerald Jewel Wasp Ampulex compressa (Fabricius is an endoparasitoid of the American cockroach Periplaneta americana (Linnaeus. Its host subjugation strategy is unusual in that envenomation is directed into the host central nervous system, eliciting a long-term behavior modification termed hypokinesia, turning stung cockroaches into a lethargic and compliant, but not paralyzed, living food supply for wasp offspring. A. compressa manipulates hypokinesic cockroaches into a burrow, where it oviposits a single egg onto a mesothoracic leg, hatching three days later. Herein we describe the life history and developmental timing of A. compressa. Using head capsule measurements and observations of mandibular morphology, we found that the larvae develop through three instars, the first two ectoparasitoid, and the third exclusively endoparasitoid. The first two instars have mandibles sufficient for piercing and cutting the cuticle respectively, while the third instar has a larger and blunter mandibular structure. During ecdysis to the third instar, the larva enters the body cavity of the cockroach, consuming internal tissues selectively, including fat body and skeletal muscle, but sparing the gut and Malpighian tubules. The developmental timing to pupation is similar between males and females, but cocoon volume and mass, and pupation duration are sexually dimorphic. Further, we show that the difference in cocoon mass and volume can be used to predict sex before eclosion, which is valuable for studies in venom pharmacology, as only females produce venom.

  12. Changes is genes coding for laccases 1 and 2 may contribute to deformation and reduction of wings in apollo butterfly (Parnassius apollo, Lepidoptera: Papilionidae) from the isolated population in Pieniny National Park (Poland).

    Science.gov (United States)

    Łukasiewicz, Kinga; Węgrzyn, Grzegorz

    2016-01-01

    An isolated population of apollo butterfly (Parnassius apollo, Lepidoptera: Papilionidae) occurs in Pieniny National Park (Poland). Deformations and reductions of wings in a relatively large number of individuals from this population is found, yet the reasons for these defects are unknown. During studies devoted to identify cause(s) of this phenomenon, we found that specific regions of genes coding of enzymes laccases 1 and 2 could not be amplified from DNA samples isolated from large fractions of malformed insects while expected PCR products were detected in almost all (with one exception) normal butterflies. Laccases (p-diphenol:dioxygen oxidoreductases) are oxidases containing several copper atoms. They catalyse single-electron oxidations of phenolic or other compounds with concomitant reduction of oxygen to water. In insects, their enzymatic activities were found previously in epidermis, midgut, Malpighian tubules, salivary glands, and reproductive tissues. Therefore, we suggest that defects in genes coding for laccases might contribute to deformation and reduction of wings in apollo butterflies, though it seems obvious that deficiency in these enzymes could not be the sole cause of these developmental improperties in P. apollo from Pieniny National Park.

  13. Molecular characterization of a peritrophic membrane protein from the silkworm, Bombyx mori.

    Science.gov (United States)

    Hu, Xiaolong; Chen, Lin; Yang, Rui; Xiang, Xingwei; Wu, Xiaofeng

    2013-02-01

    The peritrophic membrane lines the gut of most insects at one or more stages of their life cycles. It facilitates the digestive processes in the guts and protects from invasion by pathogens or food particles. In the current study, a novel PM protein, designated as BmMtch, was identified from the silkworm, Bombyx mori. The open reading frame of BmMtch is 888 bp in length, encoding 295 amino acid residues consisting of two domains (Mito_carr domains) and three transmembrane regions. They are localized on the 11th chromosome as single copy with one exon only. Quantitative real time PCR analysis (qRT-PCR) revealed that BmMtch was mainly expressed in larval fat bodies, Malpighian tubules, testis and ovaries, and could be detected through all stages of the life cycle of silkworm. Immuno-fluorescence analysis indicated that BmMtch was localized within the goblet cell of larval midgut. Western blotting analysis showed that BmMtch were detected in total proteins of PM and larval midgut. The characteristics of BmMtch indicated that BmMtch represents a novel member of insect PM proteins, without chitin-binding domains.

  14. In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae

    Directory of Open Access Journals (Sweden)

    Ruiz Lina María

    2004-01-01

    Full Text Available Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.

  15. Molecular characterization and localization of the first tyramine receptor of the American cockroach (Periplaneta americana).

    Science.gov (United States)

    Rotte, C; Krach, C; Balfanz, S; Baumann, A; Walz, B; Blenau, W

    2009-09-15

    The phenolamines octopamine and tyramine control, regulate, and modulate many physiological and behavioral processes in invertebrates. Vertebrates possess only small amounts of both substances, and thus, octopamine and tyramine, together with other biogenic amines, are referred to as "trace amines." Biogenic amines evoke cellular responses by activating G-protein-coupled receptors. We have isolated a complementary DNA (cDNA) that encodes a biogenic amine receptor from the American cockroach Periplaneta americana, viz., Peatyr1, which shares high sequence similarity to members of the invertebrate tyramine-receptor family. The PeaTYR1 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with tyramine reduces adenylyl cyclase activity in a dose-dependent manner (EC(50) approximately 350 nM). The inhibitory effect of tyramine is abolished by co-incubation with either yohimbine or chlorpromazine. Receptor expression has been investigated by reverse transcription polymerase chain reaction and immunocytochemistry. The mRNA is present in various tissues including brain, salivary glands, midgut, Malpighian tubules, and leg muscles. The effect of tyramine on salivary gland acinar cells has been investigated by intracellular recordings, which have revealed excitatory presynaptic actions of tyramine. This study marks the first comprehensive molecular, pharmacological, and functional characterization of a tyramine receptor in the cockroach.

  16. A mutualism without honeydew: what benefits for Melissotarsus emeryi ants and armored scale insects (Diaspididae?

    Directory of Open Access Journals (Sweden)

    Christian Peeters

    2017-07-01

    Full Text Available Mutualisms between ants and sap-sucking insects generally involve clear benefits for both partners: the ants provide protection in exchange for honeydew. However, a single ant genus associates with armoured scale insects (Diaspididae that do not excrete honeydew. We studied three colonies of Melissotarsus emeryi ants from two localities in Mozambique. Vast numbers of the diaspidid Morganella conspicua occupied galleries dug by the ants under the bark of living trees. Unlike free-living M. conspicua and other diaspidids, M. conspicua living with ants are known to lack shields, likely because they gain protection against enemies and desiccation. Nevertheless, we documented the occurrence of rare individuals with shields inside ant galleries, indicating that their glands continue to secrete wax and proteins as building material. This is likely to constitute a significant portion of the ants’ diet, in addition to diaspidid exuviae and excretions from the Malpighian tubules. Indeed, Melissotarsus workers cannot walk outside the galleries due to modified middle legs, forcing them to obtain all nourishment within the tree. Melissotarsus founding queens, however, must locate a suitable host tree while flying, and acquire diaspidid crawlers. This mutualism involves ants that are highly specialised to chew through living wood, and diaspidids that can also live freely outside the bark. It is extremely widespread in Africa and Madagascar, recorded from 20 tree families, and harmful effects on plant hosts require rapid study.

  17. A neurotransmitter transporter encoded by the Drosophila inebriated gene

    Science.gov (United States)

    Soehnge, Holly; Huang, Xi; Becker, Marie; Whitley, Penn; Conover, Diana; Stern, Michael

    1996-01-01

    Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this paper, we describe the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In situ hybridization of ine transcripts to developing embryos revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na+/Cl−-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function. We conclude that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron. PMID:8917579

  18. The putative Na+/Cl−-dependent neurotransmitter/osmolyte transporter inebriated in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis

    Science.gov (United States)

    Luan, Zhuo; Quigley, Caitlin; Li, Hong-Sheng

    2015-01-01

    Most organisms are able to maintain systemic water homeostasis over a wide range of external or dietary osmolarities. The excretory system, composed of the kidneys in mammals and the Malpighian tubules and hindgut in insects, can increase water conservation and absorption to maintain systemic water homeostasis, which enables organisms to tolerate external hypertonicity or desiccation. However, the mechanisms underlying the maintenance of systemic water homeostasis by the excretory system have not been fully characterized. In the present study, we found that the putative Na+/Cl−-dependent neurotransmitter/osmolyte transporter inebriated (ine) is expressed in the basolateral membrane of anterior hindgut epithelial cells. This was confirmed by comparison with a known basolateral localized protein, the α subunit of Na+-K+ ATPase (ATPα). Under external hypertonicity, loss of ine in the hindgut epithelium results in severe dehydration without damage to the hindgut epithelial cells, implicating a physiological failure of water conservation/absorption. We also found that hindgut expression of ine is required for water conservation under desiccating conditions. Importantly, specific expression of ine in the hindgut epithelium can completely restore disrupted systemic water homeostasis in ine mutants under both conditions. Therefore, ine in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis. PMID:25613130

  19. Drosophila Melanogaster as an Emerging Translational Model of Human Nephrolithiasis

    Science.gov (United States)

    Miller, Joe; Chi, Thomas; Kapahi, Pankaj; Kahn, Arnold J.; Kim, Man Su; Hirata, Taku; Romero, Michael F.; Dow, Julian A.T.; Stoller, Marshall L.

    2013-01-01

    Purpose The limitations imposed by human clinical studies and mammalian models of nephrolithiasis have hampered the development of effective medical treatments and preventative measures for decades. The simple but elegant Drosophila melanogaster is emerging as a powerful translational model of human disease, including nephrolithiasis and may provide important information essential to our understanding of stone formation. We present the current state of research using D. melanogaster as a model of human nephrolithiasis. Materials and Methods A comprehensive review of the English language literature was performed using PUBMED. When necessary, authoritative texts on relevant subtopics were consulted. Results The genetic composition, anatomic structure and physiologic function of Drosophila Malpighian tubules are remarkably similar to those of the human nephron. The direct effects of dietary manipulation, environmental alteration, and genetic variation on stone formation can be observed and quantified in a matter of days. Several Drosophila models of human nephrolithiasis, including genetically linked and environmentally induced stones, have been developed. A model of calcium oxalate stone formation is among the most recent fly models of human nephrolithiasis. Conclusions The ability to readily manipulate and quantify stone formation in D. melanogaster models of human nephrolithiasis presents the urologic community with a unique opportunity to increase our understanding of this enigmatic disease. PMID:23500641

  20. γ-glutamyl transpeptidase 1 specifically suppresses green-light avoidance via GABAA receptors in Drosophila.

    Science.gov (United States)

    Liu, Jiangqu; Gong, Zhefeng; Liu, Li

    2014-08-01

    Drosophila larvae innately show light avoidance behavior. Compared with robust blue-light avoidance, larvae exhibit relatively weaker green-light responses. In our previous screening for genes involved in larval light avoidance, compared with control w(1118) larvae, larvae with γ-glutamyl transpeptidase 1 (Ggt-1) knockdown or Ggt-1 mutation were found to exhibit higher percentage of green-light avoidance which was mediated by Rhodopsin6 (Rh6) photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt-1 in different tissues, we found that Ggt-1 in malpighian tubules was both necessary and sufficient for green-light avoidance. Our results showed that glutamate levels were lower in Ggt-1 null mutants compared with controls. Feeding Ggt-1 null mutants glutamate can normalize green-light avoidance, indicating that high glutamate concentrations suppressed larval green-light avoidance. However, rather than directly, glutamate affected green-light avoidance indirectly through GABA, the level of which was also lower in Ggt-1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABAA receptor, both exhibit elevated levels of green-light avoidance. Thus, our results elucidate the neurobiological mechanisms mediating green-light avoidance, which was inhibited in wild-type larvae. © 2014 International Society for Neurochemistry.

  1. Real-time monitoring of melittin-induced pore and tubule formation from supported lipid bilayers and its physiological relevance

    Czech Academy of Sciences Publication Activity Database

    Macháň, Radek; Miszta, Adam; Hermens, W.; Hof, Martin

    2010-01-01

    Roč. 163, č. 2 (2010), s. 200-206 ISSN 0009-3084 R&D Projects: GA MŠk(CZ) LC06063; GA AV ČR GEMEM/09/E006 Institutional research plan: CEZ:AV0Z40400503 Keywords : antimicrobiological peptides * support phospholipid builayers * ellipsometry Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.861, year: 2010

  2. The relation of germ cells per tubule in testes, serum inhibin B and FSH in cryptorchid boys

    DEFF Research Database (Denmark)

    Cortes, D; Thorup, Jørgen Mogens; Hogdall, E

    2007-01-01

    At bilateral orchiopexy bilateral biopsies may be indicated to determine fertility potential. It is currently unknown if the serum inhibin B levels at time of orchiopexy reflect the testicular status of the bilaterally cryptorchid child. The aim of this study was to relate the results of inhibin ...

  3. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi-Fen; Shyu, Huey-Wen [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chang, Yi-Chuang [Department of Nursing, Fooyin University, Kaohsiung, Taiwan (China); Tseng, Wei-Chang [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chen, Chang-Yu, E-mail: mt037@mail.fy.edu.tw [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China)

    2012-03-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  4. Development of a living membrane comprising of a functional human renal proximal tubule cell monolayer on polyethersulfone polymeric membrane

    NARCIS (Netherlands)

    Schophuizen, C.M.S.; De Napoli, Ilaria; Jansen, J.; Da Silva Teixeira, Sandra; Wilmer, M.; Hoenderop, J.G.; van den Heuvel, L.P.W.; Masereeuw, R.; Stamatialis, Dimitrios

    2015-01-01

    The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a “living membrane”, consisting of a tight kidney cell monolayer with preserved functional organic ion

  5. Grid-Free LES 3D Vortex Method for the Simulation of Tubulent Flows Over Advanced Lifting Surfaces, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Turbulent flows associated with advanced aerodynamic designs represent a considerable challenge for accurate prediction. For example, the flow past low-speed wings...

  6. Tacrolimus Modulates TGF-β Signaling to Induce Epithelial-Mesenchymal Transition in Human Renal Proximal Tubule Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jason Bennett

    2016-04-01

    Full Text Available Epithelial-mesenchymal transition (EMT, a process which describes the trans-differentiation of epithelial cells into motile mesenchymal cells, is pivotal in stem cell behavior, development and wound healing, as well as contributing to disease processes including fibrosis and cancer progression. Maintenance immunosuppression with calcineurin inhibitors (CNIs has become routine management for renal transplant patient, but unfortunately the nephrotoxicity of these drugs has been well documented. HK-2 cells were exposed to Tacrolimus (FK506 and EMT markers were assessed by RT PCR and western blot. FK506 effects on TGF-β mRNA were assessed by RT PCR and TGF-β secretion was measured by ELISA. The impact of increased TGF-β secretion on Smad signaling pathways was investigated. The impact of inhibition of TGF-β signaling on EMT processes was assessed by scratch-wound assay. The results presented in this study suggest that FK506 initiates EMT processes in the HK-2 cell line, with altered expression of epithelial and myofibroblast markers evident. Additionally, the study demonstrates that FK506 activation of the TGF-β/ SMAD pathways is an essential step in the EMT process. Overall the results demonstrate that EMT is heavily involved in renal fibrosis associated with CNI nephrotoxicity.

  7. Cell-specific expression of the glucocorticoid receptor within granular convoluted tubules of the rat submaxillary gland

    International Nuclear Information System (INIS)

    Antakly, T.; Zhang, C.X.; Sarrieau, A.; Raquidan, D.

    1991-01-01

    The submaxillary gland, a heterogeneous tissue composed essentially of two functionally distinct cell types (tubular epithelial and acinar), offers an interesting system in which to study the mechanisms of steroid-dependent growth and differentiation. One cell type, the granular convoluted tubular (GCT) cell, secretes a large number of physiologically important polypeptides, including epidermal and nerve growth factors. Two steroids, androgens and glucocorticoids, greatly influence the growth, differentiation, and secretory activity of GCT cells. Because glucocorticoids can partially mimic or potentiate androgen effects, it has been thought that glucocorticoids act via androgen receptors. Since the presence of glucocorticoid receptors is a prerequisite for glucocorticoid action, we have investigated the presence and cellular distribution of glucocorticoid receptors within the rat submaxillary gland. Binding experiments using [3H]dexamethasone revealed the presence of high affinity binding sites in rat submaxillary tissue homogenates. Most of these sites were specifically competed by dexamethasone, corticosterone, and a pure glucocorticoid agonist RU 28362. Neither testosterone nor dihydrotestosterone competed for glucocorticoid binding. The cellular distribution of glucocorticoid receptors within the submaxillary gland was investigated by immunocytochemistry, using two highly specific glucocorticoid receptor antibodies. The receptor was localized in the GCT cells, but not in the acinar cells of rat and mouse submaxillary tissue sections. In GCT cells, the glucocorticoid receptor colocalized with several secretory polypeptides, including epidermal growth factor, nerve growth factor, alpha 2u-globulin, and atrial natriuretic factor

  8. Nigella sativa EXTRACT IMPROVES SEMINIFEROUS TUBULE EPITHELIAL THICKNESS IN LEAD ACETATE-EXPOSED BALB/C MICE

    OpenAIRE

    Diana, Alis Nur; I’tishom, Reny; Sudjarwo, Sri Agus

    2017-01-01

    Lead that enters the body may lead to increased production of ROS (Reactive Oxygen Species) that may affect reproductive system. Black cumin (Nigella sativa) extract contains high antioxidant, tymoquinone, that may be used to suppress oxidative stress induced by lead in animal experiments. This study aimed to prove that black cumin (Nigella sativa) extract improves the thickness of seminiferous tubular epithelium in Balb/c mice exposed to lead (Pb) acetate. This study used post-test only cont...

  9. IGF-II receptors in luminal and basolateral membranes isolated from pars convoluta and pars recta of rabbit proximal tubule

    DEFF Research Database (Denmark)

    Jacobsen, Christian; Jessen, H; Flyvbjerg, A

    1995-01-01

    the inhibitory effect of beta-galactosidase. Analyses of 125I-IGF-II binding curves in the presence of beta-galactosidase or D-mannose 6-phosphate demonstrated that none of these compounds changed the binding affinity of 125I-IGF-II for the membrane vesicles. The IGF-II/M6P receptor content in the luminal...... membranes was in the range 0.21-0.34 pmol IGF-II/M6P receptor per mg protein and very low compared to 2.27-2.86 pmol IGF-II/M6P receptor per mg protein in basolateral membranes. Udgivelsesdato: 1995-Apr-12...

  10. Pattern of trypan blue storage in the chick embryonic kidney reflects development of absorptive capacity of the proximal tubules

    Czech Academy of Sciences Publication Activity Database

    Zemanová, Zdeňka

    2004-01-01

    Roč. 15, č. 3-4 (2004), s. 160-165 ISSN 1470-2061 R&D Projects: GA ČR GA304/04/0972 Institutional research plan: CEZ:AV0Z5011922 Keywords : chick embryo * mesonephric nephrons * trypan blue Subject RIV: ED - Physiology Impact factor: 0.724, year: 2004

  11. Characterization of the Interaction of Staphylococcal Entertoxin B with CD1d Expressed in Human Renal Proximal Tubule Epithelial Cells

    Science.gov (United States)

    2015-02-04

    underscored by its influences on the performances of natural killer T- cells and thereby mediates the innate and adaptive immune systems. Results... cells to the non- lymphoid cell types derived from kidney [13], spleen [14], lungs [15], and gut [16]. Recent findings demonstrating the translocation...pathogenesis has been in- vestigated in the past, primarily focused on the lymphoid cells [37-41], while the knowledge gap exists in compre- hending

  12. SGLT2 inhibitor lowers serum uric acid through alteration of uric acid transport activity in renal tubule by increased glycosuria

    Science.gov (United States)

    Chino, Yukihiro; Samukawa, Yoshishige; Sakai, Soichi; Nakai, Yasuhiro; Yamaguchi, Jun-ichi; Nakanishi, Takeo; Tamai, Ikumi

    2014-01-01

    Sodium glucose cotransporter 2 (SGLT2) inhibitors have been reported to lower the serum uric acid (SUA) level. To elucidate the mechanism responsible for this reduction, SUA and the urinary excretion rate of uric acid (UEUA) were analysed after the oral administration of luseogliflozin, a SGLT2 inhibitor, to healthy subjects. After dosing, SUA decreased, and a negative correlation was observed between the SUA level and the UEUA, suggesting that SUA decreased as a result of the increase in the UEUA. The increase in UEUA was correlated with an increase in urinary d-glucose excretion, but not with the plasma luseogliflozin concentration. Additionally, in vitro transport experiments showed that luseogliflozin had no direct effect on the transporters involved in renal UA reabsorption. To explain that the increase in UEUA is likely due to glycosuria, the study focused on the facilitative glucose transporter 9 isoform 2 (GLUT9ΔN, SLC2A9b), which is expressed at the apical membrane of the kidney tubular cells and transports both UA and d-glucose. It was observed that the efflux of [14C]UA in Xenopus oocytes expressing the GLUT9 isoform 2 was trans-stimulated by 10 mm d-glucose, a high concentration of glucose that existed under SGLT2 inhibition. On the other hand, the uptake of [14C]UA by oocytes was cis-inhibited by 100 mm d-glucose, a concentration assumed to exist in collecting ducts. In conclusion, it was demonstrated that the UEUA could potentially be increased by luseogliflozin-induced glycosuria, with alterations of UA transport activity because of urinary glucose. PMID:25044127

  13. The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane.

    NARCIS (Netherlands)

    Huls, M.; Brown, C.D.; Windass, A.S.; Sayer, R.; Heuvel, J.J.M.W. van den; Heemskerk, S.; Russel, F.G.M.; Masereeuw, R.

    2008-01-01

    The Breast Cancer Resistance Protein (BCRP/ABCG2) is a transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. This transporter is highly expressed in many tissues; however, in human kidney, only the mRNA was found in contrast to the mouse kidney,

  14. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    International Nuclear Information System (INIS)

    Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang; Tseng, Wei-Chang; Huang, Yeou-Lih; Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling; Chen, Chang-Yu

    2012-01-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  15. Mosquitos vetores potenciais de dirofilariose canina na Região Nordeste do Brasil Mosquitoes potential vectors of canine heartworm in the Northeast Region from Brazil

    Directory of Open Access Journals (Sweden)

    Silvia MM Ahid

    1999-12-01

    season. It accounted for 54.5% of the total, followed by Aedes albopictus (20.3%, Ae. scapularis (11% and Ae. taeniorhynchus (11%. D. immitis larvae were detected in 0.1% of the Cx. quinquefasciatus dissected (L3 in the Malpighian tubules and 0.5% of the Ae. taeniorhynchus (L2 in the Malpighian tubules. CONCLUSION: Ae. taeniorhynchus and Cx. quinquefasciatus are considered natural potential vectors of the canine heartworm in São Luís. The role of Cx. quinquefasciatus as primary vector of D. immitis, however, needs further evaluation.

  16. The Aquaporin gene family of the yellow fever mosquito, Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Lisa L Drake

    2010-12-01

    Full Text Available The mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT. Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti.Using a bioinformatics approach we screened genome databases and identified six putative AQPs in the genome of Ae. aegypti. Phylogenetic analysis showed that five of the six Ae. aegypti AQPs have high similarity to classical water-transporting AQPs of vertebrates. Using microarray, reverse transcription and real time PCR analysis we found that all six AQPs are expressed in distinct patterns in mosquito tissues/body parts. AaAQP1, 4, and 5 are strongly expressed in the adult female MT. RNAi-mediated knockdown of the MT-expressed mosquito AQPs resulted in significantly reduced diuresis.Our results support the notion that AQP1, 4, and 5 function as water transporters in the MTs of adult female Ae. aegypti mosquitoes. Our results demonstrate the importance of these AQPs for mosquito diuresis after blood ingestion and highlight their potential as targets for the development of novel vector control strategies.

  17. The Aquaporin gene family of the yellow fever mosquito, Aedes aegypti.

    Science.gov (United States)

    Drake, Lisa L; Boudko, Dmitri Y; Marinotti, Osvaldo; Carpenter, Victoria K; Dawe, Angus L; Hansen, Immo A

    2010-12-29

    The mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti. Using a bioinformatics approach we screened genome databases and identified six putative AQPs in the genome of Ae. aegypti. Phylogenetic analysis showed that five of the six Ae. aegypti AQPs have high similarity to classical water-transporting AQPs of vertebrates. Using microarray, reverse transcription and real time PCR analysis we found that all six AQPs are expressed in distinct patterns in mosquito tissues/body parts. AaAQP1, 4, and 5 are strongly expressed in the adult female MT. RNAi-mediated knockdown of the MT-expressed mosquito AQPs resulted in significantly reduced diuresis. Our results support the notion that AQP1, 4, and 5 function as water transporters in the MTs of adult female Ae. aegypti mosquitoes. Our results demonstrate the importance of these AQPs for mosquito diuresis after blood ingestion and highlight their potential as targets for the development of novel vector control strategies.

  18. Nuclear receptors HR96 and ultraspiracle from the fall armyworm (Spodoptera frugiperda), developmental expression and induction by xenobiotics.

    Science.gov (United States)

    Giraudo, Maeva; Audant, Pascaline; Feyereisen, René; Le Goff, Gaëlle

    2013-05-01

    The fall armyworm Spodoptera frugiperda is a major polyphagous pest in agriculture and little is known on how this insect can adapt to the diverse and potentially toxic plant allelochemicals that they ingest or to insecticides. To investigate the involvement of nuclear receptors in the response of S. frugiperda to its chemical environment, we cloned SfHR96, a nuclear receptor orthologous to the mammalian xenobiotic receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR). We also cloned ultraspiracle (USP), the ortholog of retinoid X receptor (RXR) that serves as partner of dimerization of PXR and CAR. Cloning of SfUSP revealed the presence of two isoforms, SfUSP-1 and SfUSP-2 in this species, that differ in their N-terminal region. The expression of these receptors as well as the ecdysone receptor was studied during specific steps of development in different tissues. SfHR96 was constitutively expressed in larval midgut, fat body and Malpighian tubules throughout the last two instars and pupal stage, as well as in Sf9 cells. EcR and SfUSP-2 showed peaks of expression before larval moults and during metamorphosis, whereas SfUSP-1 was mainly expressed in the pre-pupal stage. Receptor induction was followed after exposure of larvae or cells to 11 chemical compounds. SfHR96 was not inducible by the tested compounds. EcR was significantly induced by the 20-hydroxyecdysone agonist, methoxyfenozide, and SfUSP showed an increase expression when exposed to the juvenile hormone analog, methoprene. The cloning of these nuclear receptors is a first step in understanding the important capacities of adaptation of this insect pest. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Molecular cloning, characterization and expression analysis of ATG1 in the silkworm, Bombyx mori.

    Science.gov (United States)

    Casati, Barbara; Terova, Genciana; Cattaneo, Anna Giulia; Rimoldi, Simona; Franzetti, Eleonora; de Eguileor, Magda; Tettamanti, Gianluca

    2012-12-15

    Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process. In the present study we isolated two full-length cDNAs of 2175 (transcript variant A) and 2271 (transcript variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely related to orthologs of other insects. The encoded BmAtg1 proteins shared extensive homology with orthologs from yeast to mammals, showing high conservation at the N-terminal region where the catalytic domain and ATP- and Mg-binding sites are located. A de novo prediction of the three-dimensional structure for each protein is presented. We used real-time RT-PCR to quantify dynamic changes in mRNA copy number of BmATG1 in the midgut and fat body of fifth instar larvae undergoing starvation, as well as in other tissues of silkworm at the end of last larval instar. Our qPCR results revealed that BmATG1 expression levels at the end of larval life were comparable in the midgut, fat body and Malpighian tubules, while these were higher in the gonads; moreover, the mRNA copy number of ATG1 was very different among the anterior, middle and posterior silk glands. Real-time PCR analysis also showed that starvation significantly influenced BmATG1 mRNA copy number in the fat body of silkworm, inducing an upregulation 24h after food withdrawal, with only a slight effect in the midgut. Low expression levels of BmATG1 were observed in both tissues of control animals up to the second day of spinning phase. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.).

    Science.gov (United States)

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-04-01

    Biopesticides or transgenic crops based on Cry toxins from the soil bacterium Bacillus thuringiensis (Bt) effectively control agricultural insect pests. The sustainable use of Bt biopesticides and Bt crops is threatened, however, by the development of Cry resistance in the target pests. The diamondback moth, Plutella xylostella (L.), is the first pest that developed resistance to a Bt biopesticide in the field, and a recent study has shown that the resistance of P. xylostella to Cry1Ac is caused by a mutation in an ATP-binding cassette (ABC) transporter gene (ABCC2). In this study, we report that down-regulation of a novel ABC transporter gene from ABCG subfamily (Pxwhite) is associated with Cry1Ac resistance in P. xylostella. The full-length cDNA sequence of Pxwhite was cloned and analyzed. Spatial-temporal expression detection revealed that Pxwhite was expressed in all tissues and developmental stages, and highest expressed in Malpighian tubule tissue and in egg stage. Sequence variation analysis of Pxwhite indicated the absence of constant non-synonymous mutations between susceptible and resistant strains, whereas midgut transcript analysis showed that Pxwhite was remarkably reduced in all resistant strains and further reduced when larvae of the moderately resistant SZ-R strain were subjected to selection with Cry1Ac toxin. Furthermore, RNA interference (RNAi)-mediated suppression of Pxwhite gene expression significantly reduced larval susceptibility to Cry1Ac toxin, and genetic linkage analysis confirmed that down-regulation of Pxwhite gene is tightly linked to Cry1Ac resistance in P. xylostella. To our knowledge, this is the first report indicating that Pxwhite gene is involved in Cry1Ac resistance in P. xylostella. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Characterization and expression profiling of glutathione S-transferases in the diamondback moth, Plutella xylostella (L.).

    Science.gov (United States)

    You, Yanchun; Xie, Miao; Ren, Nana; Cheng, Xuemin; Li, Jianyu; Ma, Xiaoli; Zou, Minming; Vasseur, Liette; Gurr, Geoff M; You, Minsheng

    2015-03-05

    Glutathione S-transferases (GSTs) are multifunctional detoxification enzymes that play important roles in insects. The completion of several insect genome projects has enabled the identification and characterization of GST genes over recent years. This study presents a genome-wide investigation of the diamondback moth (DBM), Plutella xylostella, a species in which the GSTs are of special importance because this pest is highly resistant to many insecticides. A total of 22 putative cytosolic GSTs were identified from a published P. xylostella genome and grouped into 6 subclasses (with two unclassified). Delta, Epsilon and Omega GSTs were numerically superior with 5 genes for each of the subclasses. The resulting phylogenetic tree showed that the P. xylostella GSTs were all clustered into Lepidoptera-specific branches. Intron sites and phases as well as GSH binding sites were strongly conserved within each of the subclasses in the GSTs of P. xylostella. Transcriptome-, RNA-seq- and qRT-PCR-based analyses showed that the GST genes were developmental stage- and strain-specifically expressed. Most of the highly expressed genes in insecticide resistant strains were also predominantly expressed in the Malpighian tubules, midgut or epidermis. To date, this is the most comprehensive study on genome-wide identification, characterization and expression profiling of the GST family in P. xylostella. The diversified features and expression patterns of the GSTs are inferred to be associated with the capacity of this species to develop resistance to a wide range of pesticides and biological toxins. Our findings provide a base for functional research on specific GST genes, a better understanding of the evolution of insecticide resistance, and strategies for more sustainable management of the pest.

  2. Pharmacological characterization of a β-adrenergic-like octopamine receptor in Plutella xylostella.

    Science.gov (United States)

    Huang, Qing-Ting; Ma, Hai-Hao; Deng, Xi-Le; Zhu, Hang; Liu, Jia; Zhou, Yong; Zhou, Xiao-Mao

    2018-04-25

    The β-adrenergic-like octopamine receptor (OA2B2) belongs to the class of G-protein coupled receptors. It regulates important physiological functions in insects, thus is potentially a good target for insecticides. In this study, the putative open reading frame sequence of the Pxoa2b2 gene in Plutella xylostella was cloned. Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. PxOA2B2 was transiently expressed in HEK-293 cells. It was found that PxOA2B2 could be activated by both octopamine and tyramine, resulting in increased intracellular cyclic AMP (cAMP) levels, whereas dopamine and serotonin were not effective in eliciting cAMP production. Further studies with series of PxOA2B2 agonists and antagonists showed that all four tested agonists (e.g., naphazoline, clonidine, 2-phenylethylamine, and amitraz) could activate the PxOA2B2 receptor, and two of tested antagonists (e.g., phentolamine and mianserin) had significant antagonistic effects. However, antagonist of yohimbine had no effects. Quantitative real-time polymerase chain reaction analysis showed that Pxoa2b2 gene was expressed in all developmental stages of P. xylostella and that the highest expression occurred in male adults. Further analysis with fourth-instar P. xylostella larvae showed that the Pxoa2b2 gene was mainly expressed in Malpighian tubule, epidermal, and head tissues. This study provides both a pharmacological characterization and the gene expression patterns of the OA2B2 in P. xylostella, facilitating further research for insecticides using PxOA2B2 as a target. © 2018 Wiley Periodicals, Inc.

  3. Early-life inflammation, immune response and ageing.

    Science.gov (United States)

    Khan, Imroze; Agashe, Deepa; Rolff, Jens

    2017-03-15

    Age-related diseases are often attributed to immunopathology, which results in self-damage caused by an inappropriate inflammatory response. Immunopathology associated with early-life inflammation also appears to cause faster ageing, although we lack direct experimental evidence for this association. To understand the interactions between ageing, inflammation and immunopathology, we used the mealworm beetle Tenebrio molitor as a study organism. We hypothesized that phenoloxidase, an important immune effector in insect defence, may impose substantial immunopathological costs by causing tissue damage to Malpighian tubules (MTs; functionally equivalent to the human kidney), in turn accelerating ageing. In support of this hypothesis, we found that RNAi knockdown of phenoloxidase (PO) transcripts in young adults possibly reduced inflammation-induced autoreactive tissue damage to MTs, and increased adult lifespan. Our work thus suggests a causative link between immunopathological costs of early-life inflammation and faster ageing. We also reasoned that if natural selection weakens with age, older individuals should display increased immunopathological costs associated with an immune response. Indeed, we found that while old infected individuals cleared infection faster than young individuals, possibly they also displayed exacerbated immunopathological costs (larger decline in MT function) and higher post-infection mortality. RNAi-mediated knockdown of PO response partially rescued MTs function in older beetles and resulted in increased lifespan after infection. Taken together, our data are consistent with a direct role of immunopathological consequences of immune response during ageing in insects. Our work is also the first report that highlights the pervasive role of tissue damage under diverse contexts of ageing and immune response. © 2017 The Author(s).

  4. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae.

    Directory of Open Access Journals (Sweden)

    Meng Sun

    Full Text Available The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA, elongation factor 1 (EF1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ribosomal protein S13 (RPS13, ribosomal protein S20 (RPS20, tubulin (TUB, and β-actin (ACTB were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1 were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands. 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults. 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C. To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83 was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  5. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2015-01-01

    The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  6. Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae).

    Science.gov (United States)

    Xu, Gang; Wu, Shun-Fan; Teng, Zi-Wen; Yao, Hong-Wei; Fang, Qi; Huang, Jia; Ye, Gong-Yin

    2017-06-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop ligand-gated ion channel (cysLGIC) superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressalis, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs (cDNAs) via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine α-type and three β-type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species. ©2016 The Authors Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.

  7. Characterization and expression patterns of a membrane-bound trehalase from Spodoptera exigua

    Directory of Open Access Journals (Sweden)

    Xu Weihua

    2008-05-01

    Full Text Available Abstract Background The chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. Although insects possess two types, soluble trehalase (Tre-1 and membrane-bound trehalase (Tre-2, very little is known about Tre-2 and the difference in function between Tre-1 and Tre-2. Results To gain an insight into trehalase functions in insects, we investigated a putative membrane-bound trehalase from Spodoptera exigua (SeTre-2 cloned from the fat body. The deduced amino acid sequence of SeTre-2 contains 645 residues and has a predicted molecular weight of ~74 kDa and pI of 6.01. Alignment of SeTre-2 with other insect trehalases showed that it contains two trehalase signature motifs and a putative transmembrane domain, which is an important characteristic of Tre-2. Comparison of the genomic DNA and cDNA sequences demonstrated that SeTre-2 comprises 13 exons and 12 introns. Southern blot analysis revealed that S. exigua has two trehalase genes and that SeTre-2 is a single-copy gene. Northern blot analyses showed that the SeTre-2 transcript is expressed not only in the midgut, as previously reported for Bombyx mori, but also in the fat body and Malpighian tubules, although expression patterns differed between the midgut and fat body. SeTre-2 transcripts were detected in the midgut of feeding stage larvae, but not in pupae, whereas SeTre-2 mRNA was detected in the fat body of fifth instar larvae and pupae. Conclusion These findings provide new data on the tissue distribution, expression patterns and potential function of membrane-bound trehalase. The results suggest that the SeTre-2 gene may have different functions in the midgut and fat body.

  8. Immune function of a Rab-related protein by modulating the JAK-STAT signaling pathway in the silkworm, Bombyx mori.

    Science.gov (United States)

    Chen, Chen; Eldein, Salah; Zhou, Xiaosan; Sun, Yu; Gao, Jin; Sun, Yuxuan; Liu, Chaoliang; Wang, Lei

    2018-01-01

    The Rab-family GTPases mainly regulate intracellular vesicle transport, and play important roles in the innate immune response in invertebrates. However, the function and signal transduction of Rab proteins in immune reactions remain unclear in silkworms. In this study, we analyzed a Rab-related protein of silkworm Bombyx mori (BmRABRP) by raising antibodies against its bacterially expressed recombinant form. Tissue distribution analysis showed that BmRABRP mRNA and protein were high expressed in the Malpighian tubule and fat body, respectively. However, among the different stages, only the fourth instar larvae and pupae showed significant BmRABRP levels. After challenge with four pathogenic microorganisms (Escherichia coli, BmNPV, Beauveria bassiana, Micrococcus luteus), the expression of BmRABRP mRNA in the fat body was significantly upregulated. In contrast, the BmRABRP protein was significantly upregulated after infection with BmNPV, while it was downregulated by E. coli, B. bassiana, and M. luteus. A specific dsRNA was used to explore the immune function and relationship between BmRABRP and the JAK-STAT signaling pathway. After BmRABRP gene interference, significant reduction in the number of nodules and increased mortality suggested that BmRABRP plays an important role in silkworm's response to bacterial challenge. In addition, four key genes (BmHOP, BmSTAT, BmSOCS2, and BmSOCS6) of the JAK-STAT signaling pathway showed significantly altered expressions after BmRABRP silencing. BmHOP and BmSOCS6 expressions were significantly decreased, while BmSTAT and BmSOCS2 were significantly upregulated. Our results suggested that BmRABRP is involved in the innate immune response against pathogenic microorganisms through the JAK-STAT signaling pathway in silkworm. © 2017 Wiley Periodicals, Inc.

  9. Glutathione-S-transferase profiles in the emerald ash borer, Agrilus planipennis.

    Science.gov (United States)

    Rajarapu, Swapna Priya; Mittapalli, Omprakash

    2013-05-01

    The emerald ash borer, Agrilus planipennis Fairmaire is a recently discovered invasive insect pest of ash, Fraxinus spp. in North America. Glutathione-S-transferases (GST) are a multifunctional superfamily of enzymes which function in conjugating toxic compounds to less toxic and excretable forms. In this study, we report the molecular characterization and expression patterns of different classes of GST genes in different tissues and developmental stages plus their specific activity. Multiple sequence alignment of all six A. planipennis GSTs (ApGST-E1, ApGST-E2, ApGST-E3, ApGST-O1, ApGST-S1 and ApGST-μ1) revealed conserved features of insect GSTs and a phylogenetic analysis grouped the GSTs within the epsilon, sigma, omega and microsomal classes of GSTs. Real time quantitative PCR was used to study field collected samples. In larval tissues high mRNA levels for ApGST-E1, ApGST-E3 and ApGST-O1 were obtained in the midgut and Malpighian tubules. On the other hand, ApGST-E2 and ApGST-S1 showed high mRNA levels in fat body and ApGST-μ1 showed constitutive levels in all the tissues assayed. During development, mRNA levels for ApGST-E2 were observed to be the highest in feeding instars, ApGST-S1 in prepupal instars; while the others showed constitutive patterns in all the developmental stages examined. At the enzyme level, total GST activity was similar in all the tissues and developmental stages assayed. Results obtained suggest that A. planipennis is potentially primed with GST-driven detoxification to metabolize ash allelochemicals. To our knowledge this study represents the first report of GSTs in A. planipennis and also in the family of wood boring beetles. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

    Science.gov (United States)

    Li, Ting; Liu, Nannan

    2017-12-01

    This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

  11. Tissue-specific transcript profiling for ABC transporters in the sequestering larvae of the phytophagous leaf beetle Chrysomela populi.

    Directory of Open Access Journals (Sweden)

    Anja S Strauss

    Full Text Available Insects evolved ingenious adaptations to use extraordinary food sources. Particularly, the diet of herbivores enriched with noxious plant secondary metabolites requires detoxification mechanisms. Sequestration, which involves the uptake, transfer, and concentration of occasionally modified phytochemicals into specialized tissues or hemolymph, is one of the most successful detoxification strategies found in most insect orders. Due to the ability of ATP-binding cassette (ABC carriers to transport a wide range of molecules including phytochemicals and xenobiotics, it is highly likely that they play a role in this sequestration process. To shed light on the role of ABC proteins in sequestration, we describe an inventory of putative ABC transporters in various tissues in the sequestering juvenile poplar leaf beetle, Chrysomela populi.In the transcriptome of C. populi, we predicted 65 ABC transporters. To link the proteins with a possible function, we performed comparative phylogenetic analyses with ABC transporters of other insects and of humans. While tissue-specific profiling of each ABC transporter subfamily suggests that ABCB, C and G influence the plant metabolite absorption in the gut, ABCC with 14 members is the preferred subfamily responsible for the excretion of these metabolites via Malpighian tubules. Moreover, salicin, which is sequestered from poplar plants, is translocated into the defensive glands for further deterrent production. In these glands and among all identified ABC transporters, an exceptionally high transcript level was observed only for Cpabc35 (Cpmrp. RNAi revealed the deficiency of other ABC pumps to compensate the function of CpABC35, demonstrating its key role during sequestration.We provide the first comprehensive phylogenetic study of the ABC family in a phytophagous beetle species. RNA-seq data from different larval tissues propose the importance of ABC pumps to achieve a homeostasis of plant-derived compounds and

  12. Prostaglandins and their receptors in insect biology

    Directory of Open Access Journals (Sweden)

    David eStanley

    2011-12-01

    Full Text Available We treat the biological significance of prostaglandins (PGs and their known receptors in insect biology. PGs and related eicosanoids are oxygenated derivatives of arachidonic acid (AA and two other C20 polyunsaturated fatty acids. PGs are mostly appreciated in the context of biomedicine, but a growing body of literature indicates the biological significance of these compounds extends throughout the animal kingdom, and possibly beyond. PGs act in several crucial areas of insect biology. In reproduction, a specific PG, PGE2, releases oviposition behavior in most crickets and a few other insect species; PGs also mediate events in egg development in some species, which may represent all insects. PGs play major roles in modulating fluid secretion in Malpighian tubules, rectum and salivary glands, although, again, this has been studied in only a few insect species that may represent the Class. Insect immunity is a very complex defense system. PGs and other eicosanoids mediate a large number of immune reactions to infection and invasion. The actions of most PGs are mediated by specific receptors. Biomedical research has discovered a great deal of knowledge about PG receptors in mammals, including their structures, pharmacology, molecular biology and cellular locations. Studies of PG receptors in insects lag behind the biomedical background, however, recent results hold the promise of accelerated research in this area. A PG receptor has been identified in a class of lepidopteran hemocytes and experimentally linked to the release of prophenoloxidase. We conclude that research into PGs and their receptors in insects will lead to important advances in our understanding of insect biology.

  13. Purification, cDNA cloning and modification of a defensin from the coconut rhinoceros beetle, Oryctes rhinoceros.

    Science.gov (United States)

    Ishibashi, J; Saido-Sakanaka, H; Yang, J; Sagisaka, A; Yamakawa, M

    1999-12-01

    A novel member of the insect defensins, a family of antibacterial peptides, was purified from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros, immunized with Escherichia coli. A full-size cDNA was cloned by combining reverse-transcription PCR (RT-PCR), and 5'- and 3'-rapid amplification of cDNA ends (RACE). Analysis of the O. rhinoceros defensin gene expression showed it to be expressed in the fat body and hemocyte, midgut and Malpighian tubules. O. rhinoceros defensin showed strong antibacterial activity against Staphylococcus aureus. A 9-mer peptide amidated at its C-terminus, AHCLAICRK-NH2 (Ala22-Lys30-NH2), was synthesized based on the deduced amino-acid sequence, assumed to be an active site sequence by analogy with the sequence of a defensin isolated from larvae of the beetle Allomyrina dichotoma. This peptide showed antibacterial activity against S. aureus, methicillin-resistant S. aureus, E. coli and Pseudomonas aeruginosa. We further modified this oligopeptide and synthesized five 9-mer peptides, ALRLAIRKR-NH2, ALLLAIRKR-NH2, AWLLAIRKR-NH2, ALYLAIRKR-NH2 and ALWLAIRKR-NH2. These oligopeptides showed strong antibacterial activity against Gram-negative and Gram-positive bacteria. The antibacterial effect of Ala22-Lys30-NH2 analogues was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. These Ala22-Lys30-NH2 analogues did not show haemolytic activity and did not inhibit the growth of murine fibroblast cells or macrophages, except for AWLLAIRKR-NH2.

  14. Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila.

    Directory of Open Access Journals (Sweden)

    Thomas W R Harrop

    Full Text Available Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster-D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.

  15. Characterization of a β-Adrenergic-Like Octopamine Receptor in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel

    Directory of Open Access Journals (Sweden)

    Hui-Min Li

    2016-09-01

    Full Text Available The biogenic amine octopamine plays a critical role in the regulation of many physiological processes in insects. Octopamine transmits its action through a set of specific G-protein coupled receptors (GPCRs, namely octopamine receptors. Here, we report on a β-adrenergic-like octopamine receptor gene (BdOctβR1 from the oriental fruit fly, Bactrocera dorsalis (Hendel, a destructive agricultural pest that occurs in North America and the Asia-Pacific region. As indicated by RT-qPCR, BdOctβR1 was highly expressed in the central nervous system (CNS and Malpighian tubules (MT in the adult flies, suggesting it may undertake important roles in neural signaling in the CNS as well as physiological functions in the MT of this fly. Furthermore, its ligand specificities were tested in a heterologous expression system where BdOctβR1 was expressed in HEK-293 cells. Based on cyclic AMP response assays, we found that BdOctβR1 could be activated by octopamine in a concentration-dependent manner, confirming that this receptor was functional, while tyramine and dopamine had much less potency than octopamine. Naphazoline possessed the highest agonistic activity among the tested agonists. In antagonistic assays, mianserin had the strongest activity and was followed by phentolamine and chlorpromazine. Furthermore, when the flies were kept under starvation, there was a corresponding increase in the transcript level of BdOctβR1, while high or low temperature stress could not induce significant expression changes. The above results suggest that BdOctβR1 may be involved in the regulation of feeding processes in Bactrocera dorsalis and may provide new potential insecticide leads targeting octopamine receptors.

  16. Commentary

    Indian Academy of Sciences (India)

    Unknown

    genetic maps with specific segments of polytene chromosomes provided the first evidence of ... tion in Drosophila and other organisms identified the heterochromatin ... such modifications seem to provide a “histone code” in the context of gene ...

  17. Fulltext PDF

    Indian Academy of Sciences (India)

    Polytene chromosomes were first viewed under microscope in cells of ..... Confocal. Microscopy in conjunction with in situ hybridization techniques allows viewing of ... fluorochromes) simultaneously: each homolog shows paired fluorescence.

  18. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-{kappa}B p65 subunit and cytotoxicity in renal proximal tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Lee, Sang Yong [Department of Diagnostic Radiology, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Han, Myung Kwan [Department of Microbiology, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kim, Duk Hoon [Division of Forensic Medicine, National Forensic Service, Seoul (Korea, Republic of); Kim, Won, E-mail: kwon@jbnu.ac.kr [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Cisplatin increases acetylation of NF-{kappa}B p65 subunit in HK2 cells. Black-Right-Pointing-Pointer SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. Black-Right-Pointing-Pointer Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-{kappa}B (NF-{kappa}B) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD{sup +})-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-{kappa}B p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-{kappa}B during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-{kappa}B p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-{kappa}B through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  19. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-κB p65 subunit and cytotoxicity in renal proximal tubule cells

    International Nuclear Information System (INIS)

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Lee, Sang Yong; Han, Myung Kwan; Kim, Duk Hoon; Kim, Won

    2012-01-01

    Highlights: ► Cisplatin increases acetylation of NF-κB p65 subunit in HK2 cells. ► SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. ► Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-κB (NF-κB) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD + )-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-κB p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-κB during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-κB p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-κB through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  20. K+ transport in the mesonephric collecting duct system of the toad Bufo bufo : microelectrode recordings from isolated and perfused tubules

    DEFF Research Database (Denmark)

    Møbjerg, Nadja; Larsen, Erik Hviid; Novak, Ivana

    2002-01-01

    V, respectively (N=11; 11). This conductance could also be inhibited by Ba(2+), which depolarized a V(bl) of -71+/-5 mV by 9+/-3 mV (N=5). The pump inhibitor ouabain (1 mmol l(-1)) depolarized V(bl), but addition of furosemide to bath solution did not affect V(bl). The [K(+)] in urine varied from 1.3 to 22.8 mmol...

  1. The role of Omi/HtrA2 protease in neonatal postasphyxial serum-induced apoptosis in human kidney proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Zhang Yong

    2012-01-01

    Full Text Available Omi/HtrA2, a proapoptotic mitochondrial serine protease, is involved in both caspase-dependent and caspaseindependent apoptosis. A growing body of evidence indicates that Omi/HtrA2 plays an important role in the pathogenesis of a variety of ischemia-reperfusion (I/R injuries. However, the role of Omi/HtrA2 in renal injuries that occur in neonates with asphyxia remains unknown. The present study was designed to investigate whether Omi/HtrA2 plays an important role in the types of renal injuries that are induced by neonatal postasphyxial serum. Human renal proximal tubular cell line (HK-2 cells were used as targets. A 20% serum taken from neonates one day after asphyxia was applied to target cells as an attacking factor. We initially included control and postasphyxial serum-attacked groups and later included a ucf-101 group in the study. In the postasphyxial serum-treated group, cytosolic Omi/HtrA2 and caspase-3 expression in HK-2 cells was significantly higher than in the control group. Moreover, the concentration of cytosolic caspase-3 was found to be markedly decreased in HK-2 cells in the ucf-101 group. Our results suggest both that postasphyxial serum has a potent apoptosis-inducing effect on HK-2 cells and that this effect can be partially blocked by ucf-101. Taken together, our results demonstrate for the first time that postasphyxial serum from neonates results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it promotes HK-2 cell apoptosis via a protease activity-dependent, caspase-mediated pathway.

  2. Pretreatment by low-dose fibrates protects against acute free fatty acid-induced renal tubule toxicity by counteracting PPARα deterioration

    International Nuclear Information System (INIS)

    Takahashi, Kyoko; Kamijo, Yuji; Hora, Kazuhiko; Hashimoto, Koji; Higuchi, Makoto; Nakajima, Takero; Ehara, Takashi; Shigematsu, Hidekazu; Gonzalez, Frank J.; Aoyama, Toshifumi

    2011-01-01

    Development of a preventive strategy against tubular damage associated with proteinuria is of great importance. Recently, free fatty acid (FFA) toxicities accompanying proteinuria were found to be a main cause of tubular damage, which was aggravated by insufficiency of peroxisome proliferator-activated receptor alpha (PPARα), suggesting the benefit of PPARα activation. However, an earlier study using a murine acute tubular injury model, FFA-overload nephropathy, demonstrated that high-dose treatment of PPARα agonist (0.5% clofibrate diet) aggravated the tubular damage as a consequence of excess serum accumulation of clofibrate metabolites due to decreased kidney elimination. To induce the renoprotective effects of PPARα agonists without drug accumulation, we tried a pretreatment study using low-dose clofibrate (0.1% clofibrate diet) using the same murine model. Low-dose clofibrate pretreatment prevented acute tubular injuries without accumulation of its metabolites. The tubular protective effects appeared to be associated with the counteraction of PPARα deterioration, resulting in the decrease of FFAs influx to the kidney, maintenance of fatty acid oxidation, diminution of intracellular accumulation of undigested FFAs, and attenuation of disease developmental factors including oxidative stress, apoptosis, and NFκB activation. These effects are common to other fibrates and dependent on PPARα function. Interestingly, however, clofibrate pretreatment also exerted PPARα-independent tubular toxicities in PPARα-null mice with FFA-overload nephropathy. The favorable properties of fibrates are evident when PPARα-dependent tubular protective effects outweigh their PPARα-independent tubular toxicities. This delicate balance seems to be easily affected by the drug dose. It will be important to establish the appropriate dosage of fibrates for treatment against kidney disease and to develop a novel PPARα activator that has a steady serum concentration regardless of kidney dysfunction. - Graphical Abstract: Massive proteinuria introduces free fatty acid toxicity to proximal tubular epithelial cells (PTECs). PPARα activationvia clofibrate pretreatment maintains fatty acid catabolism and attenuates oxidative stress, apoptosis, and NFκB activation, resulting in protection of PTECs. The favorable properties of fibrates are evident when PPARα-dependent tubular protective effects outweigh their PPARα-independent tubular toxicities. Display Omitted Highlights: → Clofibrate pretreatment protects against acute FFA-induced tubular toxicity. → PPARα activation decreases FFA influx and maintains fatty acid catabolism. → PPARα activation attenuates oxidative stress, apoptosis, and NFκB activation. → Protective effects must outweigh PPARα-independent tubular toxicities of fibrates.

  3. Biodegradable chitin conduit tubulation combined with bone marrow mesenchymal stem cell transplantation for treatment of spinal cord injury by reducing glial scar and cavity formation

    Directory of Open Access Journals (Sweden)

    Feng Xue

    2015-01-01

    Full Text Available We examined the restorative effect of modified biodegradable chitin conduits in combination with bone marrow mesenchymal stem cell transplantation after right spinal cord hemisection injury. Immunohistochemical staining revealed that biological conduit sleeve bridging reduced glial scar formation and spinal muscular atrophy after spinal cord hemisection. Bone marrow mesenchymal stem cells survived and proliferated after transplantation in vivo, and differentiated into cells double-positive for S100 (Schwann cell marker and glial fibrillary acidic protein (glial cell marker at 8 weeks. Retrograde tracing showed that more nerve fibers had grown through the injured spinal cord at 14 weeks after combination therapy than either treatment alone. Our findings indicate that a biological conduit combined with bone marrow mesenchymal stem cell transplantation effectively prevented scar formation and provided a favorable local microenvironment for the proliferation, migration and differentiation of bone marrow mesenchymal stem cells in the spinal cord, thus promoting restoration following spinal cord hemisection injury.

  4. Biodegradable chitin conduit tubulation combined with bone marrow mesenchymal stem cell transplantation for treatment of spinal cord injury by reducing glial scar and cavity formation

    Science.gov (United States)

    Xue, Feng; Wu, Er-jun; Zhang, Pei-xun; Li-ya, A; Kou, Yu-hui; Yin, Xiao-feng; Han, Na

    2015-01-01

    We examined the restorative effect of modified biodegradable chitin conduits in combination with bone marrow mesenchymal stem cell transplantation after right spinal cord hemisection injury. Immunohistochemical staining revealed that biological conduit sleeve bridging reduced glial scar formation and spinal muscular atrophy after spinal cord hemisection. Bone marrow mesenchymal stem cells survived and proliferated after transplantation in vivo, and differentiated into cells double-positive for S100 (Schwann cell marker) and glial fibrillary acidic protein (glial cell marker) at 8 weeks. Retrograde tracing showed that more nerve fibers had grown through the injured spinal cord at 14 weeks after combination therapy than either treatment alone. Our findings indicate that a biological conduit combined with bone marrow mesenchymal stem cell transplantation effectively prevented scar formation and provided a favorable local microenvironment for the proliferation, migration and differentiation of bone marrow mesenchymal stem cells in the spinal cord, thus promoting restoration following spinal cord hemisection injury. PMID:25788929

  5. Distribution of mitochondrial DNA nucleoids inside the linear tubules vs. bulk parts of mitochondrial network as visualized by 4Pi microscopy

    Czech Academy of Sciences Publication Activity Database

    Dlasková, Andrea; Engstová, Hana; Plecitá-Hlavatá, Lydie; Lessard, M.; Alán, Lukáš; Reguera Pajuelo, David; Jabůrek, Martin; Ježek, Petr

    2015-01-01

    Roč. 47, č. 3 (2015), s. 255-263 ISSN 0145-479X R&D Projects: GA ČR(CZ) GAP305/12/1247; GA MŠk(CZ) EE2.3.30.0025; GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GA13-02033S Institutional support: RVO:67985823 Keywords : mitochondrial network * mitochondrial DNA * nucleoids * 4Pimicroscopy Subject RIV: EA - Cell Biology Impact factor: 2.080, year: 2015

  6. Chironomus alchichica sp. n. (Diptera: Chironomidae) from Lake Alchichica, Mexico.

    Science.gov (United States)

    Acosta, Raúl; Prat, Narcís; Ribera, Carles; Michailova, Paraskeva; Hernández-Fonseca, María Del Carmen; Alcocer, Javier

    2017-12-15

    Morphological analysis of all developmental stages (except female), mitochondrial DNA sequences from cytochrome c oxidase subunit I (cox1) and cytological analysis of the polytene chromosomes were used to describe a new species of Chironomus found in the littoral and profundal zones of an endorheic, warm-monomictic lake in Mexico. Male imago is distinguished by the shape of superior volsella and by an antennal and bristle ratio lower than two. The pupa is characterized by the spur morphology of abdominal segment VIII. There is also a continuous row of hooklets on abdominal segment II. The larva is distinguished by a combination of antenna, mentum, mandible, and pecten epipharyngis characteristics, and abdominal ventral tubules. Molecular and cytological analysis supported the morphological differences found. The maximum likelihood tree obtained shows that Chironomus alchichica sp. n. clusters together with Chironomus decorus-group sp. 2 Butler et al. (1995) (bootstrap support = 92%), but genetic p-distances within C. alchichica sp. n. (0.004) were lower than the p-distances between other species of the decorus-group (C. decorus-group sp. 2, Chironomus bifurcatus Wülker et al., 2009 and Chironomus maturus Johannsen, 1908) confirming that it is a different species. The new species belongs to thummi cytocomplex, (decorus-group), with chromosome set- 2n = 8 and chromosome arm combinations: AB CD EF G. Karyologically, the species is closest to Chironomus riihimaekiensis Wülker (1973). This species has very compact salivary gland chromosomes with well heterochromatinized centromere regions in chromosomes AB CD G. Several fixed homozygous inversions distinguish arm A of the species from that of C. riihimaekiensis. Arm E differs from that of C. riihimaekiensis by simple fixed homozygous inversion. Some similarities in band sequences of this arm were found with species from the decorus-group as Chironomus blaylocki Wülker et al., 2009 and C. bifurcatus (decorus

  7. Anatomy and histology of Embolyntha batesi MacLahlan, 1877 (Embiidina Anatomia e histologia de Embolyntha batesi MacLachlan, 1877 (Embiidina

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    Dyrce Lacombe

    1971-01-01

    Full Text Available The Embioptera are rather generalized insects whose internal anatomy is simple and not subject to great modifications. For this reason these insects form an ideal group for elementary anatomical and histological studies (fig. 2. The digestive tract is a long, simple tube without convolutions or diverticulae from the buccal cavity to the rectum. The buccal structures are of the chewing type. The oesophagus and ingluvia are differentiated only by slight dilation of their walls. In nymphs and females the proventriculus is very distinct due to folds which flatten as the structure becomes packed with food. The enteron is the largest in such forms and in both sexes limited caudally by the Malpighian tubules. The proctodeus has six large rectal papillae. The nervous system is complete with only the fifth abdominal segment lacking a ganglion in the metathorax includes the ganglion of the first abdominal segment. The brain exhibits very clear structure in histological sections. The tracheal system includes two pairs of thoracic spiracles and eight abdominal pairs. Only th metathoracic spiracle has an air expiration function; all others serve for inspiration. Various structures in the spiracles protect the atrium. The circulatory system includes a long, simple dorsal vessel which extends forward from the ninth abdominal segment into the cranium. It opens anteriorly near the circumoesophageal connectives. The dorsal vessel has a pair of ostia and valves corresponding to each abdominal and thoracic segment. It lacks the diverticulae or folds commonly found in more highly evolved insects. The excretory system is represented by Malphighian tubules, pericardial cells, and fat-body. The number and disposition of Malpighian tubules is variable within the order. The pericardial cells are localized around the entire dorsal vessel up to the opening of the aorta in the head. The fat-bodies form compact layers in the dorsal and ventral regions of the body. In males

  8. Target organ specific activity of drosophila MRP (ABCC1) moderates developmental toxicity of methylmercury.

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    Prince, Lisa; Korbas, Malgorzata; Davidson, Philip; Broberg, Karin; Rand, Matthew Dearborn

    2014-08-01

    Methylmercury (MeHg) is a ubiquitous and persistent neurotoxin that poses a risk to human health. Although the mechanisms of MeHg toxicity are not fully understood, factors that contribute to susceptibility are even less well known. Studies of human gene polymorphisms have identified a potential role for the multidrug resistance-like protein (MRP/ABCC) family, ATP-dependent transporters, in MeHg susceptibility. MRP transporters have been shown to be important for MeHg excretion in adult mouse models, but their role in moderating MeHg toxicity during development has not been explored. We therefore investigated effects of manipulating expression levels of MRP using a Drosophila development assay. Drosophila MRP (dMRP) is homologous to human MRP1-4 (ABCC1-4), sharing 50% identity and 67% similarity with MRP1. A greater susceptibility to MeHg is seen in dMRP mutant flies, demonstrated by reduced rates of eclosion on MeHg-containing food. Furthermore, targeted knockdown of dMRP expression using GAL4>UAS RNAi methods demonstrates a tissue-specific function for dMRP in gut, Malpighian tubules, and the nervous system in moderating developmental susceptibility to MeHg. Using X-ray synchrotron fluorescence imaging, these same tissues were also identified as the highest Hg-accumulating tissues in fly larvae. Moreover, higher levels of Hg are seen in dMRP mutant larvae compared with a control strain fed an equivalent dose of MeHg. In sum, these data demonstrate that dMRP expression, both globally and within Hg-targeted organs, has a profound effect on susceptibility to MeHg in developing flies. Our findings point to a potentially novel and specific role for dMRP in neurons in the protection against MeHg. Finally, this experimental system provides a tractable model to evaluate human polymorphic variants of MRP and other gene variants relevant to genetic studies of mercury-exposed populations. © The Author 2014. Published by Oxford University Press on behalf of the Society of

  9. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.

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    Jie Chen

    Full Text Available BACKGROUND: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1 and membrane-bound (Tre-2 trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. PRINCIPAL FINDINGS: The membrane-bound trehalase of Spodoptera exigua (SeTre-2 was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1 and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. CONCLUSIONS: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2

  10. A novel disease affecting the predatory mite Phytoseiulus persimilis (Acari, Phytoseiidae): 1. Symptoms in adult females.

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    Schütte, Conny; Kleijn, Prisca W; Dicke, Marcel

    2006-01-01

    Adult female Phytoseiulus persimilis Athias-Henriot (Acari, Phytoseiidae) of one of our laboratory populations showed a lower degree of attraction to herbivore-induced plant volatiles than other laboratory populations. We hypothesized earlier that this consistent change in foraging behavior is a symptom of a disease, as it is a contagious phenomenon. Here we describe more symptoms by comparing mated females of this population (non-responding (NR) population) with mated females of other populations that are strongly attracted to herbivore-induced plant volatiles (responding populations). The most apparent characteristic of the NR population was the presence of numerous dorso-ventrally flattened females (76% of all females). These females had a normal size after mating but shrank during adulthood. Independent of their age, shrunken females did not reproduce and died a few days after shrinking. In addition to these profound differences in short term performance, females from the NR-population showed behavioral changes, including a lower degree of attraction to herbivore-induced plant volatiles, a higher tendency to leave a prey-patch and a lower predation rate. Moreover, about half of the live females of the NR-population carried birefringent dumbbell-shaped crystals in the legs whereas live females of a responding population carried crystals only in the lumen of the Malpighian tubules and the rectum. The symptom 'crystals in the legs' was correlated with low reproduction. Energy dispersive X-ray diffraction of these crystals revealed that they contain calcium and phosphorus along with carbon and oxygen. Crystals with comparable elemental compositions and the same characteristic concentric layering are well known in insects, where they are thought to play a major role in detoxification of calcium and heavy metals, and in storage of phosphorus. The fraction of predators carrying a white spot in the distal part of the opisthosoma, due to accumulation of excretory

  11. Analysis of the predetermining effect of a sex realizer by ovary transplantations in the monogenic flyChrysomya rufifacies.

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    Ullerich, Fritz-Helmut

    1980-02-01

    In the heterozygousF'/f female-producing females of the strictly monogenic blowflyChrysomya rufifacies the gene product of the dominant or epistatic female sex realizerF' which causes sexpredetermination is thought to be synthesized either by cells of the germ line (oocytes, nurse cells or oogonia) or by somatic cells and then transferred into the oocytes. To determine the possible site of synthesis, reciprocal transplantations were made of prepupal ovaries between female-producing (thelygenic; t) and male-producing (arrhenogenic; a) females ofChrysomya rufifacies. In another series of experiments prepupal host females of the wild t-type and a-type were each provided with one additional ovary either from a0type (f/f) or a t-type (F'/f) prepupa (neither were distinguishable by their phenotypes). In all these experiments the donor females were marked by the recessive sex-linked mutation 'white' (w/w); white eyes, white Malpighian tubules). In a considerable number of cases the implanted ovaries were in contact with the host's own oviduct and grew normally, but the rate of hatched adults was somewhat reduced. Crosses between such host females andw/w males (f/f) produced female or male offspring with white eyes from the eggs of the implantedw/w ovary, as well as flies with wild-type eyes (+/w) which had developed in the host's own ovaries. In all cases so far examined, the genetically thelygenic (or arrhenogenic) host females with an additional ovary implanted from an arrhenogenic (thelygenic) donor, produced progeny of both sexes: sons (daughters) from the eggs of the donor's ovary and daugthers (sons) from the eggs of the host's own gonads.These results demonstrate that the ovaries of the t-females ofChrysomya rufifacies at least from the early prepupal stage, are autonomous for the product of theF' gene. Transplantations of the premordial germ cells (pole cells) are planned to find out whether the predeterminingF' gene product is synthesized before the prepupal

  12. Characterization and expression profiling of ATP-binding cassette transporter genes in the diamondback moth, Plutella xylostella (L.).

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    Qi, Weiping; Ma, Xiaoli; He, Weiyi; Chen, Wei; Zou, Mingmin; Gurr, Geoff M; Vasseur, Liette; You, Minsheng

    2016-09-27

    ATP-binding cassette (ABC) transporters are one of the major transmembrane protein families found in all organisms and play important roles in transporting a variety of compounds across intra and extra cellular membranes. In some species, ABC transporters may be involved in the detoxification of substances such as insecticides. The diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops worldwide, is an important species to study as it is resistant to many types of insecticides as well as biological control Bacillus thuringiensis toxins. A total of 82 ABC genes were identified from our published P. xylostella genome, and grouped into eight subfamilies (ABCA-H) based on phylogenetic analysis. Genes of subfamilies ABCA, ABCC and ABCH were found to be expanded in P. xylostella compared with those in Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens. Phylogenetic analysis indicated that many of the ABC transporters in P. xylostella are orthologous to the well-studied ABC transporter genes in the seven other species. Transcriptome- and qRT-PCR-based analysis elucidated physiological effects of ABC gene expressions of P. xylostella which were developmental stage- and tissue-specific as well as being affected by whether or not the insects were from an insecticide-resistant strain. Two ABCC and one ABCA genes were preferentially expressed in midgut of the 4th-instar larvae of a susceptible strain (Fuzhou-S) suggesting their potential roles in metabolizing plant defensive chemicals. Most of the highly expressed genes in insecticide-resistant strains were also predominantly expressed in the tissues of Malpighian tubules and midgut. This is the most comprehensive study on identification, characterization and expression profiling of ABC transporter genes in P. xylostella to date. The diversified features and expression patterns of this gene family may be associated with

  13. RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug

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    Mamidala Praveen

    2012-01-01

    Full Text Available Abstract Background Bed bugs (Cimex lectularius are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. Results We performed a next-generation RNA sequencing (RNA-Seq experiment to find differentially expressed genes between pesticide-resistant (PR and pesticide-susceptible (PS strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs and our previous 454 pyrosequenced database (21,088 ESTs. The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2 revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. Conclusions We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide

  14. Photosensitive TRPs.

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    Hardie, Roger C

    2014-01-01

    The Drosophila "transient receptor potential" channel is the prototypical TRP channel, belonging to and defining the TRPC subfamily. Together with a second TRPC channel, trp-like (TRPL), TRP mediates the transducer current in the fly's photoreceptors. TRP and TRPL are also implicated in olfaction and Malpighian tubule function. In photoreceptors, TRP and TRPL are localised in the ~30,000 packed microvilli that form the photosensitive "rhabdomere"-a light-guiding rod, housing rhodopsin and the rest of the phototransduction machinery. TRP (but not TRPL) is assembled into multimolecular signalling complexes by a PDZ-domain scaffolding protein (INAD). TRPL (but not TRP) undergoes light-regulated translocation between cell body and rhabdomere. TRP and TRPL are also found in photoreceptor synapses where they may play a role in synaptic transmission. Like other TRPC channels, TRP and TRPL are activated by a G protein-coupled phospholipase C (PLCβ4) cascade. Although still debated, recent evidence indicates the channels can be activated by a combination of PIP2 depletion and protons released by the PLC reaction. PIP2 depletion may act mechanically as membrane area is reduced by cleavage of PIP2's bulky inositol headgroup. TRP, which dominates the light-sensitive current, is Ca(2+) selective (P Ca:P Cs >50:1), whilst TRPL has a modest Ca(2+) permeability (P Ca:P Cs ~5:1). Ca(2+) influx via the channels has profound positive and negative feedback roles, required for the rapid response kinetics, with Ca(2+) rapidly facilitating TRP (but not TRPL) and also inhibiting both channels. In trp mutants, stimulation by light results in rapid depletion of microvillar PIP2 due to lack of Ca(2+) influx required to inhibit PLC. This accounts for the "transient receptor potential" phenotype that gives the family its name and, over a period of days, leads to light-dependent retinal degeneration. Gain-of-function trp mutants with uncontrolled Ca(2+) influx also undergo retinal degeneration

  15. Identification and characterization of chitin deacetylase2 from the American white moth, Hyphantria cunea (Drury).

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    Yan, Xiaoping; Zhao, Dan; Zhang, Yakun; Guo, Wei; Wang, Wei; Zhao, Kunli; Gao, Yujie; Wang, Xiaoyun

    2018-05-26

    Chitin deacetylases (CDAs) are enzymes that catalyze the conversion of chitin into chitosan, thereby influence the mechanical and permeability properties of structures such as the cuticle and peritrophic matrices. The full length cDNAs of chitin deacetylase2 (CDA2) genes from Hyphantria cunea were fully cloned by PCR amplification. Two cDNA sequences of HcCDA2 were searched from transcriptome of H. cunea and named as HcCDA2a and HcCDA2b. The deduced protein sequences showed that HaCDA2a and HaCDA2b are synthesized as preproteins of 524 and 518 amino acid residues with an 18-amino acid signal peptide, respectively. HcCDA2a and HcCDA2b contained a chitin-binding domain (ChBD), a low-density lipoprotein receptor class A domain (LDLa) and a polysaccharide deacetylase-like catalytic domain (CDA). Gene expression analyses results showed that HcCDA2a and HcCDA2b were both expressed at the head, integument, foregut, midgut, hindgut, Malpighian tubules and fat body, as well as the 1st to 5th days of fifth instar larvae. Western blot analyses revealed that HcCDA2 protein was highly abundant in the head and integument, and the developmental expression result in the fifth instars showed that HcCDA2 was highly present at the first two days. Besides, RT-PCR results showed that HcCDA2a and HcCDA2b were both expressed in integument and head, whether in molting stage or feeding stage. No visiable phenotypic changes were observed after injection of dsHcCDA2b, while lethal phenotypes of cuticle shedding failure and high mortality were resulted from injection of dsHcCDA2a. The silence of HcCDA2a leads to the ecdysis failure and death of H. cunea. These results suggest that HcCDA2 plays an important role during insect development, and provide new candidate targets and basis for developing environment-friendly pesticides. Copyright © 2017. Published by Elsevier B.V.

  16. Francisella-Like Endosymbionts and Rickettsia Species in Local and Imported Hyalomma Ticks.

    Science.gov (United States)

    Azagi, Tal; Klement, Eyal; Perlman, Gidon; Lustig, Yaniv; Mumcuoglu, Kosta Y; Apanaskevich, Dmitry A; Gottlieb, Yuval

    2017-09-15

    Hyalomma ticks (Acari: Ixodidae) are hosts for Francisella -like endosymbionts (FLE) and may serve as vectors of zoonotic disease agents. This study aimed to provide an initial characterization of the interaction between Hyalomma and FLE and to determine the prevalence of pathogenic Rickettsia in these ticks. Hyalomma marginatum , Hyalomma rufipes , Hyalomma dromedarii , Hyalomma aegyptium , and Hyalomma excavatum ticks, identified morphologically and molecularly, were collected from different hosts and locations representing the distribution of the genus Hyalomma in Israel, as well as from migratory birds. A high prevalence of FLE was found in all Hyalomma species (90.6%), as well as efficient maternal transmission of FLE (91.8%), and the localization of FLE in Malpighian tubules, ovaries, and salivary glands in H. marginatum Furthermore, we demonstrated strong cophylogeny between FLE and their host species. Contrary to FLE, the prevalence of Rickettsia ranged from 2.4% to 81.3% and was significantly different between Hyalomma species, with a higher prevalence in ticks collected from migratory birds. Using ompA gene sequences, most of the Rickettsia spp. were similar to Rickettsia aeschlimannii , while a few were similar to Rickettsia africae of the spotted fever group (SFG). Given their zoonotic importance, 249 ticks were tested for Crimean Congo hemorrhagic fever virus infection, and all were negative. The results imply that Hyalomma and FLE have obligatory symbiotic interactions, indicating a potential SFG Rickettsia zoonosis risk. A further understanding of the possible influence of FLE on Hyalomma development, as well as on its infection with Rickettsia pathogens, may lead to novel ways to control tick-borne zoonoses. IMPORTANCE This study shows that Francisella -like endosymbionts were ubiquitous in Hyalomma , were maternally transmitted, and cospeciated with their hosts. These findings imply that the interaction between FLE and Hyalomma is of an obligatory

  17. Dissecting the organ specificity of insecticide resistance candidate genes in Anopheles gambiae: known and novel candidate genes.

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    Ingham, Victoria A; Jones, Christopher M; Pignatelli, Patricia; Balabanidou, Vasileia; Vontas, John; Wagstaff, Simon C; Moore, Jonathan D; Ranson, Hilary

    2014-11-25

    The elevated expression of enzymes with insecticide metabolism activity can lead to high levels of insecticide resistance in the malaria vector, Anopheles gambiae. In this study, adult female mosquitoes from an insecticide susceptible and resistant strain were dissected into four different body parts. RNA from each of these samples was used in microarray analysis to determine the enrichment patterns of the key detoxification gene families within the mosquito and to identify additional candidate insecticide resistance genes that may have been overlooked in previous experiments on whole organisms. A general enrichment in the transcription of genes from the four major detoxification gene families (carboxylesterases, glutathione transferases, UDP glucornyltransferases and cytochrome P450s) was observed in the midgut and malpighian tubules. Yet the subset of P450 genes that have previously been implicated in insecticide resistance in An gambiae, show a surprisingly varied profile of tissue enrichment, confirmed by qPCR and, for three candidates, by immunostaining. A stringent selection process was used to define a list of 105 genes that are significantly (p ≤0.001) over expressed in body parts from the resistant versus susceptible strain. Over half of these, including all the cytochrome P450s on this list, were identified in previous whole organism comparisons between the strains, but several new candidates were detected, notably from comparisons of the transcriptomes from dissected abdomen integuments. The use of RNA extracted from the whole organism to identify candidate insecticide resistance genes has a risk of missing candidates if key genes responsible for the phenotype have restricted expression within the body and/or are over expression only in certain tissues. However, as transcription of genes implicated in metabolic resistance to insecticides is not enriched in any one single organ, comparison of the transcriptome of individual dissected body parts cannot

  18. Genetic variation associated with increased insecticide resistance in the malaria mosquito, Anopheles coluzzii.

    Science.gov (United States)

    Main, Bradley J; Everitt, Amanda; Cornel, Anthony J; Hormozdiari, Fereydoun; Lanzaro, Gregory C

    2018-04-04

    Malaria mortality rates in sub-Saharan Africa have declined significantly in recent years as a result of increased insecticide-treated bed net (ITN) usage. A major challenge to further progress is the emergence and spread of insecticide resistance alleles in the Anopheles mosquito vectors, like An. coluzzii. A non-synonymous mutation in the para voltage-gated sodium channel gene reduces pyrethroid-binding affinity, resulting in knockdown resistance (kdr). Metabolic mechanisms of insecticide resistance involving detoxification genes like cytochrome P450 genes, carboxylesterases, and glutathione S-transferases are also important. As some gene activity is tissue-specific and/or environmentally induced, gene regulatory variation may be overlooked when comparing expression from whole mosquito bodies under standard rearing conditions. We detected complex insecticide resistance in a 2014 An. coluzzii colony from southern Mali using bottle bioassays. Additional bioassays involving recombinant genotypes from a cross with a relatively susceptible 1995 An. coluzzii colony from Mali confirmed the importance of kdr and associated increased permethrin resistance to the CYP9K1 locus on the X chromosome. Significant differential expression of CYP9K1 was not observed among these colonies in Malpighian tubules. However, the P450 gene CYP6Z1 was overexpressed in resistant individuals following sublethal permethrin exposure and the carboxylesterase gene COEAE5G was constitutively overexpressed. The significant P450-related insecticide resistance observed in the 2014 An. coluzzii colony indicates that ITNs treated with the P450 inhibitor piperonyl butoxide (PBO) would be more effective in this region. The known insecticide resistance gene CYP6Z1 was differentially expressed exclusively in the context of sublethal permethrin exposure, highlighting the importance of tissue-specificity and environmental conditions in gene expression studies. The increased activity of the carboxylesterase

  19. Characteristics of six small heat shock protein genes from Bactrocera dorsalis: Diverse expression under conditions of thermal stress and normal growth.

    Science.gov (United States)

    Dou, Wei; Tian, Yi; Liu, Hong; Shi, Yan; Smagghe, Guy; Wang, Jin-Jun

    2017-11-01

    To explore the functions of small heat shock proteins (sHsps) in relation to thermal stress and development in Bactrocera dorsalis (Hendel), one of the most economically important pest species attacking a wide range of fruits and vegetables, six full-length cDNAs of sHsp genes (BdHsp17.7, 18.4, 20.4, 20.6, 21.6 and 23.8) were cloned, and the expression patterns in different developmental stages and tissues, as well as in response to both thermal and 20-hydroxyecdysone (20E) exposures, were examined using real time quantitative PCR. The open reading frames (ORFs) of six sHsps are 453, 489, 537, 543, 567 and 630bp in length, encoding proteins with molecular weights of 17.7, 18.4, 20.4, 20.6, 21.6 and 23.8kDa, respectively. BdHsp18.4 and BdHsp20.4 maintained lower expression levels in both eggs and larvae, whereas remarkably up-regulated after the larval-pupal transformation, suggesting that these two sHsps may be involved in metamorphosis. Significant tissue specificity exists among sHsps: the highest expression of BdHsp20.6 and BdHsp23.8 in the Malpighian tubules and ovary, respectively, versus a peak in the fat body for others. BdHsp20.4 and BdHsp20.6 were significantly up-regulated by thermal stress. In contrast, BdHsp18.4 and BdHsp23.8 reacted only to heat stress. BdHsp17.7 and BdHsp21.6 were insensitive to both heat and cold stresses. The degree of sHsps response depends on intensity of 20E treatment, i.e., dose and time. These results strongly suggest functional differentiation within the sHsp subfamily in B. dorsalis. The physiological function of sHsp members under thermal stress and normal growth remains the subjects of further investigation. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Registro de Nephridiophaga sp. (Protista: Nephridiophagidae en Apis mellifera (Hymenoptera: Apidae del Sur de la región Pampeana Record of Nephridiophaga sp. (Protista: Nephridiophagidae in Apis mellifera (Hymenoptera: Apidae of the southern Pampas

    Directory of Open Access Journals (Sweden)

    Santiago Plischuk

    2011-12-01

    Full Text Available Durante estudios prospectivos tendientes a la detección de protistas asociados a ápidos en la región Pampeana, se observó la presencia de esporos ovales bicóncavos y grupos de esporos (cúmulos en los túbulos de Malpighi de abejas de Dufaur, partido de Saavedra, sudoeste de la provincia de Buenos Aires. Los esporos maduros midieron 4,8 ± 0,05 x 2,4 ± 0,03 μm y la carga (intensidad promedió 5,71 ± 1,49 x 10(6 esporos/abeja. Las detecciones se efectuaron entre julio y octubre de 2006 y la prevalencia en las colmenas positivas osciló entre 1 y 16,7 %. Las características morfológicas de los esporos, el lugar de desarrollo y la especie huésped involucrada sugieren que el microorganismo en cuestión, pertenece al género Nephridiophaga y sería N. apis Ivani, especie tipo cuyo conocimiento es extremadamente limitado. El hallazgo constituye el primer registro de un nefridiofágido asociado a A mellifera fuera del continente europeo.During surveys for the detection of protists associated to Apidae in the Pampas region, biconcave oval spores, and spore clumps were observed in the Malpighian tubules of honeybees from Dufaur, Saavedra county, southwestern Buenos Aires province. Mature spores measured 4.8 ± 0.05 x 2.4 ± 0.03 μm, and mean spore load was 5.71 ± 1.49 x 10(6 per honeybee. Detections were from July to October 2006, and prevalence in positive colonies ranged from 1 to 16.7%. Morphology of the spores, the site of development, and the identity of the host species suggest that the isolated microorganism belongs to the genus Nephridiophaga and would be N apis Ivani, the type species, knowledge on which is extremely limited. The finding constitutes the first record of a nephridiophagid in honeybees outside of Europe.

  1. Detección de Malpighamoeba mellifcae (Protista: Amoebozoa en Apis mellifera (Hymenoptera: Apidaede Argentina Detection of Malpighamoeba mellifcae (Protista: Amoebozoa in Apis mellifera (Hymenoptera: Apidae of Argentina

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    Santiago Plischuk

    2010-12-01

    Full Text Available Debido a su rol como polinizador y productor de miel, la abeja Apis mellifera L. es considerado un insecto beneficioso. Si bien Argentina juega un papel de liderazgo en la producción de miel, existe un considerable vacío en el conocimiento acerca de las enfermedades de etiología protista que afectan las abejas en el país. La ameba Malpighamoeba mellificae Prell es un protista entomopatógeno que invade los túbulos de Malpighi de las abejas e interfiere con el proceso de excreción, debilitando al huésped y posiblemente facilitando la acción de otros patógenos. En esta contribución se presentan los primeros hallazgos de M. mellificae en Argentina y se brindan datos iniciales acerca de su frecuencia, intensidad de las infecciones, y co-ocurrencia con Nosema sp. Malpighamoeba mellificae se halló en dos de 36 localidades prospectadas: San Cayetano, al Sur de la provincia de Buenos Aires y San Carlos de Bariloche, en el Oeste de la provincia de Río Negro.Due to its role as a pollinator and honey producer, the honey bee Apis mellifera L. is considered a beneficial insect. Although Argentina plays a leading role in honey production, there is a considerable gap in knowledge regarding protistan diseases that affect honey bees in the country. The amoeba Malpighamoeba mellificae Prell is an entomopathogenic protist that invades the Malpighian tubules of honey bees and interferes with the excretory process, debilitating the host and possibly facilitating the action of other pathogens. In this contribution, we present the first reports of M. mellificae in Argentina, and provide some initial data about its frecuency, infection intensity, and co-occurrence with Nosema sp. Malpighamoeba mellificae was found in two out of 36 localities surveyed: San Cayetano, in southern Buenos Aires province, and San Carlos de Bariloche, in western Río Negro province.

  2. Desenvolvimento pós-embrionário do intestino anterior de Dermatobia hominis (Linnaeus Jr. (Diptera, Cuterebridae Post-embryonic development of Dermatobia hominis (Linnaeus Jr. (Diptera, Cuterebridae foregut

    Directory of Open Access Journals (Sweden)

    Ana Maria Vieira

    2001-03-01

    Full Text Available Foregut in D. hominis (Linnaeus Jr., 1781 as the majority of the larval Diptera somatic tissue, is made up of polytenic cells, and grows at the expenses of the polytenization of its nuclei followed by the increase in size of each cell. The oesophagus, of ectodermic origem, is interiorly covered by a chitinous squamous epithelium that rests upon a very thin basal lamina. This sheet is surrounded by thick muscle bundles. The oesophagus intussuscepts the midgut forming the cardia. The cardia, with three epithelial layers: two internal ones, of ectodermal origin and one external of endodermic origin. At the anterior portion of the cardia, between these two types of epithelium, there is a cluster of small, non polytenic cells, forming the imaginal disk of the foregut. Metamoiphosis begins at the end of the larval period with signs of nuclear degeneration of all the polytenic cells, as well as the increase in number of the imaginal disk ones. The oesophagic portion intussuscepted into the cardia, everts; its cells suffer apoptosis and are replaced by the new cells growing from the imaginal disk. The external layer cells also degenerate and are pinched off into the lumen of the very anterior portion of the midgut. The newly formed oesophagus intussuscepts "de novo" to form the two internal layers of the adult cardia. At the same time the midgut regenerative cells grow anteriorly to form the new external layer of the adult cardia.

  3. Drosophila Model for the Analysis of Genesis of LIM-kinase 1-Dependent Williams-Beuren Syndrome Cognitive Phenotypes: INDELs, Transposable Elements of the Tc1/Mariner Superfamily and MicroRNAs

    Czech Academy of Sciences Publication Activity Database

    Savvateeva-Popova, E.V.; Zhuravlev, A.V.; Brázda, Václav; Zakharov, G.A.; Kaminskaya, A.N.; Medvedeva, A.V.; Nikitina, E.; Tokmatcheva, E.V.; Dolgaya, J.F.; Kulikova, D.A.; Zatsepina, O.; Funikov, S.Y.; Ryazansky, S.S.; Evgen'ev, Michail B.

    2017-01-01

    Roč. 8, SEP2017 (2017), č. článku 123. ISSN 1664-8021 Institutional support: RVO:68081707 Keywords : b dna conformations * swiss-model * copy number * polytene chromosomes Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 3.789, year: 2016

  4. Journal of Genetics | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    We report novel findings on the cytogenetic location, functional complexity and maternal and germline roles of the stambh A locus of Drosophila melanogaster. stmA is localized to polytene bands 44D1.2 on 2R. stmA mutations are of two types: temperature-sensitive (ts) adult and larval paralytic or unconditional embryonic ...

  5. [Chromomeric organization of interphase chromosomes in Drosophila melanogaster].

    Science.gov (United States)

    Zhuimulev, I F; Beliaeva, E S; Zykova, T Iu; Semeshin, V F; Demakov, S A; Demakova, O V; Goncharov, F P; Khoroshko, V A; Boldyreva, L V; Kokoza, E B; Pokholkiova, G V

    2013-01-01

    As a result of treatment of bioinformatic data on the genome localization of structural proteins, histone modifications, DNase-hypersensitive regions, replication origins (taken from modENCODE) and their cytological localization to polytene chromosome structures, it is shown here that two types of interphase chromosomes -polytene chromosomes from salivary glands and from mitotically dividing cells cultures - demonstrate identical pictures of interband/band, i. e. the same localization and length on physical map and the same sets of proteins. In the interbands of both chromosome types we find the proteins that control initiation of transcription (RNA-polymerase II, transcription factors), replication (ORC2) as well as proteins modifying nucleosome structure (WDS, NURF) and proteins of insulators (BEAF). The nucleosome density and H1 histone concentration in the interbands are depleted; localization of DNase-hypersensitive regions corresponds strictly to the interbands. So, we conclude that both polytene and cell line interphase chromosomes are arranged according to general principle and polytene chromosomes represent precise model of interphase chromosomes. The interbands play a critical role in the initiation of transcription and replication. The interbands of interphase chromosomes are the sites of 5' parts of genes, while the 3' gene ends are located in the adjacent bands. The constancy of interbands decondensation results in the conclusion that the "interbands" genes are constantly active, i. e. they contain "house-keeping" genes. The large late replicating bands contain genes that do not have direct contact to the adjoining interbands are usually polygenic and contain tissue-specific genes.

  6. Comparative study of unilateral renal tubule function using 131I-o-hippuran and sup(99m)Tc-dimercaptosuccinic acid with regard to renal depth and excretion relations

    International Nuclear Information System (INIS)

    Moser, E.A.

    1980-01-01

    Good agreement was found between sonographic and nuclear renal depth data. In patients with undisturbed postrenal urodynamics, the data of unilateral renal clearance obtained by DMSA and OIH are in good agreement after depth correction. With OIH, the activity measured for unilateral congestion kidneys was higher than with DMSA. However, both methods may overestimate unilateral congestion kidneys. The OIH method should be favoured in nuclear renal diagnostics. In patients with mobile kidneys, the lower function calculated for the ptotic kidney can be evaluated only after depth correction. To reduce the radiation exposure, renal depth data required for depth correction should be determined by sonographic methods. The peak/scatter method of renal depth determination cannot be employed in practice in the 131 J hippurane test; in the sup(99m)Tc-DMSA test, sufficient agreement between peak/scatter quotient and renal depth is only obtained after background correction. The result does not warrant the tedious procedure. DMSA studies of the kidneys are appropriate in the following cases: 1. Emergency studies of unilateral renal function in cases of acute anuria due to postrenal stoppage. 2. Assessment of unilateral parenchymal function in patients with mobile kidneys if the ptotic kidney cannot be imaged by sonographic processes. 3. Search for extremely displaced renal tissue. 4. Unilateral renal function studies in patients with unilateral kidney diseases if the postrenal situation and the global renal function can be assessed by other methods. (orig./MG) [de

  7. Effects of Persea americana Mill (Lauraceae) ["Avocado"] ethanolic leaf extract on blood glucose and kidney function in streptozotocin-induced diabetic rats and on kidney cell lines of the proximal (LLCPK1) and distal tubules (MDBK).

    Science.gov (United States)

    Gondwe, M; Kamadyaapa, D R; Tufts, M A; Chuturgoon, A A; Ojewole, J A O; Musabayane, C T

    2008-01-01

    Extracts of Persea americana Mill (Lauraceae) ("Avocado") have been traditionally used to treat hypertension and diabetes mellitus. Accordingly, we studied the hypoglycaemic and renal function effects of P. americana leaf ethanolic extracts (PAE) in STZ-induced diabetic rats. Oral glucose tolerance responses to various doses of PAE were monitored in fasted rats following a glucose load. Rats treated with deionized water or standard hypoglycaemic drugs acted as untreated and treated positive controls, respectively. Acute renal effects of PAE were investigated in anesthetized rats challenged with 0.077 M NaCl after a 3.5-h equilibration for 4 h comprising 1 h control, 1.5 h treatment and 1.5 h recovery periods. PAE was added to the infusate during the treatment period. Hepatic glycogen concentration was measured after 6 weeks of daily treatment with PAE. PAE induced dose-dependent hypoglycaemic responses in STZ-induced diabetic rats while subchronic PAE treatment additionally increased hepatic glycogen concentrations. Acute PAE infusion decreased urine flow and electrolyte excretion rates, whilst subchronic treatment reduced plasma creatinine and urea concentrations. These results indicate not only the basis of the ethnomedicinal use of P. americana leaf extract in diabetes management, but also of need for further studies to identify and evaluate the safety of PAE's bioactive compounds. (c) 2008 Prous Science, S.A.U. or its licensors. All rights reserved.

  8. Could grape seed extract modulate nephritic damage induced by ...

    African Journals Online (AJOL)

    RBCs). Preadministration of GSO to Metho-induced rats revealed apparent normal renal parenchyma. The proximal convoluted tubules and collecting tubules appeared near to normal with their narrow lumen. Preadministration with GSO ...

  9. Methomyl is a water-soluble crystalline solid that gives off a ...

    African Journals Online (AJOL)

    NBU

    RBCs). Pre- administration of GSO to Metho-induced rats revealed apparent normal renal parenchyma. The proximal convoluted tubules and collecting tubules appeared near to normal with their narrow lumen. Pre- administration with GSO ...

  10. Medline Plus

    Full Text Available ... and penis. The testes are composed of coiled structures called seminiferous tubules, which are the sites of sperm production. The structure on top of the seminiferous tubules in the ...

  11. 76 FR 36349 - Diethylene Glycol MonoEthyl Ether (DEGEE); Exemption From the Requirement of a Tolerance

    Science.gov (United States)

    2011-06-22

    ..., based on decreased growth, epithelial necrosis of renal tubules and cloudy swelling of hepatic tissue... growth, FQPA SF = 1x epithelial necrosis of renal tubules and cloudy swelling of hepatic tissue... calculi, epithelial necrosis of the renal tubules and cloudy swelling of hepatic tissue were observed in...

  12. Cytogenetics of a parthenogenetic Arctic species of Micropsectra (Diptera, Chironomidae)

    Science.gov (United States)

    Porter, David L.; Martin, Jon

    2011-01-01

    Abstract Micropsectra sedna (Oliver, 1976) is a parthenogenetic midge from the Canadian Arctic. The parthenogenetic mechanism is apomictic thelytoky, with a restitutional division during oogenesis, as found in other parthenogenetic Chironomidae. It is triploid, with two similar chromosome sets, and the third is relatively dissimilar, pairing little with the diploid set. Two karyotypes were observed: a single individual with eight polytene elements in the salivary glands (3n=12), considered standard, while the majority of larvae showed only seven polytene chromosomes (3n=11). Hybrid speciation is considered likely, although chromosomal recombination following the origin of thelytoky has played some part in karyotype evolution. A single morphologically distinct larva was also found, which might be the donor of the haploid chromosome set. The apomictic restitutional system is compared to that of the other, independently derived, parthenogenetic Chironomids to assess the extent of similarity between species. PMID:24260638

  13. Progress and problems in the cytology of Ceratitis capitata

    International Nuclear Information System (INIS)

    Bedo, D.G.

    1989-01-01

    In recent years of cytological work with Ceratitis capitata has expanded rapidly in concert with increased interest in the application of genetics and molecular biology in the control of this species. Previous studies using mitotic chromosomes produced essential basic karyotypic data but resulted in a confused situation in which several chromosome nomenclature systems were in use. The need for better cytological methods and a standardization of nomenclature was recognised by the IAEA which recommended a system based on that used for Drosophila melanogaster. Polytene chromosome analysis was soon available with the development of techniques for preparing chromosome squashes from male pupal orbital trichogen cells and larval salivary glands. The application of chromosome banding techniques quickly provided detailed morphological information on mitotic chromosomes, particularly the sex chromosomes, and mapping of chromosome rearrangement break-points on newly prepared polytene chromosome maps commenced. This report describes recent unpublished results and offers comments on the still unresolved nomenclature problems in C. capitata

  14. Karyotypical characteristics of two allopatric African populations of anhydrobiotic Polypedilum Kieffer, 1912 (Diptera, Chironomidae) originating from Nigeria and Malawi.

    Science.gov (United States)

    Petrova, Ninel A; Cornette, Richard; Shimura, Sachiko; Gusev, Oleg A; Pemba, Dylo; Kikawada, Takahiro; Zhirov, Sergey V; Okuda, Takashi

    2015-01-01

    The African chironomid Polypedilumvanderplanki Hinton, 1951 is the only chironomid able to withstand almost complete desiccation in an ametabolic state known as anhydrobiosis. The karyotypes of two allopatric populations of this anhydrobiotic chironomid, one from Nigeria and another from Malawi, were described according to the polytene giant chromosomes. The karyotype from the Nigerian population was presented as the reference chromosome map for Polypedilumvanderplanki. Both populations, Nigerian and Malawian, showed the same number of chromosomes (2n=8), but important differences were found in the band sequences of polytene chromosomes, and in the number and the arrangement of active regions between the two populations. Such important differences raise the possibility that the Malawian population could constitute a distinct new species of anhydrobiotic chironomid.

  15. Chironomus in the investigation of the genetic influence of radiation

    International Nuclear Information System (INIS)

    Tarasyuk, A.N.; Kovalevich, N.F.

    2000-01-01

    The influence of γ-radiation in different doses on the structure and functional activity of polytene chromosomes of chironomus has been explored. There have been shown the increase of frequency and change of spectrum of chromosome aberrations, the induction of puffs formation. The description of the revealed chromosome aberrations is given. Possible reasons and mechanisms of the observable effects and the further research program are being discussed. (authors)

  16. A Molecular Phylogeny of Anopheles Annulipes (Diptera: Culicidae) sensu lato: The Most Species-Rich Anopheline Complex

    Science.gov (United States)

    2006-09-27

    Maximum parsimony; Sibling species; Species complex; Myxomatosis ; DNA barcoding; Australia; Papua New Guinea; ITS2; COI; COII; EF-11. Introduction... myxomatosis to con- trol rabbits (Fenner and RatcliVe, 1965). Chris Green used data from cross-matings and the band- ing pattern of polytene chromosomes to... myxomatosis based on distribution but more sam- pling is required to conWrm this. Many of the sampling locations in this study and the allozyme study of

  17. Large-scale isolation and fractionation of organs of Drosophila melanogaster larvae.

    Science.gov (United States)

    Zweidler, A; Cohen, L H

    1971-10-01

    Methods for the mass isolation of diverse organs from small animals are described. They involve novel devices: a mechanical dissecting system, a centrifugal agitator for the separation of fibrillar from globular particles, and a settling chamber for the fractionation at unit gravity of particles with sedimentation velocities above the useful range for centrifugation. The application of these methods to the isolation of polytene and nonpolytene nuclei from Drosophila melanogaster larvae is described.

  18. Reassignment of Drosophila willistoni Genome Scaffolds to Chromosome II Arms

    OpenAIRE

    Garcia, Carolina; Delprat, Alejandra; Ruiz, Alfredo; Valente, Vera L. S.

    2015-01-01

    Drosophila willistoni is a geographically widespread Neotropical species. The genome of strain Gd-H4-1 from Guadeloupe Island (Caribbean) was sequenced in 2007 as part of the 12 Drosophila Genomes Project. The assembled scaffolds were joined based on conserved linkage and assigned to polytene chromosomes based on a handful of genetic and physical markers. This paucity of markers was particularly striking in the metacentric chromosome II, comprised two similarly sized arms, IIL and IIR, tradit...

  19. Genetic organization of interphase chromosome bands and interbands in Drosophila melanogaster.

    Science.gov (United States)

    Zhimulev, Igor F; Zykova, Tatyana Yu; Goncharov, Fyodor P; Khoroshko, Varvara A; Demakova, Olga V; Semeshin, Valeriy F; Pokholkova, Galina V; Boldyreva, Lidiya V; Demidova, Darya S; Babenko, Vladimir N; Demakov, Sergey A; Belyaeva, Elena S

    2014-01-01

    Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. In the present paper, we analyze 32 cytology-mapped polytene chromosome interbands. We estimated molecular locations of these interbands, described their molecular and genetic organization and demonstrate that polytene chromosome interbands contain the 5' ends of housekeeping genes. As a rule, interbands display preferential "head-to-head" orientation of genes. They are enriched for "broad" class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC) components. In two regions, 10A and 100B, coding sequences of genes whose 5'-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called "grey" bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase) indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. We developed a special algorithm to computationally process protein localization data generated by the modENCODE project and show that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date.

  20. Topography of multi-locus deletions induced by gamma-rays and neutrons in the black, cinnabar and vestigial regions of drosophila melanogaster

    International Nuclear Information System (INIS)

    Alexandrov, I.V.; Lapidus, I.L.; Alexandrova, M.V.

    1997-01-01

    The extend and breakpoint location of 85 chromosomal-scale deletions induced by gamma-rays or fission neutrons in the black, cinnabar and vestigial regions of Drosophila genome have been examined by conventional cytogenetic analysis of the polytene chromosomes. It was found that the topographies of deletions are similar for both type of radiation and for all regions under study: the largest deletions have 3.5 Mb length, i.e. more than 2 divisions of the polytene chromosome; the breakpoints of deletions are located within the inter-bands and mapped more often in the centro-metric directions; the sizes of deletions are multiple to one, two or more visible chromomeres of polytene chromosome. These findings seem to be very well explained within the framework of the rosette-loopy model of higher (super-chromosome) level of the chromatin organization and of the notions about the illegitimate recombination promoted by the clustered damages of the core DNA resulting from the one-hit events of energy deposition at this target supported by the linear relationship observed between the delation yield and the dose of radiations studied. (authors)

  1. Reassignment of Drosophila willistoni Genome Scaffolds to Chromosome II Arms.

    Science.gov (United States)

    Garcia, Carolina; Delprat, Alejandra; Ruiz, Alfredo; Valente, Vera L S

    2015-10-04

    Drosophila willistoni is a geographically widespread Neotropical species. The genome of strain Gd-H4-1 from Guadeloupe Island (Caribbean) was sequenced in 2007 as part of the 12 Drosophila Genomes Project. The assembled scaffolds were joined based on conserved linkage and assigned to polytene chromosomes based on a handful of genetic and physical markers. This paucity of markers was particularly striking in the metacentric chromosome II, comprised two similarly sized arms, IIL and IIR, traditionally considered homologous to Muller elements C and B, respectively. In this paper we present the cytological mapping of 22 new gene markers to increase the number of markers mapped by in situ hybridization and to test the assignment of scaffolds to the polytene chromosome II arms. For this purpose, we generated, by polymerase chain reaction amplification, one or two gene probes from each scaffold assigned to the chromosome II arms and mapped these probes to the Gd-H4-1 strain's polytene chromosomes by nonfluorescent in situ hybridization. Our findings show that chromosome arms IIL and IIR correspond to Muller elements B and C, respectively, directly contrasting the current homology assignments in D. willistoni and constituting a major reassignment of the scaffolds to chromosome II arms. Copyright © 2015 Garcia et al.

  2. Genetic organization of interphase chromosome bands and interbands in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Igor F Zhimulev

    Full Text Available Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. In the present paper, we analyze 32 cytology-mapped polytene chromosome interbands. We estimated molecular locations of these interbands, described their molecular and genetic organization and demonstrate that polytene chromosome interbands contain the 5' ends of housekeeping genes. As a rule, interbands display preferential "head-to-head" orientation of genes. They are enriched for "broad" class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC components. In two regions, 10A and 100B, coding sequences of genes whose 5'-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called "grey" bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. We developed a special algorithm to computationally process protein localization data generated by the modENCODE project and show that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date.

  3. Henneguya laterocapsulata Landsberg, 1987 (Myxosporea, Myxozoa) in cultured hybrid African catfish: Ultrastructure of the parasite-host interface.

    Science.gov (United States)

    Obiekezie, A; Schmahl, G

    1993-02-19

    The ultrastructure of the host-parasite interface was studied in Henneguya laterocapsulata, parasitizing the skin of hybrid catfishes (Clarias gariepinus × Heterobranchus bidorsalis) in Nigeria. The plasmodia were located between malpighian cells, which are the main elements of the multilayered fish epidermis, and were bordered by a single cell membrane. The desmosomal junctions between the malpighian cells were forced apart by finger-like protrusions of the Plasmodium. These plasmodial protrusions finally ran into the host cell without disrupting of the host cell membrane and formed network-like extensions. At the margin of the plasmodium an extensive vacuolization occurred, leading to a wavy surface. Infections with H. laterocapsulata may be an adverse factor in the large-scale production of hybrid catfish fingerlings used for aquaculture in Africa. Copyright © 1993 Gustav Fischer Verlag · Stuttgart · Jena · New York. Published by Elsevier GmbH.. All rights reserved.

  4. Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis

    DEFF Research Database (Denmark)

    Kastner, Christian; Pohl, Marcus; Sendeski, Mauricio

    2009-01-01

    Human glomerulonephritis (GN) is characterized by sustained proteinuria, sodium retention, hypertension, and edema formation. Increasing quantities of filtered protein enter the renal tubule, where they may alter epithelial transport functions. Exaggerated endocytosis and consequent protein...... overload may affect proximal tubules, but intrinsic malfunction of distal epithelia has also been reported. A straightforward assignment to a particular tubule segment causing salt retention in GN is still controversial. We hypothesized that 1) trafficking and surface expression of major transporters...

  5. Genome mapping and characterization of the Anopheles gambiae heterochromatin

    Directory of Open Access Journals (Sweden)

    Sharakhova Maria V

    2010-08-01

    Full Text Available Abstract Background Heterochromatin plays an important role in chromosome function and gene regulation. Despite the availability of polytene chromosomes and genome sequence, the heterochromatin of the major malaria vector Anopheles gambiae has not been mapped and characterized. Results To determine the extent of heterochromatin within the An. gambiae genome, genes were physically mapped to the euchromatin-heterochromatin transition zone of polytene chromosomes. The study found that a minimum of 232 genes reside in 16.6 Mb of mapped heterochromatin. Gene ontology analysis revealed that heterochromatin is enriched in genes with DNA-binding and regulatory activities. Immunostaining of the An. gambiae chromosomes with antibodies against Drosophila melanogaster heterochromatin protein 1 (HP1 and the nuclear envelope protein lamin Dm0 identified the major invariable sites of the proteins' localization in all regions of pericentric heterochromatin, diffuse intercalary heterochromatin, and euchromatic region 9C of the 2R arm, but not in the compact intercalary heterochromatin. To better understand the molecular differences among chromatin types, novel Bayesian statistical models were developed to analyze genome features. The study found that heterochromatin and euchromatin differ in gene density and the coverage of retroelements and segmental duplications. The pericentric heterochromatin had the highest coverage of retroelements and tandem repeats, while intercalary heterochromatin was enriched with segmental duplications. We also provide evidence that the diffuse intercalary heterochromatin has a higher coverage of DNA transposable elements, minisatellites, and satellites than does the compact intercalary heterochromatin. The investigation of 42-Mb assembly of unmapped genomic scaffolds showed that it has molecular characteristics similar to cytologically mapped heterochromatin. Conclusions Our results demonstrate that Anopheles polytene chromosomes