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Sample records for midgut ph regulation

  1. Carbonic anhydrases and anion transport in mosquito midgut pH regulation

    PubMed Central

    Linser, Paul J.; Smith, Kristin E.; Seron, Terri J.; Neira Oviedo, Marco

    2009-01-01

    Summary Mosquito larvae use a digestive strategy that is relatively rare in nature. The anterior half of the larval mosquito midgut has a luminal pH that ranges between 10.5 and 11.5. Most other organisms, both large and small, initiate digestion in an acid medium. The relative uniqueness of the highly alkaline digestive strategy has been a long-standing research focus in larval lepidopterans. More recently, the disease vector potential of mosquitoes has fueled specific interest in larval mosquito biology and the alkaline digestive environment in the midgut. The probable principle anion influencing the highly alkaline gut lumen is bicarbonate/carbonate. Bicarbonate/carbonate is regulated at least in part by the activity of carbonic anhydrases. Hence, we have focused attention on the carbonic anhydrases of the mosquito larva. Anopheles gambiae, the major malaria mosquito of Africa, is an organism with a published genome which has facilitated molecular analyses of the 12 carbonic anhydrase genes annotated for this mosquito. Microarray expression analyses, tissue-specific quantitative RT-PCR, and antibody localization have been used to generate a picture of carbonic anhydrase distribution in the larval mosquito. Cytoplasmic, GPI-linked extracellular membrane-bound and soluble extracellular carbonic anhydrases have been located in the midgut and hindgut. The distribution of the enzymes is consistent with an anion regulatory system in which carbonic anhydrases provide a continuous source of bicarbonate/carbonate from the intracellular compartments of certain epithelial cells to the ectoperitrophic space between the epithelial cells and the acellular membrane separating the food bolus from the gut cells and finally into the gut lumen. Carbonic anhydrase in specialized cells of the hindgut (rectum) probably plays a final role in excretion of bicarbonate/carbonate into the aquatic environment of the larva. Detection and characterization of classic anion exchangers of the

  2. Determination of pH in Regions of the Midguts of Acaridid Mites

    PubMed Central

    Erban, Tomas; Hubert, Jan

    2010-01-01

    The pH of the guts of mites strongly affects their digestive processes. This study was carried out to determine the pH in the guts of 12 species of stored product and house dust mites. Eighteen pH indicators were chosen and offered to the mites in the feeding biotest. Based on the color changes of the indicators, the gut contents of acaridid mites were determined to be within a pH range of 4 to neutral. The gut contents showed a gradient in pH from the anterior to the posterior part. The anterior midgut (ventriculus and caeca) of most species had a pH ranging from 4.5 to 5, or slightly more alkaline for most of the species, while the middle midgut (intercolon/colon) had a pH of 5 to 6. Finally, the pH of the posterior midgut (postcolon) was between 5.5 and 7. Except for Dermatophagoides spp., no remarkable differences in the pH of the gut were observed among the tested species. Dermatophagoides spp. had a more acidic anterior midgut (a pH of 4 to 5) and colon (a pH of 5) with postcolon (a pH of below 6). The results characterizing in vivo conditions in the mite gut offer useful information to study the activity of mite digestive enzymes including their inhibitors and gut microflora. PMID:20572792

  3. Mod(mdg4) participates in hormonally regulated midgut programmed cell death during metamorphosis.

    PubMed

    Cai, Mei-Juan; Liu, Wen; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2012-12-01

    The insect midgut undergoes programmed cell death (PCD) during metamorphosis, but the molecular basis for this phenomenon has not been demonstrated. We report a mod(mdg4) protein [designated as mod(mdg4)1A] that is involved in hormonally regulated insect midgut PCD, from the lepidopteran Helicoverpa armigera. Mod(mdg4)1A is localized in the larval midgut and is highly expressed during metamorphosis. Knockdown of mod(mdg4)1a by feeding dsRNA to the larvae suppressed midgut PCD and delayed metamorphosis. The mechanism is that mod(mdg4)1a knockdown decreased the transcript levels of genes involved in PCD and metamorphosis, but increased the transcript level of inhibitor of apoptosis survivin. The transcript level of mod(mdg4)1a is independently upregulated by 20-hydroxyecdysone (20E) or juvenile hormone (JH) analog methoprene. Overlapped 20E and methoprene counteractively regulate the transcript level of mod(mdg4)1a. 20E upregulates the mod(mdg4)1a transcript level not through its nuclear receptor EcRB1. Methoprene upregulates the mod(mdg4)1a transcript level through the juvenile hormone candidate receptor Met. These findings indicate that mod(mdg4)1a participates in midgut PCD and metamorphosis by regulating the transcript levels of a network of genes via different pathways under 20E and JH regulation.

  4. MicroRNA-regulation of Anopheles gambiae immunity to Plasmodium falciparum infection and midgut microbiota.

    PubMed

    Dennison, Nathan J; BenMarzouk-Hidalgo, Omar J; Dimopoulos, George

    2015-03-01

    Invasion of the malaria vector Anopheles gambiae midgut by Plasmodium parasites triggers transcriptional changes of immune genes that mediate the antiparasitic defense. This response is largely regulated by the Toll and Immune deficiency (IMD) pathways. To determine whether A. gambiae microRNAs (miRNAs) are involved in regulating the anti-Plasmodium defense, we showed that suppression of miRNA biogenesis results in increased resistance to Plasmodium falciparum infection. In silico analysis of A. gambiae immune effector genes identified multiple transcripts with miRNA binding sites. A comparative miRNA microarray abundance analysis of P. falciparum infected and naïve mosquito midgut tissues showed elevated abundance of miRNAs aga-miR-989 and aga-miR-305 in infected midguts. Antagomir inhibition of aga-miR-305 increased resistance to P. falciparum infection and suppressed the midgut microbiota. Conversely, treatment of mosquitoes with an artificial aga-miR-305 mimic increased susceptibility to P. falciparum infection and resulted in expansion of midgut microbiota, suggesting that aga-miR-305 acts as a P. falciparum and gut microbiota agonist by negatively regulating the mosquito immune response. In silico prediction of aga-miR-305 target genes identified several anti-Plasmodium effectors. Our study shows that A. gambiae aga-miR-305 regulates the anti-Plasmodium response and midgut microbiota, likely through post-transcriptional modification of immune effector genes.

  5. A tetraspanin regulates septate junction formation in Drosophila midgut.

    PubMed

    Izumi, Yasushi; Motoishi, Minako; Furuse, Kyoko; Furuse, Mikio

    2016-03-15

    Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes through the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of septate junctions are observed; pleated (pSJs) and smooth (sSJs), which are present in ectodermally and endodermally derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and Ssk, in Drosophila indicates that the molecular compositions of sSJs and pSJs differ. A deficiency screen based on immunolocalization of Mesh identified a tetraspanin family protein, Tsp2A, as a newly discovered protein involved in sSJ formation in Drosophila Tsp2A specifically localizes at sSJs in the midgut and Malpighian tubules. Compromised Tsp2A expression caused by RNAi or the CRISPR/Cas9 system was associated with defects in the ultrastructure of sSJs, changed localization of other sSJ proteins, and impaired barrier function of the midgut. In most Tsp2A mutant cells, Mesh failed to localize to sSJs and was distributed through the cytoplasm. Tsp2A forms a complex with Mesh and Ssk and these proteins are mutually interdependent for their localization. These observations suggest that Tsp2A cooperates with Mesh and Ssk to organize sSJs. PMID:26848177

  6. The physiology of the midgut of Lutzomyia longipalpis (Lutz and Neiva 1912): pH in different physiological conditions and mechanisms involved in its control.

    PubMed

    Santos, Vânia C; Araujo, Ricardo N; Machado, Luciane A D; Pereira, Marcos H; Gontijo, Nelder F

    2008-09-01

    Nutrient digestion and absorption after blood feeding are important events for Lutzomyia longipalpis, which uses these nutrients to produce eggs. In this context, the pH inside the digestive tract is an important physiological feature as it can markedly influence the digestive process as well as interfere with Leishmania development in infected phlebotomines. It was described previously that unfed females have an acidic midgut (pH 6). In this study, the pH inside the midgut of blood-fed females was measured. The abdominal midgut (AM) pH varied from 8.15+/-0.31 in the first 10 h post-blood meal to 7.7+/-0.17 after 24 h. While the AM was alkaline during blood digestion, the pH in the thoracic midgut (TM) remained acidic (5.5-6.0). In agreement with these findings, the enzyme alpha-glucosidase, which has an optimum pH of 5.8, is mainly encountered in the acidic TM. The capacity of unfed females to maintain the acidic intestinal pH was also evaluated. Our results showed the presence of an efficient mechanism that maintains the pH almost constant at about 6 in the midgut, but not in the crop. This mechanism is promptly interrupted in the AM by blood ingestion. RT-PCR results indicated the presence of carbonic anhydrase in the midgut cells, which apparently is required to maintain the pH at 6 in the midgut of unfed females. Investigations on the phenomenon of alkalization observed after blood ingestion indicated that two mechanisms are involved: in addition to the alkalization promoted by CO2 volatilization there is a minor contribution from a second mechanism not yet characterized. Some inferences concerning Leishmania development and pH in the digestive tube are presented.

  7. Silencing of Anopheles stephensi Heme Peroxidase HPX15 Activates Diverse Immune Pathways to Regulate the Growth of Midgut Bacteria.

    PubMed

    Kajla, Mithilesh; Choudhury, Tania P; Kakani, Parik; Gupta, Kuldeep; Dhawan, Rini; Gupta, Lalita; Kumar, Sanjeev

    2016-01-01

    Anopheles mosquito midgut harbors a diverse group of endogenous bacteria that grow extensively after the blood feeding and help in food digestion and nutrition in many ways. Although, the growth of endogenous bacteria is regulated by various factors, however, the robust antibacterial immune reactions are generally suppressed in this body compartment by a heme peroxidase HPX15 crosslinked mucins barrier. This barrier is formed on the luminal side of the midgut and blocks the direct interactions and recognition of bacteria or their elicitors by the immune reactive midgut epithelium. We hypothesized that in the absence of HPX15, an increased load of exogenous bacteria will enormously induce the mosquito midgut immunity and this situation in turn, can easily regulate mosquito-pathogen interactions. In this study, we found that the blood feeding induced AsHPX15 gene in Anopheles stephensi midgut and promoted the growth of endogenous as well as exogenous fed bacteria. In addition, the mosquito midgut also efficiently regulated the number of these bacteria through the induction of classical Toll and Imd immune pathways. In case of AsHPX15 silenced midguts, the growth of midgut bacteria was largely reduced through the induction of nitric oxide synthase (NOS) gene, a downstream effector molecule of the JAK/STAT pathway. Interestingly, no significant induction of the classical immune pathways was observed in these midguts. Importantly, the NOS is a well known negative regulator of Plasmodium development, thus, we proposed that the induction of diverged immune pathways in the absence of HPX15 mediated midgut barrier might be one of the strategies to manipulate the vectorial capacity of Anopheles mosquito. PMID:27630620

  8. Silencing of Anopheles stephensi Heme Peroxidase HPX15 Activates Diverse Immune Pathways to Regulate the Growth of Midgut Bacteria

    PubMed Central

    Kajla, Mithilesh; Choudhury, Tania P.; Kakani, Parik; Gupta, Kuldeep; Dhawan, Rini; Gupta, Lalita; Kumar, Sanjeev

    2016-01-01

    Anopheles mosquito midgut harbors a diverse group of endogenous bacteria that grow extensively after the blood feeding and help in food digestion and nutrition in many ways. Although, the growth of endogenous bacteria is regulated by various factors, however, the robust antibacterial immune reactions are generally suppressed in this body compartment by a heme peroxidase HPX15 crosslinked mucins barrier. This barrier is formed on the luminal side of the midgut and blocks the direct interactions and recognition of bacteria or their elicitors by the immune reactive midgut epithelium. We hypothesized that in the absence of HPX15, an increased load of exogenous bacteria will enormously induce the mosquito midgut immunity and this situation in turn, can easily regulate mosquito-pathogen interactions. In this study, we found that the blood feeding induced AsHPX15 gene in Anopheles stephensi midgut and promoted the growth of endogenous as well as exogenous fed bacteria. In addition, the mosquito midgut also efficiently regulated the number of these bacteria through the induction of classical Toll and Imd immune pathways. In case of AsHPX15 silenced midguts, the growth of midgut bacteria was largely reduced through the induction of nitric oxide synthase (NOS) gene, a downstream effector molecule of the JAK/STAT pathway. Interestingly, no significant induction of the classical immune pathways was observed in these midguts. Importantly, the NOS is a well known negative regulator of Plasmodium development, thus, we proposed that the induction of diverged immune pathways in the absence of HPX15 mediated midgut barrier might be one of the strategies to manipulate the vectorial capacity of Anopheles mosquito.

  9. Silencing of Anopheles stephensi Heme Peroxidase HPX15 Activates Diverse Immune Pathways to Regulate the Growth of Midgut Bacteria

    PubMed Central

    Kajla, Mithilesh; Choudhury, Tania P.; Kakani, Parik; Gupta, Kuldeep; Dhawan, Rini; Gupta, Lalita; Kumar, Sanjeev

    2016-01-01

    Anopheles mosquito midgut harbors a diverse group of endogenous bacteria that grow extensively after the blood feeding and help in food digestion and nutrition in many ways. Although, the growth of endogenous bacteria is regulated by various factors, however, the robust antibacterial immune reactions are generally suppressed in this body compartment by a heme peroxidase HPX15 crosslinked mucins barrier. This barrier is formed on the luminal side of the midgut and blocks the direct interactions and recognition of bacteria or their elicitors by the immune reactive midgut epithelium. We hypothesized that in the absence of HPX15, an increased load of exogenous bacteria will enormously induce the mosquito midgut immunity and this situation in turn, can easily regulate mosquito-pathogen interactions. In this study, we found that the blood feeding induced AsHPX15 gene in Anopheles stephensi midgut and promoted the growth of endogenous as well as exogenous fed bacteria. In addition, the mosquito midgut also efficiently regulated the number of these bacteria through the induction of classical Toll and Imd immune pathways. In case of AsHPX15 silenced midguts, the growth of midgut bacteria was largely reduced through the induction of nitric oxide synthase (NOS) gene, a downstream effector molecule of the JAK/STAT pathway. Interestingly, no significant induction of the classical immune pathways was observed in these midguts. Importantly, the NOS is a well known negative regulator of Plasmodium development, thus, we proposed that the induction of diverged immune pathways in the absence of HPX15 mediated midgut barrier might be one of the strategies to manipulate the vectorial capacity of Anopheles mosquito. PMID:27630620

  10. GATAe regulates intestinal stem cell maintenance and differentiation in Drosophila adult midgut.

    PubMed

    Okumura, Takashi; Takeda, Koji; Kuchiki, Megumi; Akaishi, Marie; Taniguchi, Kiichiro; Adachi-Yamada, Takashi

    2016-02-01

    Adult intestinal tissues, exposed to the external environment, play important roles including barrier and nutrient-absorption functions. These functions are ensured by adequately controlled rapid-cell metabolism. GATA transcription factors play essential roles in the development and maintenance of adult intestinal tissues both in vertebrates and invertebrates. We investigated the roles of GATAe, the Drosophila intestinal GATA factor, in adult midgut homeostasis with its first-generated knock-out mutant as well as cell type-specific RNAi and overexpression experiments. Our results indicate that GATAe is essential for proliferation and maintenance of intestinal stem cells (ISCs). Also, GATAe is involved in the differentiation of enterocyte (EC) and enteroendocrine (ee) cells in both Notch (N)-dependent and -independent manner. The results also indicate that GATAe has pivotal roles in maintaining normal epithelial homeostasis of the Drosophila adult midgut through interaction of N signaling. Since recent reports showed that mammalian GATA-6 regulates normal and cancer stem cells in the adult intestinal tract, our data also provide information on the evolutionally conserved roles of GATA factors in stem-cell regulation. PMID:26719127

  11. Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus.

    PubMed

    Crava, Cristina M; Jakubowska, Agata K; Escriche, Baltasar; Herrero, Salvador; Bel, Yolanda

    2015-01-01

    Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity.

  12. Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus

    PubMed Central

    Crava, Cristina M.; Jakubowska, Agata K.; Escriche, Baltasar; Herrero, Salvador; Bel, Yolanda

    2015-01-01

    Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity. PMID:25993013

  13. Human IGF1 Regulates Midgut Oxidative Stress and Epithelial Homeostasis to Balance Lifespan and Plasmodium falciparum resistance in Anopheles stephensi

    PubMed Central

    Drexler, Anna L.; Pietri, Jose E.; Pakpour, Nazzy; Hauck, Eric; Wang, Bo; Glennon, Elizabeth K. K.; Georgis, Martha; Riehle, Michael A.; Luckhart, Shirley

    2014-01-01

    Insulin and insulin-like growth factor signaling (IIS) regulates cell death, repair, autophagy, and renewal in response to stress, damage, and pathogen challenge. Therefore, IIS is fundamental to lifespan and disease resistance. Previously, we showed that insulin-like growth factor 1 (IGF1) within a physiologically relevant range (0.013–0.13 µM) in human blood reduced development of the human parasite Plasmodium falciparum in the Indian malaria mosquito Anopheles stephensi. Low IGF1 (0.013 µM) induced FOXO and p70S6K activation in the midgut and extended mosquito lifespan, whereas high IGF1 (0.13 µM) did not. In this study the physiological effects of low and high IGF1 were examined in detail to infer mechanisms for their dichotomous effects on mosquito resistance and lifespan. Following ingestion, low IGF1 induced phosphorylation of midgut c-Jun-N-terminal kinase (JNK), a critical regulator of epithelial homeostasis, but high IGF1 did not. Low and high IGF1 induced midgut mitochondrial reactive oxygen species (ROS) synthesis and nitric oxide (NO) synthase gene expression, responses which were necessary and sufficient to mediate IGF1 inhibition of P. falciparum development. However, increased ROS and apoptosis-associated caspase-3 activity returned to baseline levels following low IGF1 treatment, but were sustained with high IGF1 treatment and accompanied by aberrant expression of biomarkers for mitophagy, stem cell division and proliferation. Low IGF1-induced ROS are likely moderated by JNK-induced epithelial cytoprotection as well as p70S6K-mediated growth and inhibition of apoptosis over the lifetime of A. stephensi to facilitate midgut homeostasis and enhanced survivorship. Hence, mitochondrial integrity and homeostasis in the midgut, a key signaling center for IIS, can be targeted to coordinately optimize mosquito fitness and anti-pathogen resistance for improved control strategies for malaria and other vector-borne diseases. PMID:24968248

  14. Cadmium Accumulation and Pathological Alterations in the Midgut Gland of Terrestrial Snail Helix pomatia L. from a Zinc Smelter Area: Role of Soil pH.

    PubMed

    Włostowski, Tadeusz; Kozłowski, Paweł; Łaszkiewicz-Tiszczenko, Barbara; Oleńska, Ewa

    2016-04-01

    The purpose of this study was to determine whether cadmium (Cd) accumulation and toxicity in the midgut gland of Helix pomatia snails living in a Cd-contaminated area were related to soil pH. Toxic responses in the midgut gland (i.e., increased vacuolization and lipid peroxidation) occurred in H. pomatia snails exhibiting the highest Cd levels in the gland (265-274 µg/g dry wt) and living on acidic soil (pH 5.3-5.5), while no toxicity was observed in snails accumulating less Cd (90 µg/g) and ranging on neutral soil (pH 7.0), despite the fact that total soil Cd was similar in the two cases. The accumulation of Cd in the gland was directly related to the water extractable Cd in soil, which in turn correlated inversely with soil pH, indicating that this factor had a significant effect on tissue Cd. It appeared further that the occurrence of Cd toxicity was associated with low levels of metallothionein in the gland of snails ranging on acidic soil.

  15. Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis.

    PubMed

    Romanelli, Davide; Casartelli, Morena; Cappellozza, Silvia; de Eguileor, Magda; Tettamanti, Gianluca

    2016-01-01

    We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting. PMID:27609527

  16. Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis

    NASA Astrophysics Data System (ADS)

    Romanelli, Davide; Casartelli, Morena; Cappellozza, Silvia; de Eguileor, Magda; Tettamanti, Gianluca

    2016-09-01

    We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting.

  17. Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis

    PubMed Central

    Romanelli, Davide; Casartelli, Morena; Cappellozza, Silvia; de Eguileor, Magda; Tettamanti, Gianluca

    2016-01-01

    We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting. PMID:27609527

  18. Characterization of Plasmodium developmental transcriptomes in Anopheles gambiae midgut reveals novel regulators of malaria transmission.

    PubMed

    Akinosoglou, Karolina A; Bushell, Ellen S C; Ukegbu, Chiamaka Valerie; Schlegelmilch, Timm; Cho, Jee-Sun; Redmond, Seth; Sala, Katarzyna; Christophides, George K; Vlachou, Dina

    2015-02-01

    The passage through the mosquito is a major bottleneck for malaria parasite populations and a target of interventions aiming to block disease transmission. Here, we used DNA microarrays to profile the developmental transcriptomes of the rodent malaria parasite Plasmodium berghei in vivo, in the midgut of Anopheles gambiae mosquitoes, from parasite stages in the midgut blood bolus to sporulating oocysts on the basal gut wall. Data analysis identified several distinct transcriptional programmes encompassing genes putatively involved in developmental processes or in interactions with the mosquito. At least two of these programmes are associated with the ookinete development that is linked to mosquito midgut invasion and establishment of infection. Targeted disruption by homologous recombination of two of these genes resulted in mutant parasites exhibiting notable infection phenotypes. GAMER encodes a short polypeptide with granular localization in the gametocyte cytoplasm and shows a highly penetrant loss-of-function phenotype manifested as greatly reduced ookinete numbers, linked to impaired male gamete release. HADO encodes a putative magnesium phosphatase with distinctive cortical localization along the concave ookinete periphery. Disruption of HADO compromises ookinete development leading to significant reduction of oocyst numbers. Our data provide important insights into the molecular framework underpinning Plasmodium development in the mosquito and identifies two genes with important functions at initial stages of parasite development in the mosquito midgut.

  19. Molting-associated suppression of symbiont population and up-regulation of antimicrobial activity in the midgut symbiotic organ of the Riptortus-Burkholderia symbiosis.

    PubMed

    Kim, Jiyeun Kate; Han, Sang Heum; Kim, Chan-Hee; Jo, Yong Hun; Futahashi, Ryo; Kikuchi, Yoshitomo; Fukatsu, Takema; Lee, Bok Luel

    2014-03-01

    The majority of insects possess symbiotic bacteria. Since symbiont titers can affect host phenotypes of biological importance, host insects are expected to evolve some mechanisms for regulating symbiont population. Here we report that, in the Riptortus-Burkholderia gut symbiosis, titers of the beneficial symbiont transiently decrease at the pre-molt stages in host development. This molting-associated suppression of the symbiont population is coincident with the increase of antimicrobial activity in the symbiotic midgut, which is observed in both symbiotic and aposymbiotic insects. Two genes, pyrrhocoricin-like antimicrobial peptide and c-type lysozyme, exhibit significantly increased expression in the symbiotic midgut at the pre-molt stages. These results suggest that the molting-associated up-regulation of antimicrobial activity in the symbiotic midgut represents a physiological mechanism of the host insect to regulate symbiosis, which is presumably for defending molting insects against injury and infection and/or for allocating symbiont-derived energy and resources to host molting.

  20. THAP and ATF-2 Regulated Sterol Carrier Protein-2 Promoter Activities in the Larval Midgut of the Yellow Fever Mosquito, Aedes aegypti

    PubMed Central

    Peng, Rong; Fu, Qiang; Hong, Huazhu; Schwaegler, Tyler; Lan, Que

    2012-01-01

    Expression of sterol carrier protein-2 (SCP-2) in Aedes aegypti shows a distinct temporal/spatial pattern throughout the life cycle. In order to identify the transcription factors responsible for the larval temporal/spatial regulation of AeSCP-2 transcription, AeSCP-2 promoter activities were studied in vivo via transient transfection of promoter/reporter gene assays. Regulatory sequences upstream −1.3 kb of the transcription start site of AeSCP-2 were found to be critical for the in vivo temporal/spatial promoter activity. Interestingly, the −1.6 kb promoter sequence efficiently drove the larval midgut-specific siRNA expression, indicating that the −1.6 kb upstream sequence is sufficient for temporal/spatial AeSCP-2 transcriptional activity. Four transcription factors were identified in the midgut nuclear extract from feeding larvae via labeled −1.6/−1.3 kb DNA probe pull-down and proteomic analysis. Co-transfection of the promoter/reporter gene with inducible siRNA expression of each transcription factor was performed to confirm the regulatory function of individual transcription factor on AeSCP-2 transcriptional activities in the larval midgut. The results indicate that two of the identified transcription factors, Thanatos-associated protein (THAP) and activating transcription factor-2 (ATF-2), antagonistically control AeSCP-2 transcriptional activity in the midgut of feeding larvae via the regulatory sequences between −1.6 to −1.3 kb 5′ upstream of the transcription start site. In vivo expression knockdown of THAP and ATF-2 resulted in significant changes in developmental progression, which may be partially due to their effects on AeSCP-2 expression. PMID:23056538

  1. THAP and ATF-2 regulated sterol carrier protein-2 promoter activities in the larval midgut of the yellow fever mosquito, Aedes aegypti.

    PubMed

    Peng, Rong; Fu, Qiang; Hong, Huazhu; Schwaegler, Tyler; Lan, Que

    2012-01-01

    Expression of sterol carrier protein-2 (SCP-2) in Aedes aegypti shows a distinct temporal/spatial pattern throughout the life cycle. In order to identify the transcription factors responsible for the larval temporal/spatial regulation of AeSCP-2 transcription, AeSCP-2 promoter activities were studied in vivo via transient transfection of promoter/reporter gene assays. Regulatory sequences upstream -1.3 kb of the transcription start site of AeSCP-2 were found to be critical for the in vivo temporal/spatial promoter activity. Interestingly, the -1.6 kb promoter sequence efficiently drove the larval midgut-specific siRNA expression, indicating that the -1.6 kb upstream sequence is sufficient for temporal/spatial AeSCP-2 transcriptional activity. Four transcription factors were identified in the midgut nuclear extract from feeding larvae via labeled -1.6/-1.3 kb DNA probe pull-down and proteomic analysis. Co-transfection of the promoter/reporter gene with inducible siRNA expression of each transcription factor was performed to confirm the regulatory function of individual transcription factor on AeSCP-2 transcriptional activities in the larval midgut. The results indicate that two of the identified transcription factors, Thanatos-associated protein (THAP) and activating transcription factor-2 (ATF-2), antagonistically control AeSCP-2 transcriptional activity in the midgut of feeding larvae via the regulatory sequences between -1.6 to -1.3 kb 5' upstream of the transcription start site. In vivo expression knockdown of THAP and ATF-2 resulted in significant changes in developmental progression, which may be partially due to their effects on AeSCP-2 expression. PMID:23056538

  2. Amino acids trigger down-regulation of superoxide via TORC pathway in the midgut of Rhodnius prolixus

    PubMed Central

    Gandara, Ana Caroline P.; Oliveira, José Henrique M.; Nunes, Rodrigo D.; Goncalves, Renata L.S.; Dias, Felipe A.; Hecht, Fabio; Fernandes, Denise C.; Genta, Fernando A.; Laurindo, Francisco R.M.; Oliveira, Marcus F.; Oliveira, Pedro L.

    2016-01-01

    Sensing incoming nutrients is an important and critical event for intestinal cells to sustain life of the whole organism. The TORC is a major protein complex involved in monitoring the nutritional status and is activated by elevated amino acid concentrations. An important feature of haematophagy is that huge amounts of blood are ingested in a single meal, which results in the release of large quantities of amino acids, together with the haemoglobin prosthetic group, haem, which decomposes hydroperoxides and propagates oxygen-derived free radicals. Our previous studies demonstrated that reactive oxygen species (ROS) levels were diminished in the mitochondria and midgut of the Dengue fever mosquito, Aedes aegypti, immediately after a blood meal. We proposed that this mechanism serves to avoid oxidative damage that would otherwise be induced by haem following a blood meal. Studies also performed in mosquitoes have shown that blood or amino acids controls protein synthesis through TORC activation. It was already proposed, in different models, a link between ROS and TOR, however, little is known about TOR signalling in insect midgut nor about the involvement of ROS in this pathway. Here, we studied the effect of a blood meal on ROS production in the midgut of Rhodnius prolixus. We observed that blood meal amino acids decreased ROS levels in the R. prolixus midgut immediately after feeding, via lowering mitochondrial superoxide production and involving the amino acid-sensing TORC pathway. PMID:26945025

  3. The Role of pH Regulation in Cancer Progression.

    PubMed

    McIntyre, Alan; Harris, Adrian L

    2016-01-01

    Frequently observed phenotypes of tumours include high metabolic activity, hypoxia and poor perfusion; these act to produce an acidic microenvironment. Cellular function depends on pH homoeostasis, and thus, tumours become dependent on pH regulatory mechanisms. Many of the proteins involved in pH regulation are highly expressed in tumours, and their expression is often of prognostic significance. The more acidic tumour microenvironment also has important implications with regard to chemotherapeutic and radiotherapeutic interventions. In addition, we review pH-sensing mechanisms, the role of pH regulation in tumour phenotype and the use of pH regulatory mechanisms as therapeutic targets. PMID:27557536

  4. Preferential intracellular pH regulation: hypotheses and perspectives.

    PubMed

    Shartau, Ryan B; Baker, Daniel W; Crossley, Dane A; Brauner, Colin J

    2016-08-01

    The regulation of vertebrate acid-base balance during acute episodes of elevated internal PCO2  is typically characterized by extracellular pH (pHe) regulation. Changes in pHe are associated with qualitatively similar changes in intracellular tissue pH (pHi) as the two are typically coupled, referred to as 'coupled pH regulation'. However, not all vertebrates rely on coupled pH regulation; instead, some preferentially regulate pHi against severe and maintained reductions in pHe Preferential pHi regulation has been identified in several adult fish species and an aquatic amphibian, but never in adult amniotes. Recently, common snapping turtles were observed to preferentially regulate pHi during development; the pattern of acid-base regulation in these species shifts from preferential pHi regulation in embryos to coupled pH regulation in adults. In this Commentary, we discuss the hypothesis that preferential pHi regulation may be a general strategy employed by vertebrate embryos in order to maintain acid-base homeostasis during severe acute acid-base disturbances. In adult vertebrates, the retention or loss of preferential pHi regulation may depend on selection pressures associated with the environment inhabited and/or the severity of acid-base regulatory challenges to which they are exposed. We also consider the idea that the retention of preferential pHi regulation into adulthood may have been a key event in vertebrate evolution, with implications for the invasion of freshwater habitats, the evolution of air breathing and the transition of vertebrates from water to land. PMID:27489212

  5. [Regulation effects of tourmaline on seawater pH value].

    PubMed

    Xia, Meisheng; Zhang, Hongmei; Hu, Caihong; Xu, Zirong

    2005-10-01

    In this paper, chemical analysis, X-ray diffraction and atomic force microscopy were employed to examine the characteristics of tourmaline produced in east Inner Mongolia Autonomous Region, and batch experiments were conducted to study its regulation effects on seawater pH value. The factors affecting the regulation, such as the dosage of tourmaline and the salinity and initial pH value of seawater, were also studied. The results showed that tourmaline could regulate the seawater pH value from its initial 3 and 10 to 7.1 and 8.9, respectively, and the regulation effect was greater in the seawater with lower salinity, e.g., after 120 minutes treatment, the initial pH value (5.0) of the seawater with a salinity of 5, 10, 15, 20 and 35 was increased by 3.24, 3.16, 3.06, 2.99 and 2.85 unit, respectively. Tourmaline had little effect on seawater conductivity. This study would provide an experimental base for the application of tourmaline in aquaculture. PMID:16422525

  6. [Regulation effects of tourmaline on seawater pH value].

    PubMed

    Xia, Meisheng; Zhang, Hongmei; Hu, Caihong; Xu, Zirong

    2005-10-01

    In this paper, chemical analysis, X-ray diffraction and atomic force microscopy were employed to examine the characteristics of tourmaline produced in east Inner Mongolia Autonomous Region, and batch experiments were conducted to study its regulation effects on seawater pH value. The factors affecting the regulation, such as the dosage of tourmaline and the salinity and initial pH value of seawater, were also studied. The results showed that tourmaline could regulate the seawater pH value from its initial 3 and 10 to 7.1 and 8.9, respectively, and the regulation effect was greater in the seawater with lower salinity, e.g., after 120 minutes treatment, the initial pH value (5.0) of the seawater with a salinity of 5, 10, 15, 20 and 35 was increased by 3.24, 3.16, 3.06, 2.99 and 2.85 unit, respectively. Tourmaline had little effect on seawater conductivity. This study would provide an experimental base for the application of tourmaline in aquaculture.

  7. A novel arrangement of midgut epithelium and hepatic cells implies a novel regulation of the insulin signaling pathway in long-lived millipedes.

    PubMed

    Nardi, James B; Miller, Lou Ann; Bee, Charles M

    2016-01-01

    Nutrients absorbed by the epithelial cells of the millipede midgut are channeled to a contiguous population of hepatic cells where sugars are stored as glycogen. In insects and other arthropods, however, nutrients absorbed by midgut epithelia are first passed across the epithelial basal surface to the hemolymph before storage in fat body. The inter-digitation of cellular processes at the interface of hepatic and midgut epithelial cells offers a vast surface area for exchange of nutrients. At this interface, numerous small vesicles with the dimensions of exosomes (∼30nm) may represent the mediators of nutrient exchange. Longevity and the developmental arrest of diapause are associated with reduced insulin signaling. The long lifespans for which millipedes are known may be attributable to a novel pathway with reduced insulin signaling represented by the novel arrangement of hepatic storage cells and midgut epithelial absorbing cells. PMID:27373842

  8. Regulation of Vacuolar pH in Citrus limon

    SciTech Connect

    Lincoln Taiz

    2005-06-22

    The primary objective of this grant was to characterize the vacuolar V-ATPase of lemon fruits. Lemon fruit vacuoles have an internal pH of about 2.5. Since a typical plant vacuole has a luminal pH of around 5.5, the lemon fruit V-APTase must have special properties which allow it to acidify the lumen to such a low pH: (1) it might have a different structure; (2) it might have a different H{sup +}/ATP stoichiometry; and (3) it might be regulated differently. During the course of the investigations (which began in 1996) they characterized these aspects of the V-ATPases of both lemon fruits and lime fruits. They examined lime fruits because of the availability of both acidic limes with a low vacuolar pH and sweet limes, which have a much higher vacuolar pH. The existence of two types of lime fruits allowed a comparison of the V-ATPases of the two varieties. In this report they are including two publications from 1996 and 1997 as background for the later publications. A review article with Heven Sze on V-ATPase nomenclature was also generated during the funding period. In addition to the studies on citrus fruit vacuoles, they also initiated studies in two new areas: polar auxin transport and the regulation of stomatal opening by UV-B irradiation. These studies were intended to serve as a basis of future separate grants, but the proposals they submitted on these topics were not funded.

  9. Identification and profiling of Manduca sexta microRNAs and their possible roles in regulating specific transcripts in fat body, hemocytes, and midgut

    PubMed Central

    Zhang, Xiufeng; Zheng, Yun; Cao, Xiaolong; Ren, Ren; Yu, Xiao-Qiang; Jiang, Haobo

    2014-01-01

    Significance of microRNA-mediated posttranscriptional regulation has been appreciated ever since its discovery. In the tobacco hornworm Manduca sexta, 164 conserved and 16 novel microRNAs have been identified experimentally (Zhang et al., 2012, 2014). To extend the list of microRNAs in this lepidopteran model species and further explore their possible regulatory roles, we constructed and sequenced small RNA libraries of M. sexta fat body, hemocytes and midgut, since transcriptomes of these tissues from the 5th instar larvae had been studied quite extensively. Each library represented a mixture of the same tissues from larvae that were naïve or induced by three different pathogens. From a total of 167 million reads obtained, we identified two new variants of conserved miR-281 and miR-305 and six novel microRNAs. Abundances of all microRNAs were normalized and compared to reveal their differential expression in these three tissues. Star strands of ten microRNAs were present at higher levels than the corresponding mature strands. From a list of tissue-specific transcripts, we predicted target sites in 3′-UTRs using preferentially expressed microRNA groups in each tissue and suggested possible regulatory roles of these microRNAs in energy metabolism, insecticide resistance, and some mitochondrial and immune gene expression. Examining manifold targets, microRNA regulations were suggested of multiple physiological processes. This study has enriched our knowledge of M. sexta microRNAs and how microRNAs potentially coordinate different physiological processes. PMID:25196249

  10. Intracellular pH regulation in chicken enterocytes: the importance of extracellular pH.

    PubMed

    Peral, M J; Calonge, M L; Ilundáin, A A

    1995-11-01

    The present work reports the effect of pHo on pHi and Na(+)-H+ exchanger activity. Intracellular pH tended to follow pHo, but the proton distribution across the cell membrane is not at electrochemical equilibrium. Removal of external Na+ acidified the cells by both reversing the direction of the Na(+)-H+ exchanger and hyperpolarizing the cell membrane potential. The relationship between pHo and the rate of Na(+)-dependent proton efflux following an acid load suggests that external protons interact with the Na(+)-H+ exchanger at a single site with an apparent pK (-log of the dissociation constant) of 7.22. The results demonstrate that maintenance of pHo in the physiological range is essential for maintenance of normal cell pH and that the activity of the Na(+)-H+ exchanger involved in pHi regulation is affected by external protons. The results also suggest that, at least at low pHo, some intracellular mechanism is involved in pHi regulation. PMID:8962700

  11. CPB1 of Aedes aegypti interacts with DENV2 E protein and regulates intracellular viral accumulation and release from midgut cells.

    PubMed

    Tham, Hong-Wai; Balasubramaniam, Vinod R M T; Tejo, Bimo Ario; Ahmad, Hamdan; Hassan, Sharifah Syed

    2014-12-01

    Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV). To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H) screenings against DENV2 envelope (E) protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1) was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER) of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network. PMID:25521592

  12. CPB1 of Aedes aegypti interacts with DENV2 E protein and regulates intracellular viral accumulation and release from midgut cells.

    PubMed

    Tham, Hong-Wai; Balasubramaniam, Vinod R M T; Tejo, Bimo Ario; Ahmad, Hamdan; Hassan, Sharifah Syed

    2014-12-16

    Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV). To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H) screenings against DENV2 envelope (E) protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1) was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER) of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network.

  13. Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae.

    PubMed

    Johnson, D E; Brookhart, G L; Kramer, K J; Barnett, B D; McGaughey, W H

    1990-03-01

    Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.

  14. Ventilatory regulation of arterial H(+) (pH) during exercise.

    PubMed

    Wasserman, Karlman; Cox, Timothy A; Sietsema, Kathy E

    2014-01-01

    We hypothesized that exercise ventilation and arterial H(+) ([H(+)]a) are mutually interactive, [H(+)]a stimulating V(E) and V(E) regulating [H(+)]a increase. Fifty-five patients were studied, 10 normal and 45 with cardio-respiratory disorders. Each patient underwent cardiopulmonary exercise testing with simultaneous serial arterial blood gas and pH measurements. Subsequently, they were classified into one of 7 clinical groups: (1) normal, (2) exercise-induced hypoxemia (PaO2<50mmHg), (3) exercise-induced myocardial ischemia, (4) heart failure, (5) COPD, (6) interstitial lung disease, and (7) pulmonary vasculopathy. The average resting pHa was 7.42 or 7.43 for each group. At anaerobic (lactic acidosis) threshold (AT), [H(+)]a increased due to PaCO2 increase (+2mmHg), primarily. At peak exercise, [H(+)]a increased further due to arterial HCO3(-) decrease. In summary, [H(+)]a appears to be closely regulated at rest to AT and further to peak exercise by CO2 elimination from the venous return. No evidence was observed for over-ventilation of CO2, causing the arterial blood to become more alkaline during exercise in the patient groups studied.

  15. pH regulation in adult cardiac myocytes

    SciTech Connect

    Wallert, M.A.

    1989-01-01

    The purpose of this study is to examine the pH{sub i} regulatory mechanisms of adult ventricular myocytes, the cells that perform the pumping work of the heart. The cell system for this study was the ventricular myocyte, isolated by enzymatic dissociation from adult rate heart. In agreement with the findings on other cardiac model cells, I demonstrated the existence of a Cl{sup {minus}}/HCO{sub 3}{sup {minus}} exchanger and a Na{sup +}/H{sup +} exchanger in ventricular myocytes. The existence of the anion exchanger was demonstrated in {sup 36}Cl{sup {minus}} flux experiments and as stilbene disulfonate-inhibitable and Cl{sup {minus}} gradient-dependent intracellular pH shifts in the presence of bicarbonate. The fluorescein derivative BCECF served as a fluorescent probe of intracellular pH in the these experiments. The existence of the Na{sup +}/H{sup +} exchanger was demonstrated in pH{sub i} experiments using BCECF. Further experiments characterized the kinetics of the Na{sup +}/H{sup +} exchanger and its regulation. The steady-state pH{sub i} of ventricular myocytes was 7.16 {+-} 0.11 at pH{sub 0} = 7.4. Several agonists caused a rise in steady-state pH{sub i}: the protein kinase stimulator phorbol myristate acetate (PMA), the {alpha}{sub 1}-adrenergic agonist 6-fluoro-norepinephrine (6F-NE) and the {beta}-agonist UK14304, and ATP.

  16. Influence of pH Regulation Mode in Glucose Fermentation on Product Selection and Process Stability.

    PubMed

    Mohd-Zaki, Zuhaida; Bastidas-Oyanedel, Juan R; Lu, Yang; Hoelzle, Robert; Pratt, Steven; Slater, Fran R; Batstone, Damien J

    2016-01-01

    Mixed culture anaerobic fermentation generates a wide range of products from simple sugars, and is potentially an effective process for producing renewable commodity chemicals. However it is difficult to predict product spectrum, and to control the process. One of the key control handles is pH, but the response is commonly dependent on culture history. In this work, we assess the impact of pH regulation mode on the product spectrum. Two regulation modes were applied: in the first, pH was adjusted from 4.5 to 8.5 in progressive steps of 0.5 and in the second, covered the same pH range, but the pH was reset to 5.5 before each change. Acetate, butyrate, and ethanol were produced throughout all pH ranges, but there was a shift from butyrate at pH < 6.5 to ethanol at pH > 6.5, as well as a strong and consistent shift from hydrogen to formate as pH increased. Microbial analysis indicated that progressive pH resulted in dominance by Klebsiella, while reset pH resulted in a bias towards Clostridium spp., particularly at low pH, with higher variance in community between different pH levels. Reset pH was more responsive to changes in pH, and analysis of Gibbs free energy indicated that the reset pH experiments operated closer to thermodynamic equilibrium, particularly with respect to the formate/hydrogen balance. This may indicate that periodically resetting pH conforms better to thermodynamic expectations. PMID:27681895

  17. Influence of pH Regulation Mode in Glucose Fermentation on Product Selection and Process Stability

    PubMed Central

    Mohd-Zaki, Zuhaida; Bastidas-Oyanedel, Juan R.; Lu, Yang; Hoelzle, Robert; Pratt, Steven; Slater, Fran R.; Batstone, Damien J.

    2016-01-01

    Mixed culture anaerobic fermentation generates a wide range of products from simple sugars, and is potentially an effective process for producing renewable commodity chemicals. However it is difficult to predict product spectrum, and to control the process. One of the key control handles is pH, but the response is commonly dependent on culture history. In this work, we assess the impact of pH regulation mode on the product spectrum. Two regulation modes were applied: in the first, pH was adjusted from 4.5 to 8.5 in progressive steps of 0.5 and in the second, covered the same pH range, but the pH was reset to 5.5 before each change. Acetate, butyrate, and ethanol were produced throughout all pH ranges, but there was a shift from butyrate at pH < 6.5 to ethanol at pH > 6.5, as well as a strong and consistent shift from hydrogen to formate as pH increased. Microbial analysis indicated that progressive pH resulted in dominance by Klebsiella, while reset pH resulted in a bias towards Clostridium spp., particularly at low pH, with higher variance in community between different pH levels. Reset pH was more responsive to changes in pH, and analysis of Gibbs free energy indicated that the reset pH experiments operated closer to thermodynamic equilibrium, particularly with respect to the formate/hydrogen balance. This may indicate that periodically resetting pH conforms better to thermodynamic expectations. PMID:27681895

  18. Influence of pH Regulation Mode in Glucose Fermentation on Product Selection and Process Stability

    PubMed Central

    Mohd-Zaki, Zuhaida; Bastidas-Oyanedel, Juan R.; Lu, Yang; Hoelzle, Robert; Pratt, Steven; Slater, Fran R.; Batstone, Damien J.

    2016-01-01

    Mixed culture anaerobic fermentation generates a wide range of products from simple sugars, and is potentially an effective process for producing renewable commodity chemicals. However it is difficult to predict product spectrum, and to control the process. One of the key control handles is pH, but the response is commonly dependent on culture history. In this work, we assess the impact of pH regulation mode on the product spectrum. Two regulation modes were applied: in the first, pH was adjusted from 4.5 to 8.5 in progressive steps of 0.5 and in the second, covered the same pH range, but the pH was reset to 5.5 before each change. Acetate, butyrate, and ethanol were produced throughout all pH ranges, but there was a shift from butyrate at pH < 6.5 to ethanol at pH > 6.5, as well as a strong and consistent shift from hydrogen to formate as pH increased. Microbial analysis indicated that progressive pH resulted in dominance by Klebsiella, while reset pH resulted in a bias towards Clostridium spp., particularly at low pH, with higher variance in community between different pH levels. Reset pH was more responsive to changes in pH, and analysis of Gibbs free energy indicated that the reset pH experiments operated closer to thermodynamic equilibrium, particularly with respect to the formate/hydrogen balance. This may indicate that periodically resetting pH conforms better to thermodynamic expectations.

  19. Extracellular pH regulates excitability of vomeronasal sensory neurons.

    PubMed

    Cichy, Annika; Ackels, Tobias; Tsitoura, Chryssanthi; Kahan, Anat; Gronloh, Nina; Söchtig, Melanie; Engelhardt, Corinna H; Ben-Shaul, Yoram; Müller, Frank; Spehr, Jennifer; Spehr, Marc

    2015-03-01

    The mouse vomeronasal organ (VNO) plays a critical role in semiochemical detection and social communication. Vomeronasal stimuli are typically secreted in various body fluids. Following direct contact with urine deposits or other secretions, a peristaltic vascular pump mediates fluid entry into the recipient's VNO. Therefore, while vomeronasal sensory neurons (VSNs) sample various stimulatory semiochemicals dissolved in the intraluminal mucus, they might also be affected by the general physicochemical properties of the "solvent." Here, we report cycle stage-correlated variations in urinary pH among female mice. Estrus-specific pH decline is observed exclusively in urine samples from sexually experienced females. Moreover, patch-clamp recordings in acute VNO slices reveal that mouse VSNs reliably detect extracellular acidosis. Acid-evoked responses share the biophysical and pharmacological hallmarks of the hyperpolarization-activated current Ih. Mechanistically, VSN acid sensitivity depends on a pH-induced shift in the voltage-dependence of Ih activation that causes the opening of HCN channels at rest, thereby increasing VSN excitability. Together, our results identify extracellular acidification as a potent activator of vomeronasal Ih and suggest HCN channel-dependent vomeronasal gain control of social chemosignaling. Our data thus reveal a potential mechanistic basis for stimulus pH detection in rodent chemosensory communication. PMID:25740530

  20. Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut

    PubMed Central

    Boudko, Dmitri Y.; Moroz, Leonid L.; Harvey, William R.; Linser, Paul J.

    2001-01-01

    The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH ≈ 3 units⋅mm−1) in which an extremely alkaline (pH ≈ 11) anterior midgut lies between near-neutral posterior midgut and gastric cecum (pH 7–8). A plasma membrane H+ V-ATPase energizes this alkalinization but the ion carriers involved are unknown. Capillary zone electrophoresis of body samples with outlet conductivity detection showed a specific transepithelial distribution of chloride and bicarbonate/carbonate ions, with high concentrations of both anions in the midgut tissue: 68.3 ± 5.64 and 50.8 ± 4.21 mM, respectively. Chloride was higher in the hemolymph, 57.6 ± 7.84, than in the lumen, 3.51 ± 2.58, whereas bicarbonate was higher in the lumen, 58.1 ± 7.34, than the hemolymph, 3.96 ± 2.89. Time-lapse video assays of pH profiles in vivo revealed that ingestion of the carbonic anhydrase inhibitor acetazolamide and the ion exchange inhibitor DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid), at 10−4 M eliminates lumen alkalinization. Basal application of these inhibitors in situ also reduced gradients recorded with self-referencing pH-sensitive microelectrodes near the basal membrane by ≈65% and 85% respectively. Self-referencing chloride-selective microelectrodes revealed a specific spatial profile of transepithelial chloride transport with an efflux maximum in anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes. These data suggest that an H+ V-ATPase-energized anion exchange occurs across the apical membrane of the epithelial cells and implicate an electrophoretic Cl−/HCO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{3}^{-}}}\\end{equation*}\\end{document} exchanger and carbonic anhydrase as

  1. A regulatory network controls nephrocan expression and midgut patterning

    PubMed Central

    Hou, Juan; Wei, Wei; Saund, Ranajeet S.; Xiang, Ping; Cunningham, Thomas J.; Yi, Yuyin; Alder, Olivia; Lu, Daphne Y. D.; Savory, Joanne G. A.; Krentz, Nicole A. J.; Montpetit, Rachel; Cullum, Rebecca; Hofs, Nicole; Lohnes, David; Humphries, R. Keith; Yamanaka, Yojiro; Duester, Gregg; Saijoh, Yukio; Hoodless, Pamela A.

    2014-01-01

    Although many regulatory networks involved in defining definitive endoderm have been identified, the mechanisms through which these networks interact to pattern the endoderm are less well understood. To explore the mechanisms involved in midgut patterning, we dissected the transcriptional regulatory elements of nephrocan (Nepn), the earliest known midgut specific gene in mice. We observed that Nepn expression is dramatically reduced in Sox17−/− and Raldh2−/− embryos compared with wild-type embryos. We further show that Nepn is directly regulated by Sox17 and the retinoic acid (RA) receptor via two enhancer elements located upstream of the gene. Moreover, Nepn expression is modulated by Activin signaling, with high levels inhibiting and low levels enhancing RA-dependent expression. In Foxh1−/− embryos in which Nodal signaling is reduced, the Nepn expression domain is expanded into the anterior gut region, confirming that Nodal signaling can modulate its expression in vivo. Together, Sox17 is required for Nepn expression in the definitive endoderm, while RA signaling restricts expression to the midgut region. A balance of Nodal/Activin signaling regulates the anterior boundary of the midgut expression domain. PMID:25209250

  2. Regulation of lung surfactant secretion by intracellular pH.

    PubMed

    Chander, A

    1989-12-01

    We investigated secretion of lung surfactant phosphatidylcholine (PC) using isolated perfused rat lung preparation after labeling the lung lipids in vitro with [methyl-3H]choline. The perfusion medium was Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM glucose and 3% fatty acid-poor bovine serum albumin. After ventilation of lungs with air containing 5% CO2 (control) for 1 h, 0.91% +/- 0.04 (mean +/- SE, n = 6) of total lung lipid radioactivity (greater than 95% in PC) was recovered in the cell-free lavage fluid. The secretion of PC was increased with terbutaline (50 microM), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 100 microM), phorbol L2-myristate 13-acetate (30 ng/ml), and ATP (1 mM), in each case by approximately 150%. Secretion of PC was also increased by 160% if the lungs were ventilated with air containing 0% CO2. The low CO2-mediated PC secretion was time and concentration dependent. The dose-response curve for 0-10% CO2 was S-shaped. The low CO2-induced increase in PC secretion could be largely reversed with diffusible weak acids (25 mM, acetate or butyrate) in the perfusion medium. An increase (70%) in secretion was also induced with 10 mM NH4Cl, suggesting a role for intracellular alkalosis. These observations suggest that intracellular alkalosis stimulates lung surfactant secretion. Alkalosis-stimulated secretion of PC was additive with that with terbutaline (5 X 10(-7) to 5 X 10(-4) M) or 10(-4) M 8-BrcAMP, suggesting that alkalosis effect was not mediated through the beta-adrenergic pathway of surfactant secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2514603

  3. Intestinal malrotation and midgut volvulus.

    PubMed

    Hamidi, Hidayatullah; Obaidy, Yalda; Maroof, Sahar

    2016-09-01

    A four-day-old boy presented with persistent bilious vomiting, bloody stained stool, and mild abdominal distension. Transabdominal ultrasound demonstrated a round soft-tissue mass-like structure in the right upper quadrant. With color Doppler ultrasound, the whirlpool sign was observed. Abdominal radiograph showed nonspecific findings. Upper gastrointestinal series revealed upper gastrointestinal tract obstruction at the level of distal duodenum. The diagnosis of intestinal malrotation with midgut volvulus was established and the treated surgically. Intestinal malrotation is congenital abnormal positioning of the bowel loops within the peritoneal cavity resulting in abnormal shortening of mesenteric root that is predisposed to midgut volvulus. Neonates and infants with persistent bilious vomiting should undergo diagnostic workup and preferably ultrasound as the first step. With classic sonographic appearance of whirlpool sign, even further imaging investigations is often not needed, and the surgeon should be alerted to plan surgery. PMID:27594965

  4. pH sensing via bicarbonate-regulated “soluble” adenylyl cyclase (sAC)

    PubMed Central

    Rahman, Nawreen; Buck, Jochen; Levin, Lonny R.

    2013-01-01

    Soluble adenylyl cyclase (sAC) is a source of the second messenger cyclic adenosine 3′, 5′ monophosphate (cAMP). sAC is directly regulated by bicarbonate (HCO−3) ions. In living cells, HCO−3 ions are in nearly instantaneous equilibrium with carbon dioxide (CO2) and pH due to the ubiquitous presence of carbonic anhydrases. Numerous biological processes are regulated by CO2, HCO−3, and/or pH, and in a number of these, sAC has been shown to function as a physiological CO2/HCO3/pH sensor. In this review, we detail the known pH sensing functions of sAC, and we discuss two highly-studied, pH-dependent pathways in which sAC might play a role. PMID:24324443

  5. Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation

    PubMed Central

    Elferich, Johannes; Williamson, Danielle M.; Krishnamoorthy, Bala; Shinde, Ujwal

    2013-01-01

    Eukaryotic cells maintain strict control over protein secretion, in part by using the pH gradient maintained within their secretory pathway. How eukaryotic proteins evolved from prokaryotic orthologs to exploit the pH gradient for biological functions remains a fundamental question in cell biology. Our laboratory previously demonstrated that protein domains located within precursor proteins, propeptides, encode histidine-driven pH sensors to regulate organelle-specific activation of the eukaryotic proteases furin and proprotein convertase-1/3. Similar findings have been reported in other unrelated protease families. By analyzing >10,000 unique proteases within evolutionarily unrelated families, we show that eukaryotic propeptides are enriched in histidines compared with prokaryotic orthologs. On this basis, we hypothesize that eukaryotic proteins evolved to enrich histidines within their propeptides to exploit the tightly controlled pH gradient of the secretory pathway, thereby regulating activation within specific organelles. Enrichment of histidines in propeptides may therefore be used to predict the presence of pH sensors in other proteases or even protease substrates.—Elferich, J., Williamson, D. M., Krishnamoorthy, B., Shinde, U. Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation. PMID:23585398

  6. A semipermeable enzymatic nanoreactor as an efficient modulator for reversible pH regulation

    NASA Astrophysics Data System (ADS)

    Huang, Yanyan; Lin, Youhui; Ran, Xiang; Ren, Jinsong; Qu, Xiaogang

    2014-09-01

    Here we propose a new concept for the fabrication of a semipermeable enzymatic nanoreactor as an efficient modulator to reversibly switch the pH of an aqueous environment. We used amino-functionalized, expanded mesoporous silica nanoparticles (EMSN) as a model nanocarrier to load enzymes. In order to protect enzymes from the interference of a complicated environment, polyelectrolyte multilayers (PEMs) were coated on the surface of the EMSN through layer by layer (LbL) assembly. These PEMs can serve as semipermeable membranes, allowing small molecules to diffuse in and out freely while trapping the enzymes in the nanoreactors. Compared with traditional electrochemical stimulation or optical control methods, our enzymatic regulation platform is easy to operate without complicated instruments. In addition, this system can cover a wide range of pH values and conveniently regulate pH values by simply controlling the concentrations of catalysts or reactants. Meanwhile, this strategy could be generalized to other enzymes or nanocarriers to achieve reversible pH regulation for different purposes. The switched pH values can be implemented for the modulation of the conformational changes of nucleic acids and activation of the charge conversion in drug delivery applications.Here we propose a new concept for the fabrication of a semipermeable enzymatic nanoreactor as an efficient modulator to reversibly switch the pH of an aqueous environment. We used amino-functionalized, expanded mesoporous silica nanoparticles (EMSN) as a model nanocarrier to load enzymes. In order to protect enzymes from the interference of a complicated environment, polyelectrolyte multilayers (PEMs) were coated on the surface of the EMSN through layer by layer (LbL) assembly. These PEMs can serve as semipermeable membranes, allowing small molecules to diffuse in and out freely while trapping the enzymes in the nanoreactors. Compared with traditional electrochemical stimulation or optical control methods

  7. Mechanisms of pH regulation in lamprey (Lampetra fluviatilis) red blood cells.

    PubMed

    Nikinmaa, M; Kunnamo-Ojala, T; Railo, E

    1986-05-01

    Mechanisms regulating the red cell pH in lamprey (Lampetra fluviatilis) were studied using the ammonium chloride prepulse technique. The cells were initially incubated in a physiological saline containing 20 mmol l-1 ammonium chloride, and intracellular pH measured with the DMO technique. Ammonium chloride was then rapidly removed by centrifugation, and the changes in the intracellular pH followed. The intraerythrocytic pH is primarily regulated by an amiloride-sensitive sodium/proton exchange. When sodium is present in the incubation medium, the intracellular pH rapidly recovers from the acidification associated with the removal of ammonium chloride from the incubation. When sodium is removed from the incubation medium, intracellular pH does not recover, and when the cells are treated with 10(-3) mol l-1 amiloride in the presence of sodium, carbon dioxide and bicarbonate, the intracellular pH recovery is drastically reduced. The movements of carbon dioxide, its consecutive catalysed hydration and dissociation to protons and bicarbonate and, possibly, movements of bicarbonate out of the cell acidify the cell contents. This is shown by the observation that the steady-state intracellular pH is higher in a HEPES-buffered medium than in a CO2/HCO3(-)-buffered medium at the same extracellular pH. The acidification is dependent on cellular carbonic anhydrase activity, present in lamprey red cells, which speeds up the hydration reaction. When the action of carbonic anhydrase is inhibited by acetazolamide, removal of ammonium chloride from the incubation medium does not cause intracellular acidification. PMID:3088194

  8. Comparison of midgut bacterial diversity in tropical caterpillars (Lepidoptera: Saturniidae) fed on different diets.

    PubMed

    Pinto-Tomás, Adrián A; Sittenfeld, Ana; Uribe-Lorío, Lorena; Chavarría, Felipe; Mora, Marielos; Janzen, Daniel H; Goodman, Robert M; Simon, Holly M

    2011-10-01

    As primary consumers of foliage, caterpillars play essential roles in shaping the trophic structure of tropical forests. The caterpillar midgut is specialized in plant tissue processing; its pH is exceptionally alkaline and contains high concentrations of toxic compounds derived from the ingested plant material (secondary compounds or allelochemicals) and from the insect itself. The midgut, therefore, represents an extreme environment for microbial life. Isolates from different bacterial taxa have been recovered from caterpillar midguts, but little is known about the impact of these microorganisms on caterpillar biology. Our long-term goals are to identify midgut symbionts and to investigate their functions. As a first step, different diet formulations were evaluated for rearing two species of tropical saturniid caterpillars. Using the polymerase chain reaction (PCR) with primers hybridizing broadly to sequences from the bacterial domain, 16S rRNA gene libraries were constructed with midgut DNA extracted from caterpillars reared on different diets. Amplified rDNA restriction analysis indicated that bacterial sequences recovered from the midguts of caterpillars fed on foliage were more diverse than those from caterpillars fed on artificial diet. Sequences related to Methylobacterium sp., Bradyrhizobium sp., and Propionibacterium sp. were detected in all caterpillar libraries regardless of diet, but were not detected in a library constructed from the diet itself. Furthermore, libraries constructed with DNA recovered from surface-sterilized eggs indicated potential for vertical transmission of midgut symbionts. Taken together, these results suggest that microorganisms associated with the tropical caterpillar midgut may engage in symbiotic interactions with these ecologically important insects.

  9. Effects of local pH on the formation and regulation of cristae morphologies

    NASA Astrophysics Data System (ADS)

    Song, Dong Hoon; Park, Jonghyun; Philbert, Martin A.; Sastry, Ann Marie; Lu, Wei

    2014-08-01

    Cristae, folded subcompartments of the inner mitochondrial membrane (IMM), have complex and dynamic morphologies. Since cristae are the major site of adenosine triphosphate synthesis, morphological changes of cristae have been studied in relation to functional states of mitochondria. In this sense, investigating the functional and structural significance of cristae may be critical for understanding progressive mitochondrial dysfunction. However, the detailed mechanisms of the formation and regulation of these cristae structures have not been fully elucidated. Among the hypotheses concerning the regulation of cristae morphologies, we exclusively investigate the effects of the local pH gradient on the cristae morphologies by using a numerical model. An area-difference induced curvature of the membrane is modeled as a function of local pH. This curvature is then applied to the finite element model of a closed lipid bilayer in order to find the energetically favorable membrane configuration. From this study, we substantiate the hypothesis that a tubular crista structure can be formed and regulated by the local pH gradient. Through the simulations with various initial conditions, we further demonstrate that the diameter of a crista is mainly determined by the local pH gradient, and the energetically favorable direction of crista growth is perpendicular to the longitudinal axis of a mitochondrion. Finally, the simulation results at the mitochondrial scale suggest that the cristae membrane may have a lower local pH value and/or a higher cardiolipin composition than the other parts of the IMM.

  10. Intestinal stem cells in the adult Drosophila midgut

    SciTech Connect

    Jiang, Huaqi; Edgar, Bruce A.

    2011-11-15

    Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury. -- Highlights: Black-Right-Pointing-Pointer The homeostasis and regeneration of adult fly midguts are mediated by ISCs. Black-Right-Pointing-Pointer Damaged enterocytes induce the proliferation of intestinal stem cells (ISC). Black-Right-Pointing-Pointer EGFR and Jak/Stat signalings mediate compensatory ISC proliferation. Black-Right-Pointing-Pointer Notch signaling regulates ISC self-renewal and differentiation.

  11. Preferential intracellular pH regulation represents a general pattern of pH homeostasis during acid-base disturbances in the armoured catfish, Pterygoplichthys pardalis.

    PubMed

    Harter, T S; Shartau, R B; Baker, D W; Jackson, D C; Val, A L; Brauner, C J

    2014-08-01

    Preferential intracellular pH (pHi) regulation, where pHi is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO2. To determine whether preferential pHi regulation may represent a general pattern for acid-base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2-3 h following the respective treatments and extracellular blood pH, pHi of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO2 were measured. All treatments resulted in significant changes in extracellular pH and RBC pHi that likely cover a large portion of the pH tolerance limits of this species (pH 7.15-7.86). In all tissues other than RBC, pHi remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pHi after anoxia and an increase in liver pHi following a metabolic alkalosis. Thus preferential pHi regulation appears to be a general pattern for acid-base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.

  12. Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis.

    PubMed

    Laurent, Julien; Venn, Alexander; Tambutté, Éric; Ganot, Philippe; Allemand, Denis; Tambutté, Sylvie

    2014-02-01

    The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO₂-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH₄Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na⁺-free seawater indicate a potential role of Na⁺/H⁺ plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited. PMID:24256552

  13. Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis.

    PubMed

    Laurent, Julien; Venn, Alexander; Tambutté, Éric; Ganot, Philippe; Allemand, Denis; Tambutté, Sylvie

    2014-02-01

    The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO₂-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH₄Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na⁺-free seawater indicate a potential role of Na⁺/H⁺ plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited.

  14. Regulating Emotions and Aiming for a Ph.D.: Excerpts from "Anthropology Matters"

    ERIC Educational Resources Information Center

    Hovland, Ingie

    2012-01-01

    In this article I will present a range of experiences of graduate socialisation that have been discussed in past articles in the journal "Anthropology Matters". These are the experiences of social anthropology Ph.D. students in the United Kingdom. The overarching theme for the article is "regulating emotions", and the excerpts presented illustrate…

  15. Regulating Glucose and pH, and Monitoring Oxygen in a Bioreactor

    NASA Technical Reports Server (NTRS)

    Anderson, Melody M.; Pellis, Neat R.; Jeevarajan, Antony S.; Taylor, Thomas D.; Xu, Yuanhang; Gao, Frank

    2006-01-01

    A system that automatically regulates the concentration of glucose or pH in a liquid culture medium that is circulated through a rotating-wall perfused bioreactor is described. Another system monitors the concentration of oxygen in the culture medium.

  16. Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor

    PubMed Central

    Virgilio, Stela; Cupertino, Fernanda Barbosa; Bernardes, Natália Elisa; Freitas, Fernanda Zanolli; Takeda, Agnes Alessandra Sekijima; Fontes, Marcos Roberto de Mattos; Bertolini, Maria Célia

    2016-01-01

    Environmental pH induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. Neurospora crassa shares all six components of the Aspergillus nidulans pH signaling pathway, and we investigate here their regulation during an alkaline pH stress response. We show that the N. crassa pal mutant strains, with the exception of Δpal-9, which is the A. nidulans palI homolog, exhibit low conidiation and are unable to grow at alkaline pH. Moreover, they accumulate the pigment melanin, most likely via regulation of the tyrosinase gene by the pH signaling components. The PAC-3 transcription factor binds to the tyrosinase promoter and negatively regulates its gene expression. PAC-3 also binds to all pal gene promoters, regulating their expression at normal growth pH and/or alkaline pH, which indicates a feedback regulation of PAC-3 in the pal gene expression. In addition, PAC-3 binds to the pac-3 promoter only at alkaline pH, most likely influencing the pac-3 expression at this pH suggesting that the activation of PAC-3 in N. crassa results from proteolytic processing and gene expression regulation by the pH signaling components. In N. crassa, PAC-3 is proteolytically processed in a single cleavage step predominately at alkaline pH; however, low levels of the processed protein can be observed at normal growth pH. We also demonstrate that PAC-3 preferentially localizes in the nucleus at alkaline pH stress and that the translocation may require the N. crassa importin-α since the PAC-3 nuclear localization signal (NLS) has a strong in vitro affinity with importin-α. The data presented here show that the pH signaling pathway in N. crassa shares all the components with the A. nidulans and S. cerevisiae pathways; however, it exhibits some properties not previously described in either organism. PMID:27557053

  17. Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor.

    PubMed

    Virgilio, Stela; Cupertino, Fernanda Barbosa; Bernardes, Natália Elisa; Freitas, Fernanda Zanolli; Takeda, Agnes Alessandra Sekijima; Fontes, Marcos Roberto de Mattos; Bertolini, Maria Célia

    2016-01-01

    Environmental pH induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. Neurospora crassa shares all six components of the Aspergillus nidulans pH signaling pathway, and we investigate here their regulation during an alkaline pH stress response. We show that the N. crassa pal mutant strains, with the exception of Δpal-9, which is the A. nidulans palI homolog, exhibit low conidiation and are unable to grow at alkaline pH. Moreover, they accumulate the pigment melanin, most likely via regulation of the tyrosinase gene by the pH signaling components. The PAC-3 transcription factor binds to the tyrosinase promoter and negatively regulates its gene expression. PAC-3 also binds to all pal gene promoters, regulating their expression at normal growth pH and/or alkaline pH, which indicates a feedback regulation of PAC-3 in the pal gene expression. In addition, PAC-3 binds to the pac-3 promoter only at alkaline pH, most likely influencing the pac-3 expression at this pH suggesting that the activation of PAC-3 in N. crassa results from proteolytic processing and gene expression regulation by the pH signaling components. In N. crassa, PAC-3 is proteolytically processed in a single cleavage step predominately at alkaline pH; however, low levels of the processed protein can be observed at normal growth pH. We also demonstrate that PAC-3 preferentially localizes in the nucleus at alkaline pH stress and that the translocation may require the N. crassa importin-α since the PAC-3 nuclear localization signal (NLS) has a strong in vitro affinity with importin-α. The data presented here show that the pH signaling pathway in N. crassa shares all the components with the A. nidulans and S. cerevisiae pathways; however, it exhibits some properties not previously described in either organism. PMID:27557053

  18. Prostaglandin biosynthesis by midgut tissue isolated from the tobacco hornworm, Manduca sexta.

    PubMed

    Büyükgüzel, K; Tunaz, H; Putnam, S M; Stanley, D

    2002-04-01

    We describe prostaglandin (PG) biosynthesis by isolated midgut preparations from tobacco hornworms, Manduca sexta. Microsomal-enriched midgut preparations yielded four PGs, PGA/B(2), PGD(2), PGE(2) and PGF(2alpha), all of which were confirmed by analysis on gas chromatography--mass spectrometry (GC--MS). PGA and PGB are double bond isomers which do not resolve on TLC but do resolve by GC; for convenience, we use the single term PGA(2) for this product. PGA(2) was the major product under most conditions. The midgut preparations were sensitive to reaction conditions, including radioactive substrate, protein concentration (optimal at 1mg/reaction), reaction time (optimal at 0.5 min), temperature (optimal at 22 degrees C), buffer pH (highest at pH 6), and the presence of a co-factor cocktail composed of reduced glutathione, hydroquinine and hemoglobin. In vitro PG biosynthesis was inhibited by two cyclooxygenase inhibitors, indomethacin and naproxen. Subcellular localization of PG biosynthetic activity in midgut preparations, determined by ultracentrifugation, revealed the presence of PG biosynthetic activity in the cytosolic and microsomal fractions, although most activity was found in the cytosolic fractions. This is similar to other invertebrates, and different from mammalian preparations, in which the activity is exclusively associated with the microsomal fractions. Midgut preparations from M. sexta pupae, adult cockroach, Periplaneta americana, and corn ear worms, Helicoverpa zea, also produced the same four major PG products. We infer that insect midguts are competent to biosynthesize PGs, and speculate they exert important, albeit unrevealed, actions in midgut physiology.

  19. Factors regulating nitrification in aquatic sediments: Effects of organic carbon, nitrogen availability, and pH

    USGS Publications Warehouse

    Strauss, E.A.; Mitchell, N.L.; Lamberti, G.A.

    2002-01-01

    We investigated the response in nitrification to organic carbon (C) availability, the interactive effects of the C: nitrogen (N) ratio and organic N availability, and differing pH in sediments from several streams in the upper midwestern United States. In addition, we surveyed 36 streams to assess variability in sediment nitrification rates. Labile dissolved organic carbon (DOC) additions of 30 mg C??L-1 (as acetate) to stream sediments reduced nitrification rates (P < 0.003), but lower concentration additions or dilution of ambient DOC concentration had no effect on nitrification. C:N and organic N availability strongly interacted to affect nitrification (P < 0.0001), with N availability increasing nitrification most at lower C:N. Nitrification was also strongly influenced by pH (P < 0.002), with maximum rates occurring at pH 7.5. A multiple regression model developed from the stream survey consisted of five variables (stream temperature, pH, conductivity, DOC concentration, and total extractable NH4+) and explained 60% of the variation observed in nitrification. Our results suggest that nitrification is regulated by several variables, with NH4+ availability and pH being the most important. Organic C is likely important at regulating nitrification only under high environmental C:N conditions and if most available C is relatively labile.

  20. Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation.

    PubMed

    Elferich, Johannes; Williamson, Danielle M; Krishnamoorthy, Bala; Shinde, Ujwal

    2013-08-01

    Eukaryotic cells maintain strict control over protein secretion, in part by using the pH gradient maintained within their secretory pathway. How eukaryotic proteins evolved from prokaryotic orthologs to exploit the pH gradient for biological functions remains a fundamental question in cell biology. Our laboratory previously demonstrated that protein domains located within precursor proteins, propeptides, encode histidine-driven pH sensors to regulate organelle-specific activation of the eukaryotic proteases furin and proprotein convertase-1/3. Similar findings have been reported in other unrelated protease families. By analyzing >10,000 unique proteases within evolutionarily unrelated families, we show that eukaryotic propeptides are enriched in histidines compared with prokaryotic orthologs. On this basis, we hypothesize that eukaryotic proteins evolved to enrich histidines within their propeptides to exploit the tightly controlled pH gradient of the secretory pathway, thereby regulating activation within specific organelles. Enrichment of histidines in propeptides may therefore be used to predict the presence of pH sensors in other proteases or even protease substrates.

  1. On enzymatic pH oscillations in CSTR with outlet regulator

    NASA Astrophysics Data System (ADS)

    Ohmori, Takao; Yu, Weifang; Yamamoto, Takuji; Endo, Akira; Nakaiwa, Masaru; Amemiya, Takashi; Yamaguchi, Tomohiko

    2005-05-01

    The possibility of enzymatic pH oscillations is investigated for a CSTR with an outlet regulator. A linear stability analysis shows that no oscillation is possible in a CSTR without the regulator, using a proton-producing pH-dependent enzymatic reaction. However, self-sustained oscillations are found to occur in a CSTR, where the discharge of substrate is regulated at the outlet. The regions of oscillations in the parameter space are determined using a hydrolysis of N-α-benzoyl- L-arginine ethyl ester with papain. It is found that the region is quite large only when the substrate concentration in the outflow is kept at zero.

  2. Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut.

    PubMed

    Elpidina, E N; Vinokurov, K S; Gromenko, V A; Rudenskaya, Y A; Dunaevsky, Y E; Zhuzhikov, D P

    2001-12-01

    Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.

  3. An automated system for monitoring and regulating the pH of bicarbonate buffers.

    PubMed

    Garbacz, Grzegorz; Kołodziej, Bartosz; Koziolek, Mirko; Weitschies, Werner; Klein, Sandra

    2013-06-01

    The bicarbonate buffer is considered as the most biorelevant buffer system for the simulation of intestinal conditions. However, its use in dissolution testing of solid oral dosage forms is very limited. The reason for this is the thermodynamic instability of the solution containing hydrogen carbonate ions and carbonic acid. The spontaneous loss of carbon dioxide (CO(2)) from the solution results in an uncontrolled increase of the pH. In order to maintain the pH on the desired level, either a CO(2) loss must be completely avoided or the escaped CO(2) has to be replaced by quantitative substitution, i.e. feeding the solution with the respective amount of gas, which re-acidifies the buffer after dissociation. The present work aimed at the development of a device enabling an automatic pH monitoring and regulation of hydrogen carbonate buffers during dissolution tests.

  4. pH Regulates White-Opaque Switching and Sexual Mating in Candida albicans.

    PubMed

    Sun, Yuan; Cao, Chengjun; Jia, Wei; Tao, Li; Guan, Guobo; Huang, Guanghua

    2015-11-01

    As a successful commensal and pathogen of humans, Candida albicans encounters a wide range of environmental conditions. Among them, ambient pH, which changes frequently and affects many biological processes in this species, is an important factor, and the ability to adapt to pH changes is tightly linked with pathogenesis and morphogenesis. In this study, we report that pH has a profound effect on white-opaque switching and sexual mating in C. albicans. Acidic pH promotes white-to-opaque switching under certain culture conditions but represses sexual mating. The Rim101-mediated pH-sensing pathway is involved in the control of pH-regulated white-opaque switching and the mating response. Phr2 and Rim101 could play a major role in acidic pH-induced opaque cell formation. Despite the fact that the cyclic AMP (cAMP) signaling pathway does not play a major role in pH-regulated white-opaque switching and mating, white and opaque cells of the cyr1/cyr1 mutant, which is defective in the production of cAMP, showed distinct growth defects under acidic and alkaline conditions. We further discovered that acidic pH conditions repressed sexual mating due to the failure of activation of the Ste2-mediated α-pheromone response pathway in opaque A: cells. The effects of pH changes on phenotypic switching and sexual mating could involve a balance of host adaptation and sexual reproduction in C. albicans.

  5. Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity.

    PubMed

    Bin, Bum-Ho; Bhin, Jinhyuk; Yang, Seung Ha; Shin, Misun; Nam, Yeon-Ju; Choi, Dong-Hwa; Shin, Dong Wook; Lee, Ai-Young; Hwang, Daehee; Cho, Eun-Gyung; Lee, Tae Ryong

    2015-01-01

    The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH. PMID:26057890

  6. pH regulation of amphotericin B channels activity in the bilayer lipid membrane

    PubMed Central

    Shahmoradi, Tahereh; Sepehry, Hamid; Ashrafpour, Manuchehr

    2016-01-01

    Background: Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of systemic fungal infections in spite of its secondary effects. The pH plays a crucial role in modulating biophysical features of ion channels in the bilayer lipid membranes. Aim: In this study, the role of pH in the regulation of AmB channel was assessed by single channel recording of ion channel incorporated in the artificial membrane. Materials and Methods: Bilayer lipid membrane was formed by phosphatidylcholine in a 350 μm diameter aperture between two chambers, cis and trans contained 200/50 mMKCl solutions, respectively; then AmB was incorporated into the bilayer lipid membrane. Single channel recordings were used to indicate the effects of pH changes on AmB channels activity. The records were analyzed by Clamp fit 10 software. Results: A kinetic analysis of single channel currents indicated a cation ion channel with 500 pS conductance and voltage-dependence of the open probability of the AmB channel (Po). A reduction of cis pH to 6 decreased Po and conductance. This effect was also voltage-dependent, being greater at a more positive above −40. The pH changes in the range of 6-8 had no effect on the reversal potential and ion selectivity. Conclusion: Our data indicated that extracellular acidity can reduce AmB activity. PMID:27003977

  7. EVIDENCE OF DIFFERENTIAL PH REGULATION OF THE ARABIDOPSIS VACUOLAR CA2+/H+ ANTIPORTERS CAX1 AND CAX2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis Ca(2+)/H(+) antiporters cation exchanger (CAX) 1 and 2 utilise an electrochemical gradient to transport Ca(2+) into the vacuole to help mediate Ca(2+) homeostasis. Previous whole plant studies indicate that activity of Ca(2+)/H(+) antiporters is regulated by pH. However, the pH regul...

  8. Sucrose hydrolases from the midgut of the sugarcane stalk borer Diatraea saccharalis.

    PubMed

    Carneiro, Cíntia N B; Isejima, Eliza M; Samuels, Richard I; Silva, Carlos P

    2004-11-01

    A beta-fructosidase (EC 3.2.1.26) was isolated from the midgut of larval sugar cane stalk borer Diatraea saccharalis by mild-denaturing electrophoresis and further purified to near homogeneity by gel filtration. beta-Fructosidase hydrolysed sucrose, raffinose and the fructosyl-trisaccharide isokestose, but it had no activity against maltose, melibiose and synthetic substrates for alpha-glucosidases. Two other sucrose hydrolases, one resembling a alpha-glucosidase (EC 3.2.1.20) and the other one active specifically against sucrose (sucrase) were detected in the larval midgut of D. saccharalis. All three sucrose hydrolases were associated with the midgut epithelium of larval D. saccharalis. Relative molecular mass (M(r)) of the beta-fructosidase was estimated around 45,000 (by gel filtration). The other two sucrose hydrolases had M(r) of 54,000 (alpha-glucosidase) and 59,000 (sucrase). The pH optima of the sucrose hydrolases were 5-10 for both alpha-glucosidase and sucrase and 7-8 for beta-fructosidase. Considering V(max)/K(m) ratios, beta-fructosidase preferentially cleaves isokestose rather than raffinose and sucrose. In order to evaluate the possible contribution of microorganisms isolated from the midgut to the pool of sucrose hydrolases, washed midgut epithelia were homogenised and plated onto appropriate media. Seven bacterial and one yeast species were isolated. None of the sucrose hydrolases extracted from the microorganisms corresponded to the enzymes isolated from midgut tissue homogenates. This result suggests that the major sucrose hydrolases found in the midgut of larval D. saccharalis were probably produced by the insect themselves not by the gut microflora.

  9. Alpha-galactosidases from the larval midgut of Tenebrio molitor (Coleoptera) and Spodoptera frugiperda (Lepidoptera).

    PubMed

    Grossmann, G A; Terra, W R

    2001-01-01

    There are three midgut alpha-galactosidases (TG1, TG2, TG3) from Tenebrio molitor larvae that are partially resolved by ion-exchange chromatography. The enzymes have approximately the same pH optimum (5.0), pl value (4.6) and Mr value (46000-49000) as determined by gel filtration or native electrophoresis run in polyacrylamide gels with different concentrations. Substrate specificities and functions were proposed for the major T. molitor midgut alpha-galactosidases (TG2 and TG3) based on chromatographic, carbodiimide inactivation, Tris inhibition, and on substrate competition data. Thus, TG2 would hydrolyse alpha-1,6-galactosaccharides, exemplified by raffinose, whereas TG3 would act on melibiose and apparently also on digalactosyldiglyceride, the most important compound in the thylacoid membranes of chloroplasts. Most galactoside digestion should occur in the lumen of the first two thirds of T. molitor larval midguts, since alpha-galactosidase activity predominates there. Spodoptera frugiperda larvae have three midgut alpha-galactosidases (SG1, SG2, SG3) partially resolved by ion-exchange chromatography. The enzymes have similar pH optimum (5.8), pl value (7.2) and Mr value (46000-52000), and at least the major alpha-galactosidase must have an active carboxyl group in the active site. Based on data similar to those described for T. molitor, SG1 and SG3 should hydrolyse melibiose and SG3 should digest raffinose and, perhaps, also digalactosyldiglyceride. The midgut distribution of alpha-galactosidase activity supports the proposal that alpha-galactosidase digestion occurs at the surface of anterior midgut cells in Spodoptera frugiperda larvae.

  10. Histochemical analysis of the goblet cell matrix in the larval midgut of Manduca sexta

    SciTech Connect

    Schultz, T.W.; Lozano, G.; Cajina-Quezada, M.

    1981-01-01

    Experimental analyses were made to histochemically determine the composition of the goblet cell matrix material in the larval midgut of the tobacco hornworm, Manduca sexta. Techniques employed following fixation in Carnoy fluid were the periodic acid-Schiff reaction and the alcian blue stain at pH 1.0 and pH 2.5 and following methylation and subsequent saponification. The cumulative evidence suggests that the plug material is an acid mucosubstance.

  11. Evaluation the anaerobic hydrolysis acidification stage of kitchen waste by pH regulation.

    PubMed

    Wang, Yaya; Zang, Bing; Li, Guoxue; Liu, Yu

    2016-07-01

    This study analyzed the composition and characteristic of kitchen waste (KW) from closed cleaning station of Chaoyang District, Beijing. It was featured by high vegetables and peels contents. This study investigated effect of pH regulation and uncontrolled pH (CK) on the lab-scale anaerobic hydrolysis acidification stage of KW. The optimal adjusting mode by NaOH (including dosage and frequency) was evaluated according to indexes of pH, VFAs, NH4(+)-N, TS, VS, TS/VS, TS and VS removal rate. The treatment 4 as first two days adjusting per 16h and then one time per day at pH 7 was chosen as the optimal mode with high VFAs content(47.31g/L), TS and VS removal rate (42.95% and 54.01%, respectively), low adjusting frequency, fewer dosage and practical operability. Thus, adjusting mode of treatment 4 could be considered using in anaerobic hydrolysis acidification stage on engineering.

  12. Role of metal oxide nanostructures in extracellular pH regulations

    NASA Astrophysics Data System (ADS)

    Lozhkomoev, Aleksandr S.

    2016-08-01

    A research area of great promise is the cancer treatment by regulating microenvironmental parameters of tumor cells using MgO and AlOOH. Magnesium hydroxide and aluminum oxyhydroxide (boehmite) are in the form of nanoplates and nanosheets. The morphology, structure, phases and electrokinetic properties of synthesized samples are analyzed using complex physical and chemical methods. We study how the pH of the culture medium—different when in contact with synthesized nanoplates—affects the viability of tumor cells. It is shown that MgO is more efficient in decreasing the tumor cell viability than AlOOH. In the case of magnesium hydroxide, the pH of the culture medium increases to 10.1; in the case of boehmite, to 7.7.

  13. TPC2 controls pigmentation by regulating melanosome pH and size.

    PubMed

    Ambrosio, Andrea L; Boyle, Judith A; Aradi, Al E; Christian, Keith A; Di Pietro, Santiago M

    2016-05-17

    Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca(2+) release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca(2+) sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size. PMID:27140606

  14. TPC2 controls pigmentation by regulating melanosome pH and size.

    PubMed

    Ambrosio, Andrea L; Boyle, Judith A; Aradi, Al E; Christian, Keith A; Di Pietro, Santiago M

    2016-05-17

    Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca(2+) release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca(2+) sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.

  15. Ambient pH Controls Glycogen Levels by Regulating Glycogen Synthase Gene Expression in Neurospora crassa. New Insights into the pH Signaling Pathway

    PubMed Central

    Cupertino, Fernanda Barbosa; Freitas, Fernanda Zanolli; de Paula, Renato Magalhães; Bertolini, Maria Célia

    2012-01-01

    Glycogen is a polysaccharide widely distributed in microorganisms and animal cells and its metabolism is under intricate regulation. Its accumulation in a specific situation results from the balance between glycogen synthase and glycogen phosphorylase activities that control synthesis and degradation, respectively. These enzymes are highly regulated at transcriptional and post-translational levels. The existence of a DNA motif for the Aspergillus nidulans pH responsive transcription factor PacC in the promoter of the gene encoding glycogen synthase (gsn) in Neurospora crassa prompted us to investigate whether this transcription factor regulates glycogen accumulation. Transcription factors such as PacC in A. nidulans and Rim101p in Saccharomyces cerevisiae play a role in the signaling pathway that mediates adaptation to ambient pH by inducing the expression of alkaline genes and repressing acidic genes. We showed here that at pH 7.8 pacC was over-expressed and gsn was down-regulated in wild-type N. crassa coinciding with low glycogen accumulation. In the pacCKO strain the glycogen levels and gsn expression at alkaline pH were, respectively, similar to and higher than the wild-type strain at normal pH (5.8). These results characterize gsn as an acidic gene and suggest a regulatory role for PACC in gsn expression. The truncated recombinant protein, containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH and confirmed by ChIP-PCR analysis. The PACC present in these extracts showed equal molecular mass, indicating that the protein is already processed at normal pH, in contrast to A. nidulans. Together, these results show that the pH signaling pathway controls glycogen accumulation by regulating gsn expression and suggest the existence of a different mechanism for PACC activation in N. crassa. PMID:22952943

  16. Alterations in the fat body and midgut of Culex quinquefasciatus larvae following exposure to different insecticides.

    PubMed

    Alves, Stênio Nunes; Serrão, José Eduardo; Melo, Alan Lane

    2010-08-01

    This study describes morphological alterations in the fat body and midgut of Culex quinquefasciatus larvae following exposure to different insecticides. To this end, both third and fourth instars of C. quinquefasciatus larvae were exposed for 30 and 60 min to organophosphate (50 ppb), pyrethroids (20 and 30 ppb), and avermectin derivates (1.5 and 54 ppb). Following incubation, pH measurements of the larvae gut were recorded. The fat body and midgut were also analyzed by light and transmission electron microscopy. These studies demonstrate a decrease in the pH of the larvae anterior midgut following exposure to all of the tested insecticides. Histochemical tests revealed a strong reaction for neutral lipids in the control group and a marked decrease in the group exposed to cypermethrin. Furthermore, a weak reaction with acidic lipids in larvae exposed to deltamethrin, temephos, ivermectin and abamectin was also observed. Insecticide-exposed larvae also exhibited cytoplasm granule differences, relative to control larvae. Finally, we noted a small reduction in microvilli size in the apex of digestive cells, although vesicles were found to be present. The destructive changes in the larvae were very similar regardless of the type of insecticide analyzed. These data suggest that alterations in the fat body and midgut are a common response to cellular intoxication.

  17. Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia).

    PubMed

    Manzl, Claudia; Krumschnabel, Gerhard; Schwarzbaum, Pablo J; Chabicovsky, Monika; Dallinger, Reinhard

    2004-01-01

    The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined. PMID:14695690

  18. Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals

    SciTech Connect

    Wu, Lan.

    1989-01-01

    Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward ({sup 3}H)-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics.

  19. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    SciTech Connect

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2014-07-25

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

  20. pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12.

    PubMed

    Maurer, Lisa M; Yohannes, Elizabeth; Bondurant, Sandra S; Radmacher, Michael; Slonczewski, Joan L

    2005-01-01

    consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins.

  1. Trpac1, a pH response transcription regulator, is involved in cellulase gene expression in Trichoderma reesei.

    PubMed

    He, Ronglin; Ma, Lijuan; Li, Chen; Jia, Wendi; Li, Demao; Zhang, Dongyuan; Chen, Shulin

    2014-12-01

    Fungi grow over a relatively wide pH range and adapt to extracellular pH through a genetic regulatory system mediated by a key component PacC, which is a pH transcription regulator. The cellulase production of the filamentous fungi Trichoderma reesei is sensitive to ambient pH. To investigate the connection between cellulase expression regulation and ambient pH, an ortholog of Aspergillus nidulans pacC, Trpac1, was identified and functionally characterized using a target gene deletion strategy. Deleting Trpac1 dramatically increased the cellulase production and the transcription levels of the major cellulase genes at neutral pH, which suggested Trpac1 is involved in the regulation of cellulase production. It was further observed that the expression levels of transcription factors xyr1 and ace2 also increased in the ΔTrpac1 mutant at neutral pH. In addition, the ΔTrpac1 mutant exhibited conidiation defects under neutral and alkaline pH. These results implied that Trpac1 in involved in growth and development process and cellulase gene expression in T. reesei.

  2. Epithelial carbonic anhydrases facilitate PCO2 and pH regulation in rat duodenal mucosa

    PubMed Central

    Mizumori, Misa; Meyerowitz, Justin; Takeuchi, Tetsu; Lim, Shu; Lee, Paul; Supuran, Claudiu T; Guth, Paul H; Engel, Eli; Kaunitz, Jonathan D; Akiba, Yasutada

    2006-01-01

    The duodenum is the site of mixing of massive amounts of gastric H+ with secreted HCO3−, generating CO2 and H2O accompanied by the neutralization of H+. We examined the role of membrane-bound and soluble carbonic anhydrases (CA) by which H+ is neutralized, CO2 is absorbed, and HCO3− is secreted. Rat duodena were perfused with solutions of different pH and PCO2 with or without a cell-permeant CA inhibitor methazolamide (MTZ) or impermeant CA inhibitors. Flow-through pH and PCO2 electrodes simultaneously measured perfusate and effluent pH and PCO2. High CO2 (34.7 kPa) perfusion increased net CO2 loss from the perfusate compared with controls (pH 6.4 saline, PCO2 ≈ 0) accompanied by portal venous (PV) acidification and PCO2 increase. Impermeant CA inhibitors abolished net perfusate CO2 loss and increased net HCO3− gain, whereas all CA inhibitors inhibited PV acidification and PCO2 increase. The changes in luminal and PV pH and [CO2] were also inhibited by the Na+–H+ exchanger-1 (NHE1) inhibitor dimethylamiloride, but not by the NHE3 inhibitor S3226. Luminal acid decreased total CO2 output and increased H+ loss with PV acidification and PCO2 increase, all inhibited by all CA inhibitors. During perfusion of a 30% CO2 buffer, loss of CO2 from the lumen was CA dependent as was transepithelial transport of perfused 13CO2. H+ and CO2 loss from the perfusate were accompanied by increases of PV H+ and tracer CO2, but unchanged PV total CO2, consistent with CA-dependent transmucosal H+ and CO2 movement. Inhibition of membrane-bound CAs augments the apparent rate of net basal HCO3− secretion. Luminal H+ traverses the apical membrane as CO2, is converted back to cytosolic H+, which is extruded via NHE1. Membrane-bound and cytosolic CAs cooperatively facilitate secretion of HCO3− into the lumen and CO2 diffusion into duodenal mucosa, serving as important acid–base regulators. PMID:16556652

  3. pH regulation in horizontal cells of the skate retina.

    PubMed

    Haugh-Scheidt, L; Ripps, H

    1998-04-01

    To examine the mechanisms by which horizontal cells regulate intracellular pH (pHi), measurements were recorded from isolated cells enzymatically dissociated from the skate retina utilizing the pH-sensitive dye BCECF. In a HCO3--containing Ringer solution, steady-state pHi was 7.32+/-0.13 (mean+/-S.D., n=70). Recovery from acidification was examined using the NH4+ prepulse technique. When NH4+ was removed from the extracellular solution, pHi dropped rapidly to approximately 0.3 pH units below the initial baseline, and then recovered at an initial rate of approximately 0.072 pH units/min. During recovery of pHi after the acid load, the removal of Na+ or the addition of amiloride from a HCO3--free extracellular solution reduced the rate of recovery by 79%+/-11% and 69%+/-14%, respectively. In the presence of DIDS, which inhibits primarily anion transport, or during the removal of Na+, the recovery from acidification was reduced by 83%+/-10% and 70%+/-11%, respectively, as compared to the control value in HCO3--containing solution. These results suggest that the skate horizontal cell possesses a Na/H exchanger as well as a Na+-and HCO3--dependent mechanism for removal of excess acid. Removal of HCO3- or Cl- from the extracellular solution had little effect on pHi, but removing external Na+ induced a marked decrease in pHi that fell at an initial rate of approximately 0.3 pH units min-1. This rate of acidification was decreased by 58%+/-19% in the presence of DIDS (500 micron) and reduced by 28%+/-13% with the addition of amiloride (2 mm). Thus, Na- and HCO3-dependent transport was about 2-fold more active than Na/H exchange during low Na+-induced acidification. The intrinsic pH-buffer capacity, determined from the pHi change induced by incremental reductions in the [NH4+] of the extracellular solution, was 24.2 mm/pH unit at the horizontal cell's resting pHi. Moreover, pHi was relatively insensitive to changes in membrane potential; in experiments under whole

  4. Localization of post-proline cleaving peptidases in Tenebrio molitor larval midgut.

    PubMed

    Goptar, Irina A; Filippova, Irina Yu; Lysogorskaya, Elena N; Oksenoit, Elena S; Vinokurov, Konstantin S; Zhuzhikov, Dmitry P; Bulushova, Natalja V; Zalunin, Igor A; Dunaevsky, Yakov E; Belozersky, Mikhail A; Oppert, Brenda; Elpidina, Elena N

    2008-03-01

    Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in Tenebrio molitor larval midgut with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activity at pH 5.3, and was located mainly in the more acidic anterior midgut lumen. The dynamics of PPCP1 activity and the total activity of soluble digestive peptidases in the course of food digestion were similar, suggesting that the enzyme participates in protein digestion. PPCP2 is a nondigestive soluble tissue enzyme evenly distributed along the midgut. An increase in the activity of PPCP2 was observed in buffers of pH 5.6-8.6 and was maximal at pH 7.4. The sensitivity of PPCP2 to inhibitors and the effect of pH are similar to prolyl oligopeptidases with a cysteine residue near the substrate binding site.

  5. Carbon Cycling and pH regulation on the Scotian Shelf, NW Atlantic

    NASA Astrophysics Data System (ADS)

    Thomas, Helmuth

    2015-04-01

    This presentation intends to describe the biogeochemical context for ocean acidification studies on the Scotian Shelf. The seasonality of the dominant processes, regulating surface ocean CO2 conditions, including pH, will be assessed as well as cross-shelf transports of CO2, acidity and nutrient, the latter ones exerting the "subsurface control" of CO2 air-sea fluxes and surface pH. Methods summary: The seasonal variability of inorganic carbon in the surface waters of the Scotian Shelf region of the Canadian northwestern Atlantic Ocean was assessed using hourly measurements of the partial pressure of CO2 (pCO2), and hydrographic variables obtained by an autonomous moored instrument (44.3°N and 63.3°W). These measurements were complemented by seasonal shipboard sampling of dissolved inorganic carbon (DIC), total alkalinity (TA), and pCO2, at the mooring site, and over the larger spatial scale. The Scotian Shelf is a 700 km long section of the continental shelf off Nova Scotia. Bounded by the Laurentian Channel to the northeast, and by the Northeast Channel and the Gulf of Maine to the southwest, it varies in width from 120 to 240 km covering roughly 120,000 km2 with an average depth of 90 m . Convective mixin in winter time and coastal upwelling and the associated favorable wind conditions on the Scotian Shelf have long been recognized. Strong winds of speeds greater than 10 m s-1, blowing to the northeast, and persisting for several days force relatively cold, saline, water toward the surface, displacing the warmer, fresher water offshore. Upwelling events have frequently been observed in the region in winter, and modeling studies have reproduced these observed events. Furthermore, these events may play a role in initiating and sustaining the spring phytoplankton bloom by displacing nutrient-depleted surface water and bring nutrient-rich waters up to the surface. Biological processes were found to be the dominant control on mixed-layer DIC, with the delivery of

  6. Nucleoporin 35 regulates cardiomyocyte pH homeostasis by controlling Na+-H+ exchanger-1 expression.

    PubMed

    Xu, Liang; Pan, Lei; Li, Jun; Huang, Bijun; Feng, Jing; Li, Changming; Wang, Shiyi; The, Erlinda; Liu, Yuan; Yuan, Tianyou; Zhen, Lixiao; Liang, Dandan; Liu, Yi; Li, Li; Cui, Yingyu; Jiang, Xiaoyan; Peng, Luying; Chen, Yi-Han

    2015-10-01

    The mammalian nuclear pore complex is comprised of ∼ 30 different nucleoporins (Nups). It governs the nuclear import of gene expression modulators and the export of mRNAs. In cardiomyocytes, Na(+)-H(+) exchanger-1 (NHE1) is an integral membrane protein that exclusively regulates intracellular pH (pHi) by exchanging one intracellular H(+) for one extracellular Na(+). However, the role of Nups in cardiac NHE1 expression remains unknown. We herein report that Nup35 regulates cardiomyocyte NHE1 expression by controlling the nucleo-cytoplasmic trafficking of nhe1 mRNA. The N-terminal domain of Nup35 determines nhe1 mRNA nuclear export by targeting the 5'-UTR (-412 to -213 nt) of nhe1 mRNA. Nup35 ablation weakens the resistance of cardiomyocytes to an acid challenge by depressing NHE1 expression. Moreover, we identify that Nup35 and NHE1 are simultaneously downregulated in ischemic cardiomyocytes both in vivo and in vitro. Enforced expression of Nup35 effectively counteracts the anoxia-induced intracellular acidification. We conclude that Nup35 selectively regulates cardiomyocyte pHi homeostasis by posttranscriptionally controlling NHE1 expression. This finding reveals a novel regulatory mechanism of cardiomyocyte pHi, and may provide insight into the therapeutic strategy for ischemic cardiac diseases.

  7. A DNA Fragment Mapped within the Submicroscopic Deletion of Ph1, a Chromosome Pairing Regulator Gene in Polyploid Wheat

    PubMed Central

    Gill, K. S.; Gill, B. S.

    1991-01-01

    Bread wheat is an allohexaploid consisting of three genetically related (homoeologous) genomes. The homoeologous chromosomes are capable of pairing but strict homologous pairing is observed at metaphase 1. The diploid-like pairing is regulated predominantly by Ph1, a gene mapped on long arm of chromosome 5B. We report direct evidence that a mutant of the gene (ph1b) arose from a submicroscopic deletion. A probe (XksuS1-5) detects the same missing fragment in two independent mutants ph1b and ph1c and a higher intensity fragment in a duplication of the Ph1 gene. It is likely that XksuS1-5 lies adjacent to Ph1 on the same chromosome fragment that is deleted in ph1b and ph1c. XksuS1-5 can be used to tag Ph1 gene to facilitate incorporation of genetic material from homoeologous genomes of the Triticeae. It may also be a useful marker in cloning Ph1 gene by chromosome walking. PMID:1936962

  8. Learning to Write a Research Article: Ph.D. Students' Transitions toward Disciplinary Writing Regulation

    ERIC Educational Resources Information Center

    Castello, Montserrat; Inesta, Anna; Corcelles, Mariona

    2013-01-01

    This paper presents a study designed from a socially situated and activity theory perspective aimed at gaining a deeper understanding of how Ph.D. students regulate their academic writing activity. Writing regulation is a complex activity of a highly situated and social nature, involving cyclical thought-action-emotion dynamics and the…

  9. FoxK mediates TGF-β signalling during midgut differentiation in flies

    PubMed Central

    Casas-Tinto, Sergio; Gomez-Velazquez, Melisa; Granadino, Begoña; Fernandez-Funez, Pedro

    2008-01-01

    Inductive signals across germ layers are important for the development of the endoderm in vertebrates and invertebrates (Tam, P.P., M. Kanai-Azuma, and Y. Kanai. 2003. Curr. Opin. Genet. Dev. 13:393–400; Nakagoshi, H. 2005. Dev. Growth Differ. 47:383–392). In flies, the visceral mesoderm secretes signaling molecules that diffuse into the underlying midgut endoderm, where conserved signaling cascades activate the Hox gene labial, which is important for the differentiation of copper cells (Bienz, M. 1997. Curr. Opin. Genet. Dev. 7:683–688). We present here a Drosophila melanogaster gene of the Fox family of transcription factors, FoxK, that mediates transforming growth factor β (TGF-β) signaling in the embryonic midgut endoderm. FoxK mutant embryos fail to generate midgut constrictions and lack Labial in the endoderm. Our observations suggest that TGF-β signaling directly regulates FoxK through functional Smad/Mad-binding sites, whereas FoxK, in turn, regulates labial expression. We also describe a new cooperative activity of the transcription factors FoxK and Dfos/AP-1 that regulates labial expression in the midgut endoderm. This regulatory activity does not require direct labial activation by the TGF-β effector Mad. Thus, we propose that the combined activity of the TGF-β target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm. PMID:19075113

  10. Streptococcus pyogenes malate degradation pathway links pH regulation and virulence.

    PubMed

    Paluscio, Elyse; Caparon, Michael G

    2015-03-01

    The ability of Streptococcus pyogenes to infect different niches within its human host most likely relies on its ability to utilize alternative carbon sources. In examining this question, we discovered that all sequenced S. pyogenes strains possess the genes for the malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised of four genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding a malate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression of maeP and maeE is induced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Furthermore, both maePE and maeKR are repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malate utilization requires the PTS transporter EI enzyme (PtsI), as a PtsI(-) mutant fails to express the ME genes and is unable to utilize malate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP(-), MaeK(-), and MaeR(-) mutants were attenuated for virulence, whereas a MaeE(-) mutant showed enhanced virulence compared to that of the wild type. Taken together, these data show that ME contributes to S. pyogenes' carbon source repertory, that malate utilization is a highly regulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptake and utilization contribute to the adaptive pH response, and ME can influence the outcome of infection.

  11. pH regulation in isolated in vitro perfused rat colonic crypts.

    PubMed

    Hasselblatt, P; Warth, R; Schulz-Baldes, A; Greger, R; Bleich, M

    2000-11-01

    We investigated disorders and regulation of cytosolic pH (pHi) in isolated perfused crypts from rat distal colon using the pH-sensitive dye BCECF. This preparation allows distinct examination of either luminal or basolateral transport. The effects of luminal weak organic acids and bases on pHi were examined. The physiological concentrations of both luminal CO2/HCO3- and acetic acid/acetate acidified pHi significantly, but less than when applied from the basolateral side. Corresponding changes (luminal versus basolateral) in pHi were -0.17+/-0.04 versus -0.39+/-0.04, (n=8) and -0.15+/-0.02 versus -0.41+/-0.04, (n=8), respectively. Basolateral versus luminal application of NH3/NH4+ led to a more marked change in pHi, namely 0.35+/-0.03 versus 0.008+/-0.007 pH units, (n=19). The luminal perfusion of NH3/NH4+ was controlled by applying fura-2 acid to the luminal side and at the same time recording fura-2-specific fluorescence. Hence, the influence of luminal acid/base on colonic pHi homeostasis was limited. To examine pHi regulation, we investigated the recovery from an intracellular acid load using the NH3/NH4+ pulse method. Recovery was completely dependent on basolateral Na+, indicating that luminal acid/base transport does not play a major role in pHi homeostasis. The basolateral transporters involved in pHi recovery are probably the EIPA- and HOE694-inhibitable (IC50=0.2 and 2 micromol/l, respectively) Na+/H+ exchanger NHE1 and the DIDS-inhibitable Na+-dependent HCO3- importer. PMID:11205049

  12. Streptococcus pyogenes Malate Degradation Pathway Links pH Regulation and Virulence

    PubMed Central

    Paluscio, Elyse

    2015-01-01

    The ability of Streptococcus pyogenes to infect different niches within its human host most likely relies on its ability to utilize alternative carbon sources. In examining this question, we discovered that all sequenced S. pyogenes strains possess the genes for the malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised of four genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding a malate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression of maeP and maeE is induced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Furthermore, both maePE and maeKR are repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malate utilization requires the PTS transporter EI enzyme (PtsI), as a PtsI– mutant fails to express the ME genes and is unable to utilize malate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP–, MaeK–, and MaeR– mutants were attenuated for virulence, whereas a MaeE– mutant showed enhanced virulence compared to that of the wild type. Taken together, these data show that ME contributes to S. pyogenes' carbon source repertory, that malate utilization is a highly regulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptake and utilization contribute to the adaptive pH response, and ME can influence the outcome of infection. PMID:25583521

  13. Role of "active" potassium transport in the regulation of cytoplasmic pH by nonanimal cells.

    PubMed Central

    Blatt, M R; Slayman, C L

    1987-01-01

    High-affinity potassium uptake in Neurospora occurs by symport with protons [Km (apparent) = 15 microM at pH 5.8], for which a large inward gradient (approximately 400 mV) is generated by the H+-extruding ATPase of the plasma membrane. Operating in parallel, the two transport systems yield a net 1:1 exchange of K+ for cytoplasmic H+. Since this exchange could play a role in cytoplasmic pH (pHi) regulation, the coordinated functioning of the K+-H+ symport and H+ pump has been examined during acid stress. Cytoplasmic acid loads were imposed by injection and by exposure to extracellular permeant weak acid. Multibarrelled microelectrodes were used to monitor membrane potential (Vm), pHi, and the current-voltage (I-V) characteristics of the cells. The behaviors of the H+ pump and K+-H+ symport were resolved, respectively, by fitting whole membrane I-V curves to an explicit kinetic model of the Neurospora membrane and by subtracting I-V curves obtained in the absence from those obtained in the presence of 5-200 microM K+ outside. Proton pumping accelerates nearly in proportion with the cytoplasmic H+ concentration, but pHi recovery from imposed acid loads is dependent on micromolar K+ outside. Potassium import via the symport leads to a measurable alkalinization of the cytoplasm in accordance with stoichiometric (1:1) K+/H+ exchange. Potassium transport is accelerated at low pHi, but in a manner consistent with its inherent voltage sensitivity and changes in Vm resulting from an increased rate of H+ extrusion by the pump. The primary response to acid stress thus rests with the H+ pump, but K+ transport introduces an essential kinetic "valve" that can regulate net H+ export. Images PMID:3472234

  14. Recurrent Midgut Bleeding due to Jejunal Angioleiomyoma

    PubMed Central

    Mityushin, Petr

    2016-01-01

    Angioleiomyoma being a type of true smooth muscle gastrointestinal tumors can lead to serious life-threatening gastrointestinal bleeding. We report a case of 21-year-old male patient with recurrent midgut bleeding. Contrast-enhanced CT revealed highly vascular small bowel neoplasm. The patient underwent laparotomy with bowel resection and recovered uneventfully. Histopathology revealed jejunal angioleiomyoma. PMID:27668116

  15. Recurrent Midgut Bleeding due to Jejunal Angioleiomyoma.

    PubMed

    Gachabayov, Mahir; Mityushin, Petr

    2016-01-01

    Angioleiomyoma being a type of true smooth muscle gastrointestinal tumors can lead to serious life-threatening gastrointestinal bleeding. We report a case of 21-year-old male patient with recurrent midgut bleeding. Contrast-enhanced CT revealed highly vascular small bowel neoplasm. The patient underwent laparotomy with bowel resection and recovered uneventfully. Histopathology revealed jejunal angioleiomyoma. PMID:27668116

  16. Recurrent Midgut Bleeding due to Jejunal Angioleiomyoma

    PubMed Central

    Mityushin, Petr

    2016-01-01

    Angioleiomyoma being a type of true smooth muscle gastrointestinal tumors can lead to serious life-threatening gastrointestinal bleeding. We report a case of 21-year-old male patient with recurrent midgut bleeding. Contrast-enhanced CT revealed highly vascular small bowel neoplasm. The patient underwent laparotomy with bowel resection and recovered uneventfully. Histopathology revealed jejunal angioleiomyoma.

  17. Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

    PubMed

    Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

    2016-07-01

    The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general. PMID:27091863

  18. Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

    PubMed

    Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

    2016-07-01

    The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general.

  19. Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator.

    PubMed Central

    Poulsen, J H; Fischer, H; Illek, B; Machen, T E

    1994-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel regulated by protein kinase A. The most common mutation in cystic fibrosis (CF), deletion of Phe-508 (delta F508-CFTR), reduces Cl- secretion, but the fatal consequences of CF have been difficult to rationalize solely in terms of this defect. The aim of this study was to determine the role of CFTR in HCO3- transport across cell membranes. HCO3- permeability was assessed from measurements of intracellular pH [pHi; from spectrofluorimetry of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein] and of channel activity (patch clamp; cell attached and isolated, inside-out patches) on NIH 3T3 fibroblasts and C127 mammary epithelial cells transfected with wild-type CFTR (WT-CFTR) or delta F508-CFTR, and also on mock-transfected cells. When WT-CFTR-transfected cells were acidified (pulsed with NH4Cl) and incubated in Na(+)-free (N-methyl-D-glucamine substitution) solutions (to block Na(+)-dependent pHi regulatory mechanisms), pHi remained acidic (pH approximately 6.5) until the cells were treated with 20 microM forskolin (increases cellular [cAMP]); pHi then increased toward (but not completely to) control level (pHi 7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pHi recovery in delta F508 and mock-transfected cells. This Na(+)-independent, forskolin-dependent pHi recovery was not observed in HCO3-/CO2-free medium. Forskolin-treated WT-CFTR-transfected (but not delta F508-CFTR or mock-transfected) cells in Cl(-)-containing, HCO3(-)-free solutions showed Cl- channels with a linear I/V relationship and a conductance of 10.4 +/- 0.5 pS in symmetrical 150 mM Cl-. When channels were incubated with different [Cl-] and [HCO3-] on the inside and outside, the Cl-/HCO3- permeability ratio (determined from reversal potentials of I/V curves) was 3.8 +/- 1.0 (mean +/- SEM; n = 9); the ratio of conductances was 3.9 +/- 0.5 (at 150 mM Cl- and 127 m

  20. SLC26A Gene Family Participate in pH Regulation during Enamel Maturation.

    PubMed

    Yin, Kaifeng; Lei, Yuejuan; Wen, Xin; Lacruz, Rodrigo S; Soleimani, Manoocher; Kurtz, Ira; Snead, Malcolm L; White, Shane N; Paine, Michael L

    2015-01-01

    The bicarbonate transport activities of Slc26a1, Slc26a6 and Slc26a7 are essential to physiological processes in multiple organs. Although mutations of Slc26a1, Slc26a6 and Slc26a7 have not been linked to any human diseases, disruption of Slc26a1, Slc26a6 or Slc26a7 expression in animals causes severe dysregulation of acid-base balance and disorder of anion homeostasis. Amelogenesis, especially the enamel formation during maturation stage, requires complex pH regulation mechanisms based on ion transport. The disruption of stage-specific ion transporters frequently results in enamel pathosis in animals. Here we present evidence that Slc26a1, Slc26a6 and Slc26a7 are highly expressed in rodent incisor ameloblasts during maturation-stage tooth development. In maturation-stage ameloblasts, Slc26a1, Slc26a6 and Slc26a7 show a similar cellular distribution as the cystic fibrosis transmembrane conductance regulator (Cftr) to the apical region of cytoplasmic membrane, and the distribution of Slc26a7 is also seen in the cytoplasmic/subapical region, presumably on the lysosomal membrane. We have also examined Slc26a1 and Slc26a7 null mice, and although no overt abnormal enamel phenotypes were observed in Slc26a1-/- or Slc26a7-/- animals, absence of Slc26a1 or Slc26a7 results in up-regulation of Cftr, Ca2, Slc4a4, Slc4a9 and Slc26a9, all of which are involved in pH homeostasis, indicating that this might be a compensatory mechanism used by ameloblasts cells in the absence of Slc26 genes. Together, our data show that Slc26a1, Slc26a6 and Slc26a7 are novel participants in the extracellular transport of bicarbonate during enamel maturation, and that their functional roles may be achieved by forming interaction units with Cftr. PMID:26671068

  1. SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

    PubMed Central

    Yin, Kaifeng; Lei, Yuejuan; Wen, Xin; Lacruz, Rodrigo S.; Soleimani, Manoocher; Kurtz, Ira; Snead, Malcolm L.; White, Shane N.; Paine, Michael L.

    2015-01-01

    The bicarbonate transport activities of Slc26a1, Slc26a6 and Slc26a7 are essential to physiological processes in multiple organs. Although mutations of Slc26a1, Slc26a6 and Slc26a7 have not been linked to any human diseases, disruption of Slc26a1, Slc26a6 or Slc26a7 expression in animals causes severe dysregulation of acid-base balance and disorder of anion homeostasis. Amelogenesis, especially the enamel formation during maturation stage, requires complex pH regulation mechanisms based on ion transport. The disruption of stage-specific ion transporters frequently results in enamel pathosis in animals. Here we present evidence that Slc26a1, Slc26a6 and Slc26a7 are highly expressed in rodent incisor ameloblasts during maturation-stage tooth development. In maturation-stage ameloblasts, Slc26a1, Slc26a6 and Slc26a7 show a similar cellular distribution as the cystic fibrosis transmembrane conductance regulator (Cftr) to the apical region of cytoplasmic membrane, and the distribution of Slc26a7 is also seen in the cytoplasmic/subapical region, presumably on the lysosomal membrane. We have also examined Slc26a1 and Slc26a7 null mice, and although no overt abnormal enamel phenotypes were observed in Slc26a1-/- or Slc26a7-/- animals, absence of Slc26a1 or Slc26a7 results in up-regulation of Cftr, Ca2, Slc4a4, Slc4a9 and Slc26a9, all of which are involved in pH homeostasis, indicating that this might be a compensatory mechanism used by ameloblasts cells in the absence of Slc26 genes. Together, our data show that Slc26a1, Slc26a6 and Slc26a7 are novel participants in the extracellular transport of bicarbonate during enamel maturation, and that their functional roles may be achieved by forming interaction units with Cftr. PMID:26671068

  2. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    PubMed Central

    Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

    2013-01-01

    Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania. PMID:24037187

  3. Altered intracellular pH regulation in neutrophils from patients with cystic fibrosis.

    PubMed

    Coakley, R J; Taggart, C; Canny, G; Greally, P; O'Neill, S J; McElvaney, N G

    2000-07-01

    Cystic fibrosis (CF) is a condition characterized by neutrophil-mediated lung damage and bacterial colonization. The physiological basis for reported functional alterations in CF neutrophils, including increased release of neutrophil elastase, myeloperoxidase, and oxidants, is unknown. These processes are, however, regulated by intracellular pH (pH(i)). We demonstrate here that pH(i) regulation is altered in neutrophils from CF patients. Although resting pH(i) is similar, pH(i) after acid loading and activation (N-formyl-methionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate) is more acidic in CF cells than in normal cells. Furthermore, patients with non-CF-related bronchiectasis handle acid loading and activation in a fashion similar to subjects with normal neutrophils, suggesting that chronic pulmonary inflammation alone does not explain the difference in pH(i). This is further supported by data showing that normal neutrophils exposed to the CF pulmonary milieu respond by increasing pH(i) as opposed to decreasing pH(i) as seen in activated CF neutrophils. These pH(i) differences in activated or acid-loaded CF neutrophils are abrogated by ZnCl(2) but not by amiloride and bafilomycin A(1), suggesting that passive proton conductance is abnormal in CF. In addition, DIDS, which inhibits HCO(3)(-)/Cl(-) exchange, causes alkalinization of control but not of CF neutrophils, suggesting that anion transport is also abnormal in CF neutrophils. In summary, we have shown that pH(i) regulation in CF neutrophils is intrinsically abnormal, potentially contributing to the pulmonary manifestations of the condition.

  4. Tsetse EP Protein Protects the Fly Midgut from Trypanosome Establishment

    PubMed Central

    Haines, Lee R.; Lehane, Stella M.; Pearson, Terry W.; Lehane, Michael J.

    2010-01-01

    African trypanosomes undergo a complex developmental process in their tsetse fly vector before transmission back to a vertebrate host. Typically, 90% of fly infections fail, most during initial establishment of the parasite in the fly midgut. The specific mechanism(s) underpinning this failure are unknown. We have previously shown that a Glossina-specific, immunoresponsive molecule, tsetse EP protein, is up regulated by the fly in response to gram-negative microbial challenge. Here we show by knockdown using RNA interference that this tsetse EP protein acts as a powerful antagonist of establishment in the fly midgut for both Trypanosoma brucei brucei and T. congolense. We demonstrate that this phenomenon exists in two species of tsetse, Glossina morsitans morsitans and G. palpalis palpalis, suggesting tsetse EP protein may be a major determinant of vector competence in all Glossina species. Tsetse EP protein levels also decline in response to starvation of the fly, providing a possible explanation for increased susceptibility of starved flies to trypanosome infection. As starvation is a common field event, this fact may be of considerable importance in the epidemiology of African trypanosomiasis. PMID:20221444

  5. Bacterial Infection and Immune Responses in Lutzomyia longipalpis Sand Fly Larvae Midgut.

    PubMed

    Heerman, Matthew; Weng, Ju-Lin; Hurwitz, Ivy; Durvasula, Ravi; Ramalho-Ortigao, Marcelo

    2015-01-01

    The midgut microbial community in insect vectors of disease is crucial for an effective immune response against infection with various human and animal pathogens. Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants. Sand flies are major vectors of leishmaniasis, and shown to harbor a wide variety of Gram-negative and Gram-positive bacteria. Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site. Here, we assess the distribution of two bacteria commonly found within the gut of sand flies, Pantoea agglomerans and Bacillus subtilis. We demonstrate that these bacteria are able to differentially infect the larval digestive tract, and regulate the immune response in sand fly larvae. Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut.

  6. Intracellular pH regulation by acid-base transporters in mammalian neurons

    PubMed Central

    Ruffin, Vernon A.; Salameh, Ahlam I.; Boron, Walter F.; Parker, Mark D.

    2014-01-01

    Intracellular pH (pHi) regulation in the brain is important in both physiological and physiopathological conditions because changes in pHi generally result in altered neuronal excitability. In this review, we will cover 4 major areas: (1) The effect of pHi on cellular processes in the brain, including channel activity and neuronal excitability. (2) pHi homeostasis and how it is determined by the balance between rates of acid loading (JL) and extrusion (JE). The balance between JE and JL determine steady-state pHi, as well as the ability of the cell to defend pHi in the face of extracellular acid-base disturbances (e.g., metabolic acidosis). (3) The properties and importance of members of the SLC4 and SLC9 families of acid-base transporters expressed in the brain that contribute to JL (namely the Cl-HCO3 exchanger AE3) and JE (the Na-H exchangers NHE1, NHE3, and NHE5 as well as the Na+- coupled HCO3− transporters NBCe1, NBCn1, NDCBE, and NBCn2). (4) The effect of acid-base disturbances on neuronal function and the roles of acid-base transporters in defending neuronal pHi under physiopathologic conditions. PMID:24592239

  7. Differential Protein Modulation in Midguts of Aedes aegypti Infected with Chikungunya and Dengue 2 Viruses

    PubMed Central

    Tchankouo-Nguetcheu, Stéphane; Khun, Huot; Pincet, Laurence; Roux, Pascal; Bahut, Muriel; Huerre, Michel; Guette, Catherine; Choumet, Valérie

    2010-01-01

    Background Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place. Methodology and Principal Findings Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE), we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI) with dengue 2 (DENV-2) and chikungunya (CHIKV) viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification. Conclusion/Significance Our study constitutes the first analysis of the protein response of Aedes aegypti's midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha glucosidase, may favour

  8. Ruminant Nutrition Symposium: Role of fermentation acid absorption in the regulation of ruminal pH.

    PubMed

    Aschenbach, J R; Penner, G B; Stumpff, F; Gäbel, G

    2011-04-01

    , SCFA absorption also accelerates urea transport into the rumen, which via ammonium recycling, may remove protons from rumen to the blood. Ammonium absorption into the blood is also stimulated by luminal SCFA. It is suggested that the interacting transport processes for SCFA, urea, and ammonia represent evolutionary adaptations of ruminants to actively coordinate energy fermentation, protein assimilation, and pH regulation in the rumen.

  9. Midgut morphophysiology in Sitophilus zeamais Motschulsky, 1855 (Coleoptera: Curculionidae).

    PubMed

    de Sousa, Géssica; Conte, Helio

    2013-08-01

    Sitophilus zeamais is one of the most aggressive pests of stored grains, causing a significant decrease in the nutritional quality of the grains and major losses in economic trade. The foraging capacity of this pest is assigned to its highly efficient digestive system. Investigations on the morphofunctional features of the midgut, which is the most active region of the alimentary canal, are fundamental to understand the feeding habits of this species. In this study, the midgut of adult insects was isolated, processed, and analyzed on light microscopy, scanning and transmission electron microscopy, protein and enzymatic activities determination, including analyses of the starch hydrolysis products. In S. zeamais, the midgut was differentiated into anterior midgut and posterior midgut, and consisted of digestive, regenerative and endocrine cells. The anterior midgut showed high density of regenerative crypts. Cells containing organelles associated with protein synthesis and presence of amylases and lipases indicated that majority of the digestion process occurred in the anterior midgut. The posterior midgut exhibited numerous gastric caeca and peritrophic membrane. Cells with poorly differentiated cytoplasmic into organelles, elongated microvilli, and low enzymatic activities indicated that the posterior midgut was mainly involved in absorption.

  10. A macrophage cell model for pH and volume regulation.

    PubMed

    Luo, C; Clark, J W; Heming, T A; Bidani, A

    2006-01-21

    A whole-cell model of a macrophage (mphi) is developed to simulate pH and volume regulation during a NH4Cl prepulse challenge. The cell is assumed spherical, with a plasma membrane that separates the cytosolic and extracellular bathing media. The membrane contains background currents for Na+, K+ and Cl-, a Na(+)-K+ pump, a V-type H(+)-extruder (V-ATPase), and a leak pathway for NH4+. Cell volume is controlled by instantaneous osmotic balance between cytosolic and extracellular osmolytes. Simulations reveal that the mphi model can mimic alterations in measured pH(i) and cell volume (Vol(i)) data during and after delivery of an ammonia prepulse, which induces an acid load within the cell. Our analysis indicates that there are substantial problems in quantifying transporter-mediated H+ efflux solely from experimental observations of pH(i) recovery, as is commonly done in practice. Problems stemming from the separation of effects arise, since there is residual NH4+ dissociation to H+ inside the mphi during pH(i) recovery, as well as, proton extrusion via the V-ATPase. The core assumption of conventional measurement techniques used to estimate the H+ extrusion current (I(H)) is that the recovery phase is solely dependent on transporter-mediated H+ extrusion. However, our model predictions suggest that there are major problems in using this approach, due to the complex interactions between I(H), NH3/NH4+ buffering and NH3/NH4+ efflux during the active acid extrusion phase. That is, the conventional buffer capacity-based I(H) estimation must also take into account the perturbation that a prepulse challenge brings to the cytoplasmic acid buffer itself. The importance of this whole-cell model of mphipH(i) and volume regulation lies in its potential for extension to the characterization of several other types of non-excitable cells, such as the microglia (brain macrophage) and the T-lymphocyte. PMID:16043192

  11. Mechanisms of microenvironmental pH regulation in the cuticle of Ascaris suum.

    PubMed

    Sims, S M; Magas, L T; Barsuhn, C L; Ho, N F; Geary, T G; Thompson, D P

    1992-07-01

    The excretion kinetics of various organic acids by Ascaris suum were quantified to determine if the excretion of these metabolic end-products could generate and maintain a microclimate pH within the aqueous compartment of the cuticle. Ligated and nonligated A. suum were incubated in media buffered with 0.25 or 2.5 mM Hepes (initial pH 7.5) or 0.5 or 5 mM glycine (initial pH 3.25). The concentration of organic acids and the pH of the media were followed for 24 h. Several volatile fatty acids, including acetic, 2-methylbutyric, 2-methylvaleric, n-valeric, and n-butyric, were excreted at relatively high rates. Propionic, n-caproic, 2-methylcaproic, tiglic acid, and the non-volatile organic acids, lactic and succinic, were excreted more slowly. The organic acids were excreted at a constant rate and in apparently fixed molar concentration ratios. The accumulation of organic acids was associated with changes in pH of the medium until a limiting constant pH, in the vicinity of the pKa of the volatile fatty acids, was reached. The rate of organic acid excretion was not affected by initial medium pH, buffer capacity, or parasite ligation. The rate of pH change induced by the excretion of organic acids was also insensitive to whether ligated or nonligated A. suum were used, but was dependent on the initial buffer capacity of the medium. These results suggest that A. suum excrete the end-products of carbohydrate metabolism across the cuticle. The presence of organic acids in the aqueous pores of the cuticle creates and maintains a microclimate pH of about 5.0 +/- 0.3. This pH will influence the transport properties of weak acids and bases and should be considered in the design of delivery systems for anthelmintics. PMID:1501633

  12. Regulation of arsenic mobility on basaltic glass surfaces by speciation and pH.

    PubMed

    Sigfusson, Bergur; Meharg, Andrew A; Gislason, Sigurdur R

    2008-12-01

    The importance of geothermal energy as a source for electricity generation and district heating has increased over recent decades. Arsenic can be a significant constituent of the geothermal fluids pumped to the surface during power generation. Dissolved As exists in different oxidation states, mainly as As(III) and As(V), and the charge of individual species varies with pH. Basaltic glass is one of the most important rock types in many high-temperature geothermal fields. Static batch and dynamic column experiments were combined to generate and validate sorption coefficients for As(III) and As(V) in contact with basaltic glass at pH 3-10. Validation was carried out by two empirical kinetic models and a surface complexation model (SCM). The SCM provided a better fit to the experimental column data than kinetic models at high pH values. However, in certain circumstances, an adequate estimation of As transport in the column could not be attained without incorporation of kinetic reactions. The varying mobility with pH was due to the combined effects of the variable charge of the basaltic glass with the pH point of zero charge at 6.8 and the individual As species as pH shifted, respectively. The mobility of As(III) decreased with increasing pH. The opposite was true for As(V), being nearly immobile at pH 3 to being highly mobile at pH 10. Incorporation of appropriate sorption constants, based on the measured pH and Eh of geothermal fluids, into regional groundwater-flow models should allow prediction of the As(III) and As(V) transport from geothermal systems to adjacent drinking water sources and ecosystems.

  13. Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes.

    PubMed

    Isoe, Jun; Rascón, Alberto A; Kunz, Susan; Miesfeld, Roger L

    2009-12-01

    Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post-blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles (P < 0.001). These results demonstrate that AaLT, AaSPVI, and AaSPVII all contribute to blood protein digestion and oocyte maturation, even though AaSPVI is the only abundant midgut late phase serine protease that appears to function as a classic trypsin enzyme. PMID:19883761

  14. Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells

    PubMed Central

    Ramphul, Urvashi N.; Garver, Lindsey S.; Molina-Cruz, Alvaro; Canepa, Gaspar E.; Barillas-Mury, Carolina

    2015-01-01

    The malaria parasite, Plasmodium, must survive and develop in the mosquito vector to be successfully transmitted to a new host. The Plasmodium falciparum Pfs47 gene is critical for malaria transmission. Parasites that express Pfs47 (NF54 WT) evade mosquito immunity and survive, whereas Pfs47 knockouts (KO) are efficiently eliminated by the complement-like system. Two alternative approaches were used to investigate the mechanism of action of Pfs47 on immune evasion. First, we examined whether Pfs47 affected signal transduction pathways mediating mosquito immune responses, and show that the Jun-N-terminal kinase (JNK) pathway is a key mediator of Anopheles gambiae antiplasmodial responses to P. falciparum infection and that Pfs47 disrupts JNK signaling. Second, we used microarrays to compare the global transcriptional responses of A. gambiae midguts to infection with WT and KO parasites. The presence of Pfs47 results in broad and profound changes in gene expression in response to infection that are already evident 12 h postfeeding, but become most prominent at 26 h postfeeding, the time when ookinetes invade the mosquito midgut. Silencing of 15 differentially expressed candidate genes identified caspase-S2 as a key effector of Plasmodium elimination in parasites lacking Pfs47. We provide experimental evidence that JNK pathway regulates activation of caspases in Plasmodium-invaded midgut cells, and that caspase activation is required to trigger midgut epithelial nitration. Pfs47 alters the cell death pathway of invaded midgut cells by disrupting JNK signaling and prevents the activation of several caspases, resulting in an ineffective nitration response that makes the parasite undetectable by the mosquito complement-like system. PMID:25552553

  15. Effect of low pH exposure on Na(+) regulation in two cichlid fish species of the Amazon.

    PubMed

    Duarte, Rafael M; Ferreira, Marcio S; Wood, Chris M; Val, Adalberto L

    2013-11-01

    We evaluated the effects of acute exposure to low pH on Na(+) regulation in two Amazon cichlids collected from natural ion-poor "blackwaters", angelfish (Pterophyllum scalare) and discus (Symphysodon discus). Na(+) uptake kinetic parameters, unidirectional Na(+) fluxes, and net Cl(-) fluxes were determined at pH6.0 and 3.6. At pH6.0, both species presented low unidirectional Na(+) flux rates, with kinetics showing a relatively low affinity for Na(+) (angelfish Km=79, discus Km=268μmolL(-1)), with similar maximum transport capacities (Jmax~535nmolg(-1)h(-1)). Overall, there appeared to be high sensitivity to inhibition by low pH, yet low intrinsic branchial permeability limiting diffusive ion effluxes, resulting in relatively low net loss rates of Na(+), the same strategy as seen previously in other blackwater cichlids, and very different from the strategy of blackwater characids. At low pH, Na(+) uptake in angelfish was inhibited competitively (increased Km=166μmolL(-1)) and non-competitively (decreased Jmax=106nmolg(-1)h(-1)), whereas in discus, only a decrease in Jmax (112nmolg(-1)h(-1)) was statistically significant. An acute reduction in H(+)-ATPase activity, but not in Na(+)/K(+)-ATPase activity, in the gills of angelfish suggests a possible mechanism for this non-competitive inhibition at low pH. Discus fish were more tolerant to low pH than angelfish, as seen by lesser effects of exposure to pH3.6 on unidirectional Na(+) uptake and efflux rates and net Na(+) and Cl(-) loss rates. Overall, discus are better than angelfish in maintaining ionic balance under acidic, ion-poor conditions. PMID:23911980

  16. The pH regulated phycobiliproteins loading and releasing of polyelectrolytes multilayer microcapsules.

    PubMed

    Li, Ye; Lu, Liying; Zhang, Hengjian; Wang, Jin

    2012-05-01

    The polyelectrolytes multilayer microcapsules considered as a good matrix can meet the requirements of protein encapsulation and release. It is important to understand the factors affecting the encapsulation and release of proteins in capsules. In this study, the eight layers hollow capsules (PSS/PAH)(4) and nine layers hollow capsules (PSS/PAH)(4)PSS are fabricated. The protein, R-Phycoerythrins (R-PEs) is employed as a probe instead of fluorescein isothiocyanate labeled proteins to investigate protein loading capacities on capsules as a function of pH, since R-PEs demonstrate an excellent stability over a broad pH range. The loading capacities of R-PEs on capsules (PSS/PAH)(4) or (PSS/PAH)(4)PSS are demonstrated to be sensitive to pH. The R-PE encapsulated in capsules exhibit the largest load capacity around isoelectric point of the protein independent of outer most layer of polyelectrolytes. However, if the pH of buffer is far away from the isoelectric point of the protein, they are absorbed on the surface of capsules. Based on a Freundlich model, capsules take up proteins on their surface by monolayer adsorption. The release process of R-PEs from microcapsules to solution is also shown to be sensitive to pH. Proteins show a faster release process around isoelectric point. Therefore, the pH sensitive polyelectrolyte microcapsules may offer a promising delivery system for loading and releasing proteins in biological systems depending on environment.

  17. Fine Physical Mapping of Ph1, a Chromosome Pairing Regulator Gene in Polyploid Wheat

    PubMed Central

    Gill, K. S.; Gill, B. S.; Endo, T. R.; Mukai, Y.

    1993-01-01

    The diploid-like chromosome pairing in polyploid wheat is controlled by the Ph1 (pairing homoeologous) gene that is located on chromosome arm 5BL. By using a combination of cytogenetic and molecular techniques, we report the physical location of the Ph1 gene to a submicroscopic chromosome region (Ph1 gene region) that is flanked by the breakpoints of two deletions (5BL-1 and ph1c) and is marked by a DNA probe (XksuS1). The Ph1 gene region is present distal to the breakpoint of deletion 5BL-1 but proximal to the C-band 5BL2.1. Two other DNA probes (Xpsr128 and Xksu75) flank the region-Xpsr128 being proximal and Xksu75 being distal. The estimated size of the region is less than 3 Mb. The chromosome region around the Ph1 gene is high in recombination as the genetic distance of the region between 5BL-1 breakpoint and C-band 5BL2.1 (not resolved by the microscope) is at least 9.3 cM. PMID:8375657

  18. Overcoming Hypoxia-Mediated Tumor Progression: Combinatorial Approaches Targeting pH Regulation, Angiogenesis and Immune Dysfunction

    PubMed Central

    McDonald, Paul C.; Chafe, Shawn C.; Dedhar, Shoukat

    2016-01-01

    Hypoxia is an important contributor to the heterogeneity of the microenvironment of solid tumors and is a significant environmental stressor that drives adaptations which are essential for the survival and metastatic capabilities of tumor cells. Critical adaptive mechanisms include altered metabolism, pH regulation, epithelial-mesenchymal transition, angiogenesis, migration/invasion, diminished response to immune cells and resistance to chemotherapy and radiation therapy. In particular, pH regulation by hypoxic tumor cells, through the modulation of cell surface molecules such as extracellular carbonic anhydrases (CAIX and CAXII) and monocarboxylate transporters (MCT-1 and MCT-4) functions to increase cancer cell survival and enhance cell invasion while also contributing to immune evasion. Indeed, CAIX is a vital regulator of hypoxia mediated tumor progression, and targeted inhibition of its function results in reduced tumor growth, metastasis, and cancer stem cell function. However, the integrated contributions of the repertoire of hypoxia-induced effectors of pH regulation for tumor survival and invasion remain to be fully explored and exploited as therapeutic avenues. For example, the clinical use of anti-angiogenic agents has identified a conundrum whereby this treatment increases hypoxia and cancer stem cell components of tumors, and accelerates metastasis. Furthermore, hypoxia results in the infiltration of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Treg) and Tumor Associated Macrophages (TAMs), and also stimulates the expression of PD-L1 on tumor cells, which collectively suppress T-cell mediated tumor cell killing. Therefore, combinatorial targeting of angiogenesis, the immune system and pH regulation in the context of hypoxia may lead to more effective strategies for curbing tumor progression and therapeutic resistance, thereby increasing therapeutic efficacy and leading to more effective strategies for the treatment of patients with

  19. pH Regulation of Electrogenic Sugar/H+ Symport in MFS Sugar Permeases

    PubMed Central

    Bazzone, Andre; Madej, M. Gregor; Kaback, H. Ronald

    2016-01-01

    Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ≥ 9.4 and at acidic to neutral pH = 4.6–7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters. PMID:27227677

  20. Sites of pH regulation of the urea channel of Helicobacter pylori.

    PubMed

    Weeks, D L; Sachs, G

    2001-06-01

    Helicobacter pylori (Hp) and Streptococcus salivarius (Ss) require intrabacterial urease for acid resistance and express a urea channel, UreI. The presence of UreI was shown to increase urea permeability approximately 300-fold over that of a non-polar ureI deletion mutant. Expression of SsUreI in Xenopus oocytes increased urea uptake pH independently, whereas HpUreI shows an acidic pH dependence, half-maximal at pH 6.0. Mutagenesis of all histidines, aspartates, glutamates and the lysine in the periplasmic domain of HpUreI showed that His-123, His-131, Asp-129, Asp-140, Glu-138 and Lys-132 in the second periplasmic loop (PL2) and His-193 in the C-terminus (Ct) were important for activation of transport. With the exception of a lysine that was shown to substitute for His-193 in HpUreI, these charged amino acids are absent in SsUreI. A chimera in which PL1 of HpUreI was replaced by PL1 of SsUreI retained activity at acidic pH and gained partial activity at neutral pH. Exchange of PL2 inactivated transport, whereas exchange of Ct had no effect. Chimeras, in which either PL1 or PL2 of HpUreI replaced those of SsUreI, retained wild-type transport, but replacement of the Ct or both loops inactivated transport. PL1 appears to be important for restricting transport through HpUreI at neutral pH, whereas protonation of three histidines in PL2 and Ct and the presence of three dicarboxylic amino acids in PL2 appears to be necessary to activate HpUreI at acidic pH.

  1. pH Regulation of Electrogenic Sugar/H+ Symport in MFS Sugar Permeases.

    PubMed

    Bazzone, Andre; Madej, M Gregor; Kaback, H Ronald; Fendler, Klaus

    2016-01-01

    Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ≥ 9.4 and at acidic to neutral pH = 4.6-7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters. PMID:27227677

  2. Suppressor of Deltex mediates Pez degradation and modulates Drosophila midgut homeostasis.

    PubMed

    Wang, Chao; Zhang, Wenxiang; Yin, Meng-Xin; Hu, Lianxin; Li, Peixue; Xu, Jiajun; Huang, Hongling; Wang, Shimin; Lu, Yi; Wu, Wenqing; Ho, Margaret S; Li, Lin; Zhao, Yun; Zhang, Lei

    2015-01-01

    Pez functions as an upstream negative regulator of Yorkie (Yki) to regulate intestinal stem cell (ISC) proliferation and is essential for the activity of the Hippo pathway specifically in the Drosophila midgut epithelium. Here we report that Suppressor of Deltex (Su(dx)) acts as a negative regulator of Pez. We show that Su(dx) targets Pez for degradation both in vitro and in vivo. Overexpression of Su(dx) induces proliferation in the fly midgut epithelium, which can be rescued by overexpressed Pez. We also demonstrate that the interaction between Su(dx) and Pez, bridged by WW domains and PY/PPxY motifs, is required for Su(dx)-mediated Pez degradation. Furthermore, we find that Kibra, a binding partner of Pez, stabilizes Pez via WW-PY/PPxY interaction. Moreover, PTPN14, a Pez mammalian homolog, is degraded by overexpressed Su(dx) or Su(dx) homologue WWP1 in mammalian cells. These results reveal a previously unrecognized mechanism of Pez degradation in maintaining the homeostasis of Drosophila midgut. PMID:25814387

  3. Bursicon-α subunit modulates dLGR2 activity in the adult Drosophila melanogaster midgut independently to Bursicon-β.

    PubMed

    Scopelliti, Alessandro; Bauer, Christin; Cordero, Julia B; Vidal, Marcos

    2016-06-17

    Bursicon is the main regulator of post molting and post eclosion processes during arthropod development. The active Bursicon hormone is a heterodimer of Burs-α and Burs-β. However, adult midguts express Burs-α to regulate the intestinal stem cell niche. Here, we examined the potential expression and function of its heterodimeric partner, Burs-β in the adult midgut. Unexpectedly, our evidence suggests that Burs-β is not significantly expressed in the adult midgut. burs-β mutants displayed the characteristic developmental defects but showed wild type-like adult midguts, thus uncoupling the developmental and adult phenotypes seen in burs-α mutants. Gain of function data and ex vivo experiments using a cAMP biosensor, demonstrated that Burs-α is sufficient to drive stem cell quiescence and to activate dLGR2 in the adult midgut. Our evidence suggests that the post developmental transactivation of dLGR2 in the adult midgut is mediated by Burs-α and that the β subunit of Bursicon is dispensable for these activities. PMID:27191973

  4. Bursicon-α subunit modulates dLGR2 activity in the adult Drosophila melanogaster midgut independently to Bursicon-β

    PubMed Central

    Scopelliti, Alessandro; Bauer, Christin; Cordero, Julia B.; Vidal, Marcos

    2016-01-01

    ABSTRACT Bursicon is the main regulator of post molting and post eclosion processes during arthropod development. The active Bursicon hormone is a heterodimer of Burs-α and Burs-β. However, adult midguts express Burs-α to regulate the intestinal stem cell niche. Here, we examined the potential expression and function of its heterodimeric partner, Burs-β in the adult midgut. Unexpectedly, our evidence suggests that Burs-β is not significantly expressed in the adult midgut. burs-β mutants displayed the characteristic developmental defects but showed wild type-like adult midguts, thus uncoupling the developmental and adult phenotypes seen in burs-α mutants. Gain of function data and ex vivo experiments using a cAMP biosensor, demonstrated that Burs-α is sufficient to drive stem cell quiescence and to activate dLGR2 in the adult midgut. Our evidence suggests that the post developmental transactivation of dLGR2 in the adult midgut is mediated by Burs-α and that the β subunit of Bursicon is dispensable for these activities. PMID:27191973

  5. Developmental adaptations in cytosolic phosphate content and pH regulation in the sheep heart in vivo.

    PubMed Central

    Portman, M A; Ning, X H

    1990-01-01

    This study examines adaptations in myocardial cytosolic phosphate content and buffering capacity that occur in vivo as a function of development. Phosphate metabolites were monitored in an open chest sheep preparation using a 31P magnetic resonance surface coil over the left ventricle. Newborn lambs (aged 4-9 d, n = 5) underwent exchange transfusion with adult blood to reduce blood-borne 2,3-diphosphoglycerate contamination of the heart monophosphate and phosphomonoester resonances, thus allowing determination of these phosphate concentrations. The blood-exchanged newborns and mature controls (aged 30-60 d, n = 5) were infused with 0.4 N hydrochloric acid to decrease pH from greater than 7.35 to less than 7.00. Simultaneously, intracellular and extracellular pH were determined from the chemical shifts of the respective phosphate peaks and compared to arterial blood pH. Findings were as follows: (a) diphosphoglycerate contribution to the cardiac spectrum was found to be negligible, (b) significant decreases in cytosolic phosphate (P less than 0.03) and phosphomonoester (P less than 0.01) content occurred with maturation, and (c) large decreases in extracellular pH (greater than 0.5 U) in both groups were similarly associated with only small changes in intracellular pH (less than 0.1 U). Change in cytosolic phosphate content implies that alterations occur in the phosphorylation potential with resulting effects on regulation of myocardial respiration, and cardiac energetics. PMID:2254447

  6. Gene expression analysis of the endosymbiont-bearing midgut tissue during ontogeny of the carpenter ant Camponotus floridanus.

    PubMed

    Ratzka, Carolin; Gross, Roy; Feldhaar, Heike

    2013-06-01

    Insects have frequently evolved mutualistic relationships with extracellular and/or intracellular bacterial endosymbionts. Infection with endosymbionts seems to affect several cellular functions of the host such as immune pathways, oxidative stress regulation and autophagy. Our current knowledge about specific host factors leading to endosymbiont tolerance and/or control is still scarce and is based on very few associations between insect hosts and bacteria only. Camponotus floridanus ants harbour the obligate intracellular bacterium Blochmannia floridanus within specialized midgut cells called bacteriocytes. The number of Blochmannia endosymbionts within the midgut tissue increases strongly during host development and reaches a maximum at the late pupal stage, where the entire midgut is transformed into a symbiotic organ. After eclosion of workers the number of Blochmannia strongly decreases again. We chose 15 candidate genes from C. floridanus likely to be involved in host-symbiont interactions based on their significant homology to previously investigated symbiosis-relevant genes from other insects. We determined the expression of these genes in the endosymbiont-bearing midgut tissue in comparison to the residual body tissue at different developmental stages of C. floridanus in order to reveal changes in gene expression correlating with changes in endosymbiont number per host. Strikingly, two pattern recognition receptors (amidase PGRP-LB and PGRP-SC2) were highly expressed in the midgut tissue at the pupal stage, potentially down-modulating the IMD pathway to enable endosymbiont tolerance. Moreover, we investigated the immune gene expression in response to bacterial challenge at the pupal stage. Results showed that the midgut tissue differs in expression pattern in contrast to the residual body. Our results support a key role for amidase PGRPs, especially PGRP-LB, in regulation of the immune response towards endosymbionts in C. floridanus and suggest an

  7. Gene expression analysis of the endosymbiont-bearing midgut tissue during ontogeny of the carpenter ant Camponotus floridanus.

    PubMed

    Ratzka, Carolin; Gross, Roy; Feldhaar, Heike

    2013-06-01

    Insects have frequently evolved mutualistic relationships with extracellular and/or intracellular bacterial endosymbionts. Infection with endosymbionts seems to affect several cellular functions of the host such as immune pathways, oxidative stress regulation and autophagy. Our current knowledge about specific host factors leading to endosymbiont tolerance and/or control is still scarce and is based on very few associations between insect hosts and bacteria only. Camponotus floridanus ants harbour the obligate intracellular bacterium Blochmannia floridanus within specialized midgut cells called bacteriocytes. The number of Blochmannia endosymbionts within the midgut tissue increases strongly during host development and reaches a maximum at the late pupal stage, where the entire midgut is transformed into a symbiotic organ. After eclosion of workers the number of Blochmannia strongly decreases again. We chose 15 candidate genes from C. floridanus likely to be involved in host-symbiont interactions based on their significant homology to previously investigated symbiosis-relevant genes from other insects. We determined the expression of these genes in the endosymbiont-bearing midgut tissue in comparison to the residual body tissue at different developmental stages of C. floridanus in order to reveal changes in gene expression correlating with changes in endosymbiont number per host. Strikingly, two pattern recognition receptors (amidase PGRP-LB and PGRP-SC2) were highly expressed in the midgut tissue at the pupal stage, potentially down-modulating the IMD pathway to enable endosymbiont tolerance. Moreover, we investigated the immune gene expression in response to bacterial challenge at the pupal stage. Results showed that the midgut tissue differs in expression pattern in contrast to the residual body. Our results support a key role for amidase PGRPs, especially PGRP-LB, in regulation of the immune response towards endosymbionts in C. floridanus and suggest an

  8. The midgut of the silkmoth Bombyx mori is able to recycle molecules derived from degeneration of the larval midgut epithelium.

    PubMed

    Franzetti, Eleonora; Romanelli, Davide; Caccia, Silvia; Cappellozza, Silvia; Congiu, Terenzio; Rajagopalan, Muthukumaran; Grimaldi, Annalisa; de Eguileor, Magda; Casartelli, Morena; Tettamanti, Gianluca

    2015-08-01

    The midgut represents the middle part of the alimentary canal and is responsible for nutrient digestion and absorption in insect larva. Despite the growing interest in this organ for different purposes, such as studies on morphogenesis and differentiation, stem cell biology, cell death processes and transport mechanisms, basic information on midgut development is still lacking for a large proportion of insect species. Undoubtedly, this lack of data could hinder the full exploitation of practical applications that involve midgut as their primary target. This may represent in particular a significant problem for Lepidoptera, an insect order that includes some of the most important species of high economic importance. With the aim of overcoming this fragmentation of knowledge, we performed a detailed morphofunctional analysis of the midgut of the silkworm, Bombyx mori, a representative model among Lepidoptera, during its development from the larval up to the adult stage, focusing attention on stem cells. Our data demonstrate stem cell proliferation and differentiation, not only in the larval midgut but also in the pupal and adult midgut epithelium. Moreover, we present evidence for a complex trophic relationship between the dying larval epithelium and the new adult one, which is established during metamorphosis. This study, besides representing the first morphological and functional characterization of the changes that occur in the midgut of a lepidopteron during the transition from the larva to the moth, provides a detailed analysis of the midgut of the adult insect, a stage that has been neglected up to now.

  9. CsrRS and Environmental pH Regulate Group B Streptococcus Adherence to Human Epithelial Cells and Extracellular Matrix

    PubMed Central

    Park, Su Eun; Jiang, Shengmei

    2012-01-01

    Streptococcus agalactiae (group B Streptococcus or GBS) is a common colonizer of the gastrointestinal and genital tracts and an important cause of invasive infections in newborn infants and in adults with predisposing chronic conditions or advanced age. Attachment to epithelial surfaces at mucosal sites is a critical step in the successful colonization of a human host, and regulation of this process is likely to play an important role in both commensalism and dissemination to cause invasive disease. We found that inactivation of the CsrRS (or CovRS) two-component system increased GBS adherence to epithelial cells derived from human vaginal, cervical, and respiratory epithelium, as well as increasing adherence to extracellular matrix proteins and increasing biofilm formation on polystyrene. Neutral (as opposed to acidic) pH enhanced GBS binding to vaginal epithelial cells and to fibrinogen and fibronectin, effects that were partially dependent on CsrRS. The regulatory effects of CsrRS and environmental pH on bacterial adherence correlated with their effects on the expression of multiple surface adhesins, as assessed by quantitative reverse transcription-PCR. We conclude that GBS adherence to epithelial and abiotic surfaces is regulated by the CsrRS two-component system and by environmental pH through their regulatory effects on the expression of bacterial surface adhesins. Dynamic regulation of GBS adherence enhances the organism's adaptability to survival in multiple niches in the human host. PMID:22949550

  10. Binding-protein-dependent alanine transport in Rhodobacter sphaeroides is regulated by the internal pH.

    PubMed

    Abee, T; van der Wal, F J; Hellingwerf, K J; Konings, W N

    1989-09-01

    The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R. sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F. E. Nano, Ph.D. thesis, University of Illinois, Urbana, 1984). Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M. G. L. Elferink, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1986). The results presented in this paper offer an explanation for these findings. Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system. The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3). The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system. Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles. This indicates that the H+/lactose symport system is active without electron transfer. Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport. Uptake of TMG and AIB is regulated by the internal pH. The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB. At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8. In a previous study, we found that K+ plays an essential role in regulating the internal pH (T. Abee, K. J. Hellingwerf, and W

  11. Tissue-specific PhBPBT expression is differentially regulated in response to endogenous ethylene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethylene is a gaseous plant hormone involved in many physiological processes including senescence, fruit ripening, and defense. Here we show the effects of pollination and wound-induced ethylene signals on transcript accumulation of benzoyl CoA:benzyl alcohol/phenylethanol benzoyltransferase (PhBPBT...

  12. Dynamin GTPase Regulation is Altered by PH Domain Mutations Found in Centronuclear Myopathy Patients

    SciTech Connect

    Kenniston, J.; Lemmon, M

    2010-01-01

    The large GTPase dynamin has an important membrane scission function in receptor-mediated endocytosis and other cellular processes. Self-assembly on phosphoinositide-containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin-homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C-terminal {alpha}-helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either 'sensitize' dynamin to lipid stimulation or elevate basal GTPase rates by promoting self-assembly and thus rendering dynamin no longer lipid responsive. We also describe a low-resolution structure of dimeric dynamin from small-angle X-ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self-assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.

  13. Rewiring global regulator cAMP receptor protein (CRP) to improve E. coli tolerance towards low pH.

    PubMed

    Basak, Souvik; Geng, Hefang; Jiang, Rongrong

    2014-03-10

    Bioprocesses such as production of organic acids or acid hydrolysis of bioresources during biofuel production often suffer limitations due to microbial sensitivity under acidic conditions. Approaches for improving the acid tolerance of these microbes have mainly focused on using metabolic engineering tools. Here, we tried to improve strain acidic tolerance from its transcription level, i.e. we adopted error-prone PCR method to engineer global regulator cAMP receptor protein (CRP) of Escherichia coli to improve its performance at low pH. The best mutant AcM1 was identified from random mutagenesis libraries based on its growth performance. AcM1 almost doubled (0.113h(-1)) the growth rate of the control (0.062h(-1)) at pH 4.24. It also demonstrated better thermotolerance than the control at 48°C, whose growth was completely inhibited at this temperature. Quantitative real time reverse transcription PCR results revealed a stress response overlap among low pH stress-, oxidative stress- and osmotic stress-related genes. The chief enzyme responsible for cell acid tolerance, glutamate decarboxylase, demonstrated over twofold activity in AcM1 compared to the control. Differential binding properties of AcM1 mutant CRP with Class-I, II, and III CRP-dependent promoters suggested that modifications to native CRP may lead to transcription profile changes. Hence, we believe that transcriptional engineering of global regulator CRP can provide a new strain engineering alternative for E. coli.

  14. pH Dependence of the Stress Regulator DksA

    PubMed Central

    Furman, Ran; Danhart, Eric M.; NandyMazumdar, Monali; Yuan, Chunhua; Foster, Mark P.; Artsimovitch, Irina

    2015-01-01

    DksA controls transcription of genes associated with diverse stress responses, such as amino acid and carbon starvation, oxidative stress, and iron starvation. DksA binds within the secondary channel of RNA polymerase, extending its long coiled-coil domain towards the active site. The cellular expression of DksA remains constant due to a negative feedback autoregulation, raising the question of whether DksA activity is directly modulated during stress. Here, we show that Escherichia coli DksA is essential for survival in acidic conditions and that, while its cellular levels do not change significantly, DksA activity and binding to RNA polymerase are increased at lower pH, with a concomitant decrease in its stability. NMR data reveal pH-dependent structural changes centered at the interface of the N and C-terminal regions of DksA. Consistently, we show that a partial deletion of the N-terminal region and substitutions of a histidine 39 residue at the domain interface abolish pH sensitivity in vitro. Together, these data suggest that DksA responds to changes in pH by shifting between alternate conformations, in which competing interactions between the N- and C-terminal regions modify the protein activity. PMID:25799498

  15. Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

    PubMed

    Namkoong, Eun; Shin, Yong-Hwan; Bae, Jun-Seok; Choi, Seulki; Kim, Minkyoung; Kim, Nahyun; Hwang, Sung-Min; Park, Kyungpyo

    2015-01-01

    Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.

  16. Carbon and nitrogen dynamics across a bedrock-regulated subarctic pH gradient

    NASA Astrophysics Data System (ADS)

    Tomczyk, N.; Heim, E. W.; Sadowsky, J.; Remiszewski, K.; Varner, R. K.; Bryce, J. G.; Frey, S. D.

    2014-12-01

    Bedrock geochemistry has been shown to influence landscape evolution due to nutrient limitation on primary production. There may also be less direct interactions between bedrock-derived chemicals and ecosystem function. Effects of calcium (Ca) and pH on soil carbon (C) and nitrogen (N) cycling have been shown in acid impacted forests o f North America. Understanding intrinsic factors that affect C and nutrient dynamics in subarctic ecosystems has implications for how these ecosystems will respond to a changing climate. How the soil microbial community allocates enzymes to acquire resources from the environment can indicate whether a system is nutrient or energy limited. This study examined whether bedrock geochemistry exerts pressure on nutrient cycles in the overlying soils. In thin, weakly developed soils, bedrock is the primary mineral material and is a source of vital nutrients. Nitrogen (N) and C are not derived from bedrock, but their cycling is still affected by reactions with geologically-derived chemicals. Our study sites near Abisko, Sweden (~68°N) were selected adjacent to five distinct bedrock outcrops (quartzite, slate, carbonate, and two different metasedimenty units). All sites were at a similar elevation (~700 m a.s.l.) and had similar vegetation (subarctic heath). Nutrient concentrations in bedrock and soils were measured in addition to soil microbial biomass and extracellular enzyme activity. We found a statistically significant correlation between soil Ca concentrations and soil pH (r = 0.88, p < 0.01). There were also significant relationships between soil pH and the ratio of C-acquiring to N-acquiring enzyme activity (r = -0.89, p < 0.01), soil pH and soil C-to-N ratio (r = -0.76, p < 0.01), and the ratio of C-acquiring to N-acquiring enzyme activity and soil C-to-N ratio (r = 0.78, p < 0.01). These results suggest that soil Ca concentrations influence C and N cycling dynamics in these soils through their effect on soil pH.

  17. Tyramine biosynthesis in Enterococcus durans is transcriptionally regulated by the extracellular pH and tyrosine concentration

    PubMed Central

    Linares, Daniel M.; Fernández, María; Martín, M. Cruz; Álvarez, Miguel A.

    2009-01-01

    Summary The microbial decarboxylation of some amino acids leads to the undesirable presence of biogenic amines in foods. One of the most abundant and frequent biogenic amines found in fermented foods is tyramine, which is produced by the decarboxylation of tyrosine. In the present work, transcriptional analysis of tyramine biosynthesis in Enterococcus durans IPLA655, a strain isolated from cheese, was studied. The gene coding for the tyrosine decarboxylase (tdcA) and that coding for the tyrosine‐tyramine antiporter (tyrP) form an operon transcribed from the promoter PtdcA, the expression of which is regulated by the extracellular pH and tyrosine concentration. Quantification of gene expression during the log phase of growth showed high concentrations of tyrosine and acidic pH conditions to induce tdcA‐tyrP polycistronic messenger transcription. PMID:21255297

  18. pH regulation in anoxic rice coleoptiles at pH 3.5: biochemical pHstats and net H+ influx in the absence and presence of NO3−

    PubMed Central

    Greenway, Hank; Kulichikhin, Konstantin Y.; Cawthray, Gregory R.; Colmer, Timothy D.

    2012-01-01

    During anoxia, cytoplasmic pH regulation is crucial. Mechanisms of pH regulation were studied in the coleoptile of rice exposed to anoxia and pH 3.5, resulting in H+ influx. Germinating rice seedlings survived a combination of anoxia and exposure to pH 3.5 for at least 4 d, although development was retarded and net K+ efflux was continuous. Further experiments used excised coleoptile tips (7–10 mm) in anoxia at pH 6.5 or 3.5, either without or with 0.2 mM NO3−, which distinguished two processes involved in pH regulation. Net H+ influx (μmol g−1 fresh weight h−1) for coleoptiles with NO3− was ∼1.55 over the first 24 h, being about twice that in the absence of NO3−, but then decreased to 0.5–0.9 as net NO3− uptake declined from ∼1.3 to 0.5, indicating reduced uptake via H+–NO3− symports. NO3− reduction presumably functioned as a biochemical pHstat. A second biochemical pHstat consisted of malate and succinate, and their concentrations decreased substantially with time after exposure to pH 3.5. In anoxic coleoptiles, K+ balancing the organic anions was effluxed to the medium as organic anions declined, and this efflux rate was independent of NO3− supply. Thus, biochemical pHstats and reduced net H+ influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5. PMID:22174442

  19. Regulation of intracellular pH in neuronal and glial tumour cells, studied by multinuclear NMR spectroscopy.

    PubMed

    Flögel, U; Willker, W; Leibfritz, D

    1994-06-01

    The effect of extracellular pH (pHe) on intracellular pH (pHi) and cellular metabolism was examined by multinuclear NMR spectroscopy of cells in vivo and in vitro. A decrease in pHe from 7.4 to 6.4 led to a significant drop in pHi, in both neuronal and glial tumour cells, as detected by in vivo 31P NMR of cells embedded in basement membrane gel threads. A more than 50% decrease in both the phosphocreatine (PCr) level and derivatives of glycolysis (i.e., glycerol 3-phosphate) was observed, concomitantly to the fall in pHi. A 50% decrease in intracellular lactate levels was seen in in vivo 1H NMR spectra under these conditions. Reperfusion with fresh medium (pHe 7.4) resulted in the full recovery of pHi, simultaneously with an increase in both PCr and intracellular lactate back to their control levels. Perchloric acid and lipid extract measurements confirmed the observations made by in vivo 31P and 1H NMR spectroscopy and further showed a decrease both in tricarboxylic acid cycle activity and phospholipid synthesis. The data revealed no significant differences between the neuronal and glial tumour cells investigated. pHi measurements in the presence of inhibitors of the various pH regulatory mechanisms showed that the Na+/H+ exchanger, the carbonic anhydrase and at least one of the bicarbonate-transport systems are involved in pH regulation of both cell types. The results suggest that Na+/H+ exchange is the preferred mechanism by which both neuronal and glial cells regulate their pHi after extracellular acidification.

  20. Lysosomal pH Plays a Key Role in Regulation of mTOR Activity in Osteoclasts.

    PubMed

    Hu, Yingwei; Carraro-Lacroix, Luciene R; Wang, Andrew; Owen, Celeste; Bajenova, Elena; Corey, Paul N; Brumell, John H; Voronov, Irina

    2016-02-01

    Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth. It has been shown to play an important role in osteoclast differentiation, particularly at the earlier stages of osteoclastogenesis. mTOR activation and function, as part of mTORC1 complex, is dependent on lysosomal localization and the vacuolar H(+) -ATPase (V-ATPase) activity; however, the precise mechanism is still not well understood. Using primary mouse osteoclasts that are known to have higher lysosomal pH due to R740S mutation in the V-ATPase a3 subunit, we investigated the role of lysosomal pH in mTORC1 signaling. Our results demonstrated that +/R740S cells had increased basal mTOR protein levels and mTORC1 activity compared to +/+ osteoclasts, while mTOR gene expression was decreased. Treatment with lysosomal inhibitors chloroquine and ammonium chloride, compounds known to raise lysosomal pH, significantly increased mTOR protein levels in +/+ cells, confirming the importance of lysosomal pH in mTOR signaling. These results also suggested that mTOR could be degraded in the lysosome. To test this hypothesis, we cultured osteoclasts with chloroquine or proteasomal inhibitor MG132. Both chloroquine and MG132 increased mTOR and p-mTOR protein levels in +/+ osteoclasts, suggesting that mTOR undergoes both lysosomal and proteasomal degradation. Treatment with cycloheximide, an inhibitor of new protein synthesis, confirmed that mTOR is constitutively expressed and degraded. These results show that, in osteoclasts, the lysosome plays a key role not only in mTOR activation but also in its deactivation through protein degradation, representing a novel molecular mechanism of mTOR regulation.

  1. pH regulates ammonia-oxidizing bacteria and archaea in paddy soils in Southern China.

    PubMed

    Li, Hu; Weng, Bo-Sen; Huang, Fu-Yi; Su, Jian-Qiang; Yang, Xiao-Ru

    2015-07-01

    Ammonia-oxidizing archaea (AOA) and bacteria (AOB) play important roles in nitrogen cycling. However, the effects of environmental factors on the activity, abundance, and diversity of AOA and AOB and the relative contributions of these two groups to nitrification in paddy soils are not well explained. In this study, potential nitrification activity (PNA), abundance, and diversity of amoA genes from 12 paddy soils in Southern China were determined by potential nitrification assay, quantitative PCR, and cloning. The results showed that PNA was highly variable between paddy soils, ranging from 4.05 ± 0.21 to 9.81 ± 1.09 mg NOx-N kg(-1) dry soil day(-1), and no significant correlation with soil parameters was found. The abundance of AOA was predominant over AOB, indicating that AOA may be the major members in aerobic ammonia oxidation in these paddy soils. Community compositions of AOA and AOB were highly variable among samples, but the variations were best explained by pH. AOA sequences were affiliated to the Nitrosopumilus cluster and Nitrososphaera cluster, and AOB were classified into the lineages of Nitrosospira and Nitrosomonas, with Nitrosospira being predominant over Nitrosomonas, accounting for 83.6 % of the AOB community. Moreover, the majority of Nitrosomonas was determined in neutral soils. Canonical correspondence analysis (CCA) analysis further demonstrated that AOA and AOB community structures were significantly affected by pH, soil total organic carbon, total nitrogen, and C/N ratio, suggesting that these factors exert strong effects on the distribution of AOB and AOA in paddy soils in Southern China. In conclusion, our results imply that soil pH was a key explanatory variable for both AOA and AOB community structure and nitrification activity.

  2. Intracellular pH regulation in isolated rat bile duct epithelial cells.

    PubMed Central

    Strazzabosco, M; Mennone, A; Boyer, J L

    1991-01-01

    To evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport. Images PMID:2022723

  3. The zebrafish merovingian mutant reveals a role for pH regulation in hair cell toxicity and function.

    PubMed

    Stawicki, Tamara M; Owens, Kelly N; Linbo, Tor; Reinhart, Katherine E; Rubel, Edwin W; Raible, David W

    2014-07-01

    Control of the extracellular environment of inner ear hair cells by ionic transporters is crucial for hair cell function. In addition to inner ear hair cells, aquatic vertebrates have hair cells on the surface of their body in the lateral line system. The ionic environment of these cells also appears to be regulated, although the mechanisms of this regulation are less understood than those of the mammalian inner ear. We identified the merovingian mutant through genetic screening in zebrafish for genes involved in drug-induced hair cell death. Mutants show complete resistance to neomycin-induced hair cell death and partial resistance to cisplatin-induced hair cell death. This resistance is probably due to impaired drug uptake as a result of reduced mechanotransduction ability, suggesting that the mutants have defects in hair cell function independent of drug treatment. Through genetic mapping we found that merovingian mutants contain a mutation in the transcription factor gcm2. This gene is important for the production of ionocytes, which are cells crucial for whole body pH regulation in fish. We found that merovingian mutants showed an acidified extracellular environment in the vicinity of both inner ear and lateral line hair cells. We believe that this acidified extracellular environment is responsible for the defects seen in hair cells of merovingian mutants, and that these mutants would serve as a valuable model for further study of the role of pH in hair cell function. PMID:24973752

  4. The zebrafish merovingian mutant reveals a role for pH regulation in hair cell toxicity and function

    PubMed Central

    Stawicki, Tamara M.; Owens, Kelly N.; Linbo, Tor; Reinhart, Katherine E.; Rubel, Edwin W.; Raible, David W.

    2014-01-01

    Control of the extracellular environment of inner ear hair cells by ionic transporters is crucial for hair cell function. In addition to inner ear hair cells, aquatic vertebrates have hair cells on the surface of their body in the lateral line system. The ionic environment of these cells also appears to be regulated, although the mechanisms of this regulation are less understood than those of the mammalian inner ear. We identified the merovingian mutant through genetic screening in zebrafish for genes involved in drug-induced hair cell death. Mutants show complete resistance to neomycin-induced hair cell death and partial resistance to cisplatin-induced hair cell death. This resistance is probably due to impaired drug uptake as a result of reduced mechanotransduction ability, suggesting that the mutants have defects in hair cell function independent of drug treatment. Through genetic mapping we found that merovingian mutants contain a mutation in the transcription factor gcm2. This gene is important for the production of ionocytes, which are cells crucial for whole body pH regulation in fish. We found that merovingian mutants showed an acidified extracellular environment in the vicinity of both inner ear and lateral line hair cells. We believe that this acidified extracellular environment is responsible for the defects seen in hair cells of merovingian mutants, and that these mutants would serve as a valuable model for further study of the role of pH in hair cell function. PMID:24973752

  5. HCO3(-)-independent pH regulation in astrocytes in situ is dominated by V-ATPase.

    PubMed

    Hansen, Daniel Bloch; Garrido-Comas, Nestor; Salter, Mike; Fern, Robert

    2015-03-27

    The mechanisms of HCO3(-)-independent intracellular pH (pHi) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H(+) pump by Cl(-) removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pHi in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3-6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3(-)-independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.

  6. Partial characterization of a lipase from gypsy moth (lymantria dispar L.) larval midgut.

    PubMed

    Mrdaković, Marija; Lazarević, Jelica; Perić-Mataruga, Vesna; Ilijin, Larisa; Vlahović, Milena

    2008-01-01

    Lipase activity of the gypsy moth (Lymantria dispar L.) was studied by the spectrophotometric method using crude homogenate of fifth-instar larval midgut tissues as the enzyme source and p-nitrophenyl caprylate (pNPC) as substrate. A Km value of 0.310 mM and a Vmax value of 1.479 U/mg prot. were obtained for this substrate. Among various p-nitrophenyl esters tested, maximum activity was obtained for p-nitrophenyl caprylate and p-nitrophenyl caprate. The enzyme was most active at alkaline pH, with maximum at pH 8.2. Decreased activity was detected after preincubation in buffers of pH below 7.0 and above 8.2. The enzyme was unstable at room temperature. The enzyme was Ca2+ independent. Its activity was inhibited by PMSF, Fe2+, Ag+ and Pb2+, while Fe3+ inhibited enzyme activity by about 40%.

  7. Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

    SciTech Connect

    Lee, Shin-Hae; Park, Joung-Sun; Kim, Young-Shin; Chung, Hae-Young; Yoo, Mi-Ae

    2012-03-10

    Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. In the present study, we utilize the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed in the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or Timp-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. Our data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis. -- Highlights: Black-Right-Pointing-Pointer Mmp1 is expressed in the adult midgut. Black-Right-Pointing-Pointer Mmp1 is involved in the regulation of ISC proliferation activity. Black-Right-Pointing-Pointer Mmp1-related ISC proliferation is associated with EGFR signaling. Black-Right-Pointing-Pointer Mmp1 in the gut is required for the intestinal homeostasis and longevity.

  8. Escherichia coli Response to Uranyl Exposure at Low pH and Associated Protein Regulations

    PubMed Central

    Khemiri, Arbia; Carrière, Marie; Bremond, Nicolas; Ben Mlouka, Mohamed Amine; Coquet, Laurent; Llorens, Isabelle; Chapon, Virginie; Jouenne, Thierry; Cosette, Pascal; Berthomieu, Catherine

    2014-01-01

    Better understanding of uranyl toxicity in bacteria is necessary to optimize strains for bioremediation purposes or for using bacteria as biodetectors for bioavailable uranyl. In this study, after different steps of optimization, Escherichia colicells were exposed to uranyl at low pH to minimize uranyl precipitation and to increase its bioavailability. Bacteria were adapted to mid acidic pH before exposure to 50 or 80 µM uranyl acetate for two hours at pH≈3. To evaluate the impact of uranium, growth in these conditions were compared and the same rates of cells survival were observed in control and uranyl exposed cultures. Additionally, this impact was analyzedby two-dimensional differential gel electrophoresis proteomics to discover protein actors specifically present or accumulated in contact with uranium.Exposure to uranium resulted in differential accumulation of proteins associated with oxidative stress and in the accumulation of the NADH/quinone oxidoreductase WrbA. This FMN dependent protein performs obligate two-electron reduction of quinones, and may be involved in cells response to oxidative stress. Interestingly, this WrbA protein presents similarities with the chromate reductase from E. coli, which was shown to reduce uranyl in vitro. PMID:24587082

  9. Midgut tissue of male pine engraver , Ips pini, synthesizes monoterpenoid pheromone component ipsdienol de novo

    NASA Astrophysics Data System (ADS)

    Hall, Gregory M.; Tittiger, Claus; Andrews, Gracie L.; Mastick, Grant S.; Kuenzli, Marilyn; Luo, Xin; Seybold, Steven J.; Blomquist, Gary J.

    2002-02-01

    For over three decades the site and pathways of bark beetle aggregation pheromone production have remained elusive. Studies on pheromone production in Ips spp. bark beetles have recently shown de novo biosynthesis of pheromone components via the mevalonate pathway. The gene encoding a key regulated enzyme in this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase ( HMG-R), showed high transcript levels in the anterior midgut of male pine engravers, Ips pini (Say) (Coleoptera:Scolytidae). HMG-R expression in the midgut was sex, juvenile hormone, and feeding dependent, providing strong evidence that this is the site of acyclic monoterpenoid (ipsdienol) pheromone production in male beetles. Additionally, isolated midgut tissue from fed or juvenile hormone III (JH III)-treated males converted radiolabeled acetate to ipsdienol, as assayed by radio-HPLC. These data support the de novo production of this frass-associated aggregation pheromone component by the mevalonate pathway. The induction of a metazoan HMG-R in this process does not support the postulated role of microorganisms in ipsdienol production.

  10. Responses of midgut amylases of Helicoverpa armigera to feeding on various host plants.

    PubMed

    Kotkar, Hemlata M; Sarate, Priya J; Tamhane, Vaijayanti A; Gupta, Vidya S; Giri, Ashok P

    2009-08-01

    Midgut digestive amylases and proteinases of Helicoverpa armigera, a polyphagous and devastating insect pest of economic importance have been studied. We also identified the potential of a sorghum amylase inhibitor against H. armigera midgut amylase. Amylase activities were detected in all the larval instars, pupae, moths and eggs; early instars had lower amylase levels which steadily increased up to the sixth larval instar. Qualitative and quantitative differences in midgut amylases of H. armigera upon feeding on natural and artificial diets were evident. Natural diets were categorized as one or more members of legumes, vegetables, flowers and cereals belonging to different plant families. Amylase activity and isoform patterns varied depending on host plant and/or artificial diet. Artificial diet-fed H. armigera larvae had comparatively high amylase activity and several unique amylase isoforms. Correlation of amylase and proteinase activities of H. armigera with the protein and carbohydrate content of various diets suggested that H. armigera regulates the levels of these digestive enzymes in response to macromolecular composition of the diet. These adjustments in the digestive enzymes of H. armigera may be to obtain better nourishment from the diet and avoid toxicity due to nutritional imbalance. H. armigera, a generalist feeder experiences a great degree of nutritional heterogeneity in its diet. An investigation of the differences in enzyme levels in response to macronutrient balance and imbalance highlight their importance in insect nutrition.

  11. Improved volatile fatty acids anaerobic production from waste activated sludge by pH regulation: Alkaline or neutral pH?

    PubMed

    Ma, Huijun; Chen, Xingchun; Liu, He; Liu, Hongbo; Fu, Bo

    2016-02-01

    In this study, the anaerobic fermentation was carried out for volatile fatty acids (VFAs) production at different pH (between 7.0 and 10.0) conditions with untreated sludge and heat-alkaline pretreated waste activated sludge. In the fermentation with untreated sludge, the extent of hydrolysis of organic matters and extent of acidification at alkaline pH are 54.37% and 30.37%, respectively, resulting in the highest VFAs yield at 235.46mg COD/gVS of three pH conditions. In the fermentation with heat-alkaline pretreated sludge, the acidification rate and VFAs yield at neutral pH are 30.98% and 240.14mg COD/gVS, respectively, which are higher than that at other pH conditions. With the glucose or bovine serum albumin as substrate for VFAs production, the neutral pH showed a higher VFAs concentration than the alkaline pH condition. The results of terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that the alkaline pH caused low microbial richness. Based on the results in this study, we demonstrated that the alkaline pH is favor of hydrolysis of organic matter in sludge while neutral pH improved the acidogenesis for the VFAs production from sludge. Our finding is obvious different to the previous research and helpful for the understanding of how heat-alkaline pretreatment and alkaline fermentation influence the VFAs production, and beneficial to the development of VFAs production process.

  12. CsrRS Regulates Group B Streptococcus Virulence Gene Expression in Response to Environmental pH: a New Perspective on Vaccine Development▿ †

    PubMed Central

    Santi, Isabella; Grifantini, Renata; Jiang, Sheng-Mei; Brettoni, Cecilia; Grandi, Guido; Wessels, Michael R.; Soriani, Marco

    2009-01-01

    To identify factors involved in the response of group B streptococci (GBS) to environmental pH, we performed a comparative global gene expression analysis of GBS at acidic and neutral pHs. We found that the transcription of 317 genes was increased at pH 5.5 relative to that at pH 7.0, while 61 genes were downregulated. The global response to acid stress included the differential expression of genes involved in transport, metabolism, stress response, and virulence. Known vaccine candidates, such as BibA and pilus components, were also regulated by pH. We observed that many of the genes involved in the GBS response to pH are known to be controlled by the CsrRS two-component system. Comparison of the regulon of wild-type strain 2603 V/R with that of a csrRS deletion mutant strain revealed that the pH-dependent regulation of 90% of the downregulated genes and 59.3% of the up-regulated genes in strain 2603 V/R was CsrRS dependent and that many virulence factors were overexpressed at high pH. Beta-hemolysin regulation was abrogated by selective inactivation of csrS, suggesting the implication of the CsrS protein in pH sensing. These results imply that the translocation of GBS from the acidic milieu of the vagina to the neutral pH of the neonatal lung signals the up-regulation of GBS virulence factors and conversion from a colonizing to an invasive phenotype. In addition, the fact that increased exposure of BibA on the bacterial surface at pH 7.0 induced opsonophagocytic killing of GBS in immune serum highlights the importance of pH regulation in the protective efficacy of specific antibodies to surface-exposed GBS proteins. PMID:19542277

  13. The cathepsin L-like proteinases from the midgut of Tenebrio molitor larvae: sequence, properties, immunocytochemical localization and function.

    PubMed

    Cristofoletti, Plínio T; Ribeiro, Alberto F; Terra, Walter R

    2005-08-01

    CDNAs coding for five procathepsin L-like proteinases (pCALs) were cloned and sequenced from a cDNA library prepared from Tenebrio molitor larval midguts: pCAL1a (with the isoforms pCAL1b and pCAL1c), pCAL2, and pCAL3. All the pCALs have the active residues Cys 25, His 169, Asn 175, and Gln 19 (papain numbering), the ERFNIN motif of papain-like enzymes and their sequences are homologous to cathepsin L enzymes. pCAL1a was expressed in bacterial systems. It is auto-catalytically activated at low pH, has kinetic properties and N-terminal sequence identical to hemocyte cathepsin L-like proteinase (CAL) and was used to raise antibodies. Semi-quantitative RT-PCR data showed that mRNAs for pCAL2 and pCAL3 were transcribed in midgut and in lesser amounts in hemolymph, whereas that for pCAL1a was transcribed in these tissues and also in fat body, Malpighian tubules, and carcass. Imunochemical detection recognized pCAL1a translation in all tissue homogenates, except anterior midgut. At this region, the presence of pCAL2 is suggested on the grounds of electrophoretical migration and high recovery of CAL2 activity from anterior midgut cells and from isolated midgut contents. Immunocytochemical localization data revealed that pCAL1a occurs in lysosome-like vesicles in all tissues, except anterior midgut, where a labelling considered to correspond to pCAL2 is found in large acidic granules being released by apocrine secretion. Putative pCAL2 was also detected in midgut contents, probably in the form of CAL2, the major luminal CAL, which was purified to homogeneity. A cladogram of insect CALs result in a monophyletic branch with lysosomal T. molitor enzymes and enzymes from five insect orders and in a polyphyletic array of coleopteran sequences, including digestive CALs from T. molitor. The data suggest that only Coleoptera have digestive CALs that may originate by gene duplication and independent evolution relative to the gene encoding the lysosomal enzyme.

  14. Kinetic transport model for cellular regulation of pH and solute concentration in the renal proximal tubule.

    PubMed Central

    Verkman, A S; Alpern, R J

    1987-01-01

    An open circuit kinetic model was developed to calculate the time course of proximal tubule cell pH, solute concentrations, and volume in response to induced perturbations in luminal or peritubular fluid composition. Solute fluxes were calculated from electrokinetic equations containing terms for known carrier saturabilities, allosteric dependences, and ion coupling ratios. Apical and basolateral membrane potentials were determined iteratively from the requirements of cell electroneutrality and equal opposing transcellular and paracellular currents. The model converged to membrane potentials accurate to 0.05% in one to four iterations. Model variables included cell concentrations of Na, K, HCO3, glucose, pH (uniform CO2), volume, and apical and basolateral membrane potentials. The basic model contained passive apical membrane transport of Na/H, Na/glucose, H and K, basolateral transport of Na/3HCO3, K, H, and glucose, and paracellular transport of Na, K, Cl, and HCO3; apical H and basolateral 3Na/2K-ATPases were present. Apical Na/H and basolateral K transport were regulated allosterically by pH. Apical Na/H transport, basolateral Na/3HCO3 transport, and the 3Na/2K-ATPase were saturable. Model parameters were chosen from data in the rat proximal tubule. Model predictions for the magnitude and time course of cell pH, Na, and membrane potential in response to rapid changes in apical and peritubular Na and HCO3 were in excellent agreement with experiment. In addition, the model requires that there exist an apical H-ATPase, basolateral Na/3HCO3 transport saturable with HCO3, and electroneutral basolateral K transport. PMID:3580482

  15. Outward K+ channels in Brassica chinensis pollen protoplasts are regulated by external and internal pH.

    PubMed

    Fan, L-M; Wang, Y-F; Wu, W-H

    2003-03-01

    Patch-clamp whole-cell and single-channel recording techniques were used to investigate the regulation of outward K(+) channels by external and internal protons in Brassica chinensispollen protoplasts. Outward K(+) currents and conductance were insensitive to external pH (pH(o)) except at pH 4.5. Maximal conductance (G(max)) for the outward K(+) currents was inhibited at acidic external pH. Half-activation voltage ( E(1/2)) for the outward K(+) currents shifted to more positive voltages along with the decrease in pH(o). E(1/2) can be described by a modified Henderson-Hasselbalch equation expected from a single titratable binding site. The activation kinetics of the outward K(+) channels was largely insensitive to pH(o). An internal pH (pH(i)) of 4.5 significantly increased outward K(+) currents and conductance. G(max) for the outward K(+) currents decreased with elevations in pH(i). In contrast to the effect of pH(o), E(1/2) was shifted to more positive voltages with elevations in pH(i). The outward K(+) currents, G(max) and E(1/2) can be described by the modified Henderson-Hasselbalch equation. Furthermore, acidifying pH(i) accelerated the activation of the outward K(+) currents significantly. The differences in electro-physiological properties among previously reported and currently described plant outward K(+) channels may reflect differences in the structure of these channels.

  16. Regulating proton-coupled electron transfer for efficient water splitting by manganese oxides at neutral pH

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Akira; Inuzuka, Riko; Takashima, Toshihiro; Hayashi, Toru; Hashimoto, Kazuhito; Nakamura, Ryuhei

    2014-06-01

    Manganese oxides have been extensively investigated as model systems for the oxygen-evolving complex of photosystem II. However, most bioinspired catalysts are inefficient at neutral pH and functional similarity to the oxygen-evolving complex has been rarely achieved with manganese. Here we report the regulation of proton-coupled electron transfer involved in water oxidation by manganese oxides. Pyridine and its derivatives, which have pKa values intermediate to the water ligand bound to manganese(II) and manganese(III), are used as proton-coupled electron transfer induction reagents. The induction of concerted proton-coupled electron transfer is demonstrated by the detection of deuterium kinetic isotope effects and compliance of the reactions with the libido rule. Although proton-coupled electron transfer regulation is essential for the facial redox change of manganese in photosystem II, most manganese oxides impair these regulatory mechanisms. Thus, the present findings may provide a new design rationale for functional analogues of the oxygen-evolving complex for efficient water splitting at neutral pH.

  17. Regulating proton-coupled electron transfer for efficient water splitting by manganese oxides at neutral pH

    PubMed Central

    Yamaguchi, Akira; Inuzuka, Riko; Takashima, Toshihiro; Hayashi, Toru; Hashimoto, Kazuhito; Nakamura, Ryuhei

    2014-01-01

    Manganese oxides have been extensively investigated as model systems for the oxygen-evolving complex of photosystem II. However, most bioinspired catalysts are inefficient at neutral pH and functional similarity to the oxygen-evolving complex has been rarely achieved with manganese. Here we report the regulation of proton-coupled electron transfer involved in water oxidation by manganese oxides. Pyridine and its derivatives, which have pKa values intermediate to the water ligand bound to manganese(II) and manganese(III), are used as proton-coupled electron transfer induction reagents. The induction of concerted proton-coupled electron transfer is demonstrated by the detection of deuterium kinetic isotope effects and compliance of the reactions with the libido rule. Although proton-coupled electron transfer regulation is essential for the facial redox change of manganese in photosystem II, most manganese oxides impair these regulatory mechanisms. Thus, the present findings may provide a new design rationale for functional analogues of the oxygen-evolving complex for efficient water splitting at neutral pH. PMID:24977746

  18. PIP Water Transport and Its pH Dependence Are Regulated by Tetramer Stoichiometry.

    PubMed

    Jozefkowicz, Cintia; Sigaut, Lorena; Scochera, Florencia; Soto, Gabriela; Ayub, Nicolás; Pietrasanta, Lía Isabel; Amodeo, Gabriela; González Flecha, F Luis; Alleva, Karina

    2016-03-29

    Many plasma membrane channels form oligomeric assemblies, and heterooligomerization has been described as a distinctive feature of some protein families. In the particular case of plant plasma membrane aquaporins (PIPs), PIP1 and PIP2 monomers interact to form heterotetramers. However, the biological properties of the different heterotetrameric configurations formed by PIP1 and PIP2 subunits have not been addressed yet. Upon coexpression of tandem PIP2-PIP1 dimers in Xenopus oocytes, we can address, for the first time to our knowledge, the functional properties of single heterotetrameric species having 2:2 stoichiometry. We have also coexpressed PIP2-PIP1 dimers with PIP1 and PIP2 monomers to experimentally investigate the localization and biological activity of each tetrameric assembly. Our results show that PIP2-PIP1 heterotetramers can assemble with 3:1, 1:3, or 2:2 stoichiometry, depending on PIP1 and PIP2 relative expression in the cell. All PIP2-PIP1 heterotetrameric species localize at the plasma membrane and present the same water transport capacity. Furthermore, the contribution of any heterotetrameric assembly to the total water transport through the plasma membrane doubles the contribution of PIP2 homotetramers. Our results also indicate that plasma membrane water transport can be modulated by the coexistence of different tetrameric species and by intracellular pH. Moreover, all the tetrameric species present similar cooperativity behavior for proton sensing. These findings throw light on the functional properties of PIP tetramers, showing that they have flexible stoichiometry dependent on the quantity of PIP1 and PIP2 molecules available. This represents, to our knowledge, a novel regulatory mechanism to adjust water transport across the plasma membrane.

  19. A pH Switch Regulates the Inverse Relationship between Membranolytic and Chaperone-like Activities of HSP-1/2, a Major Protein of Horse Seminal Plasma.

    PubMed

    Kumar, C Sudheer; Swamy, Musti J

    2016-07-01

    HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch. PMID:27292547

  20. LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

    SciTech Connect

    Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh; Brosens, Jan; Lang, Florian

    2015-05-08

    Objective: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H{sup +} and lactate. H{sup +} extrusion is in part accomplished by Na{sup +}/H{sup +} exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. Methods: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pH{sub i} estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na{sup +}/H{sup +} exchanger activity from Na{sup +} dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. Results: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na{sup +}/H{sup +} exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). Conclusions: LeftyA markedly down-regulates NHE1 expression, Na{sup +}/H{sup +} exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition. - Highlights: • LeftyA, an inhibitor of tumor growth, reduces Na{sup +}/H{sup +}-exchanger activity by 55%. • LeftyA decreases NHE1 transcripts by 99.6% and NHE1 protein by 71%. • LeftyA decreases cytosolic pH from 7.22 ± 0.02 to 7.05 ± 0.02. • Cytosolic acidification by Lefty A decreases glycolysis by 41%. • Cytosolic acidification by Lefty A compromises energy production of tumor cells.

  1. Tetramerization of the LexA repressor in solution: implications for gene regulation of the E.coli SOS system at acidic pH.

    PubMed

    Sousa, Francisco J R; Lima, Luis M T R; Pacheco, Ana B F; Oliveira, Cristiano L P; Torriani, Iris; Almeida, Darcy F; Foguel, Debora; Silva, Jerson L; Mohana-Borges, Ronaldo

    2006-06-16

    Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon. PMID:16701697

  2. Direct Sensing of Intracellular pH by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel*♦

    PubMed Central

    Chen, Jeng-Haur; Cai, Zhiwei; Sheppard, David N.

    2009-01-01

    In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pHi). CFTR modulates pHi through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pHi. Here, we investigate the direct effects of pHi on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pHi increased the open probability (Po) of wild-type CFTR, whereas alkaline pHi decreased Po and inhibited Cl− flow through the channel. Acidic pHi potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pHi inhibited the MgATP dependence of wild-type CFTR by decreasing channel activity. Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pHi and inhibition by alkaline pHi, suggesting that site 2 is a critical determinant of the pHi sensitivity of CFTR. The effects of pHi also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl− channel senses directly pHi. The direct regulation of CFTR by pHi has important implications for the regulation of epithelial ion transport. PMID:19837660

  3. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

    SciTech Connect

    Galkina, Svetlana I. . E-mail: galkina@genebee.msu.su; Sud'ina, Galina F.; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na{sup +} and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl{sup -} efflux through chloride channels and Na{sup +} influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  4. Lipid production from hemicellulose with Lipomyces starkeyi in a pH regulated fed-batch cultivation.

    PubMed

    Brandenburg, Jule; Blomqvist, Johanna; Pickova, Jana; Bonturi, Nemailla; Sandgren, Mats; Passoth, Volkmar

    2016-08-01

    This study investigated lipid production from the hemicellulosic fraction of birch wood by the oleaginous yeast Lipomyces starkeyi. Birch wood chips were thermochemically pretreated by hot water extraction, and the liquid phase, containing 45.1 g/l xylose as the major sugar, 13.1 g/l acetic acid and 4.7 g/l furfural, was used for cultivations of L. starkeyi CBS1807. The hydrolysate strongly inhibited yeast growth; the strain could only grow in medium containing 30% hydrolysate at pH 6. At pH 5, growth stopped already upon the addition of about 10% hydrolysate. In fed-batch cultures fed with hydrolysate or a model xylose-acetic acid mixture, co-consumption of xylose and acetic acid was observed, which resulted in a pH increase. This phenomenon was utilized to establish a pH-stat fed-batch cultivation in which, after an initial feeding, hydrolysate or model mixture was connected to the pH-regulation system of the bioreactor. Under these conditions we obtained growth and lipid production in cultures grown on either xylose or glucose during the batch phase. In cultivations fed with model mixture, a maximum lipid content of 60.5% of the cell dry weight (CDW) was obtained; however, not all xylose was consumed. When feeding hydrolysate, growth was promoted and carbon sources were completely consumed, resulting in higher CDW with maximum lipid content of 51.3%. In both cultures the lipid concentration was 8 g/l and a lipid yield of 0.1 g/g carbon source was obtained. Lipid composition was similar in all cultivations, with C18:1 and C16:0 being the most abundant fatty acids. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Netrins and Frazzled/DCC promote the migration and mesenchymal to epithelial transition of Drosophila midgut cells

    PubMed Central

    Pert, Melissa; Gan, Miao; Saint, Robert; Murray, Michael J.

    2015-01-01

    ABSTRACT Mesenchymal-epithelial transitions (METs) are important in both development and the growth of secondary tumours. Although the molecular basis for epithelial polarity is well studied, less is known about the cues that induce MET. Here we show that Netrins, well known as chemotropic guidance factors, provide a basal polarising cue during the Drosophila midgut MET. Both netrinA and netrinB are expressed in the visceral mesoderm, the substrate upon which midgut cells migrate, while their receptor frazzled (fra) is expressed in midgut cells. Netrins are required to polarise Fra to the basal surface, and Netrins and Fra undergo mutually-dependent endocytosis, with Fra subsequently trafficking to late endosomes. Mutations to fra and netrins affect both migration and MET but to different degrees. Loss of fra strongly delays migration, midgut cells fail to extend protrusions, and apico-basal polarisation of proteins and epithelium formation is inhibited. In netrin mutants, the migration phenotype is weaker and cells still extend protrusions. However, apico-basal polarisation of proteins, including Fra, and FActin is greatly disrupted and a monolayer fails to form. Delocalised accumulations of FActin are prevalent in netrin mutants but not fra mutants suggesting delocalised Fra may disrupt the MET. βPS localisation is also affected in netrin mutants in that a basal gradient is reduced while localisation to the midgut/VM interface is increased. Since a similar effect is seen when endocytosis is inhibited, Netrin and Fra may regulate Integrin turnover. The results suggest Netrin-dependent basal polarisation of Fra is critical for the formation of an epithelium. PMID:25617422

  6. Netrins and Frazzled/DCC promote the migration and mesenchymal to epithelial transition of Drosophila midgut cells.

    PubMed

    Pert, Melissa; Gan, Miao; Saint, Robert; Murray, Michael J

    2015-01-23

    Mesenchymal-epithelial transitions (METs) are important in both development and the growth of secondary tumours. Although the molecular basis for epithelial polarity is well studied, less is known about the cues that induce MET. Here we show that Netrins, well known as chemotropic guidance factors, provide a basal polarising cue during the Drosophila midgut MET. Both netrinA and netrinB are expressed in the visceral mesoderm, the substrate upon which midgut cells migrate, while their receptor frazzled (fra) is expressed in midgut cells. Netrins are required to polarise Fra to the basal surface, and Netrins and Fra undergo mutually-dependent endocytosis, with Fra subsequently trafficking to late endosomes. Mutations to fra and netrins affect both migration and MET but to different degrees. Loss of fra strongly delays migration, midgut cells fail to extend protrusions, and apico-basal polarisation of proteins and epithelium formation is inhibited. In netrin mutants, the migration phenotype is weaker and cells still extend protrusions. However, apico-basal polarisation of proteins, including Fra, and FActin is greatly disrupted and a monolayer fails to form. Delocalised accumulations of FActin are prevalent in netrin mutants but not fra mutants suggesting delocalised Fra may disrupt the MET. βPS localisation is also affected in netrin mutants in that a basal gradient is reduced while localisation to the midgut/VM interface is increased. Since a similar effect is seen when endocytosis is inhibited, Netrin and Fra may regulate Integrin turnover. The results suggest Netrin-dependent basal polarisation of Fra is critical for the formation of an epithelium.

  7. A subset of enteroendocrine cells is activated by amino acids in the Drosophila midgut.

    PubMed

    Park, Jeong-Ho; Chen, Ji; Jang, Sooin; Ahn, Tae Jung; Kang, KyeongJin; Choi, Min Sung; Kwon, Jae Young

    2016-02-01

    The intestine is involved in digestion and absorption, as well as the regulation of metabolism upon sensation of the internal intestinal environment. Enteroendocrine cells are thought to mediate these internal intestinal chemosensory functions. Using the CaLexA (calcium-dependent nuclear import of LexA) method, we examined the enteroendocrine cell populations that are activated when flies are subjected to various dietary conditions such as starvation, sugar, high fat, protein, or pathogen exposure. We find that a specific subpopulation of enteroendocrine cells in the posterior midgut which express Dh31 and tachykinin are activated by the presence of proteins and amino acids. PMID:26801353

  8. Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

    SciTech Connect

    Buarque, Diego S.; Spindola, Leticia M.N.; Martins, Rafael M.; Braz, Gloria R.C.; Tanaka, Aparecida S.

    2011-09-23

    Highlights: {yields} Tigutcystatin inhibits Trypanosoma cruzi cysteine proteases with high specificity. {yields} Tigutcystatin expression is up-regulated in response to T. cruzi infection. {yields} It is the first cysteine proteases inhibitor characterized from a triatomine insect. -- Abstract: The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K{sub i} = 3.29 nM) and human cathepsin L (K{sub i} = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.

  9. Circadian Regulation of the PhCCD1 Carotenoid Cleavage Dioxygenase Controls Emission of β-Ionone, a Fragrance Volatile of Petunia Flowers1

    PubMed Central

    Simkin, Andrew J.; Underwood, Beverly A.; Auldridge, Michele; Loucas, Holly M.; Shibuya, Kenichi; Schmelz, Eric; Clark, David G.; Klee, Harry J.

    2004-01-01

    Carotenoids are thought to be the precursors of terpenoid volatile compounds that contribute to flavor and aroma. One such volatile, β-ionone, is important to fragrance in many flowers, including petunia (Petunia hybrida). However, little is known about the factors regulating its synthesis in vivo. The petunia genome contains a gene encoding a 9,10(9′,10′) carotenoid cleavage dioxygenase, PhCCD1. The PhCCD1 is 94% identical to LeCCD1A, an enzyme responsible for formation of β-ionone in tomato (Lycopersicon esculentum; Simkin AJ, Schwartz SH, Auldridge M, Taylor MG, Klee HJ [2004] Plant J [in press]). Reduction of PhCCD1 transcript levels in transgenic plants led to a 58% to 76% decrease in β-ionone synthesis in the corollas of selected petunia lines, indicating a significant role for this enzyme in volatile synthesis. Quantitative reverse transcription-PCR analysis revealed that PhCCD1 is highly expressed in corollas and leaves, where it constitutes approximately 0.04% and 0.02% of total RNA, respectively. PhCCD1 is light-inducible and exhibits a circadian rhythm in both leaves and flowers. β-Ionone emission by flowers occurred principally during daylight hours, paralleling PhCCD1 expression in corollas. The results indicate that PhCCD1 activity and β-ionone emission are likely regulated at the level of transcript. PMID:15516502

  10. A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae.

    PubMed

    Elpidina, E N; Tsybina, T A; Dunaevsky, Y E; Belozersky, M A; Zhuzhikov, D P; Oppert, B

    2005-08-01

    A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.

  11. Sequence variation and differential splicing of the midgut cadherin gene in Trichoplusia ni.

    PubMed

    Zhang, Xin; Kain, Wendy; Wang, Ping

    2013-08-01

    The insect midgut cadherin serves as an important receptor for the Cry toxins from Bacillus thuringiensis (Bt). Variation of the cadherin in insect populations provides a genetic potential for development of cadherin-based Bt resistance in insect populations. Sequence analysis of the cadherin from the cabbage looper, Trichoplusia ni, together with cadherins from 18 other lepidopterans showed a similar phylogenetic relationship of the cadherins to the phylogeny of Lepidoptera. The midgut cadherin in three laboratory populations of T. ni exhibited high variability, although the resistance to Bt toxin Cry1Ac in the T. ni strain is not genetically associated with cadherin gene mutations. A total of 142 single nucleotide polymorphisms (SNPs) were identified in the cadherin cDNAs from the T. ni strains, including 20 missense mutations. In addition, insertion and deletion polymorphisms (indels) were also identified in the cadherin alleles in T. ni. More interestingly, the results from this study reveal that differential splicing of mRNA also occurs in the cadherin gene expression. Therefore, variation of the midgut cadherin in insects may not only be caused by cadherin gene mutations, but could also result from alternative splicing of its mRNA regulated by factors acting in trans. Analysis of cadherin gene alleles in F2, F3 and F4 progenies from the cross between the Cry1Ac resistant and the susceptible strain after consecutive selections with Cry1Ac for three generations showed that selection with Cry1Ac did not result in an increase of frequencies of the cadherin alleles originated from the resistant strain. PMID:23743444

  12. Molecular cloning and characterization of a midgut chymotrypsin-like enzyme from the lesser grain borer, Rhyzopertha dominica.

    PubMed

    Zhu, Y C; Baker, J E

    2000-04-01

    A cDNA encoding a chymotrypsinogen-like protein in midguts of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) was cloned and sequenced. The 901 bp cDNA contains an 816-nucleotide open reading frame encoding 272-amino acids. The predicted molecular mass and pI of the mature enzyme are 23.7 kDa and 4.64, respectively. The encoded protein includes amino acid sequence motifs that are conserved with 5 homologous chymotrypsinogen proteins from other insects. Features of the putative chymotrypsin-like protein from R. dominica include the serine proteinase active site (His(90), Asp(133), Ser(226)), conserved cysteine residues for disulfide bridges, the residues (Gly(220), Gly(243), Asp(252)) that determine chymotrypsin specificity, and both zymogen activation and signal peptides. A TPCK-sensitive caseinolytic protein (P6) with an estimated molecular mass of 24 kDa is present in midgut extracts of R. dominica and can be resolved by electrophoresis on 4-16% polyacrylamide gels. The molecular mass of this caseinolytic enzyme is similar to that of the chymotrypsin deduced from cDNA. Midgut extracts of R. dominica readily hydrolyzed azocasein and N-succinyl-alanine-alanine-proline-phenylalanine-p- nitroanilide (SAAPFpNA), a chymotrypsin-specific substrate. Properties of the enzymes responsible for these activities were partially characterized with respect to distribution in the gut, optimum pH, and sensitivity toward selected proteinase inhibitors. Optimal activity against both azocasein and SAAPFpNA occurs in a broad pH range from about 7 to 10. Both azocasein and SAAPFpNA activities, located primarily in the anterior midgut region, are inhibited by aprotinin, phenylmethyl sulphonylfluoride (PMSF), and soybean trypsin inhibitor (STI). TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) and chymostatin inhibited more than 60% of SAAPFpNA but only about 10-20% of azocasein activity. These results provide additional evidence for the presence of

  13. Left-Sided Appendicitis in an Elderly Patient with Midgut Malrotation.

    PubMed

    Chuang, Pei Wen; Huang, Bo-Ming; Liu, Chung Hsien; Chen, Chien-Chin; Tsai, Ming-Jen

    2015-12-01

    Appendicitis is a common surgical abdominal disease with various presentations. Its diagnosis may be obscured by asymptomatic congenital anatomical anomalies like midgut malrotation. Midgut malrotation is a rare fetal anomaly resulting from incomplete or failure of midgut rotation and fixation. It is mostly presented with bowel obstruction or volvulus in early life. Presentation in adult is rare. Here, we report an elderly patient presented with left lower abdominal pain and urinary tract infection. Abdominal computed tomography revealed left-sided appendicitis with non-rotational-type midgut malrotation. Clinicians should bear in mind the possibility of underlying midgut malrotation, as appendicitis could be the first presentation of this rare congenital condition. PMID:27011586

  14. Trichomonas vaginalis haemolysis: pH regulates a contact-independent mechanism based on pore-forming proteins.

    PubMed

    Fiori, P L; Rappelli, P; Addis, M F; Sechi, A; Cappuccinelli, P

    1996-02-01

    There is a controversy in literature about involvement of secreted factors in the pathogenetic mechanisms of Trichomonas vaginalis, described mostly as contact-dependent. We found that the protozoan, under triggering conditions, is able to release molecules that lead to lysis without direct contact between parasite and target cells as a prerequisite. In this paper we characterize contact-independent cytotoxicity using the red blood cell as a cellular model. Contact-independent haemolysis is a phenomenon were pH exerts a key role, triggering the secretion of a lytic molecule and regulating its activity. A partial physicochemical characterization of the haemolytic factor suggests that a protein of M(r) > 30 kDa could be the effector responsible for damage. Furthermore, the parasite-induced membrane permeabilization, detected by measuring potassium escape from the target cell, and an effective osmotic protection by carbohydrates allowed us to relate the previously described pore-forming mechanism involved in contact-dependent cytotoxicity with the contact-independent lysis. PMID:8722099

  15. Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism.

    PubMed

    Caccia, Silvia; Di Lelio, Ilaria; La Storia, Antonietta; Marinelli, Adriana; Varricchio, Paola; Franzetti, Eleonora; Banyuls, Núria; Tettamanti, Gianluca; Casartelli, Morena; Giordana, Barbara; Ferré, Juan; Gigliotti, Silvia; Ercolini, Danilo; Pennacchio, Francesco

    2016-08-23

    Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host. PMID:27506800

  16. More Frequent than Desired: Midgut Stem Cell Somatic Mutations.

    PubMed

    Li, Qi; Ip, Y Tony

    2015-12-01

    The accumulation of somatic mutations in adult stem cells contributes to the decline of tissue functions and cancer initiation. In this issue of Cell Stem Cell, Siudeja et al. (2015) investigate the rate and mechanism of naturally occurring mutations in Drosophila midgut intestinal stem cells during aging and find high-frequency mutations arising from multiple mechanisms. PMID:26637937

  17. Adult midgut malrotation presented with acute bowel obstruction and ischemia

    PubMed Central

    Zengin, Akile; Uçar, Bercis İmge; Düzgün, Şükrü Aydın; Bayhan, Zülfü; Zeren, Sezgin; Yaylak, Faik; Şanal, Bekir; Bayhan, Nilüfer Araz

    2016-01-01

    Introduction Intestinal malrotation refers to the partial or complete failure of rotation of midgut around the superior mesenteric vessels in embryonic life. Arrested midgut rotation results due to narrow-based mesentery and increases the risk of twisting midgut and subsequent obstruction and necrosis. Presentation of case 40 years old female patient admitted to emergency service with acute abdomen and computerized tomography scan showed dilated large and small intestine segments with air-fluid levels and twisted mesentery around superior mesenteric artery and vein indicating “whirpool sign”. Discussion Malrotation in adults is a rare cause of midgut volvulus as though it should be considered in differential diagnosis in patients presented with acute abdomen and intestinal ischemia. Even though clinical symptoms are obscure, adult patients usually present with vomiting and recurrent abdominal pain due to chronic partial obstruction. Contrast enhanced radiograph has been shown to be the most accurate method. Typical radiological signs are corkscrew sign, which is caused by the dilatation of various duodenal segments at different levels and the relocation of duodenojejunal junction due to jejunum folding. As malrotation commonly causes intestinal obstruction, patients deserve an elective laparotomy. Conclusion Malrotation should be considered in differential diagnosis in patients presented with acute abdomen and intestinal ischemia. Surgical intervention should be prompt to limit morbidity and mortality. PMID:27015011

  18. Aspen defense chemicals influence midgut bacterial community composition of gypsy moth.

    PubMed

    Mason, Charles J; Rubert-Nason, Kennedy F; Lindroth, Richard L; Raffa, Kenneth F

    2015-01-01

    Microbial symbionts are becoming increasingly recognized as mediators of many aspects of plant - herbivore interactions. However, the influence of plant chemical defenses on gut associates of insect herbivores is less well understood. We used gypsy moth (Lymantria dispar L.), and differing trembling aspen (Populus tremuloides Michx.) genotypes that vary in chemical defenses, to assess the influence of foliar chemistry on bacterial communities of larval midguts. We evaluated the bacterial community composition of foliage, and of midguts of larvae feeding on those leaves, using next-generation high-throughput sequencing. Plant defense chemicals did not influence the composition of foliar communities. In contrast, both phenolic glycosides and condensed tannins affected the bacterial consortia of gypsy moth midguts. The two most abundant operational taxonomic units were classified as Ralstonia and Acinetobacter. The relative abundance of Ralstonia was higher in midguts than in foliage when phenolic glycoside concentrations were low, but lower in midguts when phenolic glycosides were high. In contrast, the relative abundance of Ralstonia was lower in midguts than in foliage when condensed tannin concentrations were low, but higher in midguts when condensed tannins were high. Acinetobacter showed a different relationship with host chemistry, being relatively more abundant in midguts than with foliage when condensed tannin concentrations were low, but lower in midguts when condensed tannins were high. Acinetobacter tended to have a greater relative abundance in midguts of insects feeding on genotypes with high phenolic glycoside concentrations. These results show that plant defense chemicals influence herbivore midgut communities, which may in turn influence host utilization.

  19. Midgut Microbial Community of Culex quinquefasciatus Mosquito Populations from India

    PubMed Central

    Chandel, Kshitij; Mendki, Murlidhar J.; Parikh, Rasesh Y.; Kulkarni, Girish; Tikar, Sachin N.; Sukumaran, Devanathan; Prakash, Shri; Parashar, Brahma D.; Shouche, Yogesh S.; Veer, Vijay

    2013-01-01

    The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a

  20. A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia

    PubMed Central

    Yin, Jing; Chang, Xiaoxiao; Kasuga, Takao; Bui, Mai; Reid, Michael S; Jiang, Cai-Zhong

    2015-01-01

    The basic helix-loop-helix (bHLH) transcription factors (TFs) play important roles in regulating multiple biological processes in plants. However, there are few reports about the function of bHLHs in flower senescence. In this study, a bHLH TF, PhFBH4, was found to be dramatically upregulated during flower senescence. Transcription of PhFBH4 is induced by plant hormones and abiotic stress treatments. Silencing of PhFBH4 using virus-induced gene silencing or an antisense approach extended flower longevity, while transgenic petunia flowers with an overexpression construct showed a reduction in flower lifespan. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was significantly changed in petunia PhFBH4 transgenic flowers. Furthermore, silencing or overexpression of PhFBH4 reduced or increased, respectively, transcript abundances of important ethylene biosynthesis-related genes, ACS1 and ACO1, thereby influencing ethylene production. An electrophoretic mobility shift assay showed that the PhFBH4 protein physically interacted with the G-box cis-element in the promoter of ACS1, suggesting that ACS1 was a direct target of the PhFBH4 protein. In addition, ectopic expression of this gene altered plant development including plant height, internode length, and size of leaves and flowers, accompanied by alteration of transcript abundance of the gibberellin biosynthesis-related gene GA2OX3. Our results indicate that PhFBH4 plays an important role in regulating plant growth and development through modulating the ethylene biosynthesis pathway. PMID:26715989

  1. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    NASA Astrophysics Data System (ADS)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  2. A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The basic helix-loop-helix (bHLH) transcription factors (TFs) play important roles in regulating multiple biological processes in plants. However, there are few reports about the function of bHLHs in flower senescence. In this study, a bHLH TF, PhFBH4, was found to be dramatically upregulated during...

  3. Self-assembly of NH₂-(α,L-lysine)₅-COOH and SDS into nanodiscs or nanoribbons regulated by pH.

    PubMed

    Zhang, S; Yang, S; Zang, J; Yang, R; Zhao, G; Xu, C

    2014-09-01

    NH2-(α,l-lysine)5-COOH and SDS can self-assemble into nanodiscs or nanoribbons. We show that pH can regulate not only the diameter of nanodiscs but also the conversion between nanodiscs and nanoribbons. This system can be used as two different templates for fabricating platinum nanowires and nanodiscs.

  4. Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface*

    PubMed Central

    Jinadasa, Tushare; Szabó, Elöd Z.; Numata, Masayuki; Orlowski, John

    2014-01-01

    Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H+-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na+/H+ exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress. PMID:24936055

  5. Intracellular pH of giant salivary gland cells of the leech Haementeria ghilianii: regulation and effects on secretion.

    PubMed

    Wuttke, W A; Munsch, T; Berry, M S

    1994-04-01

    1. Intracellular pH (pHi) and membrane potential (Em) of giant salivary gland cells of the leech, Haementeria ghilianii, were measured with double-barrelled, neutral-carrier, pH-sensitive microelectrodes. 2. Em was -51 +/- 11.2 mV and pHi was 6.98 +/- 0.1 (mean +/- S.D., N = 41) in Hepes-buffered saline (nominally HCO3(-)-free; extracellular pH, pHe = 7.4). pHi was independent of Em. 3. Amiloride (2 mmol l-1) had no effect on resting pHi or on pHi recovery from an acid load (induced by the NH4+ pre-pulse technique). Removal of external Na+ produced a progressive acidification which was blocked by amiloride, and the drug also slowed the recovery of pHi on reintroduction of Na+. The results indicate the presence of an electroneutral Na+/H+ exchanger whose access to amiloride is competitively blocked by Na+. 4. In certain smaller cells of the gland, which probably form a separate population, removal of external Na+ did not affect pHi, and recovery from an acid load was blocked by amiloride. There may, therefore, be two types of Na+/H+ exchanger, differing in reversibility and sensitivity to amiloride. 5. Recovery of pHi from NH4(+)-induced acid loading was not affected by bicarbonate-buffered saline (2% CO2; 11 mmol l-1 HCO3-) or by addition of the anion-exchange blocker SITS (10(-4) mol l-1). This suggests that there is no significant contribution of a HCO3(-)-dependent transport mechanism to pHi regulation in the gland cells. 6. Removal of external Cl- slowly reduced pHi and there was a transient increase (overshoot) in pHi when Cl- was reintroduced. These effects of Cl- are probably explained by changes in the Na+ gradient. Intracellular Na+ and Cl- activities were measured with ion-selective microelectrodes. 7. Acidification with NH4+ was difficult, probably because of the cells' poor permeability to this ion. Attempts to introduce NH4+ via the Na+ pump or Na+/Cl- transporter were not successful. The H+/K+ ionophore nigericin (1 microgram ml-1), however, produced

  6. Internal pH regulation facilitates in situ long-term acclimation of massive corals to end-of-century carbon dioxide conditions.

    PubMed

    Wall, M; Fietzke, J; Schmidt, G M; Fink, A; Hofmann, L C; de Beer, D; Fabricius, K E

    2016-01-01

    The resilience of tropical corals to ocean acidification depends on their ability to regulate the pH within their calcifying fluid (pHcf). Recent work suggests pHcf homeostasis under short-term exposure to pCO2 conditions predicted for 2100, but it is still unclear if pHcf homeostasis can be maintained throughout a corals lifetime. At CO2 seeps in Papua New Guinea, massive Porites corals have grown along a natural seawater pH gradient for decades. This natural gradient, ranging from pH 8.1-7.4, provides an ideal platform to determine corals' pHcf (using boron isotopes). Porites maintained a similar pHcf (~8.24) at both a control (pH 8.1) and seep-influenced site (pH 7.9). Internal pHcf was slightly reduced (8.12) at seawater pH 7.6, and decreased to 7.94 at a site with a seawater pH of 7.4. A growth response model based on pHcf mirrors the observed distribution patterns of this species in the field. We suggest Porites has the capacity to acclimate after long-time exposure to end-of-century reduced seawater pH conditions and that strong control over pHcf represents a key mechanism to persist in future oceans. Only beyond end-of-century pCO2 conditions do they face their current physiological limit of pH homeostasis and pHcf begins to decrease. PMID:27477963

  7. Internal pH regulation facilitates in situ long-term acclimation of massive corals to end-of-century carbon dioxide conditions

    PubMed Central

    Wall, M.; Fietzke, J.; Schmidt, G. M.; Fink, A; Hofmann, L. C.; de Beer, D.; Fabricius, K. E.

    2016-01-01

    The resilience of tropical corals to ocean acidification depends on their ability to regulate the pH within their calcifying fluid (pHcf). Recent work suggests pHcf homeostasis under short-term exposure to pCO2 conditions predicted for 2100, but it is still unclear if pHcf homeostasis can be maintained throughout a corals lifetime. At CO2 seeps in Papua New Guinea, massive Porites corals have grown along a natural seawater pH gradient for decades. This natural gradient, ranging from pH 8.1–7.4, provides an ideal platform to determine corals’ pHcf (using boron isotopes). Porites maintained a similar pHcf (~8.24) at both a control (pH 8.1) and seep-influenced site (pH 7.9). Internal pHcf was slightly reduced (8.12) at seawater pH 7.6, and decreased to 7.94 at a site with a seawater pH of 7.4. A growth response model based on pHcf mirrors the observed distribution patterns of this species in the field. We suggest Porites has the capacity to acclimate after long-time exposure to end-of-century reduced seawater pH conditions and that strong control over pHcf represents a key mechanism to persist in future oceans. Only beyond end-of-century pCO2 conditions do they face their current physiological limit of pH homeostasis and pHcf begins to decrease. PMID:27477963

  8. Preparation of DNA-adsorbed TiO2 particles--augmentation of performance for environmental purification by increasing DNA adsorption by external pH regulation.

    PubMed

    Amano, Takeharu; Toyooka, Tatsushi; Ibuki, Yuko

    2010-01-01

    We have previously developed a novel photocatalyst, DNA-attached titanium dioxide (DNA-TiO(2)), useful for the recovery and decomposition of chemicals [Suzuki et al. Environ. Sci. Technol. 42, 8076, 2008]. Chemicals accumulated in DNA near the surface of TiO(2) and were degraded under UV light. The efficiency of their removal was dependent on the amount of DNA adsorbed on TiO(2), indicating the attachment of larger amounts of DNA to result in higher efficiency. In this study, we succeeded in improving the performance of DNA-TiO(2) by increasing the amount of DNA adsorbed by regulating the external pH. The adsorption of DNA by TiO(2) dramatically increased at pH2, to about fourfold that at other pH values (pH4-10). Repeating the process of DNA addition increased the adsorption further. The attached DNA was stable on the surface of TiO(2) at pH2-10 and 4-56 degrees C, the same as DNA-TiO(2) prepared at pH7. As the DNA-TiO(2) prepared at pH2 retained much DNA on its surface, chemicals (methylene blue, ethidium bromide, etc.) which could intercalate or react with DNA were effectively removed from solutions. The photocatalytic degradation was slow at first, but the final degradation rate was higher than for non-adsorbed TiO(2) and DNA-TiO(2) prepared at pH7. These results indicated that preparation of DNA-TiO(2) at pH2 has advantages in that much DNA can be attached and large amounts of chemicals can be concentrated in the DNA, resulting in extensive decomposition under UV light.

  9. Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation

    PubMed Central

    1991-01-01

    The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin- permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control." PMID:1719125

  10. Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating machinery in tumors

    PubMed Central

    Sedlakova, Olga; Svastova, Eliska; Takacova, Martina; Kopacek, Juraj; Pastorek, Jaromir; Pastorekova, Silvia

    2013-01-01

    Acidic tissue microenvironment contributes to tumor progression via multiple effects including the activation of angiogenic factors and proteases, reduced cell-cell adhesion, increased migration and invasion, etc. In addition, intratumoral acidosis can influence the uptake of anticancer drugs and modulate the response of tumors to conventional therapy. Acidification of the tumor microenvironment often develops due to hypoxia-triggered oncogenic metabolism, which leads to the extensive production of lactate, protons, and carbon dioxide. In order to avoid intracellular accumulation of the acidic metabolic products, which is incompatible with the survival and proliferation, tumor cells activate molecular machinery that regulates pH by driving transmembrane inside-out and outside-in ion fluxes. Carbonic anhydrase IX (CA IX) is a hypoxia-induced catalytic component of the bicarbonate import arm of this machinery. Through its catalytic activity, CA IX directly participates in many acidosis-induced features of tumor phenotype as demonstrated by manipulating its expression and/or by in vitro mutagenesis. CA IX can function as a survival factor protecting tumor cells from hypoxia and acidosis, as a pro-migratory factor facilitating cell movement and invasion, as a signaling molecule transducing extracellular signals to intracellular pathways (including major signaling and metabolic cascades) and converting intracellular signals to extracellular effects on adhesion, proteolysis, and other processes. These functional implications of CA IX in cancer are supported by numerous clinical studies demonstrating the association of CA IX with various clinical correlates and markers of aggressive tumor behavior. Although our understanding of the many faces of CA IX is still incomplete, existing knowledge supports the view that CA IX is a biologically and clinically relevant molecule, exploitable in anticancer strategies aimed at targeting adaptive responses to hypoxia and/or acidosis

  11. Differential proteomic analysis of midguts from Nosema ceranae-infected honeybees reveals manipulation of key host functions.

    PubMed

    Vidau, Cyril; Panek, Johan; Texier, Catherine; Biron, David G; Belzunces, Luc P; Le Gall, Morgane; Broussard, Cédric; Delbac, Frédéric; El Alaoui, Hicham

    2014-09-01

    Many invasive pathogens effectively bypass the insect defenses to ensure the completion of their life cycle. Among those, an invasive microsporidian species, Nosema ceranae, can cause nosemosis in honeybees. N. ceranae was first described in the Asian honeybee Apis cerana and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the first barrier against N. ceranae attacks. To bring proteomics data on honeybee/N. ceranae crosstalk and more precisely to decipher the worker honeybee midgut response after an oral inoculation of N. ceranae (10days post-infection), we used 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) combined with mass spectrometry. Forty-five protein spots produced by the infected worker honeybee group were shown to be differentially expressed when compared to the uninfected group; 14 were subsequently identified by mass spectrometry. N. ceranae mainly caused a modulation of proteins involved in three key host biological functions: (i) energy production, (ii) innate immunity (reactive oxygen stress) and (iii) protein regulation. The modulation of these host biological functions suggests that N. ceranae creates a zone of "metabolic habitat modification" in the honeybee midgut favoring its development by enhancing availability of nutrients and reducing the worker honeybee defense.

  12. Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan

    PubMed Central

    Nunes, Rodrigo Dutra; Ventura-Martins, Guilherme; Moretti, Débora Monteiro; Medeiros-Castro, Priscilla; Rocha-Santos, Carlucio; Daumas-Filho, Carlos Renato de Oliveira; Bittencourt-Cunha, Paula Rego Barros; Martins-Cardoso, Karina; Cudischevitch, Cecília Oliveira; Menna-Barreto, Rubem Figueiredo Sadok; Oliveira, José Henrique Maia; Gusmão, Desiely Silva; Alves Lemos, Francisco José; Alviano, Daniela Sales; Oliveira, Pedro Lagerblad; Lowenberger, Carl; Majerowicz, David; Oliveira, Ricardo Melo; Mesquita, Rafael Dias; Atella, Georgia Correa

    2016-01-01

    Background Mosquitoes feed on plant-derived fluids such as nectar and sap and are exposed to bioactive molecules found in this dietary source. However, the role of such molecules on mosquito vectorial capacity is unknown. Weather has been recognized as a major determinant of the spread of dengue, and plants under abiotic stress increase their production of polyphenols. Results Here, we show that including polyphenols in mosquito meals promoted the activation of AMP-dependent protein kinase (AMPK). AMPK positively regulated midgut autophagy leading to a decrease in bacterial proliferation and an increase in vector lifespan. Suppression of AMPK activity resulted in a 6-fold increase in midgut microbiota. Similarly, inhibition of polyphenol-induced autophagy induced an 8-fold increase in bacterial proliferation. Mosquitoes maintained on the polyphenol diet were readily infected by dengue virus. Conclusion The present findings uncover a new direct route by which exacerbation of autophagy through activation of the AMPK pathway leads to a more efficient control of mosquito midgut microbiota and increases the average mosquito lifespan. Our results suggest for the first time that the polyphenol content and availability of the surrounding vegetation may increase the population of mosquitoes prone to infection with arboviruses. PMID:27732590

  13. Regulation of Serratia marcescens ompF and ompC porin genes in response to osmotic stress, salicylate, temperature and pH.

    PubMed

    Begic, Sanela; Worobec, Elizabeth A

    2006-02-01

    Serratia marcescens is a Gram-negative enterobacterium that has become an important opportunistic pathogen, largely due to its high degree of natural antibiotic resistance. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, which is controlled in part by OmpC and OmpF porin proteins. OmpF expression is regulated by micF, an RNA transcript encoded upstream of the ompC gene, which hybridizes with the ompF transcript to inhibit its translation. Regulation of S. marcescens porin gene expression, as well as that of micF, was investigated using beta-galactosidase reporter gene fusions in response to 5, 8 and 10 % sucrose, 1, 5 and 8 mM salicylate, and different pH and temperature values. beta-Galactosidase activity assays revealed that a lower growth temperature (28 degrees C), a more basic pH (pH 8), and an absence of sucrose and salicylate induce the transcription of the ompF gene, whereas the induction of ompC is stimulated at a higher growth temperature (42 degrees C), acidic pH (pH 6), and maximum concentrations of sucrose (10 %) and salicylate (8 mM). In addition, when multiple conditions were tested, temperature had the predominant effect, followed by pH. In this study, it was found that the MicF regulatory mechanism does not play a role in the osmoregulation of the ompF and ompC genes, whereas MicF does repress OmpF expression in the presence of salicylate and high growth temperature, and under low pH conditions.

  14. Insect midgut α-mannosidases from family 38 and 47 with emphasis on those of Tenebrio molitor.

    PubMed

    Moreira, Nathalia R; Cardoso, Christiane; Ribeiro, Alberto F; Ferreira, Clelia; Terra, Walter R

    2015-12-01

    α-Mannosidases are enzymes which remove non-reducing terminal residues from glycoconjugates. Data on both GH47 and GH38 (Golgi and lysosomal) enzymes are available. Data on insect midgut α-mannosidases acting in digestion are preliminary and do not include enzyme sequences. Tenebrio molitor midgut α-mannosidases were separated by chromatography into two activity peaks: a major (Man1) and a minor (Man2). An antibody generated against a synthetic peptide corresponding to a sequence of α-mannosidase fragment recognizes Man2 but not Man1. That fragment was later found to correspond to TmMan2 (GenBank access KP892646), showing that the cDNA coding for Man2 is actually TmMan2. TmMan2 codes for a mature α-mannosidase with 107.5 kDa. Purified Man2 originates after SDS-PAGE one band of about 72 kDa and another of 51 kDa, which sums 123 kDa, in agreement with gel filtration (123 kDa) data. These results suggest that Man2 is processed into peptides that remain noncovalently linked within the functional enzyme. The physical and kinetical properties of purified Man1 and Man2 are similar. They have a molecular mass of 123 kDa (gel filtration), pH optimum (5.6) and response to inhibitors like swainsonine (Man1 Ki, 68 nM; Man2 Ki, 63 nM) and deoxymannojirimycin (Man1 Ki, 0.12 mM; Man2 Ki, 0.15 mM). Their substrate specificities are a little different as Man2 hydrolyzes α-1,3 and α-1,6 bonds better than α-1,2, whereas the contrary is true for Man1. Thus, they pertain to Class II (GH38 α-mannosidases), that are catabolic α-mannosidases similar to lysosomal α-mannosidase. However, Man2, in contrast to true lysosomal α-mannosidase, is secreted (immunocytolocalization data) into the midgut contents. There, Man2 may participate in digestion of fungal cell walls, known to have α-mannosides in their outermost layer. The amount of family 38 α-mannosidase sequences found in the transcriptome (454 pyrosequencing) of the midgut of 9 insects pertaining to 5 orders is

  15. Insect midgut α-mannosidases from family 38 and 47 with emphasis on those of Tenebrio molitor.

    PubMed

    Moreira, Nathalia R; Cardoso, Christiane; Ribeiro, Alberto F; Ferreira, Clelia; Terra, Walter R

    2015-12-01

    α-Mannosidases are enzymes which remove non-reducing terminal residues from glycoconjugates. Data on both GH47 and GH38 (Golgi and lysosomal) enzymes are available. Data on insect midgut α-mannosidases acting in digestion are preliminary and do not include enzyme sequences. Tenebrio molitor midgut α-mannosidases were separated by chromatography into two activity peaks: a major (Man1) and a minor (Man2). An antibody generated against a synthetic peptide corresponding to a sequence of α-mannosidase fragment recognizes Man2 but not Man1. That fragment was later found to correspond to TmMan2 (GenBank access KP892646), showing that the cDNA coding for Man2 is actually TmMan2. TmMan2 codes for a mature α-mannosidase with 107.5 kDa. Purified Man2 originates after SDS-PAGE one band of about 72 kDa and another of 51 kDa, which sums 123 kDa, in agreement with gel filtration (123 kDa) data. These results suggest that Man2 is processed into peptides that remain noncovalently linked within the functional enzyme. The physical and kinetical properties of purified Man1 and Man2 are similar. They have a molecular mass of 123 kDa (gel filtration), pH optimum (5.6) and response to inhibitors like swainsonine (Man1 Ki, 68 nM; Man2 Ki, 63 nM) and deoxymannojirimycin (Man1 Ki, 0.12 mM; Man2 Ki, 0.15 mM). Their substrate specificities are a little different as Man2 hydrolyzes α-1,3 and α-1,6 bonds better than α-1,2, whereas the contrary is true for Man1. Thus, they pertain to Class II (GH38 α-mannosidases), that are catabolic α-mannosidases similar to lysosomal α-mannosidase. However, Man2, in contrast to true lysosomal α-mannosidase, is secreted (immunocytolocalization data) into the midgut contents. There, Man2 may participate in digestion of fungal cell walls, known to have α-mannosides in their outermost layer. The amount of family 38 α-mannosidase sequences found in the transcriptome (454 pyrosequencing) of the midgut of 9 insects pertaining to 5 orders is

  16. Na/sup +/ requirement for growth, photosynthesis, and pH regulation of the alkalotolerant cyanobacterium Synechococcus leopoliensis

    SciTech Connect

    Miller, A.G.; Turpin, D.H.; Canvin, D.T.

    1984-07-01

    It was found that Na/sup +/ is required for all the alkalotolerance of the cyanobacterium Synechococcus leopoliensis. Cell division did not occur at any pH in the absence of Na/sup +/, but cells inoculated into Na/sup +/-free growth medium at pH 6.8 did continue metabolic activity, and over a period of 48 h, the cells became twice their normal size. Many of these cells remained viable for at least 59 h and formed colonies on Na/sup +/-containing medium. Cells grown in the presence of Na/sup +/ and inoculated into Na/sup +/-free growth medium at pH 9.6 rapidly lost viability. An Na/sup +/ concentration of ca. 0.5 milliequivalents x liter/sup -1/ was required for sustained growth above pH 9.0. The Na/sup +/ requirement could be only partially met by Li/sup +/ and not at all by K/sup +/ or Rb/sup +/. Cells incubated in darkness in growth medium at pH 6.8 had an intracellular pH near neutrality in the presence or absence of Na/sup +/. When the external pH was shifted to 9.6, only cells in the presence of Na/sup +/ were able to maintain an intracellular pH near 7.0. The membrane potential, however, remained high (-120mV) in the absence or presence of Na/sup +/ unless collapsed by the addition of gramicidin. Thus, the inability to maintain a neutral intracellular pH at pH 9.6 in the absence of Na/sup +/ was not due to a generalized disruption of membrane integrity. Even cells containing Na/sup +/ still required added Na/sup +/ to restore photosynthetic rates to normal after the cells had been washed in Na/sup +/-free buffer at pH 9.6. This requirement was only partially met by Li/sup +/ and was not met at all by K/sup +/, Rb/sup +/, Cs/sup +/, Mg/sup 2 +/, or Ca/sup 2 +/. The restoration of photosynthesis by added Na/sup +/ occurred within 30 s and suggests a role for extracellular Na/sup +/. Part of our results can be explained in terms of the operation of an Na/sup +//H/sup +/ antiporter activity in the plasma membrane, but some results would seem to require other

  17. Analysis of the regulation of the Ustilago maydis proteome by dimorphism, pH or MAPK and GCN5 genes.

    PubMed

    Martínez-Salgado, José L; León-Ramírez, Claudia G; Pacheco, Alberto Barrera; Ruiz-Herrera, José; de la Rosa, Ana P Barba

    2013-02-21

    Ustilago maydis is a dimorphic corn pathogenic basidiomycota whose haploid cells grow in yeast form at pH7, while at pH3 they grow in the mycelial form. Two-dimensional gel electrophoresis (2-DE) coupled with LC-ESI/MS-MS was used to analyze the differential accumulation of proteins in yeast against mycelial morphologies. 2-DE maps were obtained in the pH range of 5-8 and 404 total protein spots were separated. From these, 43 were differentially accumulated when comparing strains FB2wt, constitutive yeast CL211, and constitutive mycelial GP25 growing at pH7 against pH3. Differentially accumulated proteins in response to pH are related with defense against reactive oxygen species or toxic compounds. Up-accumulation of CipC and down-accumulation of Hmp1 were specifically related with mycelial growth. Changes in proteins that were affected by mutation in the gene encoding the adaptor of a MAPK pathway (CL211 strain) were UM521* and transcription factors Btf3, Sol1 and Sti1. Mutation of GCN5 (GP25 strain) affected the accumulation of Rps19-ribosomal protein, Mge1-heath shock protein, and Lpd1-dihydrolipoamide dehydrogenase. Our results complement the information about the genes and proteins related with the dimorphic transition in U. maydis and changes in proteins affected by mutations in a MAPK pathway and GCN5 gene.

  18. Superior mesenteric vein rotation: a CT sign of midgut malrotation

    SciTech Connect

    Nichols, D.M.; Li, D.K.

    1983-10-01

    Computed tomography (CT) of the pancreas, with its excellent display of peripancreatic anatomy, allows visualization of the major vessels entering the mesenteric root. In scans of the normal upper abdomen obtained at or just below the level of the uncinate process of the pancreas, the proximal superior mesenteric vein (SMV) easily can be identified lying on the right ventral aspect of the superior mesenteric artery (SMA). The authors have observed a characteristic abnormality in this normal vascular arrangement on CT scans of the pancreas in three adult patients with suspected chronic pancreatitis who were subsequently proved to have midgut malrotation. They called this the SMV rotation sign and believe that its detection even on CT scans limited to the level of the pancreas should alert the radiologist to the presence of a midgut malrotation that may have been unsuspected.

  19. Regulation of intracellular pH during H+-coupled oligopeptide absorption in enterocytes from guinea-pig ileum

    PubMed Central

    Hayashi, Hisayoshi; Suzuki, Yuichi

    1998-01-01

    The mechanisms for regulating the intracellular pH (pHi) level during oligopeptide absorption were investigated in the enterocytes from guinea-pig ileum by identifying the acid-base transporters responsible for extruding H+ that enters the cell through the H+-oligopeptide cotransporter. The pHi level was measured by microfluorometry in an isolated villus tip loaded with the pH-sensitive fluoroprobe 2′7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oligopeptide-induced increment in the short-circuit current (Isc) was determined in a mucosal sheet in Ussing chambers. A CO2/HCO3−-buffered solution was used. The superfusion of glycylglycine (Gly-Gly, l0 mM) caused a decrease in pHi level, which returned to the basal level after removing Gly-Gly. This pHi recovery was strongly dependent on extracellular Na+. Amiloride partially inhibited the pHi recovery rate with an IC50 value of 41 μM, the maximum inhibition being approximately 70%. In the presence of amiloride at its maximum concentration (0.3 mM), the addition of 0.6 mM DIDS caused a further decrease, but did not abolish the pHi recovery rate. In the absence of CO2 and HCO3−, the pHi recovery was almost completely abolished by 0.3 mM amiloride. The intracellular H+ accumulation induced by 0.3 mM amiloride or by 0.6 mM DIDS, as estimated from the pHi decrease and buffer capacity, was significantly greater during Gly-Gly superfusion than under resting conditions. The increase in Isc induced by luminal glycylproline was attenuated by either removing serosal Na+ or by adding 0.5 mM amiloride or 0.6 mM DIDS to the serosal side. We conclude that both Na+-dependent, amiloride-sensitive acid extrusion, probably by the Na+-H+ exchanger, and Na+- and HCO3−-dependent, DIDS-sensitive acid extrusion, possibly by the Na+-HCO3− cotransporter, are involved in extruding H+ that enters cells by the H+-oligopeptide cotransport. It is proposed that these acid extrusion (or base loading) mechanisms are present

  20. Cultivation of Chlorella zofingiensis in bench-scale outdoor ponds by regulation of pH using dairy wastewater in winter, South China.

    PubMed

    Huo, Shuhao; Wang, Zhongming; Zhu, Shunni; Zhou, Weizheng; Dong, Renjie; Yuan, Zhenhong

    2012-10-01

    Cultivation of Chlorella zofingiensis and nutrients removal in dairy wastewater were investigated in bench-scale outdoor ponds in winter, South China. The impacts of the two types of pH regulations, 5 ≈ 6% CO(2) and acetic acid (HAc) on this process were studied. After 6 days cultivation, the removal rates of total nitrogen (TN) and orthophosphate (PO(4)(3-)) using CO(2) regulation were better than those using HAc. The removal rates of PO(4)(3-) and TN were 97.5% and 51.7%, respectively using CO(2) regulation; 79.6% (TN) and 42.0% (PO(4)(3-)) were obtained using HAc regulation. Higher biomass, protein, sugar content, and stable pH control were found using CO(2) regulation. However, significantly higher lipid content (31.8%) was observed using HAc regulation. The dominant differences of fatty acids were the content of C18:1 and C18:3. The growth characteristics and environmental conditions especially during the typical logarithmic phase were also analyzed. PMID:22858469

  1. Brain-midgut cross-talk and autocrine metabolastat via the sNPF/CCAP negative feed-back loop in the American cockroach, Periplaneta americana.

    PubMed

    Mikani, Azam; Watari, Yasuhiko; Takeda, Makio

    2015-12-01

    Immunohistochemical reactivities against short neuropeptide F (sNPF-ir) and crustacean cardioactive peptide (CCAP-ir) were detected in both the brain-subesophageal ganglion (Br-SOG) and midgut epithelial cells of the male American cockroach, Periplaneta americana. Four weeks of starvation increased the number of sNPF-ir cells and decreased the CCAP-ir cells in the Br-SOG, whereas refeeding reversed these effects. The contents of sNPF in the Br-SOG, midgut and hemolymph titer decreased in response to an injection of CCAP into the hemocoel of normally fed male cockroaches, while CCAP titers/contents decreased in response to an injection of sNPF. The results of a double-labeling experiment demonstrated that sNPF-ir co-existed in CCAP-ir cells in the pars intercerebralis (PI), dorsolateral region of protocerebrum (DL), deutocerebrum (De) and SOG. sNPF-ir and CCAP-ir were also colocalized in the midgut. sNPF and CCAP are neuropeptides and midgut factors that interact with each other. Since the two peptides are known to be secreted by identical cells that affect each other, this constitutes autocrine negative feedback regulation for a quick response to food accessibility/inaccessibility. These peptides not only constitute the switch in the digestive mechanism but also couple digestive adaptation with behavior. A CCAP injection suppressed locomotor activity when cockroaches were starved, whereas sNPF activated it when they were fed.

  2. Expression of a sugar clade gustatory receptor, BmGr6, in the oral sensory organs, midgut, and central nervous system of larvae of the silkworm Bombyx mori.

    PubMed

    Mang, Dingze; Shu, Min; Endo, Haruka; Yoshizawa, Yasutaka; Nagata, Shinji; Kikuta, Shingo; Sato, Ryoichi

    2016-03-01

    Insects taste nonvolatile chemicals through gustatory receptors (Grs) and make choices for feeding, mating, and oviposition. To date, genome projects have identified 69 Gr genes in the silkworm, Bombyx mori; however, the expression sites of these Grs remain to be explored. In this study, we used reverse transcription (RT)-PCR to investigate expression of the B. mori Gr-6 (BmGr6) gene, a member of the putative sugar clade gene family in various tissues. BmGr6 is expressed in the midgut, central nervous system (CNS), and oral sensory organs. Moreover, immunohistochemistry using an anti-BmGr6 antiserum demonstrated that BmGr6 is expressed in cells by oral sensory organs, midgut and nervous system. Furthermore, double-immunohistochemistry indicated that BmGr6 is expressed in midgut enteroendocrine cells, also in CNS neurosecretory cells. In particular, a portion of BmGr6-expressing cells, in both midgut and CNS, secretes FMRFamide-related peptides (FaRPs). These results suggest that BmGr6 functions not only as a taste receptor, but also as a chemical sensor such as for the regulation of gut movement, physiological conditions, and feeding behavior of larvae. PMID:26721200

  3. Bacillus thuringiensis pore-forming toxins trigger massive shedding of GPI-anchored aminopeptidase N from gypsy moth midgut epithelial cells.

    PubMed

    Valaitis, Algimantas P

    2008-06-01

    The insecticidal Cry proteins produced by Bacillus thuringiensis strains are pore-forming toxins (PFTs) that bind to the midgut brush border membrane and cause extensive damage to the midgut epithelial cells of susceptible insect larvae. Force-feeding B. thuringiensis PFTs to Lymantria dispar larvae elicited rapid and massive shedding of a glycosylphosphatidylinositol (GPI)-anchored aminopeptidase N (APN) from midgut epithelial cells into the luminal fluid, and depletion of the membrane-anchored enzyme on the midgut epithelial cells. The amount of APN released into the luminal fluid of intoxicated larvae was dose- and time-dependent, and directly related to insecticidal potency of the PFTs. The induction of toxin-induced shedding of APN was inhibited by cyclic AMP and MAPK kinase (MEK) inhibitors PD98059 and U0126, indicating that signal transduction in the MEK/ERK pathway is involved in the regulation of the shedding process. APN released from epithelial cells appears to be generated by the action of a phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of the GPI anchor based upon detection of a cross-reacting determinant (CRD) on the protein shed into the luminal fluid. Alkaline phosphatase was also released from the gut epithelial cells, supporting the conclusion that other GPI-anchored proteins are released as a consequence of the activation PI-PLC. These observations are the basis of a novel and highly sensitive tool for evaluating the insecticidal activity of new Cry proteins obtained though discovery or protein engineering.

  4. Whole-genome expression analysis in the third instar larval midgut of Drosophila melanogaster.

    PubMed

    Harrop, Thomas W R; Pearce, Stephen L; Daborn, Phillip J; Batterham, Philip

    2014-09-05

    Survival of insects on a substrate containing toxic substances such as plant secondary metabolites or insecticides is dependent on the metabolism or excretion of those xenobiotics. The primary sites of xenobiotic metabolism are the midgut, Malpighian tubules, and fat body. In general, gene expression in these organs is reported for the entire tissue by online databases, but several studies have shown that gene expression within the midgut is compartmentalized. Here, RNA sequencing is used to investigate whole-genome expression in subsections of third instar larval midguts of Drosophila melanogaster. The data support functional diversification in subsections of the midgut. Analysis of the expression of gene families that are implicated in the metabolism of xenobiotics suggests that metabolism may not be uniform along the midgut. These data provide a starting point for investigating gene expression and xenobiotic metabolism and other functions of the larval midgut.

  5. Whole-Genome Expression Analysis in the Third Instar Larval Midgut of Drosophila melanogaster

    PubMed Central

    Harrop, Thomas W. R.; Pearce, Stephen L.; Daborn, Phillip J.; Batterham, Philip

    2014-01-01

    Survival of insects on a substrate containing toxic substances such as plant secondary metabolites or insecticides is dependent on the metabolism or excretion of those xenobiotics. The primary sites of xenobiotic metabolism are the midgut, Malpighian tubules, and fat body. In general, gene expression in these organs is reported for the entire tissue by online databases, but several studies have shown that gene expression within the midgut is compartmentalized. Here, RNA sequencing is used to investigate whole-genome expression in subsections of third instar larval midguts of Drosophila melanogaster. The data support functional diversification in subsections of the midgut. Analysis of the expression of gene families that are implicated in the metabolism of xenobiotics suggests that metabolism may not be uniform along the midgut. These data provide a starting point for investigating gene expression and xenobiotic metabolism and other functions of the larval midgut. PMID:25193493

  6. Low pH, Aluminum, and Phosphorus Coordinately Regulate Malate Exudation through GmALMT1 to Improve Soybean Adaptation to Acid Soils1[W][OA

    PubMed Central

    Liang, Cuiyue; Piñeros, Miguel A.; Tian, Jiang; Yao, Zhufang; Sun, Lili; Liu, Jiping; Shaff, Jon; Coluccio, Alison; Kochian, Leon V.; Liao, Hong

    2013-01-01

    Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function. PMID:23341359

  7. The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH.

    PubMed

    Hao, Meng-Shu; Jensen, Anna M; Boquist, Ann-Sofie; Liu, Yun-Jun; Rasmusson, Allan G

    2015-01-01

    NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene. PMID:26413894

  8. Cellulolytic environment in the midgut of the wood-feeding higher termite Nasutitermes takasagoensis.

    PubMed

    Tokuda, Gaku; Watanabe, Hirofumi; Hojo, Masaru; Fujita, Ai; Makiya, Hiromi; Miyagi, Mio; Arakawa, Gaku; Arioka, Manabu

    2012-01-01

    Unlike lower termites, xylophagous higher termites thrive on wood without the aid of symbiotic protists. In the higher termite Nasutitermes takasagoensis, both endogenous endo-β-1,4-glucanase and β-glucosidase genes are expressed in the midgut, which is believed to be the main site of cellulose digestion. To further explore the detailed cellulolytic system in the midgut of N. takasagoensis, we performed immunohistochemistry and digital light microscopy to determine distributions of cellulolytic enzymes in the salivary glands and the midgut as well as the total cellulolytic activity in the midgut. Although cellulolytic enzymes were uniformly produced in the midgut epithelium, the concentration of endo-β-1,4-glucanase activity and luminal volume in the midgut were comparable to those of the wood-feeding lower termite Coptotermes formosanus, which digests cellulose with the aid of hindgut protists. However, the size of ingested wood particles was considerably larger in N. takasagoensis than that in C. formosanus. Nevertheless, it is possible that the cellulolytic system in the midgut of N. takasagoensis hydrolyzes highly crystalline cellulose to a certain extent. The glucose produced did not accumulate in the midgut lumen. Therefore, the present study suggests that the midgut of the higher termite provides the necessary conditions for cellulolysis.

  9. A hypothetical model of crossing Bombyx mori nucleopolyhedrovirus through its host midgut physical barrier.

    PubMed

    Cheng, Yang; Wang, Xue-Yang; Hu, Hao; Killiny, Nabil; Xu, Jia-Ping

    2014-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV.

  10. Ligninolytic peroxidase gene expression by Pleurotus ostreatus: differential regulation in lignocellulose medium and effect of temperature and pH.

    PubMed

    Fernández-Fueyo, Elena; Castanera, Raul; Ruiz-Dueñas, Francisco J; López-Lucendo, María F; Ramírez, Lucía; Pisabarro, Antonio G; Martínez, Angel T

    2014-11-01

    Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25°C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25°C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn(2+) (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3>vp2/vp3/mnp1/mnp2/mnp6>mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10°C or 37°C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10°C treatment, while many of them were alternatively upregulated (often 6h after the thermal shock) and downregulated (12h) at 37°C. Interestingly, mnp4 and

  11. Ligninolytic peroxidase gene expression by Pleurotus ostreatus: differential regulation in lignocellulose medium and effect of temperature and pH.

    PubMed

    Fernández-Fueyo, Elena; Castanera, Raul; Ruiz-Dueñas, Francisco J; López-Lucendo, María F; Ramírez, Lucía; Pisabarro, Antonio G; Martínez, Angel T

    2014-11-01

    Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25°C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25°C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn(2+) (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3>vp2/vp3/mnp1/mnp2/mnp6>mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10°C or 37°C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10°C treatment, while many of them were alternatively upregulated (often 6h after the thermal shock) and downregulated (12h) at 37°C. Interestingly, mnp4 and

  12. pH up-regulation as a potential mechanism for the cold-water coral Lophelia pertusa to sustain growth in aragonite undersaturated conditions

    NASA Astrophysics Data System (ADS)

    Wall, M.; Ragazzola, F.; Foster, L. C.; Form, A.; Schmidt, D. N.

    2015-12-01

    Cold-water corals are important habitat formers in deep-water ecosystems and at high latitudes. Ocean acidification and the resulting change in aragonite saturation are expected to affect these habitats and impact coral growth. Counter to expectations, the deep water coral Lophelia pertusa has been found to be able to sustain growth even in undersaturated conditions. However, it is important to know whether such undersaturation modifies the skeleton and thus its ecosystem functioning. Here we used Synchrotron X-Ray Tomography and Raman spectroscopy to examine changes in skeleton morphology and fibre orientation. We combined the morphological assessment with boron isotope analysis to determine if changes in growth are related to changes in control of calcification pH. We compared the isotopic composition and structure formed in their natural environment to material grown in culture at lower pH conditions. Skeletal morphology is highly variable but shows no distinctive differences between natural and low pH conditions. Raman investigations found no difference in macromorphological skeletal arrangement of early mineralization zones and secondary thickening between the treatments. The δ11B analyses show that L. pertusa up-regulates the internal calcifying fluid pH (pHcf) during calcification compared to ambient seawater pH and maintains a similar elevated pHcf at increased pCO2 conditions. We suggest that as long as the energy is available to sustain the up-regulation, i.e. individuals are well fed, there is no detrimental effect to the skeletal morphology.

  13. Modulation of Phagosomal pH by Candida albicans Promotes Hyphal Morphogenesis and Requires Stp2p, a Regulator of Amino Acid Transport

    PubMed Central

    Vylkova, Slavena; Lorenz, Michael C.

    2014-01-01

    Candida albicans, the most important fungal pathogen of humans, has a unique interaction with macrophages in which phagocytosis induces a switch from the yeast to hyphal form, allowing it to escape by rupturing the immune cell. While a variety of factors induce this switch in vitro, including neutral pH, it is not clear what triggers morphogenesis within the macrophage where the acidic environment should inhibit this transition. In vitro, C. albicans grown in similar conditions in which amino acids are the primary carbon source generate large quantities of ammonia to raise the extracellular pH and induce the hyphal switch. We show here that C. albicans cells neutralize the macrophage phagosome and that neutral pH is a key inducer of germination in phagocytosed cells by using a mutant lacking STP2, a transcription factor that regulates the expression of multiple amino acid permeases, that is completely deficient in alkalinization in vitro. Phagocytosed stp2Δ mutant cells showed significant reduction in hypha formation and escaped from macrophages less readily compared to wild type cells; as a result stp2Δ mutant cells were killed at a higher rate and caused less damage to RAW264.7 macrophages. Stp2p-regulated import leads to alkalinization of the phagosome, since the majority of the wild type cells fail to co-localize with acidophilic dyes, whereas the stp2Δ mutant cells were located in acidic phagosomes. Furthermore, stp2Δ mutant cells were able to form hyphae and escape from neutral phagosomes, indicating that the survival defect in these cells was pH dependent. Finally, these defects are reflected in an attenuation of virulence in a mouse model of disseminated candidiasis. Altogether our results suggest that C. albicans utilizes amino acids to promote neutralization of the phagosomal pH, hyphal morphogenesis, and escape from macrophages. PMID:24626429

  14. Lactate and glucose concomitant consumption as a self-regulated pH detoxification mechanism in HEK293 cell cultures.

    PubMed

    Liste-Calleja, Leticia; Lecina, Martí; Lopez-Repullo, Jonatan; Albiol, Joan; Solà, Carles; Cairó, Jordi Joan

    2015-12-01

    One of the most important limitations of mammalian cell-based processes is the secretion and accumulation of lactate as a by-product of their metabolism. Among the cell lines commonly used in industrial bioprocesses, HEK293 has been gaining importance over the last years. Up recently, HEK293 cells were known to consume lactate in late stages of cell culture usually when glucose and/or glutamine were depleted from media. Remarkably, in both scenarios, no significant cell growth was reported. However, we have observed a different metabolic behavior regarding lactate production and consumption in HEK293 cultures. HEK293 cells were able to co-metabolize glucose and lactate simultaneously, even in exponentially growing cell cultures. Our deep study of the effects of environmental conditions on lactate metabolism revealed that pH was the key to trigger the metabolic shift from lactate production to lactate and glucose concomitant consumption. Remarkably, this shift could be triggered at will when pH was set at 6.8. Even more interesting was the fact that lowering pH to 6.6 and supplementing media with exogenous lactate resulted in co-consumption of glucose and lactate from the beginning of cell culture, without affecting cell growth or protein productivity. On the contrary, cell growth was clearly hampered at this low pH if extracellular lactate was lacking. From our results, we hypothesize that HEK293 cells metabolize extracellular lactate as a strategy for pH detoxification, by means of co-transporting extracellular protons together with lactate into the cytosol. This novel hypothesis for unraveling lactate metabolism in HEK293 cells could open a door to re-direct genetic engineering strategies in order to obtain more efficient cell lines and also to further develop animal cell technology applications.

  15. Single mutation (A162H) in human cardiac troponin I corrects acid pH sensitivity of Ca2+-regulated actomyosin S1 ATPase.

    PubMed

    Dargis, Roland; Pearlstone, Joyce R; Barrette-Ng, Isabelle; Edwards, Helena; Smillie, Lawrence B

    2002-09-20

    In contrast to skeletal muscle, the efficiency of the contractile apparatus of cardiac tissue has long been known to be severely compromised by acid pH as in the ischemia of myocardial infarction and other cardiac myopathies. Recent reports (Westfall, M. V., and Metzger, J. M. (2001) News Physiol. Sci. 16, 278-281; Li, G., Martin, A. F., and Solaro, R. J. (2001) J. Mol. Cell. Cardiol. 33, 1309-1320) have indicated that the reduced Ca(2+) sensitivity of cardiac contractility at low pH (regulated human cardiac troponin-tropomyosin actomyosin S1 ATPase assay, we report that a single TnI mutation, A162H, restores Ca(2+) sensitivity at pH 6.5 to that at pH 7.0. Levels of inhibition (pCa 7.0), activation (pCa 4.0), and cooperativity of ATPase activity were minimally affected. Two other mutations (Q155R and E164V) also previously suggested by us (Pearlstone, J. R., Sykes, B. D., and Smillie, L. B. (1997) Biochemistry 36, 7601-7606) and involving charged residues showed no such effects. With fast skeletal muscle troponin, a single TnI H130A mutation reduced Ca(2+) sensitivity at pH 6.5 to levels approaching the cardiac system at pH 6.5. These observations provide structural insight into long-standing physiological and clinical phenomena and are of potential relevance to therapeutic treatments of heart disease by gene transfer, stem cell, and cell transplantation approaches. PMID:12151382

  16. Aspen defense chemicals influence midgut bacterial community composition of gypsy moth.

    PubMed

    Mason, Charles J; Rubert-Nason, Kennedy F; Lindroth, Richard L; Raffa, Kenneth F

    2015-01-01

    Microbial symbionts are becoming increasingly recognized as mediators of many aspects of plant - herbivore interactions. However, the influence of plant chemical defenses on gut associates of insect herbivores is less well understood. We used gypsy moth (Lymantria dispar L.), and differing trembling aspen (Populus tremuloides Michx.) genotypes that vary in chemical defenses, to assess the influence of foliar chemistry on bacterial communities of larval midguts. We evaluated the bacterial community composition of foliage, and of midguts of larvae feeding on those leaves, using next-generation high-throughput sequencing. Plant defense chemicals did not influence the composition of foliar communities. In contrast, both phenolic glycosides and condensed tannins affected the bacterial consortia of gypsy moth midguts. The two most abundant operational taxonomic units were classified as Ralstonia and Acinetobacter. The relative abundance of Ralstonia was higher in midguts than in foliage when phenolic glycoside concentrations were low, but lower in midguts when phenolic glycosides were high. In contrast, the relative abundance of Ralstonia was lower in midguts than in foliage when condensed tannin concentrations were low, but higher in midguts when condensed tannins were high. Acinetobacter showed a different relationship with host chemistry, being relatively more abundant in midguts than with foliage when condensed tannin concentrations were low, but lower in midguts when condensed tannins were high. Acinetobacter tended to have a greater relative abundance in midguts of insects feeding on genotypes with high phenolic glycoside concentrations. These results show that plant defense chemicals influence herbivore midgut communities, which may in turn influence host utilization. PMID:25475786

  17. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment.

  18. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment. PMID:27480055

  19. Hsp70 and small Hsps are the major heat shock protein members involved in midgut metamorphosis in the common cutworm, Spodoptera litura.

    PubMed

    Gu, J; Huang, L-X; Shen, Y; Huang, L-H; Feng, Q-L

    2012-10-01

    Heat shock proteins (Hsps) are important chaperones, which are involved in various signal pathways and regulate lots of physiological processes. Early research suggested that some Hsps are involved in insect development. However, few studies have been carried out to explore the roles of Hsps, especially in larval-pupal metamorphosis. In the present study, 49 Hsp unigenes were identified in the Spodoptera litura transcriptome and their mRNA expression profiles during midgut metamorphosis were examined using a tag-based digital gene expression system. The genes with the most different levels of expression were then cloned and their expression patterns in midguts from sixth instar larvae to pupae were analysed using real time quantitative PCR. The responses of these genes to juvenile hormone (JH) and 20-hydroxyecdysone (20E) were also studied. The results showed that the mRNA levels of 22 Hsp unigenes changed significantly during midgut metamorphosis. Amongst these 22 unigenes, hsp70, hsp20.4 and hsp20.8 were the most up-regulated members, and hsp15.9, hsp19.3 and hsp22.0 were the most down-regulated ones. Further studies showed that hsp70, hsp20.4 and hsp20.8 were remarkably up-regulated by JH. In addition, 20E slightly increased the mRNA levels of both hsp20.4 and hsp20.8. However, hsp15.9, hsp19.3 and hsp22.0 did not respond to either JH or 20E. These results indicate that Hsp70 and small Hsps (sHsps) are probably the major players in midgut metamorphosis in S. litura. The current findings provide valuable insights into the roles of the Hsp superfamily in insect metamorphosis.

  20. Enteritis necroticans with midgut necrosis caused by Clostridium perfringens.

    PubMed

    Clarke, L E; Diekmann-Guiroy, B; McNamee, W; Java, D J; Weiss, S M

    1994-05-01

    Enteritis necroticans is a necrotizing process manifesting as segmental gangrene of the bowel, triggered by Clostridium perfringens toxins under specific dietary conditions. It is a rare disease in developed countries and is probably underdiagnosed. A case of enteritis necroticans presenting with midgut necrosis with sepsis and hemolysis is reported herein. Bacteriologic culture of blood and peritoneal content revealed C perfringens. Dietary history, including the ingestion of meat together with sweet potatoes, should increase clinical suspicion of enteritis necroticans. Early recognition and timely surgical intervention are required for successful treatment. Clinicians are encouraged to be aware of this clinically fulminant yet rarely recognized surgical entity.

  1. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells.

    PubMed

    Lara, Flavio Alves; Pohl, Paula C; Gandara, Ana Caroline; Ferreira, Jessica da Silva; Nascimento-Silva, Maria Clara; Bechara, Gervásio Henrique; Sorgine, Marcos H F; Almeida, Igor C; Vaz, Itabajara da Silva; Oliveira, Pedro L

    2015-01-01

    In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new

  2. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells

    PubMed Central

    Lara, Flavio Alves; Pohl, Paula C.; Gandara, Ana Caroline; Ferreira, Jessica da Silva; Nascimento-Silva, Maria Clara; Bechara, Gervásio Henrique; Sorgine, Marcos H. F.; Almeida, Igor C.; Vaz, Itabajara da Silva; Oliveira, Pedro L.

    2015-01-01

    In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new

  3. Host plant-specific remodeling of midgut physiology in the generalist insect herbivore Trichoplusia ni.

    PubMed

    Herde, Marco; Howe, Gregg A

    2014-07-01

    Species diversity in terrestrial ecosystems is influenced by plant defense compounds that alter the behavior, physiology, and host preference of insect herbivores. Although it is established that insects evolved the ability to detoxify specific allelochemicals, the mechanisms by which polyphagous insects cope with toxic compounds in diverse host plants are not well understood. Here, we used defended and non-defended plant genotypes to study how variation in chemical defense affects midgut responses of the lepidopteran herbivore Trichoplusia ni, which is a pest of a wide variety of native and cultivated plants. The genome-wide midgut transcriptional response of T. ni larvae to glucosinolate-based defenses in the crucifer Arabidopsis thaliana was characterized by strong induction of genes encoding Phase I and II detoxification enzymes. In contrast, the response of T. ni to proteinase inhibitors and other jasmonate-regulated defenses in tomato (Solanum lycopersicum) was dominated by changes in the expression of digestive enzymes and, strikingly, concomitant repression of transcripts encoding detoxification enzymes. Unbiased proteomic analyses of T. ni feces demonstrated that tomato defenses remodel the complement of T.ni digestive enzymes, which was associated with increased amounts of serine proteases and decreased lipase protein abundance upon encountering tomato defense chemistry. These collective results indicate that T. ni adjusts its gut physiology to the presence of host plant-specific chemical defenses, and further suggest that plants may exploit this digestive flexibility as a defensive strategy to suppress the production of enzymes that detoxify allelochemicals. PMID:24727019

  4. Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH.

    PubMed

    Pinelli, Laurent; Nissant, Antoine; Edwards, Aurélie; Lourdel, Stéphane; Teulon, Jacques; Paulais, Marc

    2016-09-01

    ClC-K2, a member of the ClC family of Cl(-) channels and transporters, forms the major basolateral Cl(-) conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl(-) absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl(-), and Ca(2+) on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca(2+) strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl(-) has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl(-)/HCO3 (-) exchange in type B intercalated cells. PMID:27574292

  5. Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi.

    PubMed

    Bi, Fangcheng; Barad, Shiri; Ment, Dana; Luria, Neta; Dubey, Amit; Casado, Virginia; Glam, Nofar; Mínguez, Jose Diaz; Espeso, Eduardo A; Fluhr, Robert; Prusky, Dov

    2016-10-01

    Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens-Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum-secrete small pH-affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC-dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non-preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high-carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host-dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization. PMID:26666972

  6. Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi.

    PubMed

    Bi, Fangcheng; Barad, Shiri; Ment, Dana; Luria, Neta; Dubey, Amit; Casado, Virginia; Glam, Nofar; Mínguez, Jose Diaz; Espeso, Eduardo A; Fluhr, Robert; Prusky, Dov

    2016-10-01

    Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens-Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum-secrete small pH-affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC-dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non-preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high-carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host-dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization.

  7. Plastidial transporters KEA1, -2, and -3 are essential for chloroplast osmoregulation, integrity, and pH regulation in Arabidopsis

    PubMed Central

    Kunz, Hans-Henning; Gierth, Markus; Herdean, Andrei; Satoh-Cruz, Mio; Kramer, David M.; Spetea, Cornelia; Schroeder, Julian I.

    2014-01-01

    Multiple K+ transporters and channels and the corresponding mutants have been described and studied in the plasma membrane and organelle membranes of plant cells. However, knowledge about the molecular identity of chloroplast K+ transporters is limited. Potassium transport and a well-balanced K+ homeostasis were suggested to play important roles in chloroplast function. Because no loss-of-function mutants have been identified, the importance of K+ transporters for chloroplast function and photosynthesis remains to be determined. Here, we report single and higher-order loss-of-function mutants in members of the cation/proton antiporters-2 antiporter superfamily KEA1, KEA2, and KEA3. KEA1 and KEA2 proteins are targeted to the inner envelope membrane of chloroplasts, whereas KEA3 is targeted to the thylakoid membrane. Higher-order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in the kea1kea2 mutants, but increased in the kea3 mutant, indicating an altered chloroplast pH homeostasis. Electron microscopy of kea1kea2 leaf cells revealed dramatically swollen chloroplasts with disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed the observed phenotypes. Furthermore, the kea1kea2 background enables genetic analyses of the functional significance of other chloroplast transporters as exemplified here in kea1kea2Na+/H+ antiporter1 (nhd1) triple mutants. Taken together, the presented data demonstrate a fundamental role of inner envelope KEA1 and KEA2 and thylakoid KEA3 transporters in chloroplast osmoregulation, integrity, and ion and pH homeostasis. PMID:24794527

  8. Togavirus-associated pathologic changes in the midgut of a natural mosquito vector.

    PubMed Central

    Weaver, S C; Scott, T W; Lorenz, L H; Lerdthusnee, K; Romoser, W S

    1988-01-01

    Arthropod-borne viruses were not previously believed to cause discernible pathologic changes in their natural mosquito vectors. We report cytopathologic lesions in the midgut of the mosquito, Culiseta melanura, 2 to 5 days after oral infection with eastern equine encephalomyelitis virus. Sloughing of densely staining, heavily infected epithelial cells into the midgut lumen was observed by light and transmission electron microscopy, along with degeneration of cells within the epithelium. Pathological changes in midgut epithelial cells sometimes included loss of brush border and basal lamina integrity. Disruption of the midgut basal lamina could result in bypassing of barriers to virus dissemination within the mosquito and allow rapid transmission to occur. Alternatively, luminal sloughing of heavily infected midgut epithelial cells may serve to modulate mosquito infections. These findings challenge previous beliefs regarding the benign nature of arbovirus-invertebrate host relationships. Images PMID:2896802

  9. Ultrastructure and immunofluorescence of the midgut of Bombus morio (Hymenoptera: Apidae: Bombini).

    PubMed

    Gonçalves, Wagner Gonzaga; Fernandes, Kenner Morais; Barcellos, Marcelo Silva; Silva, Fernanda Pereira; Magalhães-Junior, Marcos Jorge; Zanuncio, José Cola; Martins, Gustavo Ferreira; Serrão, José Eduardo

    2014-06-01

    Bumblebees are widely distributed across the world and have great economic and ecological importance as pollinators in the forest as well as in agriculture. The insect midgut consists of three cell types, which play various important roles in digestion, absorption, and hormone production. The present study characterized the anterior and posterior midgut regions of the bumblebee, Bombus morio. The digestive, regenerative and endocrine cells in the midgut showed regional differences in their number, nuclear size, as well as the size of the striated border. Ultrastructurally, the digestive cells contained many mitochondria and long microvilli; however, in the anterior midgut region, these cells showed dilated basal labyrinths with a few openings for the hemocoel, whereas the labyrinths of the basal posterior region remained inverse characteristics. Thus, the characterization of the midgut of B. morio supported an ecto-endoperitrophic circulation, contributing to a better understanding of the digestive process in this bee.

  10. Togavirus-associated pathologic changes in the midgut of a natural mosquito vector.

    PubMed

    Weaver, S C; Scott, T W; Lorenz, L H; Lerdthusnee, K; Romoser, W S

    1988-06-01

    Arthropod-borne viruses were not previously believed to cause discernible pathologic changes in their natural mosquito vectors. We report cytopathologic lesions in the midgut of the mosquito, Culiseta melanura, 2 to 5 days after oral infection with eastern equine encephalomyelitis virus. Sloughing of densely staining, heavily infected epithelial cells into the midgut lumen was observed by light and transmission electron microscopy, along with degeneration of cells within the epithelium. Pathological changes in midgut epithelial cells sometimes included loss of brush border and basal lamina integrity. Disruption of the midgut basal lamina could result in bypassing of barriers to virus dissemination within the mosquito and allow rapid transmission to occur. Alternatively, luminal sloughing of heavily infected midgut epithelial cells may serve to modulate mosquito infections. These findings challenge previous beliefs regarding the benign nature of arbovirus-invertebrate host relationships.

  11. cAMP/Protein Kinase A Activates Cystic Fibrosis Transmembrane Conductance Regulator for ATP Release from Rat Skeletal Muscle during Low pH or Contractions

    PubMed Central

    Cai, Weisong; Ballard, Heather J.

    2012-01-01

    We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific CFTR inhibitor, glibenclamide, or the specific CFTR inhibitor, CFTRinh-172, suggesting that CFTR was involved, and by inhibition of lactic acid entry to cells, indicating that intracellular pH depression was required. Muscle contractions significantly elevated interstitial ATP, but CFTRinh-172 abolished the increase. The cAMP/PKA pathway was involved in the signal transduction pathway for CFTR-regulated ATP release from muscle: forskolin increased CFTR phosphorylation and stimulated ATP release from muscle or myocytes; lactic acid increased intracellular cAMP, pCREB and PKA activity, whereas IBMX enhanced ATP release from myocytes. Inhibition of PKA with KT5720 abolished lactic-acid- or contraction-induced ATP release from muscle. Inhibition of either the Na+/H+-exchanger (NHE) with amiloride or the Na+/Ca2+-exchanger (NCX) with SN6 or KB-R7943 abolished lactic-acid- or contraction-induced release of ATP from muscle, suggesting that these exchange proteins may be involved in the activation of CFTR. Our data suggest that CFTR-regulated release contributes to ATP release from contracting muscle in vivo, and that cAMP and PKA are involved in the activation of CFTR during muscle contractions or acidosis; NHE and NCX may be involved in the signal transduction pathway. PMID:23226244

  12. Candida albicans Mds3p, a conserved regulator of pH responses and virulence identified through insertional mutagenesis.

    PubMed Central

    Davis, Dana A; Bruno, Vincent M; Loza, Lucio; Filler, Scott G; Mitchell, Aaron P

    2002-01-01

    Candida albicans is a commensal fungus that causes diverse infections after antibiotic use or immune debilitation. Gene discovery has been limited because the organism is an asexual diploid. We have developed a strategy that yields random homozygous insertion mutants. The strategy has permitted identification of several prospective essential genes. Many of these genes are homologous to nonessential Saccharomyces cerevisiae genes, and some have no S. cerevisiae homolog. These findings may expand the range of antifungal drug targets. We have also identified new genes required for pH-dependent filamentation, a trait previously associated with virulence. One newly identified gene, MDS3, is required for expression in alkaline media of two filamentation-associated genes, HWP1 and ECE1, but is not required for expression of other pH-response genes. In S. cerevisiae, the two MDS3 homologs are required for growth in alkaline media, thus arguing that Mds3p function in adaptation to external pH changes is conserved. Epistasis tests show that Mds3p contributes to virulence and alkaline pH responses independently of the well-characterized Rim101p pH-response pathway. PMID:12524333

  13. Ionic Specificity in pH Regulated Charged Interfaces: Fe[superscript 3+]versus La[superscript 3+

    SciTech Connect

    Wang, Wenjie; Park, Rebecca Y.; Meyer, David H.; Travesset, Alex; Vaknin, David

    2012-03-26

    We determine the distribution of two trivalent ions Fe{sup 3+} and La{sup 3+} next to two different amphiphilic charged interfaces as ions or complexes, consisting of the phosphate lipid dihexadecyl phosphate (DHDP) and the fatty acid arachidic acid (AA). These amphiphiles provide a wide range of pK{sub a} values, from 2.1 (DHDP) to 5.1 (AA), thus allowing manipulation of the surface charge over extremely low pH (pH {approx}1 or larger), and the two ions provide two limiting cases of specificity for the amphiphiles. We find that La{sup 3+} distribution is mostly sensitive to the surface charge, whereas the Fe{sup 3+} binding depends on its character in the solution and is highly specific, as indicated by the crucial role played by iron complexes (Fe(OH){sub 3} or Fe(OH){sup 2+}) forming covalent bonds even for an uncharged interface. The implications of the results to other ions and/or amphiphilic interfaces are also discussed.

  14. Respiratory compensation and blood pH regulation during variable intensity exercise in trained versus untrained subjects.

    PubMed

    Del Coso, Juan; Hamouti, Nassim; Aguado-Jimenez, Roberto; Mora-Rodriguez, Ricardo

    2009-09-01

    To determine whether endurance-trained cyclists (T; n = 10) have a superior blood-respiratory buffering for metabolic acidosis relative to untrained subjects (UT; n = 10) during variable intensity exercise (VAR). On three occasions, T and UT pedaled for 24 min alternating high- and low-intensities as percentage of their second ventilatory threshold (VT2): VAR(LOW) 87.5-37.5% VT2, VAR(MODERATE) 125-25% VT2, and VAR(HIGH) 162.5-12.5% VT2 to complete the same amount of work. Before and just after each VAR trial, maximal cycling power (P(MAX)) was assessed. For each trial, the respiratory compensation for exercise acidosis (ventilatory equivalent for CO2) and the final blood pH, lactate and bicarbonate concentrations were similar for T and UT subjects. However, after VAR(HIGH), UT reduced P(MAX) (-14 +/- 1%; P < 0.05) while T did not. Our data suggest that endurance training confers adaptations to withstand the low pH provoked by VAR without losing cycling power, although this response is not due to differences in blood-respiratory buffering.

  15. A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

    SciTech Connect

    Wieczorek, H.; Weerth, S.; Schindlbeck, M.; Klein, U.

    1989-07-05

    Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.

  16. Control of Cl− Efflux in Chara corallina by Cytosolic pH, Free Ca2+, and Phosphorylation Indicates a Role of Plasma Membrane Anion Channels in Cytosolic pH Regulation1

    PubMed Central

    Johannes, Eva; Crofts, Alan; Sanders, Dale

    1998-01-01

    Enhanced Cl− efflux during acidosis in plants is thought to play a role in cytosolic pH (pHc) homeostasis by short-circuiting the current produced by the electrogenic H+ pump, thereby facilitating enhanced H+ efflux from the cytosol. Using an intracellular perfusion technique, which enables experimental control of medium composition at the cytosolic surface of the plasma membrane of charophyte algae (Chara corallina), we show that lowered pHc activates Cl− efflux via two mechanisms. The first is a direct effect of pHc on Cl− efflux; the second mechanism comprises a pHc-induced increase in affinity for cytosolic free Ca2+ ([Ca2+]c), which also activates Cl− efflux. Cl− efflux was controlled by phosphorylation/dephosphorylation events, which override the responses to both pHc and [Ca2+]c. Whereas phosphorylation (perfusion with the catalytic subunit of protein kinase A in the presence of ATP) resulted in a complete inhibition of Cl− efflux, dephosphorylation (perfusion with alkaline phosphatase) arrested Cl− efflux at 60% of the maximal level in a manner that was both pHc and [Ca2+]c independent. These findings imply that plasma membrane anion channels play a central role in pHc regulation in plants, in addition to their established roles in turgor/volume regulation and signal transduction. PMID:9733536

  17. Regulation of nitrogen uptake and assimilation: Effects of nitrogen source, root-zone pH, and aerial CO2 concentration on growth and productivity of soybeans

    NASA Technical Reports Server (NTRS)

    Raper, C. D.; Tolley-Henry, L.

    1989-01-01

    An important feature of controlled-environment crop production systems such as those to be used for life support of crews during space exploration is the efficient utilization of nitrogen supplies. Making decisions about the best sources of these supplies requires research into the relationship between nitrogen source and the physiological processes which regulate vegetative and reproductive plant growth. Work done in four areas within this research objective is reported: (1) experiments on the effects of root-zone pH on preferential utilization of NO3(-) versus NH4(+) nitrogen; (2) investigation of processes at the whole-plant level that regulate nitrogen uptake; (3) studies of the effects of atmospheric CO2 and NO3(-) supply on the growth of soybeans; and (4) examination of the role of NO3(-) uptake in enhancement of root respiration.

  18. The Pochonia chlamydosporia Serine Protease Gene vcp1 Is Subject to Regulation by Carbon, Nitrogen and pH: Implications for Nematode Biocontrol

    PubMed Central

    Ward, Elaine; Kerry, Brian R.; Manzanilla-López, Rosa H.; Mutua, Gerald; Devonshire, Jean; Kimenju, John; Hirsch, Penny R.

    2012-01-01

    The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances. PMID:22558192

  19. Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations.

    PubMed

    Alleva, Karina; Niemietz, Christa M; Sutka, Moira; Maurel, Christophe; Parisi, Mario; Tyerman, Stephen D; Amodeo, Gabriela

    2006-01-01

    Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.

  20. NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse.

    PubMed

    Jalali, R; Guo, J; Zandieh-Doulabi, B; Bervoets, T J M; Paine, M L; Boron, W F; Parker, M D; Bijvelds, M J C; Medina, J F; DenBesten, P K; Bronckers, A L J J

    2014-11-01

    During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

  1. Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat.

    PubMed Central

    Buckler, K J; Vaughan-Jones, R D; Peers, C; Nye, P C

    1991-01-01

    1. The dual-emission pH-sensitive fluoroprobe carboxy-SNARF-1 (carboxy-seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady-state pHi in cells bathed in a HEPES-buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2-HCO3(-)-buffered Tyrode solution (pH 7.4) had a more 'normal' pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre-pulse) was in the range 7-20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES-buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM-NH4Cl pre-pulse) was inhibited by the Na(+)-H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N-methyl-D-glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES-buffered Tyrode solution to one buffered with 5% CO2-HCO3- resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2-HCO3- was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2-HCO3(-)-buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre-pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4-diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced

  2. Integrated Regulation of Acetoin Fermentation by Quorum Sensing and pH in Serratia plymuthica RVH1 ▿

    PubMed Central

    Moons, Pieter; Van Houdt, Rob; Vivijs, Bram; Michiels, Chris M.; Aertsen, Abram

    2011-01-01

    During fermentation of sugars, a number of bacterial species are able to switch from mixed acid production to acetoin and 2,3-butanediol production in order to avoid lethal acidification of their environment, although the regulation of this switch is only poorly understood. In this study, we report the identification of the budAB structural operon, involved in acetoin production in Serratia plymuthica RVH1, and its activation by a LysR-type regulator encoded by budR, immediately upstream of this operon. In addition, the regulation of budR transcription was elucidated and found to be subject to negative control by BudR itself and to positive control by external stimuli such as N-(3-oxohexanoyl)-l-homoserine lactone (OHHL) quorum sensing signaling molecules and acetate. Interestingly, however, we observed that induction of budR transcription by OHHL or acetate did not require BudR, indicating the involvement of additional regulatory factors in relaying these environmental signals to the budR promoter. PMID:21441339

  3. Integrated regulation of acetoin fermentation by quorum sensing and pH in Serratia plymuthica RVH1.

    PubMed

    Moons, Pieter; Van Houdt, Rob; Vivijs, Bram; Michiels, Chris W; Michiels, Chris M; Aertsen, Abram

    2011-05-01

    During fermentation of sugars, a number of bacterial species are able to switch from mixed acid production to acetoin and 2,3-butanediol production in order to avoid lethal acidification of their environment, although the regulation of this switch is only poorly understood. In this study, we report the identification of the budAB structural operon, involved in acetoin production in Serratia plymuthica RVH1, and its activation by a LysR-type regulator encoded by budR, immediately upstream of this operon. In addition, the regulation of budR transcription was elucidated and found to be subject to negative control by BudR itself and to positive control by external stimuli such as N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) quorum sensing signaling molecules and acetate. Interestingly, however, we observed that induction of budR transcription by OHHL or acetate did not require BudR, indicating the involvement of additional regulatory factors in relaying these environmental signals to the budR promoter.

  4. Multifunctional hybrid nanogel for integration of optical glucose sensing and self-regulated insulin release at physiological pH.

    PubMed

    Wu, Weitai; Mitra, Nivedita; Yan, Elsa C Y; Zhou, Shuiqin

    2010-08-24

    Optical detection of glucose, high drug loading capacity, and self-regulated drug delivery are simultaneously possible using a multifunctional hybrid nanogel particle under a rational design in a colloid chemistry method. Such hybrid nanogels are made of Ag nanoparticle (NP) cores covered by a copolymer gel shell of poly(4-vinylphenylboronic acid-co-2-(dimethylamino)ethyl acrylate) [p(VPBA-DMAEA)]. The introduction of the glucose sensitive p(VPBA-DMAEA) gel shell onto Ag NPs makes the polymer-bound Ag NPs responsive to glucose. While the small sized Ag cores (10 +/- 3 nm) provide fluorescence as an optical code, the responsive polymer gel shell can adapt to a surrounding medium of different glucose concentrations over a clinically relevant range (0-30 mM), convert the disruptions in homeostasis of glucose level into optical signals, and regulate release of preloaded insulin. This shows a new proof-of-concept for diabetes treatment that exploits the properties from each building block of a multifunctional nano-object. The highly versatile multifunctional hybrid nanogels could potentially be used for simultaneous optical diagnosis, self-regulated therapy, and monitoring of the response to treatment.

  5. Multifunctional hybrid nanogel for integration of optical glucose sensing and self-regulated insulin release at physiological pH.

    PubMed

    Wu, Weitai; Mitra, Nivedita; Yan, Elsa C Y; Zhou, Shuiqin

    2010-08-24

    Optical detection of glucose, high drug loading capacity, and self-regulated drug delivery are simultaneously possible using a multifunctional hybrid nanogel particle under a rational design in a colloid chemistry method. Such hybrid nanogels are made of Ag nanoparticle (NP) cores covered by a copolymer gel shell of poly(4-vinylphenylboronic acid-co-2-(dimethylamino)ethyl acrylate) [p(VPBA-DMAEA)]. The introduction of the glucose sensitive p(VPBA-DMAEA) gel shell onto Ag NPs makes the polymer-bound Ag NPs responsive to glucose. While the small sized Ag cores (10 +/- 3 nm) provide fluorescence as an optical code, the responsive polymer gel shell can adapt to a surrounding medium of different glucose concentrations over a clinically relevant range (0-30 mM), convert the disruptions in homeostasis of glucose level into optical signals, and regulate release of preloaded insulin. This shows a new proof-of-concept for diabetes treatment that exploits the properties from each building block of a multifunctional nano-object. The highly versatile multifunctional hybrid nanogels could potentially be used for simultaneous optical diagnosis, self-regulated therapy, and monitoring of the response to treatment. PMID:20731458

  6. Ultrastructural changes of the midgut epithelium in Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada: Eutardigrada) during oogenesis.

    PubMed

    Rost-Roszkowska, Magdalena M; Poprawa, Izabela; Wójtowicz, Maria; Kaczmarek, Lukasz

    2011-04-01

    The midgut epithelium of Isohypsibius granulifer granulifer (Eutardigrada) is composed of columnar digestive cells. At its anterior end, a group of cells with cytoplasm which differs from the cytoplasm of digestive cells is present. Probably, those cells respond to crescent-like cells (midgut regenerative cells) described for some tardigrade species. Their mitotic divisions have not been observed. We analyzed the ultrastructure of midgut digestive cells in relation to five different stages of oogenesis (previtellogenesis, beginning of the vitellogenesis, vitellogenesis--early choriogenesis, vitellogenesis--middle choriogenesis, late choriogenesis). In the midgut epithelium cells, the gradual accumulation of glycogen granules, lipid droplets and structures of varying electron density occurs. During vitellogenesis and choriogenesis, in the cytoplasm of midgut cells we observed the increasing number of organelles which are responsible for the intensive synthesis of lipids, proteins and saccharides such as cisterns of endoplasmic reticulum and Golgi complexes. At the end of oogenesis, autophagy also intensifies in midgut epithelial cells, which is probably caused by the great amount of reserve material. Midgut epithelium of analyzed species takes part in the yolk precursor synthesis. PMID:20661605

  7. Altered protoxin activation by midgut enzymes from a Bacillus thuringiensis resistant strain of Plodia interpunctella.

    PubMed

    Oppert, B; Kramer, K J; Johnson, D E; MacIntosh, S C; McGaughey, W H

    1994-02-15

    Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B. thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B. thuringiensis subspecies kurstaki (HD-1, Dipel). Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate alpha-N-benzoyl-DL-arginine rho-nitroanilide than extracts from susceptible or kurstaki-resistant insects. Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract. The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin. The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.

  8. Ultrastructural changes of the midgut epithelium in Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada: Eutardigrada) during oogenesis.

    PubMed

    Rost-Roszkowska, Magdalena M; Poprawa, Izabela; Wójtowicz, Maria; Kaczmarek, Lukasz

    2011-04-01

    The midgut epithelium of Isohypsibius granulifer granulifer (Eutardigrada) is composed of columnar digestive cells. At its anterior end, a group of cells with cytoplasm which differs from the cytoplasm of digestive cells is present. Probably, those cells respond to crescent-like cells (midgut regenerative cells) described for some tardigrade species. Their mitotic divisions have not been observed. We analyzed the ultrastructure of midgut digestive cells in relation to five different stages of oogenesis (previtellogenesis, beginning of the vitellogenesis, vitellogenesis--early choriogenesis, vitellogenesis--middle choriogenesis, late choriogenesis). In the midgut epithelium cells, the gradual accumulation of glycogen granules, lipid droplets and structures of varying electron density occurs. During vitellogenesis and choriogenesis, in the cytoplasm of midgut cells we observed the increasing number of organelles which are responsible for the intensive synthesis of lipids, proteins and saccharides such as cisterns of endoplasmic reticulum and Golgi complexes. At the end of oogenesis, autophagy also intensifies in midgut epithelial cells, which is probably caused by the great amount of reserve material. Midgut epithelium of analyzed species takes part in the yolk precursor synthesis.

  9. Acquisition and structuring of midgut bacterial communities in gypsy moth (Lepidoptera: Erebidae) larvae.

    PubMed

    Mason, Charles J; Raffa, Kenneth F

    2014-06-01

    Insects are associated with a diversity of bacteria that colonize their midguts. The extent to which these communities reflect maternal transmission, environmental acquisition, and subsequent structuring by the extreme conditions within the insect gut are poorly understood in many species. We used gypsy moth (Lymantria dispar L.) as a model to investigate interactions between egg mass and environmental sources of bacteria on larval midgut communities. Egg masses were collected from several wild and laboratory populations, and the effects of diet, initial egg mass community, and internal host environment were evaluated using 454 16S-rRNA gene pyrosequencing. Wild populations were highly diverse, while laboratory-maintained egg masses were associated with few operational taxonomic units. As larvae developed, their midgut bacterial communities became more similar to each other and the consumed diet despite initial differences in egg mass-associated bacteria. Subsequent experiments revealed that while midgut membership was more similar to bacteria associated with diet than with egg mass-associated bacteria, we were unable to detect distinct, persistent differences attributable to specific host plants. The differences between foliar communities and midgut communities of larvae that ingested them were owing to relative changes in populations of several bacteria phylotypes. We conclude that gypsy moth has a relatively characteristic midgut bacterial community that is reflective of, but ultimately distinct from, its foliar diet. This work demonstrates that environmental acquisition of diverse microbes can lead to similar midgut bacterial assemblages, underscoring the importance of host physiological environment in structuring bacterial communities. PMID:24780292

  10. Acquisition and structuring of midgut bacterial communities in gypsy moth (Lepidoptera: Erebidae) larvae.

    PubMed

    Mason, Charles J; Raffa, Kenneth F

    2014-06-01

    Insects are associated with a diversity of bacteria that colonize their midguts. The extent to which these communities reflect maternal transmission, environmental acquisition, and subsequent structuring by the extreme conditions within the insect gut are poorly understood in many species. We used gypsy moth (Lymantria dispar L.) as a model to investigate interactions between egg mass and environmental sources of bacteria on larval midgut communities. Egg masses were collected from several wild and laboratory populations, and the effects of diet, initial egg mass community, and internal host environment were evaluated using 454 16S-rRNA gene pyrosequencing. Wild populations were highly diverse, while laboratory-maintained egg masses were associated with few operational taxonomic units. As larvae developed, their midgut bacterial communities became more similar to each other and the consumed diet despite initial differences in egg mass-associated bacteria. Subsequent experiments revealed that while midgut membership was more similar to bacteria associated with diet than with egg mass-associated bacteria, we were unable to detect distinct, persistent differences attributable to specific host plants. The differences between foliar communities and midgut communities of larvae that ingested them were owing to relative changes in populations of several bacteria phylotypes. We conclude that gypsy moth has a relatively characteristic midgut bacterial community that is reflective of, but ultimately distinct from, its foliar diet. This work demonstrates that environmental acquisition of diverse microbes can lead to similar midgut bacterial assemblages, underscoring the importance of host physiological environment in structuring bacterial communities.

  11. Environmental pH changes, but not the LuxS signalling pathway, regulate SpeB expression in M1 group A streptococci.

    PubMed

    Chiang-Ni, Chuan; Zheng, Po-Xing; Tsai, Pei-Jane; Chuang, Woei-Jer; Lin, Yee-Shin; Liu, Ching-Chuan; Wu, Jiunn-Jong

    2012-01-01

    The autoinducer-2/LuxS signalling pathway participates in quorum sensing in diverse bacterial species. In group A streptococci (GAS), LuxS has been shown to be involved in regulating the expression of several important virulence factors. Streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that has important roles in GAS pathogenesis, is positively regulated by LuxS in M3 and M5 strains. In the present study, it was found that the supernatant harvested from an overnight culture stimulated M1 strains to express speB. However, mutation of the luxS gene in M1 strains or treating M1 strains with luxS mutant culture supernatant did not affect speB expression, indicating that the LuxS pathway is not involved in regulation of speB expression in M1 strains. In addition, the acid property of culture broth was found to be able to stimulate M1 strains to express speB in the same LuxS-independent manner. These results indicate that speB expression in M1 strains is induced by environmental pH changes but is not regulated by the LuxS signalling pathway. PMID:21890514

  12. Antioxidant defenses in caterpillars: role of the ascorbate-recycling system in the midgut lumen.

    PubMed

    Barbehenn, R V; Bumgarner, S L; Roosen, E F; Martin, M M

    2001-04-01

    This study demonstrates that an ascorbate-recycling system in the midgut lumen can act as an effective antioxidant defense in caterpillars that feed on prooxidant-rich foods. In tannin-sensitive larvae of the forest tent caterpillar, Malacosoma disstria (Lasiocampidae), ingested tannic acid is oxidized in the midgut lumen, generating significant quantities of peroxides, including hydrogen peroxide, which readily diffuses across cell membranes and is a powerful cytotoxin. By contrast, in the tannin-tolerant larvae of the white-marked tussock moth, Orgyia leucostigma (Lymantriidae), tannic acid oxidation and the generation of peroxides are suppressed. The superior defense of O. leucostigma against oxidative stress imposed by the oxidation of ingested polyphenols can be explained by the presence of higher concentrations of ascorbate and glutathione in the midgut lumen. In O. leucostigma at least 50% of the ingested ascorbate present in the anterior midgut is still present in the posterior midgut, whereas in M. disstria, only 10% of the ascorbate is present in the posterior half of the midgut. We propose that the maintenance of higher levels of ascorbate in the midgut lumen of O. leucostigma than in M. disstria is explained by the secretion of glutathione into the midgut lumen by O. leucostigma, thereby forming a complete ascorbate-recycling system. The concentration of glutathione in the midgut lumen of O. leucostigma is 3.5-fold higher than in M. disstria and more than double the concentration in the diet. Our results emphasize the importance of a defensive strategy in herbivorous insects based on the maintenance of conditions in the gut lumen that reduce or eliminate the potential prooxidant behavior of ingested phenols.

  13. Localisation of laminin within Plasmodium berghei oocysts and the midgut epithelial cells of Anopheles stephensi

    PubMed Central

    Nacer, Adéla; Walker, Karen; Hurd, Hilary

    2008-01-01

    Background Oocysts of the malaria parasite form and develop in close proximity to the mosquito midgut basal lamina and it has been proposed that components of this structure play a crucial role in the development and maturation of oocysts that produce infective sporozoites. It is further suggested that oocysts incorporate basal lamina proteins into their capsule and that this provides them with a means to evade recognition by the mosquito's immune system. The site of production of basal lamina proteins in insects is controversial and it is still unclear whether haemocytes or midgut epithelial cells are the main source of components of the mosquito midgut basal lamina. Of the multiple molecules that compose the basal lamina, laminin is known to interact with a number of Plasmodium proteins. In this study, the localisation of mosquito laminin within the capsule and cytoplasm of Plasmodium berghei oocysts and in the midgut epithelial cells of Anopheles stephensi was investigated. Results An ultrastructural examination of midgut sections from infected and uninfected An. stephensi was performed. Post-embedded immunogold labelling demonstrated the presence of laminin within the mosquito basal lamina. Laminin was also detected on the outer surface of the oocyst capsule, incorporated within the capsule and associated with sporozoites forming within the oocysts. Laminin was also found within cells of the midgut epithelium, providing support for the hypothesis that these cells contribute towards the formation of the midgut basal lamina. Conclusion We suggest that ookinetes may become coated in laminin as they pass through the midgut epithelium. Thereafter, laminin secreted by midgut epithelial cells and/or haemocytes, binds to the outer surface of the oocyst capsule and that some passes through and is incorporated into the developing oocysts. The localisation of laminin on sporozoites was unexpected and the importance of this observation is less clear. PMID:18808667

  14. Borrelia burgdorferi Proteins Whose Expression Is Similarly Affected by Culture Temperature and pH

    PubMed Central

    Ramamoorthy, Ramesh; Scholl-Meeker, Dorothy

    2001-01-01

    Previously, we had demonstrated the upregulation in the expression of several proteins, including the lipoproteins OspC and P35, of Borrelia burgdorferi in the stationary growth phase. Since the expression of OspC is also known to be affected by culture temperature and pH, we examined the effects of both variables on the expression of the remaining stationary-phase-upregulated proteins. Our study revealed that the expression of each of the remaining stationary-phase-upregulated proteins, P35 included, was also influenced by culture temperature; these proteins were selectively expressed at 34°C but not at 24°C. Significantly, the expression of a majority of these proteins was also affected by culture pH, since they were abundantly expressed at pH 7.0 (resembling the tick midgut pH of 6.8 during feeding) but only sparsely at pH 8.0 (a condition closer to that of the unfed tick midgut pH of 7.4). We propose that this group of B. burgdorferi proteins, which in culture is selectively expressed under conditions of 34°C and pH 7.0, may be induced in the tick midgut during the feeding event. Furthermore, the differential and coordinate expression of these proteins under different environmental conditions suggests that the encoding genes may be coregulated. PMID:11254645

  15. Metal ions and electrolytes regulate the dissociation of heme from human hemopexin at physiological pH.

    PubMed

    Mauk, Marcia R; Mauk, A Grant

    2010-07-01

    The stability of the hemopexin-heme (Hx-heme) complex to dissociation of the heme prosthetic group has been examined in bicarbonate buffers in the presence and absence of various divalent metal ions. In NH(4)HCO(3) buffer (pH 7.4, 20 mm, 25 degrees C) containing Zn(2+) (100 microm), 14% of the heme dissociates from this complex (4.5 microm) within 10 min, and 50% dissociates within 2 h. In the absence of metal ions, the rate of dissociation of this complex is far lower, is decreased further in KHCO(3) solution, and is minimal in NaHCO(3). In NH(4)HCO(3) buffer, dissociation of the Hx-heme complex is accelerated by addition of divalent metals with decreasing efficiency in the order Zn(2+) > Cu(2+) > Ni(2+) > Co(2+)>Mn(2+). Addition of Ca(2+) prior to addition of Zn(2+) stabilizes the Hx-heme complex to dissociation of the heme group, and addition of Ca(2+) after Zn(2+)-induced dissociation of the Hx-heme complex results in re-formation of the Hx-heme complex. These effects are greatly accelerated at 37 degrees C and diminished in other buffers. Overall, the solution conditions that promote formation of the Hx-heme complex are similar to those found in blood plasma, and conditions that promote release of heme are similar to those that the Hx-heme complex should encounter in endosomes following endocytosis of the complex formed with its hepatic receptor. PMID:20430887

  16. DNA Damage–Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

    PubMed Central

    Zhao, Rui; Oxley, David; Smith, Trevor S; Follows, George A; Green, Anthony R; Alexander, Denis R

    2007-01-01

    The pro-survival protein Bcl-xL is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-xL deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-xL deamidation remains unknown and its functional consequences unclear. We show here that rBcl-xL deamidation generates an iso-Asp52/iso-Asp66 species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-xL deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-xL deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-xL deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy. PMID:17177603

  17. Characterization of a Digestive α-Amylase in the Midgut of Pieris brassicae L. (Lepidoptera: Pieridae).

    PubMed

    Sharifloo, Ali; Zibaee, Arash; Sendi, Jalal J; Jahroumi, Khalil Talebi

    2016-01-01

    The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had V max values of 4.64 and 3.02 U/mg protein and K m values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (β-aminoethylether) N, N, N', N'-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones. PMID:27014094

  18. Characterization of a Digestive α-Amylase in the Midgut of Pieris brassicae L. (Lepidoptera: Pieridae)

    PubMed Central

    Sharifloo, Ali; Zibaee, Arash; Sendi, Jalal J.; Jahroumi, Khalil Talebi

    2016-01-01

    The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had Vmax values of 4.64 and 3.02 U/mg protein and Km values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (β-aminoethylether) N, N, N′, N′-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones. PMID:27014094

  19. Midgut proteases of the cardamom shoot and capsule borer Conogethes punctiferalis (Lepidoptera: Pyralidae) and their interaction with aprotinin.

    PubMed

    Josephrajkumar, A; Chakrabarty, R; Thomas, G

    2006-02-01

    Protease inhibitors cause mortality in a range of insects, and transgenic plants expressing protease inhibitors have been protected against pest attack, particularly internal feeders that are not amenable to control by conventional means. A study of luminal proteases in Conogethes punctiferalis Guenée was performed to identify potential targets for proteinaceous biopesticides, such as protease inhibitors. The midgut protease profile of the gut lumen from C. punctiferalis was studied to determine the conditions for optimal protein hydrolysis. Optimum conditions for peptidase activity were found to be in 50 mm Tris-HCl, pH 10 containing 20 mm CaCl2; incubation for 30 min at 40 degrees C. Four synthetic substrates, i.e. benzoyl-arg-p-nitroanilide, benzoyl-tyr-p-nitroanilide, succinyl-ala-ala-pro-leu-p-nitroanilide (SAAPLpNA) and leu-p-nitroanilide were hydrolysed by C. punctiferalis gut proteases in Tris-HCl buffer pH 10. Trypsin and elastase-like chymotrypsin were the prominent digestive proteases, and age-related modulation of midgut proteases existed for trypsin, chymotrypsin, elastase-like chymotrypsin and leucine aminopeptidase. Serine protease inhibitors such as aprotinin, soybean trypsin inhibitor and phenylmethanesulfonyl fluoride inhibited peptidase activity. Some metal ions such as Ca(2+), Mg(2+), Pb(2+) and Co(2+) enhanced BApNA-ase activity whereas others like Mn(2+), Zn(2+), Cu(2+), Fe(2+) and Hg(2+) were inhibitory at 6 mm concentration. Trypsin and elastase-like chymotrypsin were significantly inhibited by 94% and 29%, respectively, by aprotinin (150 nm) under in vitro conditions. A possible incorporation of protease inhibitors into transgenic plants is discussed.

  20. The regulation of intracellular pH studied by 31P- and 1H-NMR spectroscopy in superfused guinea-pig cerebral cortex slices.

    PubMed

    Brooks, K J; Bachelard, H S

    1992-10-01

    (1) The intracellular pH (pHi) of superfused slices of guinea-pig cerebral cortex was measured in 31P-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P). The pHi was found to be 7.30 +/- 0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20 +/- 0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHe below 7.05 resulted in pHi falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by 1H-NMR. (3) The tissues showed an ability to buffer higher pH: increasing pHe to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44 +/- 0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with O2/CO2, 80:20 (pHe 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery of pHi to 7.31 +/- 0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.88 +/- 0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 mM), or the sodium/proton exchange inhibitor, amiloride (1 mM), there was an incomplete return of pHi to the control value (pHi 6.90 +/- 0.20, n = 5, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1303163

  1. Cruzipain Promotes Trypanosoma cruzi Adhesion to Rhodnius prolixus Midgut

    PubMed Central

    Uehara, Lívia Almeida; Moreira, Otacílio C.; Oliveira, Ana Carolina; Azambuja, Patrícia; Lima, Ana Paula Cabral Araujo; Britto, Constança; dos Santos, André Luis Souza; Branquinha, Marta Helena; d'Avila-Levy, Claudia Masini

    2012-01-01

    Background Trypanosoma cruzi is the etiological agent of Chagas' disease. Cysteine peptidases are relevant to several aspects of the T. cruzi life cycle and are implicated in parasite-mammalian host relationships. However, little is known about the factors that contribute to the parasite-insect host interaction. Methodology/Principal Findings Here, we have investigated whether cruzipain could be involved in the interaction of T. cruzi with the invertebrate host. We analyzed the effect of treatment of T. cruzi epimastigotes with anti-cruzipain antibodies or with a panel of cysteine peptidase inhibitors (cystatin, antipain, E-64, leupeptin, iodocetamide or CA-074-OMe) on parasite adhesion to Rhodnius prolixus posterior midgut ex vivo. All treatments, with the exception of CA074-OMe, significantly decreased parasite adhesion to R. prolixus midgut. Cystatin presented a dose-dependent reduction on the adhesion. Comparison of the adhesion rate among several T. cruzi isolates revealed that the G isolate, which naturally possesses low levels of active cruzipain, adhered to a lesser extent in comparison to Dm28c, Y and CL Brener isolates. Transgenic epimastigotes overexpressing an endogenous cruzipain inhibitor (pCHAG), chagasin, and that have reduced levels of active cruzipain adhered to the insect gut 73% less than the wild-type parasites. The adhesion of pCHAG parasites was partially restored by the addition of exogenous cruzipain. In vivo colonization experiments revealed low levels of pCHAG parasites in comparison to wild-type. Parasites isolated after passage in the insect presented a drastic enhancement in the expression of surface cruzipain. Conclusions/Significance These data highlight, for the first time, that cruzipain contributes to the interaction of T. cruzi with the insect host. PMID:23272264

  2. Resilience of cold-water scleractinian corals to ocean acidification: Boron isotopic systematics of pH and saturation state up-regulation

    NASA Astrophysics Data System (ADS)

    McCulloch, Malcolm; Trotter, Julie; Montagna, Paolo; Falter, Jim; Dunbar, Robert; Freiwald, André; Försterra, Günter; López Correa, Matthias; Maier, Cornelia; Rüggeberg, Andres; Taviani, Marco

    2012-06-01

    The boron isotope systematics has been determined for azooxanthellate scleractinian corals from a wide range of both deep-sea and shallow-water environments. The aragonitic coral species, Caryophyllia smithii, Desmophyllum dianthus, Enallopsammia rostrata, Lophelia pertusa, and Madrepora oculata, are all found to have relatively high δ11B compositions ranging from 23.2‰ to 28.7‰. These values lie substantially above the pH-dependent inorganic seawater borate equilibrium curve, indicative of strong up-regulation of pH of the internal calcifying fluid (pHcf), being elevated by ˜0.6-0.8 units (ΔpH) relative to ambient seawater. In contrast, the deep-sea calcitic coral Corallium sp. has a significantly lower δ11B composition of 15.5‰, with a corresponding lower ΔpH value of ˜0.3 units, reflecting the importance of mineralogical control on biological pH up-regulation. The solitary coral D. dianthus was sampled over a wide range of seawater pHT and shows an approximate linear correlation with ΔpHDesmo = 6.43 - 0.71pHT (r2 = 0.79). An improved correlation is however found with the closely related parameter of seawater aragonite saturation state, where ΔpHDesmo = 1.09 - 0.14Ωarag (r2 = 0.95), indicating the important control that carbonate saturation state has on calcification. The ability to up-regulate internal pHcf, and consequently Ωcf, of the calcifying fluid is therefore a process present in both azooxanthellate and zooxanthellate aragonitic corals, and is attributed to the action of Ca2+-ATPase in modulating the proton gradient between seawater and the site of calcification. These findings also show that the boron isotopic compositions (δ11Bcarb) of aragonitic corals are highly systematic and consistent with direct uptake of the borate species within the biologically controlled extracellular calcifying medium. We also show that the relatively strong up-regulation of pH and consequent elevation of the internal carbonate saturation state (Ωcf ˜8

  3. Assembling of Holotrichia parallela (dark black chafer) midgut tissue transcriptome and identification of midgut proteins that bind to Cry8Ea toxin from Bacillus thuringiensis.

    PubMed

    Shu, Changlong; Tan, Shuqian; Yin, Jiao; Soberón, Mario; Bravo, Alejandra; Liu, Chunqing; Geng, Lili; Song, Fuping; Li, Kebin; Zhang, Jie

    2015-09-01

    Holotrichia parallela is one of the most severe crop pests in China, affecting peanut, soybean, and sweet potato crops. Previous work showed that Cry8Ea toxin is highly effective against this insect. In order to identify Cry8Ea-binding proteins in the midgut cells of H. parallela larvae, we assembled a midgut tissue transcriptome by high-throughput sequencing and used this assembled transcriptome to identify Cry8Ea-binding proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, we obtained de novo sequences of cDNAs from midgut tissue of H. parallela larvae and used available cDNA data in the GenBank. In a parallel assay, we obtained 11 Cry8Ea-binding proteins by pull-down assays performed with midgut brush border membrane vesicles. Peptide sequences from these proteins were matched to the H. parallela newly assembled midgut transcriptome, and 10 proteins were identified. Some of the proteins were shown to be intracellular proteins forming part of the cell cytoskeleton and/or vesicle transport such as actin, myosin, clathrin, dynein, and tubulin among others. In addition, an apolipophorin, which is a protein involved in lipid metabolism, and a novel membrane-bound alanyl aminopeptidase were identified. Our results suggest that Cry8Ea-binding proteins could be different from those characterized for Cry1A toxins in lepidopteran insects. PMID:26135984

  4. Nucleolar localization of SmMAK16 protein from Schistosoma mansoni is regulated by three distinct signals that function independent of pH or phosphorylation status.

    PubMed

    Hoellerich, Elisa; Dunagan, Christie; Maring, Daniel; Wong, Yun-Lan C; Shouldice, Daniel; Stripe, Jennifer; Kline, Tayah; Albert, Thomas J; Milhon, Jon L

    2014-01-01

    SmMAK16 from the trematode Schistosoma mansoni is a protein that is known to localize in the nucleolus. Recent findings show that SmMAK16 is involved in 60S ribosomal subunit synthesis. Although the SmMAK16 protein contains putative nuclear localization signals (NLS), little is known about their precise function, redundancy or regulation. The goal of the current study was to identify and characterize the presence and functional regulation of the localization signals in SmMAK16. The SmMAK16 coding sequence and specific fragments were individually cloned in-frame into the pEGFP-C2 expression vector to encode Green Fluorescent Protein (GFP) fusion proteins. Constructs were individually transfected into COS-7 cells and fluorescent microscopy used to determine the cellular location and thus the presence of signals regulating nuclear and nucleolar localization. SmMAK16 was found to contain two NLSs and one nucleolar localization signal (NoLS). One of the signals contains a sequence identical to an established nucleolar detention signal that reportedly functions only under acidic cellular conditions. The localization of the SmMAK16-GFP constructs was analyzed under acidic conditions; however, altering pH did not influence the localization of SmMAK16. It has been previously reported that casein kinase 2 (CK2) can phosphorylate SmMAK16 at serines adjacent to one of the NLSs. One of these CK2 sites and the adjacent NLS are conserved with that of the SV40 Large T Antigen (LTA) and phosphorylation of this site in the SV40 LTA regulates the kinetics of the NLS. To discover if kinetic regulation also occurs in SmMAK16, mutant and wild type SmMAK16-GFP proteins were purified and injected into individual COS-7 cells. No difference in the rate of transport was found between wt and mutant SmMAK16 proteins. Therefore, SmMAK16 localizes to the nucleolus using three separate signals, two NLSs and one NoLS, however, these signals appear to function independently of pH and

  5. Nucleolar localization of SmMAK16 protein from Schistosoma mansoni is regulated by three distinct signals that function independent of pH or phosphorylation status.

    PubMed

    Hoellerich, Elisa; Dunagan, Christie; Maring, Daniel; Wong, Yun-Lan C; Shouldice, Daniel; Stripe, Jennifer; Kline, Tayah; Albert, Thomas J; Milhon, Jon L

    2014-01-01

    SmMAK16 from the trematode Schistosoma mansoni is a protein that is known to localize in the nucleolus. Recent findings show that SmMAK16 is involved in 60S ribosomal subunit synthesis. Although the SmMAK16 protein contains putative nuclear localization signals (NLS), little is known about their precise function, redundancy or regulation. The goal of the current study was to identify and characterize the presence and functional regulation of the localization signals in SmMAK16. The SmMAK16 coding sequence and specific fragments were individually cloned in-frame into the pEGFP-C2 expression vector to encode Green Fluorescent Protein (GFP) fusion proteins. Constructs were individually transfected into COS-7 cells and fluorescent microscopy used to determine the cellular location and thus the presence of signals regulating nuclear and nucleolar localization. SmMAK16 was found to contain two NLSs and one nucleolar localization signal (NoLS). One of the signals contains a sequence identical to an established nucleolar detention signal that reportedly functions only under acidic cellular conditions. The localization of the SmMAK16-GFP constructs was analyzed under acidic conditions; however, altering pH did not influence the localization of SmMAK16. It has been previously reported that casein kinase 2 (CK2) can phosphorylate SmMAK16 at serines adjacent to one of the NLSs. One of these CK2 sites and the adjacent NLS are conserved with that of the SV40 Large T Antigen (LTA) and phosphorylation of this site in the SV40 LTA regulates the kinetics of the NLS. To discover if kinetic regulation also occurs in SmMAK16, mutant and wild type SmMAK16-GFP proteins were purified and injected into individual COS-7 cells. No difference in the rate of transport was found between wt and mutant SmMAK16 proteins. Therefore, SmMAK16 localizes to the nucleolus using three separate signals, two NLSs and one NoLS, however, these signals appear to function independently of pH and

  6. Adjustments of molecular key components of branchial ion and pH regulation in Atlantic cod (Gadus morhua) in response to ocean acidification and warming.

    PubMed

    Michael, Katharina; Kreiss, Cornelia M; Hu, Marian Y; Koschnick, Nils; Bickmeyer, Ulf; Dupont, Sam; Pörtner, Hans-O; Lucassen, Magnus

    2016-03-01

    Marine teleost fish sustain compensation of extracellular pH after exposure to hypercapnia by means of efficient ion and acid-base regulation. Elevated rates of ion and acid-base regulation under hypercapnia may be stimulated further by elevated temperature. Here, we characterized the regulation of transepithelial ion transporters (NKCC1, NBC1, SLC26A6, NHE1 and 2) and ATPases (Na(+)/K(+) ATPase and V-type H(+) ATPase) in gills of Atlantic cod (Gadus morhua) after 4 weeks of exposure to ambient and future PCO2 levels (550 μatm, 1200 μatm, 2200 μatm) at optimum (10 °C) and summer maximum temperature (18 °C), respectively. Gene expression of most branchial ion transporters revealed temperature- and dose-dependent responses to elevated PCO2. Transcriptional regulation resulted in stable protein expression at 10 °C, whereas expression of most transport proteins increased at medium PCO2 and 18 °C. mRNA and protein expression of distinct ion transport proteins were closely co-regulated, substantiating cellular functional relationships. Na(+)/K(+) ATPase capacities were PCO2 independent, but increased with acclimation temperature, whereas H(+) ATPase capacities were thermally compensated but decreased at medium PCO2 and 10 °C. When functional capacities of branchial ATPases were compared with mitochondrial F1Fo ATP-synthase strong correlations of F1Fo ATP-synthase and ATPase capacities generally indicate close coordination of branchial aerobic ATP demand and supply. Our data indicate physiological plasticity in the gills of cod to adjust to a warming, acidifying ocean within limits. In light of the interacting and non-linear, dose-dependent effects of both climate factors the role of these mechanisms in shaping resilience under climate change remains to be explored.

  7. Vitamin K2-enhanced liver regeneration is associated with oval cell expansion and up-regulation of matrilin-2 expression in 2-AAF/PH rat model.

    PubMed

    Lin, M; Sun, P; Zhang, G; Xu, X; Liu, G; Miao, H; Yang, Y; Xu, H; Zhang, L; Wu, P; Li, M

    2014-03-01

    Normal liver has a great potential of regenerative capacity after partial hepatectomy. In clinic, however, most patients receiving partial hepatectomy are usually suffering from chronic liver diseases with severely damaged hepatocyte population. Under these conditions, activation of hepatic progenitor cell (oval cell in rodents) population might be considered as an alternative mean to enhance liver functional recovery. Vitamin K2 has been shown to promote liver functional recovery in patients with liver cirrhosis. In this study, we explored the possibility of vitamin K2 treatment in activating hepatic oval cell for liver regeneration with the classic 2-acetamido-fluorene/partial hepatectomy (2-AAF/PH) model in Sprague-Dawley rats. In 2-AAF/PH animals, vitamin K2 treatment induced a dose-dependent increase of liver regeneration as assessed by the weight ratio of remnant liver versus whole body and by measuring serum albumin level. In parallel, a drastic expansion of oval cell population as assessed by anti-OV6 and anti-CK19 immunostaining was noticed in the periportal zone of the remnant liver. Since matrilin-2 was linked to oval cell proliferation and liver regeneration after partial hepatectomy, we assessed its expression at both the mRNA and protein levels. The results revealed a significant increase after vitamin K2 treatment in parallel with the expansion of oval cell population. Consistently, knocking down matrilin-2 expression in vivo largely reduced vitamin K2-induced liver regeneration and oval cell proliferation in 2-AAF/PH animals. In conclusion, these data suggest that vitamin K2 treatment enhances liver regeneration after partial hepatectomy, which is associated with oval cell expansion and matrilin-2 up-regulation.

  8. Intracellular pH and its relationship to regulation of ion transport in normal and cystic fibrosis human nasal epithelia.

    PubMed

    Willumsen, N J; Boucher, R C

    1992-09-01

    1. Intracellular pH (pHi) of cultured human airway epithelial cells from normal and cystic fibrosis (CF) subjects were measured with double-barrelled pH-sensitive liquid exchanger microelectrodes. The cells, which were grown to confluence on a permeable collagen matrix support, were mounted in a modified miniature Ussing chamber. All studies were conducted under open circuit conditions. Values are given as means +/- S.E.M. and n refers to the number of preparations. 2. Normal preparations (n = 15) were characterized by a transepithelial potential difference (Vt) of -18 +/- 2 mV, an apical membrane potential (Va) of -19 +/- 2 mV, a basolateral membrane potential (Vb) of -37 +/- 2 mV, a transepithelial resistance (Rt) of 253 +/- 15 omega cm2, a fractional apical membrane resistance (fRa) of 0.40 +/- 0.04 and an equivalent short circuit current (Ieq) of -73 +/- 7 microA cm-2. 3. CF preparations (n = 13) were characterized by a Vt of -46 +/- 7 mV, a Va of 3 +/- 5 mV, a Vb of -43 +/- 3 mV, Rt of 373 +/- 47 omega cm2, fRa of 0.44 +/- 0.04 and an Ieq of -130 +/- 16 microA cm-2. All parameters except Vb and fRa were significantly different (P < 0.025) from those of normal preparations. 4. Despite large differences in electrochemical driving force for proton flow across the apical cell membranes between normal and CF preparations (-4 +/- 3 mV and 20 +/- 7 mV, respectively), pHi was similar (7.15 +/- 0.02 and 7.11 +/- 0.05, respectively). The driving force across the basolateral membrane was similar in normal and CF preparations (22 +/- 3 and 26 +/- 3 mV, respectively). 5. Intracellular alkalinization achieved by removal of CO2 from the luminal Ringer solution or by luminal ammonium prepulse led to stimulation of Ieq in both normal (from -58 to -70 microA cm-2, n = 4; P < 0.05) and CF (from -144 to -163 microA cm-2, n = 4; P < 0.005) preparations. The increase in Ieq was associated with a reduction of Rt, increase in fRa, and hyperpolarization of Vb. All changes in

  9. Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a Model for In Vivo Gene Expression†

    PubMed Central

    Loughman, Jennifer A.; Caparon, Michael

    2006-01-01

    For a pathogen such as Streptococcus pyogenes, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analyses of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we determined that the gene encoding the SpeB cysteine protease is up-regulated over the course of infection in a murine soft-tissue model. Conditions were identified, including growth phase, acidic pH, and an NaCl concentration of <0.1 M, that were required for expression of speB in vitro. Analysis of global expression profiles in response to these conditions in vitro identified a set of coregulated genes whose expression patterns showed a significant correlation with that of speB when examined during infection of murine soft tissues. This analysis revealed that a culture medium that promotes high levels of SpeB expression in vitro produced an expression profile that showed significant correlation to the profile observed in vivo. Taken together, these studies establish culture conditions that mimic in vivo expression patterns; that growth phase, pH, and NaCl may mimic relevant cues sensed by S. pyogenes during infection; and that identification of other environmental cues that alter expression of speB in vitro may provide insight into the signals that direct global patterns of gene expression in vivo. PMID:16385029

  10. Identification of CD25 as STAT5-Dependent Growth-Regulator of Leukemic Stem Cells in Ph+ CML

    PubMed Central

    Sadovnik, Irina; Hoelbl-Kovacic, Andrea; Herrmann, Harald; Eisenwort, Gregor; Warsch, Wolfgang; Hoermann, Gregor; Greiner, Georg; Blatt, Katharina; Peter, Barbara; Stefanzl, Gabriele; Berger, Daniela; Bilban, Martin; Herndlhofer, Susanne; Sill, Heinz; Sperr, Wolfgang R.; Streubel, Berthold; Mannhalter, Christine; Holyoake, Tessa L.; Sexl, Veronika; Valent, Peter

    2015-01-01

    Purpose In chronic myeloid leukemia (CML), leukemic stem cells (LSCs) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel interesting markers of CML LSCs. Experimental Design CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25+ CML cell line KU812. Results In contrast to normal hematopoietic stem cells, CD34+/CD38− CML LSCs expressed the interleukin-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin−/Sca-1+/Kit+ stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5-depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cells in vitro and their engraftment in vivo in NOD/SCID-IL-2Rγ−/− mice. In drug-screening experiments, the PI3-Kinase/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic anti-neoplastic effects on CML cells when applied in combination with nilotinib or ponatinib. Conclusion CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth-regulator of CML LSCs, which may have biological and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML. PMID:26607600

  11. Sarcolemmal localisation of Na+/H+ exchange and Na+-HCO3- co-transport influences the spatial regulation of intracellular pH in rat ventricular myocytes.

    PubMed

    Garciarena, Carolina D; Ma, Yu-ling; Swietach, Pawel; Huc, Laurence; Vaughan-Jones, Richard D

    2013-05-01

    Membrane acid extrusion by Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC) is essential for maintaining a low cytoplasmic [H(+)] (∼60 nm, equivalent to an intracellular pH (pHi) of 7.2). This protects myocardial function from the high chemical reactivity of H(+) ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pHi regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 m formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pHi imaging reveals that NBC stimulation induces spatially uniform pHi recovery from acidosis, whereas NHE1 stimulation induces pHi non-uniformity during recovery (of ∼0.1 units, for 2-3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pHi microdomains is favoured by low cytoplasmic H(+) mobility and long H(+) diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H(+)]i regulation in ventricular myocytes, suggesting that, by guarding pHi, NHE1 preferentially protects gap junctional communication at intercalated discs, while NBC locally protects t-tubular excitation-contraction coupling.

  12. The relationship between rumen acidosis resistance and expression of genes involved in regulation of intracellular pH and butyrate metabolism of ruminal epithelial cells in steers.

    PubMed

    Schlau, N; Guan, L L; Oba, M

    2012-10-01

    Past research has focused on the prevention and management of subacute rumen acidosis by manipulating the ration; however, the severity of acidosis varies even among animals fed a common high-grain diet. The objectives of this study were to compare the ruminal volatile fatty acid (VFA) profile and expression of genes involved in the metabolism of butyrate, the VFA most extensively metabolized by the ruminal epithelium, and intracellular pH regulation in ruminal epithelial cells between acidosis-resistant (AR) and acidosis-susceptible (AS) steers. Acidosis indexes (area per day under pH 5.8 divided by dry matter intake) were measured for 17 steers fed a common high-grain diet, and the 3 steers with the lowest (1.4 ± 1.2 pH∙min/kg) and the 3 with the highest values (23.9 ± 7.4 pH∙min/kg) were classified as AR and AS, respectively, and used in the subsequent study. The steers were force-fed a diet containing 85% grain at 60% of the expected daily intake (5.8 ± 0.8 and 5.6 ± 0.6 kg for AR and AS, respectively) within 30 min. Mean ruminal pH over the postprandial 6-h period was higher for AR compared with AS (6.02 vs. 5.55), and mean total VFA concentration was 74% for AR compared with AS (122 vs. 164 mM). Molar proportion of butyrate in the ruminal fluid was 139% higher for AR compared with AS (17.5 vs. 7.33 mol/100 mol of VFA). Expression of monocarboxylate cotransporter isoform 1, sodium hydrogen exchanger isoforms 1 and 2, and anion exchangers (downregulated in adenoma and putative anion exchanger, isoform 1) did not differ between AR and AS steers. However, expression of sodium hydrogen exchanger isoform 3, which imports Na(+) to the epithelial cell and exports H(+) to the rumen, was 176% higher in AR steers than in AS steers. Higher ruminal pH for AR might be partly due to a faster rate of VFA absorption, lower VFA production, or both.

  13. Theory of gastric CO2 ventilation and its control during respiratory acidosis: implications for central chemosensitivity, pH regulation, and diseases causing chronic CO2 retention.

    PubMed

    Dean, Jay B

    2011-02-15

    The theory of gastric CO(2) ventilation describes a previously unrecognized reflex mechanism controlled by neurons in the caudal solitary complex (cSC) for non-alveolar elimination of systemic CO(2) during respiratory acidosis. Neurons in the cSC, which is a site of CO(2) chemosensitivity for cardiorespiratory control, also control various gastroesophageal reflexes that remove CO(2) from blood. CO(2) is consumed in the production of gastric acid and bicarbonate in the gastric epithelium and then reconstituted as CO(2) in the stomach lumen from the reaction between H(+) and HCO(3)(-). Respiratory acidosis and gastric CO(2) distension induce cSC/vagovagal mediated transient relaxations of the lower esophageal sphincter to vent gastric CO(2) upwards by bulk flow along an abdominal-to-esophageal (=intrapleural) pressure gradient the magnitude of which increases during abdominal (gastric) compression caused by increased contractions of respiratory muscles. Esophageal distension induces cSC/nucleus ambiguus/vagovagal reflex relaxation of the upper esophageal sphincter and CO(2) is vented into the pharynx and mixed with pulmonary gas during expiration or, alternatively, during eructation. It is proposed that gastric CO(2) ventilation provides explanations for (1) the postprandial increase in expired CO(2) and (2) the negative P(blood - expired)CO₂difference that occurs with increased inspired CO(2). Furthermore, it is postulated that gastric CO(2) ventilation and alveolar CO(2) ventilation are coordinated under dual control by CO(2) chemosensitive neurons in the cSC. This new theory, therefore, presupposes a level of neural control and coordination between two previously presumed dissimilar organ systems and supports the notion that different sites of CO(2) chemosensitivity address different aspects of whole body pH regulation. Consequently, not all sites of central chemosensitivity are equal regarding the mechanism(s) activated for CO(2) elimination. A distributed CO(2

  14. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    SciTech Connect

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  15. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus.

    PubMed

    Li-Byarlay, Hongmei; Pittendrigh, Barry R; Murdock, Larry L

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant-insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology. PMID:27594789

  16. The midgut epithelium of aquatic arthropods: a critical target organ in environmental toxicology.

    PubMed Central

    Beaty, Barry J; Mackie, Ryan S; Mattingly, Kimberly S; Carlson, Jonathan O; Rayms-Keller, Alfredo

    2002-01-01

    The midgut epithelium of aquatic arthropods is emerging as an important and toxicologically relevant organ system for monitoring environmental pollution. The peritrophic matrix of aquatic arthropods, which is secreted by the midgut epithelium cells, is perturbed by copper or cadmium. Molecular biological studies have identified and characterized two midgut genes induced by heavy metals in the midgut epithelium. Many other metal-responsive genes (MRGs) await characterization. One of the MRGs codes for an intestinal mucin, which is critical for protecting the midgut from toxins and pathogens. Another codes for a tubulin gene, which is critical for structure and function of the midgut epithelial cells. Perturbation of expression of either gene could condition aquatic arthropod survivorship. Induction of these MRGs is a more sensitive and rapid indicator of heavy-metal pollution than biological assays. Characterization of genes induced by pollutants could provide mechanistic understanding of fundamental cellular responses to pollutants and insight into determinants of aquatic arthropod population genetic structure and survivorship in nature. PMID:12634118

  17. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus

    PubMed Central

    Li-Byarlay, Hongmei; Pittendrigh, Barry R.; Murdock, Larry L.

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant–insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology. PMID:27594789

  18. Implications of Time Bomb model of ookinete invasion of midgut cells.

    PubMed

    Han, Yeon Soo; Barillas-Mury, Carolina

    2002-10-01

    In this review, we describe the experimental observations that led us to propose the Time Bomb model of ookinete midgut invasion and discuss potential implications of this model when considering malaria transmission-blocking strategies aimed at arresting parasite development within midgut cells. A detailed analysis of the molecular interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei parasites, as they migrate through midgut cells, revealed that ookinetes induce nitric oxide synthase (NOS) expression, remodeling of the actin cytoskeleton and characteristic morphological changes in the invaded epithelial cells. Parasites inflict extensive damage that ultimately leads to genome fragmentation and cell death. During their migration through the cytoplasm, ookinetes release a subtilisin-like protease (PbSub2) and the surface protein (Pbs21). The model proposes that ookinetes must escape rapidly from the invaded cells, as the responses mediating cell death could be potentially lethal to the parasites. In other words, the physical and/or chemical damage triggered by the parasite can be thought of as a 'lethal bomb'. Once this cascade of events is initiated, the parasite must leave the cellular compartment within a limited time to escape unharmed from the 'bomb' it has activated. The midgut epithelium has the ability to heal rapidly by 'budding off' the damaged cells to the midgut lumen without losing its integrity. PMID:12225921

  19. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus

    PubMed Central

    Li-Byarlay, Hongmei; Pittendrigh, Barry R.; Murdock, Larry L.

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant–insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology.

  20. Midgut of the non-hematophagous mosquito Toxorhynchites theobaldi (Diptera, Culicidae).

    PubMed

    Godoy, Raquel S M; Fernandes, Kenner M; Martins, Gustavo F

    2015-10-30

    In most mosquito species, the females require a blood-feeding for complete egg development. However, in Toxorhynchites mosquitoes, the eggs develop without blood-feeding, and both females and males exclusively feed on sugary diets. The midgut is a well-understood organ in blood-feeding mosquitoes, but little is known about it in non-blood-feeding ones. In the present study, the detailed morphology of the midgut of Toxorhynchites theobaldi were investigated using histochemical and ultrastructural methods. The midgut of female and male T. theobaldi adults consists of a long, slender anterior midgut (AMG), and a short, dilated posterior midgut (PMG). The AMG is subdivided into AMG1 (short, with folds) and AMG2 (long, without folds). Nerve branches and enteroendocrine cells are present in AMG and PMG, respectively. Compared with the PMG of blood-feeding female mosquitoes, the PMG of T. theobaldi is smaller; however, in both mosquitoes, PMG seems be the main region of food digestion and absorption, and protein secretion. The epithelial folds present in the AMG of T. theobaldi have not been reported in other mosquitoes; however, the midgut muscle organization and endocrine control of the digestion process are conserved in both T. theobaldi and blood-feeding mosquitoes.

  1. Midgut of the non-hematophagous mosquito Toxorhynchites theobaldi (Diptera, Culicidae)

    PubMed Central

    Godoy, Raquel S. M.; Fernandes, Kenner M.; Martins, Gustavo F.

    2015-01-01

    In most mosquito species, the females require a blood-feeding for complete egg development. However, in Toxorhynchites mosquitoes, the eggs develop without blood-feeding, and both females and males exclusively feed on sugary diets. The midgut is a well-understood organ in blood-feeding mosquitoes, but little is known about it in non-blood-feeding ones. In the present study, the detailed morphology of the midgut of Toxorhynchites theobaldi were investigated using histochemical and ultrastructural methods. The midgut of female and male T. theobaldi adults consists of a long, slender anterior midgut (AMG), and a short, dilated posterior midgut (PMG). The AMG is subdivided into AMG1 (short, with folds) and AMG2 (long, without folds). Nerve branches and enteroendocrine cells are present in AMG and PMG, respectively. Compared with the PMG of blood-feeding female mosquitoes, the PMG of T. theobaldi is smaller; however, in both mosquitoes, PMG seems be the main region of food digestion and absorption, and protein secretion. The epithelial folds present in the AMG of T. theobaldi have not been reported in other mosquitoes; however, the midgut muscle organization and endocrine control of the digestion process are conserved in both T. theobaldi and blood-feeding mosquitoes. PMID:26514271

  2. Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus

    PubMed Central

    Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian

    2015-01-01

    ABSTRACT Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. IMPORTANCE Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the

  3. Transcriptomic analysis provides insights on hexavalent chromium induced DNA double strand breaks and their possible repair in midgut cells of Drosophila melanogaster larvae.

    PubMed

    Mishra, Manish; Sharma, A; Shukla, A K; Pragya, P; Murthy, R C; de Pomerai, David; Dwivedi, U N; Chowdhuri, D Kar

    2013-01-01

    Hexavalent chromium [Cr(VI)] is a well known mutagen and carcinogen. Since genomic instability due to generation of double strand breaks (DSBs) is causally linked to carcinogenesis, we tested a hypothesis that Cr(VI) causes in vivo generation of DSBs and elicits DNA damage response. We fed repair proficient Drosophila melanogaster (Oregon R(+)) larvae Cr(VI) (20.0μg/ml) mixed food for 24 and 48h and observed a significant (p<0.05) induction of DSBs in their midgut cells after 48h using neutral Comet assay. Global gene expression profiling in Cr(VI)-exposed Oregon R(+) larvae unveiled mis-regulation of DSBs responsive repair genes both after 24 and 48h. In vivo generation of DSBs in exposed Drosophila was confirmed by an increased pH2Av immunostaining along with the activation of cell cycle regulation genes. Analysis of mis-regulated genes grouped under DSB response by GOEAST indicated the participation of non-homologous end joining (NHEJ) DSB repair pathway. We selected two strains, one mutant (ligIV) and another ku80-RNAi (knockdown of ku80), whose functions are essentially linked to NHEJ-DSB repair pathway. As a proof of principle, we compared the DSBs generation in larvae of these two strains with that of repair proficient Oregon R(+). Along with this, DSBs generation in spn-A and okr [essential genes in homologous recombination repair (HR) pathway] mutants was also tested for the possible involvement of HR-DSB repair. A significantly increased DSBs generation in the exposed ku80-RNAi and ligIV (mutant) larvae because of impaired repair, concomitant with an insignificant DSBs generation in okr and spn-A mutant larvae indicates an active participation of NHEJ repair pathway. The study, first of its kind to our knowledge, while providing evidences for in vivo generation of DSBs in Cr(VI) exposed Drosophila larvae, assumes significance for its relevance to higher organisms due to causal link between DSB generation and Cr(VI)-induced carcinogenesis.

  4. Microprofiles of oxygen, redox potential, and pH, and microbial fermentation products in the highly alkaline gut of the saprophagous larva of Penthetria holosericea (Diptera: Bibionidae).

    PubMed

    Šustr, Vladimír; Stingl, Ulrich; Brune, Andreas

    2014-08-01

    The saprophagous larvae of bibionid flies harbor bacteria in their alkaline intestinal tracts, but little is known about the contribution of the gut microbiota to the digestion of their recalcitrant diet. In this study, we measured oxygen and hydrogen partial pressure, redox potential and pH in the midgut, gastric caeca and hindgut of larvae of the bibionid fly Penthetria holosericea with Clark-type O2 and H2 microsensors, platinum redox microelectrodes, and LIX-type pH microelectrodes. The center of the midgut lumen was anoxic, whereas gastric caeca and hindgut were hypoxic. However, redox potential profiles indicated oxidizing conditions throughout the gut, with lowest values in the midgut (+20 to +60mV). Hydrogen production was not detected. The midgut was extremely alkaline (pH around 11), whereas hindgut and gastric caeca were neutral to slightly alkaline. While HPLC analysis showed high concentrations of glucose in the midgut (15mM) and gastric caeca (27mM), the concentrations of microbial fermentation products such as lactate (2-4mM), acetate (<1mM) and succinate (<0.5mM) were low in all gut regions, suggesting that the contribution of microorganisms to the digestive process, particularly in the alkaline midgut, is only of minor importance. We conclude that the digestive strategy of the saprophytic larva of P. holosericea, which feeds selectively on decomposed leaves and its own microbe-rich faeces, differs fundamentally from those of detritivorous and humivorous insects, which host a highly active, fermentative microbiota in their alkaline midgut or hindgut compartments. PMID:24971929

  5. In situ analysis of pH gradients in mosquito larvae using non-invasive, self-referencing, pH-sensitive microelectrodes.

    PubMed

    Boudko, D Y; Moroz, L L; Linser, P J; Trimarchi, J R; Smith, P J; Harvey, W R

    2001-02-01

    The alkaline environment, pH approximately 11, in the anterior midgut lumen of mosquito larvae is essential for normal nutrition and development. The mechanism of alkalization is, however, unknown. Although evidence from immunohistochemistry, electron microscopy and electrophysiology suggests that a V-ATPase is present in the basal membranes of the epithelial cells, its physiological role in the alkalization process has not been demonstrated. To investigate a possible role of the V-ATPase in lumen alkalization, pH gradients emanating from the hemolymph side of the midgut in semi-intact mosquito larvae were measured using non-invasive, self-referencing, ion-selective microelectrodes (SERIS). Large H+ concentration gradients, with highest concentrations close to the basal membrane (outward [H+] gradients), were found in the anterior midgut, whereas much smaller gradients, with concentrations lowest close to this membrane (inward [H+] gradients), were found in the gastric caeca and posterior midgut. Similar region-specific pH gradients, with consistent anterior-to-posterior profiles, were observed in individuals of two Aedes species, Aedes aegypti from semi-tropical Florida and Aedes canadensis from north-temperate Massachusetts. The gradients remained in a steady state for up to 6 h, the maximum duration of the recordings. Bafilomycin A1 (10(-5), 10(-7 )mol x l(-1)) on the hemolymph side greatly diminished the [H+] gradients in the anterior midgut but had no effect on the gradients in the gastric caecum and posterior midgut. These physiological data are consistent with the previous findings noted above. Together, they support the hypothesis that a basal, electrogenic H+ V-ATPase energizes luminal alkalization in the anterior midgut of larval mosquitoes.

  6. Two-Stage pH Control Strategy Based on the pH Preference of Acetoin Reductase Regulates Acetoin and 2,3-Butanediol Distribution in Bacillus subtilis

    PubMed Central

    Rao, Zhiming; Yang, Taowei; Xu, Zhenghong; Yang, Shangtian; Li, Huazhong

    2014-01-01

    Acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), which catalyzes the interconversion between acetoin and 2,3-butanediol, plays an important role in distribution of the products pools. This work characterized the Bacillus subtilis AR/BDH for the first time. The enzyme showed very different pH preferences of pH 6.5 for reduction and pH 8.5 for oxidation. Based on these above results, a two-stage pH control strategy was optimized for acetoin production, in which the pH was controlled at 6.5 for quickly converting glucose to acetoin and 2,3-butanediol, and then 8.0 for reversely transforming 2,3-butanediol to acetoin. By over-expression of AR/BDH in the wild-type B. subtilis JNA 3-10 and applying fed-batch fermentation based on the two-stage pH control strategy, acetoin yield of B. subtilis was improved to a new record of 73.6 g/l, with the productivity of 0.77 g/(l·h). The molar yield of acetoin was improved from 57.5% to 83.5% and the ratio of acetoin/2,3-butanediol was switched from 2.7∶1 to 18.0∶1. PMID:24608678

  7. Aspergillus niger mstA encodes a high-affinity sugar/H+ symporter which is regulated in response to extracellular pH.

    PubMed Central

    Vankuyk, Patricia A; Diderich, Jasper A; MacCabe, Andrew P; Hererro, Oscar; Ruijter, George J G; Visser, Jaap

    2004-01-01

    A sugar-transporter-encoding gene, mstA, which is a member of the major facilitator superfamily, has been cloned from a genomic DNA library of the filamentous fungus Aspergillus niger. To enable the functional characterization of MSTA, a full-length cDNA was expressed in a Saccharomyces cerevisiae strain deficient in hexose uptake. Uptake experiments using 14C-labelled monosaccharides demonstrated that although able to transport D-fructose ( K(m), 4.5+/-1.0 mM), D-xylose ( K(m), 0.3+/-0.1 mM) and D-mannose ( K(m), 60+/-20 microM), MSTA has a preference for D-glucose (K(m), 25+/-10 microM). pH changes associated with sugar transport indicate that MSTA catalyses monosaccharide/H+ symport. Expression of mstA in response to carbon starvation and upon transfer to poor carbon sources is consistent with a role for MSTA as a high-affinity transporter for D-glucose, D-mannose and D-xylose. Northern analysis has shown that mstA is subject to CreA-mediated carbon catabolite repression and pH regulation mediated by PacC. A. niger strains in which the mstA gene had been disrupted are phenotypically identical with isogenic reference strains when grown on 0.1-60 mM D-glucose, D-mannose, D-fructose or D-xylose. This indicates that A. niger possesses other transporters capable of compensating for the absence of MSTA. PMID:14717659

  8. Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

    PubMed Central

    Di Domenico, Fabio; Foppoli, Cesira; Blarzino, Carla; Perluigi, Marzia; Paolini, Francesca; Morici, Salvatrice; Coccia, Raffaella; Cini, Chiara; De Marco, Federico

    2009-01-01

    Background Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype. Methods The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored. Results We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs. Conclusion Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the

  9. Morphological alterations induced by boric acid and fipronil in the midgut of worker honeybee (Apis mellifera L.) larvae : Morphological alterations in the midgut of A. mellifera.

    PubMed

    da Silva Cruz, Aline; da Silva-Zacarin, Elaine C M; Bueno, Odair C; Malaspina, Osmar

    2010-04-01

    Morphological alterations, by means of histological and ultrastructural analysis, have been used to determine the effects of boric acid and fipronil on midgut tissues of honeybee worker, Apis mellifera L. larvae. In order to observe possible morphological alterations in the midgut, two groups of bioassays were performed. In the first one, the larvae were chronically treated with different concentrations of boric acid added to the food (1.0, 2.5 and 7.5 mg/g). In the second group, the larvae were fed with diets containing different concentrations of fipronil (0.1 and 1 microg/g) and compared with control groups without these chemical compounds. In the first bioassay, the larvae were collected on day 3 and in the second bioassay on day 4, when the mortality rate obtained in the toxicological bioassay was not very high. The larval midguts were removed and processed for morphological analyses using a light and transmission electron microscopy. We observed cytoplasmic vacuolizations, with the absence of autophagic vacuoles, and chromatinic compacting in most of the cells in the groups treated with pesticides. The morphological alterations were far greater in the larvae treated with boric acid than in the larvae treated with fipronil. Our data suggest that the midgut cell death observed was in response to boric acid and fipronil action. This study significantly improves the understanding of the toxicological effect of these insecticides from the ecotoxicological perspective.

  10. Characterization of a midgut-specific chitin synthase gene (LmCHS2) responsible for biosynthesis of chitin of peritrophic matrix in Locusta migratoria.

    PubMed

    Liu, Xiaojian; Zhang, Huanhuan; Li, Sheng; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2012-12-01

    Chitin, an essential component of peritrophic matrix (PM), is produced by a series of biochemical reactions. Chitin synthase plays a crucial role in chitin polymerization in chitin biosynthetic pathway. In this study, we identified and characterized a full-length cDNA of chitin synthase 2 gene (LmCHS2) from Locusta migratoria. The cDNA contains an open reading frame of 4569 nucleotides that encode 1523 amino acid residues, and 76- and 373-nucleotides for 5'- and 3'-noncoding regions, respectively. Analysis of LmCHS2 transcript in different tissues of the locust by using real-time quantitative PCR indicated that LmCHS2 was exclusively expressed in midgut and gastric caeca (a part of the midgut). The highest expression was found in the anterior midgut with a decline of the transcript level from the anterior to posterior regions. During growth and development of locusts, there was only a slight expression in eggs, but the expression gradually increased from nymphs to adults. In situ hybridization further revealed that LmCHS2 transcript mainly presented in the apical regions of brush border forming columnar cells of gastric caeca. LmCHS2 dsRNA was injected to fifth-instar nymphs to further explore biological functions of LmCHS2. Significantly down-regulated transcript of LmCHS2 resulted in a cessation of feeding and a high mortality of the insect. However, no visible abnormal morphological change of locusts was observed until insects molted to adults. After dissection, we found that the average length of midguts from the LmCHS2 dsRNA-injected locusts was shorter than that of the control insects that were injected with dsGFP. Furthermore, microsection of midguts showed that the PM of the LmCHS2 dsRNA-injected nymphs was amorphous and thin as compared with the controls. Our results demonstrate that LmCHS2 is responsible for the biosynthesis of chitin associated with PM and plays an essential role in locust growth and development. PMID:23006725

  11. Relative contribution of ruminal buffering systems to pH regulation in feedlot cattle fed either low- or high-forage diets.

    PubMed

    Chibisa, G E; Beauchemin, K A; Penner, G B

    2016-07-01

    The relative contribution of ruminal short-chain fatty acid (SCFA) absorption and salivary buffering to pH regulation could potentially change under different dietary conditions. Therefore, the objective of this study was to investigate the effects of altering the ruminal supply of rapidly fermentable carbohydrate (CHO) on absorptive function and salivation in beef cattle. Eight heifers (mean BW±SD=410±14 kg) were randomly allocated to two treatments in a crossover design with 37-day periods. Dietary treatments were barley silage at 30% low forage (LF) or 70% high forage (HF) of dietary dry matter (DM), with the remainder of the diet consisting of barley grain (65% or 25% on a DM basis) and a constant level (5%) of supplement. The LF and HF diets contained 45.3% and 30.9% starch, and 4.1% and 14.0% physically effective fiber (DM basis), respectively. Ruminal pH was continuously measured from day 17 to day 23, whereas ruminal fluid was collected on day 23 to determine SCFA concentration. Ruminal liquid passage rate was determined on day 23 using Cr-ethylenediaminetetraacetic acid. Eating or resting salivation was measured by collecting masticate (days 28 and 29) or saliva samples (days 30 and 31) at the cardia, respectively. On days 30 and 31, the temporarily isolated and washed reticulo-rumen technique was used to measure total, and Cl--competitive (an indirect measure of protein-mediated transport) absorption of acetate, propionate and butyrate. As a result of the higher dietary starch content and DM intake, the ruminal supply of rapidly fermentable CHO, total ruminal SCFA concentration (118 v. 95 mM; P<0.001) and osmolality (330 v. 306 mOsm/kg; P=0.018) were greater in cattle fed LF compared with HF. In addition, feeding LF resulted in a longer duration (2.50 v. 0.09 h/day; P=0.02) and a larger area (0.44 v. 0.01 (pH×h)/day; P=0.050) that pH was below 5.5. There was no diet effect on total and Cl--competitive absorption (mmol/h and %/h) of acetate, propionate

  12. Relative contribution of ruminal buffering systems to pH regulation in feedlot cattle fed either low- or high-forage diets.

    PubMed

    Chibisa, G E; Beauchemin, K A; Penner, G B

    2016-07-01

    The relative contribution of ruminal short-chain fatty acid (SCFA) absorption and salivary buffering to pH regulation could potentially change under different dietary conditions. Therefore, the objective of this study was to investigate the effects of altering the ruminal supply of rapidly fermentable carbohydrate (CHO) on absorptive function and salivation in beef cattle. Eight heifers (mean BW±SD=410±14 kg) were randomly allocated to two treatments in a crossover design with 37-day periods. Dietary treatments were barley silage at 30% low forage (LF) or 70% high forage (HF) of dietary dry matter (DM), with the remainder of the diet consisting of barley grain (65% or 25% on a DM basis) and a constant level (5%) of supplement. The LF and HF diets contained 45.3% and 30.9% starch, and 4.1% and 14.0% physically effective fiber (DM basis), respectively. Ruminal pH was continuously measured from day 17 to day 23, whereas ruminal fluid was collected on day 23 to determine SCFA concentration. Ruminal liquid passage rate was determined on day 23 using Cr-ethylenediaminetetraacetic acid. Eating or resting salivation was measured by collecting masticate (days 28 and 29) or saliva samples (days 30 and 31) at the cardia, respectively. On days 30 and 31, the temporarily isolated and washed reticulo-rumen technique was used to measure total, and Cl--competitive (an indirect measure of protein-mediated transport) absorption of acetate, propionate and butyrate. As a result of the higher dietary starch content and DM intake, the ruminal supply of rapidly fermentable CHO, total ruminal SCFA concentration (118 v. 95 mM; P<0.001) and osmolality (330 v. 306 mOsm/kg; P=0.018) were greater in cattle fed LF compared with HF. In addition, feeding LF resulted in a longer duration (2.50 v. 0.09 h/day; P=0.02) and a larger area (0.44 v. 0.01 (pH×h)/day; P=0.050) that pH was below 5.5. There was no diet effect on total and Cl--competitive absorption (mmol/h and %/h) of acetate, propionate

  13. Mutations in domain I interhelical loops affect the rate of pore formation by the Bacillus thuringiensis Cry1Aa toxin in insect midgut brush border membrane vesicles.

    PubMed

    Lebel, Geneviève; Vachon, Vincent; Préfontaine, Gabrielle; Girard, Frédéric; Masson, Luke; Juteau, Marc; Bah, Aliou; Larouche, Geneviève; Vincent, Charles; Laprade, Raynald; Schwartz, Jean-Louis

    2009-06-01

    Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.

  14. Regulation of the Na+/K+-ATPase Ena1 Expression by Calcineurin/Crz1 under High pH Stress: A Quantitative Study.

    PubMed

    Petrezsélyová, Silvia; López-Malo, María; Canadell, David; Roque, Alicia; Serra-Cardona, Albert; Marqués, M Carmen; Vilaprinyó, Ester; Alves, Rui; Yenush, Lynne; Ariño, Joaquín

    2016-01-01

    Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase. PMID:27362362

  15. The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules.

    PubMed

    Feingold, Daniel; Starc, Tanja; O'Donnell, Michael J; Nilson, Laura; Dent, Joseph A

    2016-09-01

    Pentameric ligand-gated ion channels (pLGICs) constitute a large protein superfamily in metazoa whose role as neurotransmitter receptors mediating rapid, ionotropic synaptic transmission has been extensively studied. Although the vast majority of pLGICs appear to be neurotransmitter receptors, the identification of pLGICs in non-neuronal tissues and homologous pLGIC-like proteins in prokaryotes points to biological functions, possibly ancestral, that are independent of neuronal signalling. Here, we report the molecular and physiological characterization of a highly divergent, orphan pLGIC subunit encoded by the pHCl-2 (CG11340) gene, in Drosophila melanogaster We show that pHCl-2 forms a channel that is insensitive to a wide array of neurotransmitters, but is instead gated by changes in extracellular pH. pHCl-2 is expressed in the Malpighian tubules, which are non-innervated renal-type secretory tissues. We demonstrate that pHCl-2 is localized to the apical membrane of the epithelial principal cells of the tubules and that loss of pHCl-2 reduces urine production during diuresis. Our data implicate pHCl-2 as an important source of chloride conductance required for proper urine production, highlighting a novel role for pLGICs in epithelial tissues regulating fluid secretion and osmotic homeostasis. PMID:27358471

  16. Regulation of the Na+/K+-ATPase Ena1 Expression by Calcineurin/Crz1 under High pH Stress: A Quantitative Study

    PubMed Central

    Petrezsélyová, Silvia; López-Malo, María; Canadell, David; Roque, Alicia; Serra-Cardona, Albert; Marqués, M. Carmen; Vilaprinyó, Ester; Alves, Rui; Yenush, Lynne

    2016-01-01

    Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase. PMID:27362362

  17. Alterations in the Helicoverpa armigera Midgut Digestive Physiology after Ingestion of Pigeon Pea Inducible Leucine Aminopeptidase

    PubMed Central

    Lomate, Purushottam R.; Jadhav, Bhakti R.; Giri, Ashok P.; Hivrale, Vandana K.

    2013-01-01

    Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory. PMID:24098675

  18. Insect midgut carboxypeptidases with emphasis on S10 hemipteran and M14 lepidopteran carboxypeptidases.

    PubMed

    Ferreira, C; Rebola, K G O; Cardoso, C; Bragatto, I; Ribeiro, A F; Terra, W R

    2015-04-01

    We compared the whole complement of midgut carboxypeptidases from 10 insects pertaining to five orders based on transcriptomes obtained by deep sequencing and biochemical data. Most of the carboxypeptidases were metallocarboxypeptidases from family M14, with carboxypeptidase A (CPA) predominating over carboxypeptidase B (CPB). They were found in all of the insects studied except for the hemipterans and a bruchid beetle. M14 carboxypeptidases were expressed only in the midgut of Spodoptera frugiperda (Lepidoptera). The most expressed CPA from this insect (SfCPA) was cloned, sequenced and expressed as a recombinant enzyme. This enzyme was used to generate antibodies used to demonstrate that SfCPA is secreted by an exocytic route. Serine carboxypeptidases from family S10 were found in all of the insects studied here. In S. frugiperda, they are expressed in all tissues besides the midgut, in accordance with their presumed lysosomal role. In the hemipteran Dysdercus peruvianus, S10 carboxypeptidases are expressed only in midgut, suggesting that they are digestive enzymes. This was confirmed by enzyme assays of midgut contents. Furthermore, the substrate specificity of D. peruvianus S10 carboxypeptidases are predicted to be one CPC (preferring hydrophobic residues) and one CPD (preferring basic residues), thus able to hydrolyse the peptides formed by their digestive cathepsin D and cathepsin L, respectively. The role of S10 carboxypeptidases in bruchid beetles are suggested to be the same as in hemipterans.

  19. DNA duplication is essential for the repair of gastrointestinal perforation in the insect midgut

    PubMed Central

    Huang, Wuren; Zhang, Jie; Yang, Bing; Beerntsen, Brenda T.; Song, Hongsheng; Ling, Erjun

    2016-01-01

    Invertebrate animals have the capacity of repairing wounds in the skin and gut via different mechanisms. Gastrointestinal perforation, a hole in the human gastrointestinal system, is a serious condition, and surgery is necessary to repair the perforation to prevent an abdominal abscess or sepsis. Here we report the repair of gastrointestinal perforation made by a needle-puncture wound in the silkworm larval midgut. Following insect gut perforation, only a weak immune response was observed because the growth of Escherichia coli alone was partially inhibited by plasma collected at 6 h after needle puncture of the larval midgut. However, circulating hemocytes did aggregate over the needle-puncture wound to form a scab. While, cell division and apoptosis were not observed at the wound site, the needle puncture significantly enhanced DNA duplication in cells surrounding the wound, which was essential to repair the midgut perforation. Due to the repair capacity and limited immune response caused by needle puncture to the midgut, this approach was successfully used for the injection of small compounds (ethanol in this study) into the insect midgut. Consequently, this needle-puncture wounding of the insect gut can be developed for screening compounds for use as gut chemotherapeutics in the future. PMID:26754166

  20. Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

    PubMed

    Lomate, Purushottam R; Jadhav, Bhakti R; Giri, Ashok P; Hivrale, Vandana K

    2013-01-01

    Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

  1. MdSOS2L1 forms a complex with MdMYB1 to control vacuolar pH by transcriptionally regulating MdVHA-B1 in apples.

    PubMed

    Sun, Cui-Hui; Zhang, Quan-Yan; Sun, Mei-Hong; Hu, Da-Gang

    2016-01-01

    Vacuolar pH is important and involves in many different physiological processes in plants. A recent paper published in Plant Physiology reveals that MdMYB1 regulates vacuolar pH by directly transcriptionally regulating proton pump genes and malate transporters genes, such as V-ATPase subunit gene MdVHA-B1. Here, we found that MdSOS2L1 in vitro did not directly interact with MdMYB1, however, in vivo formed a complex with MdMYB1 in the nucleus to regulate MdVHA-B1-mediated vacuolar acidification. This finding shed light on the role of MdSOS2L1 in transcriptionally regulating MdVHA-B1 in addition to its post-modified function in apples. PMID:26910596

  2. Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

    PubMed Central

    Kurtz, I

    1987-01-01

    The apical transport processes responsible for proton secretion were studied in the isolated perfused rabbit S3 proximal tubule. Intracellular pH (pHi) was measured with the pH dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pHi in S3 tubules in nominally HCO3(-)-free solutions was 7.08 +/- 0.03. Removal of Na+ (lumen) caused a decrease in pHi of 0.34 +/- 0.06 pH/min. The decrease in pHi was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (lumen) and Cl- removal (lumen, bath). After a brief exposure to 20 mM NH4Cl, pHi fell by approximately 0.7 and recovered at a rate of 0.89 +/- 0.15 pH/min in the nominal absence of Na+, HCO3-, organic anions, and SO4(2-) (lumen, bath). 1 mM N,N'-dicyclohexylcarbodiimide (lumen), 1 mM N-ethylmaleimide (lumen), 0.5 mM colchicine (bath), and 0.5 mM iodoacetic acid (lumen, bath) inhibited the Na+-independent pHi recovery rate by 73%, 55%, 77%, and 86%, respectively, whereas 1 mM KCN (lumen, bath) did not inhibit pHi recovery. Reduction of intracellular, but not extracellular chloride, also decreased the Na+-independent pHi recovery rate. In conclusion, the S3 proximal tubule has an apical Na+/H+ antiporter with a Michaelis constant for Na+ of 29 mM and a maximum velocity of 0.47 pH/min. S3 tubules also possess a plasma membrane H+-ATPase that can regulate pHi, has a requirement for intracellular chloride, and utilizes ATP derived primarily from glycolysis. PMID:2888787

  3. Maintenance of midgut epithelial cells from Dendroctonus valens (Coleoptera: Scolytidae) in vitro.

    PubMed

    Sánchez, L; Andrade, J L; Cisneros, R; Zúñiga, G

    2004-01-01

    This article describes the culture of epithelial cells from anterior and posterior midgut regions of adult Dendroctonus valens. Culture conditions were established, and cell adherence was improved by means of a new technique that allowed the cells to grow between two glass coverslips. Cytoplasmic projections occur as anterior midgut cells grow to confluence; these projections were not observed in cells of the posterior midgut. The optimal culture medium for the maintenance of these epithelial cells was Roswell Park Memorial Institute 1640 medium at 25 degrees C. Cells in Grace's medium died in 24 h. Cultures did not require CO(2) atmosphere, but culture development was favored by the microaerophilic environment and the dark conditions in which the cells were grown, between the coverslips.

  4. EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila

    PubMed Central

    Jiang, Huaqi; Grenley, Marc O.; Bravo, Maria-Jose; Blumhagen, Rachel Z.; Edgar, Bruce A.

    2010-01-01

    Many tissues in higher animals undergo dynamic homeostatic growth, wherein damaged or aged cells are replaced by the progeny of resident stem cells. To maintain homeostasis, stem cells must respond to tissue needs. Here we show that in response to damage or stress in the intestinal (midgut) epithelium of adult Drosophila, multiple EGFR ligands and rhomboids (intramembrane proteases that activate some EGFR ligands) are induced, leading to the activation of EGFR signaling in intestinal stem cells (ISCs). Activation of EGFR signaling promotes ISC division and midgut epithelium regeneration, thus maintaining tissue homeostasis. ISCs defective in EGFR signaling cannot grow or divide, are poorly maintained, and cannot support midgut epithelium regeneration following enteric infection by the bacterium, Pseudomonas entomophila. Furthermore, ISC proliferation induced by Jak/Stat signaling is dependent upon EGFR signaling. Thus the EGFR/Ras/MAPK signaling pathway plays central, essential roles in ISC maintenance and the feedback system that mediates intestinal homeostasis. PMID:21167805

  5. Ultrastructural analysis of midgut cells from Culex quinquefasciatus (Diptera: Culicidae) larvae resistant to Bacillus sphaericus.

    PubMed

    de Melo, Janaina Viana; Vasconcelos, Romero Henrique Teixeira; Furtado, André Freire; Peixoto, Christina Alves; Silva-Filha, Maria Helena Neves Lobo

    2008-12-01

    The larvicidal action of the entomopathogen Bacillus sphaericus towards Culex quinquefasciatus is due to the binary (Bin) toxin present in crystals, which are produced during bacterial sporulation. The Bin toxin needs to recognize and bind specifically to a single class of receptors, named Cqm1, which are 60-kDa alpha-glucosidases attached to the apical membrane of midgut cells by a glycosylphosphatidylinositol anchor. C. quinquefasciatus resistance to B. sphaericus has been often associated with the absence of the alpha-glucosidase Cqm1 in larvae midgut microvilli. In this work, we aimed to investigate, at the ultrastructural level, the midgut cells from C. quinquefasciatus larvae whose resistance relies on the lack of the Cqm1 receptor. The morphological analysis showed that midgut columnar cells from the resistant larvae are characterized by a pronounced production of lipid inclusions, throughout the 4th instar. At the end of this stage, resistant larvae had an increased size and number of these inclusions in the midgut cells, while only a small number were observed in the cells from susceptible larvae. The morphological differences in the midgut cells of resistant larvae found in this work suggested that the lack of the Cqm1 receptor, which also has a physiological role as being an alpha-glucosidase, can be related to changes in the cell metabolism. The ultrastructural effects of Bin toxin on midgut epithelial cells from susceptible and resistant larvae were also investigated. The cytopathological alterations observed in susceptible larvae treated with a lethal concentration of toxin included breakdown of the endoplasmic reticulum, mitochondrial swelling, microvillar disruption and vacuolization. Some effects were observed in cells from resistant larvae, although those alterations did not lead to larval death, indicating that the receptor Cqm1 is essential to mediate the larvicidal action of the toxin. This is the first ultrastructural study to show differences

  6. Carbonic anhydrase activity monitored in vivo by hyperpolarized 13C-magnetic resonance spectroscopy demonstrate its importance for pH regulation in tumors

    PubMed Central

    Gallagher, Ferdia A.; Sladen, Helen; Kettunen, Mikko I.; Serrao, Eva M.; Rodrigues, Tiago B.; Wright, Alan; Gill, Andrew B.; McGuire, Sarah; Booth, Thomas C.; Boren, Joan; McIntyre, Alan; Miller, Jodi L.; Lee, Shen-Han; Honess, Davina; Day, Sam E.; Hu, De-en; Howat, William J.; Harris, Adrian L.; Brindle, Kevin M.

    2015-01-01

    Carbonic anhydrase (CA) buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3−). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized 13C label between bicarbonate (H13CO3−) and carbon dioxide (13CO2), following injection of hyperpolarized H13CO3−, using 13C magnetic resonance spectroscopy (13C-MRS) magnetization transfer measurements. 31P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and 13C-MRS measurements of the H13CO3−/13CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the 13C measurements overestimated pH due to incomplete equilibration of the hyperpolarized 13C label between the H13CO3− and 13CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevates tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH, and supports the hypothesis that a major function of CAIX is to lower the extracellular pH. PMID:26249175

  7. Carbonic Anhydrase Activity Monitored In Vivo by Hyperpolarized 13C-Magnetic Resonance Spectroscopy Demonstrates Its Importance for pH Regulation in Tumors.

    PubMed

    Gallagher, Ferdia A; Sladen, Helen; Kettunen, Mikko I; Serrao, Eva M; Rodrigues, Tiago B; Wright, Alan; Gill, Andrew B; McGuire, Sarah; Booth, Thomas C; Boren, Joan; McIntyre, Alan; Miller, Jodi L; Lee, Shen-Han; Honess, Davina; Day, Sam E; Hu, De-En; Howat, William J; Harris, Adrian L; Brindle, Kevin M

    2015-10-01

    Carbonic anhydrase buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3(-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3(-), using (13)C-magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3(-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3(-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevated tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH and supports the hypothesis that a major function of CAIX is to lower the extracellular pH.

  8. Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae).

    PubMed

    Zhu, Y C; Baker, J E

    1999-12-01

    Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites

  9. Analysis of differentially expressed genes between fluoride-sensitive and fluoride-endurable individuals in midgut of silkworm, Bombyx mori.

    PubMed

    Qian, Heying; Li, Gang; He, Qingling; Zhang, Huaguang; Xu, Anying

    2016-08-15

    Fluoride tolerance is an economically important trait of silkworm. Near-isogenic lines (NILs) of the dominant endurance to fluoride (Def) gene in Bombyx mori has been constructed before. Here, we analyzed the gene expression profiles of midgut of fluoride-sensitive and fluoride-endurable individuals of Def NILs by using high-throughput Illumina sequencing technology and bioinformatics tools, and identified differentially expressed genes between these individuals. A total of 3,612,399 and 3,567,631 clean tags for the libraries of fluoride-endurable and fluoride-sensitive individuals were obtained, which corresponded to 32,933 and 43,976 distinct clean tags, respectively. Analysis of differentially expressed genes indicates that 241 genes are differentially expressed between the two libraries. Among the 241 genes, 30 are up-regulated and 211 are down-regulated in fluoride-endurable individuals. Pathway enrichment analysis demonstrates that genes related to ribosomes, pancreatic secretion, steroid biosynthesis, glutathione metabolism, steroid biosynthesis, and glycerolipid metabolism are down-regulated in fluoride-endurable individuals. qRT-PCR was conducted to confirm the results of the DGE. The present study analyzed differential expression of related genes and tried to find out whether the crucial genes were related to fluoride detoxification which might elucidate fluoride effect and provide a new way in the fluorosis research. PMID:27106117

  10. Seasonality and Locality Affect the Diversity of Anopheles gambiae and Anopheles coluzzii Midgut Microbiota from Ghana

    PubMed Central

    Gendrin, Mathilde; Pels, Nana Adjoa P.; Yeboah-Manu, Dorothy; Christophides, George K.; Wilson, Michael D.

    2016-01-01

    Symbiotic bacteria can have important implications in the development and competence of disease vectors. In Anopheles mosquitoes, the composition of the midgut microbiota is largely influenced by the larval breeding site, but the exact factors shaping this composition are currently unknown. Here, we examined whether the proximity to urban areas and seasons have an impact on the midgut microbial community of the two major malaria vectors in Africa, An. coluzzii and An. gambiae. Larvae and pupae were collected from selected habitats in two districts of Ghana during the dry and rainy season periods. The midgut microbiota of adults that emerged from these collections was determined by 454-pyrosequencing of the 16S ribosomal DNA. We show that in both mosquito species, Shewanellaceae constituted on average of 54% and 73% of the midgut microbiota from each site in the dry and rainy season, respectively. Enterobacteriaceae was found in comparatively low abundance below 1% in 22/30 samples in the dry season, and in 25/38 samples in the rainy season. Our data indicate that seasonality and locality significantly affect both the diversity of microbiota and the relative abundance of bacterial families with a positive impact of dry season and peri-urban settings. PMID:27322614

  11. Apoptosis and necrosis during the circadian cycle in the centipede midgut.

    PubMed

    Rost-Roszkowska, M M; Chajec, Ł; Vilimova, J; Tajovský, K

    2016-07-01

    Three types of cells have been distinguished in the midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata: digestive, secretory, and regenerative cells. According to the results of our previous studies, we decided to analyze the relationship between apoptosis and necrosis in their midgut epithelium and circadian rhythms. Ultrastructural analysis showed that these processes proceed in a continuous manner that is independent of the circadian rhythm in L. forficatus, while in S. cingulata necrosis is activated at midnight. Additionally, the description of apoptosis and necrosis showed no differences between males and females of both species analyzed. At the beginning of apoptosis, the cell cytoplasm becomes electron-dense, apparently in response to shrinkage of the cell. Organelles such as the mitochondria, cisterns of endoplasmic reticulum transform and degenerate. Nuclei gradually assume lobular shapes before the apoptotic cell is discharged into the midgut lumen. During necrosis, however, the cytoplasm of the cell becomes electron-lucent, and the number of organelles decreases. While the digestive cells of about 10 % of L. forficatus contain rickettsia-like pathogens, the corresponding cells in S. cingulata are free of rickettsia. As a result, we can state that apoptosis in L. forficatus is presumably responsible for protecting the organism against infections, while in S. cingulata apoptosis is not associated with the elimination of pathogens. Necrosis is attributed to mechanical damage, and the activation of this process coincides with proliferation of the midgut regenerative cells at midnight in S. cingulata.

  12. Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

    PubMed Central

    Bittencourt-Cunha, Paula Rêgo; Silva-Cardoso, Livia; de Oliveira, Giselle Almeida; da Silva, José Roberto; da Silveira, Alan Barbosa; Kluck, George Eduardo Gabriel; Souza-Lima, Michele; Gondim, Katia Calp; Dansa-Petretsky, Marilvia; Silva, Carlos Peres; Masuda, Hatisaburo; da Silva, Mario Alberto Cardoso; Atella, Georgia Correa

    2013-01-01

    In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs. PMID:23827998

  13. Gene expression in midgut tissues of Diaphorina citri: Application to biology and vector control

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We produced a gene expression dataset from the midgut tissues of the Asian citrus psyllid (AsCP), Diaphorina citri (Hemiptera: Psyllidae). The AsCP is the primary vector associated with the spread of a devastating citrus trees disease, huanglongbing (HLB). The occurrence and spread of the AsCP and H...

  14. Midgut gene expression in Asian citrus psyllid (Hemiptera: Psyllidae) Diaphorina citri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We produced a gene expression dataset from the midgut tissues of the Asian citrus psyllid (AsCP), Diaphorina citri (Hemiptera: Psyllidae). The AsCP is the primary vector of the bacterium associated with a devastating citrus disease known as huanglongbing (HLB). The occurrence and spread of the AsCP ...

  15. Seasonality and Locality Affect the Diversity of Anopheles gambiae and Anopheles coluzzii Midgut Microbiota from Ghana.

    PubMed

    Akorli, Jewelna; Gendrin, Mathilde; Pels, Nana Adjoa P; Yeboah-Manu, Dorothy; Christophides, George K; Wilson, Michael D

    2016-01-01

    Symbiotic bacteria can have important implications in the development and competence of disease vectors. In Anopheles mosquitoes, the composition of the midgut microbiota is largely influenced by the larval breeding site, but the exact factors shaping this composition are currently unknown. Here, we examined whether the proximity to urban areas and seasons have an impact on the midgut microbial community of the two major malaria vectors in Africa, An. coluzzii and An. gambiae. Larvae and pupae were collected from selected habitats in two districts of Ghana during the dry and rainy season periods. The midgut microbiota of adults that emerged from these collections was determined by 454-pyrosequencing of the 16S ribosomal DNA. We show that in both mosquito species, Shewanellaceae constituted on average of 54% and 73% of the midgut microbiota from each site in the dry and rainy season, respectively. Enterobacteriaceae was found in comparatively low abundance below 1% in 22/30 samples in the dry season, and in 25/38 samples in the rainy season. Our data indicate that seasonality and locality significantly affect both the diversity of microbiota and the relative abundance of bacterial families with a positive impact of dry season and peri-urban settings.

  16. Midgut serine proteases and alternative host plant utilization in Pieris brassicae L.

    PubMed Central

    Kumar, Rakesh; Bhardwaj, Usha; Kumar, Pawan; Mazumdar-Leighton, Sudeshna

    2015-01-01

    Pieris brassicae L. is a serious pest of cultivated crucifers in several parts of the world. Larvae of P. brassicae also feed prolifically on garden nasturtium (Tropaeolum majus L., of the family Tropaeolaceae). Proteolytic digestion was studied in larvae feeding on multiple hosts. Fourth instars were collected from cauliflower fields before transfer onto detached, aerial tissues of selected host plants in the lab. Variable levels of midgut proteases were detected in larvae fed on different hosts using protein substrates (casein and recombinant RBCL cloned from cauliflower) and diagnostic, synthetic substrates. Qualitative changes in midgut trypsin activities and quantitative changes in midgut chymotrypsin activities were implicated in physiological adaptation of larvae transferred to T. majus. Midgut proteolytic activities were inhibited to different extents by serine protease inhibitors, including putative trypsin inhibitors isolated from herbivore-attacked and herbivore-free leaves of cauliflower (CfTI) and T. majus (TpTI). Transfer of larvae to T. majus significantly influenced feeding parameters but not necessarily when transferred to different tissues of the same host. Results obtained are relevant for devising sustainable pest management strategies, including transgenic approaches using genes encoding plant protease inhibitors. PMID:25873901

  17. Apoptosis and necrosis during the circadian cycle in the centipede midgut.

    PubMed

    Rost-Roszkowska, M M; Chajec, Ł; Vilimova, J; Tajovský, K

    2016-07-01

    Three types of cells have been distinguished in the midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata: digestive, secretory, and regenerative cells. According to the results of our previous studies, we decided to analyze the relationship between apoptosis and necrosis in their midgut epithelium and circadian rhythms. Ultrastructural analysis showed that these processes proceed in a continuous manner that is independent of the circadian rhythm in L. forficatus, while in S. cingulata necrosis is activated at midnight. Additionally, the description of apoptosis and necrosis showed no differences between males and females of both species analyzed. At the beginning of apoptosis, the cell cytoplasm becomes electron-dense, apparently in response to shrinkage of the cell. Organelles such as the mitochondria, cisterns of endoplasmic reticulum transform and degenerate. Nuclei gradually assume lobular shapes before the apoptotic cell is discharged into the midgut lumen. During necrosis, however, the cytoplasm of the cell becomes electron-lucent, and the number of organelles decreases. While the digestive cells of about 10 % of L. forficatus contain rickettsia-like pathogens, the corresponding cells in S. cingulata are free of rickettsia. As a result, we can state that apoptosis in L. forficatus is presumably responsible for protecting the organism against infections, while in S. cingulata apoptosis is not associated with the elimination of pathogens. Necrosis is attributed to mechanical damage, and the activation of this process coincides with proliferation of the midgut regenerative cells at midnight in S. cingulata. PMID:26277351

  18. Does autophagy in the midgut epithelium of centipedes depend on the day/night cycle?

    PubMed

    Rost-Roszkowska, M M; Chajec, Ł; Vilimova, J; Tajovský, K; Kszuk-Jendrysik, M

    2015-01-01

    The midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata, is composed of digestive, secretory and regenerative cells. In L. forficatus, the autophagy occurred only in the cytoplasm of the digestive cells as a sporadic process, while in S. cingulata, it occurred intensively in the digestive, secretory and regenerative cells of the midgut epithelium. In both of the species that were analyzed, this process proceeded in a continuous manner and did not depend on the day/night cycle. Ultrastructural analysis showed that the autophagosomes and autolysosomes were located mainly in the apical and perinuclear cytoplasm of the digestive cells in L. forficatus. However, in S. cingulata, the entire cytoplasm was filled with autophagosomes and autolysosomes. Initially the membranes of phagophores surround organelles during autophagosome formation. Autolysosomes result from the fusion of autophagosomes and lysosomes. Residual bodies which are the last stage of autophagy were released into the midgut lumen due to necrosis. Autophagy in the midgut epithelia that were analyzed was confirmed using acid phosphatase and mono-dansyl-cadaverine stainings. PMID:25464151

  19. Intravital imaging of Bacillus thuringiensis Cry1A toxin binding sites in the midgut of silkworm.

    PubMed

    Li, Na; Wang, Jing; Han, Heyou; Huang, Liang; Shao, Feng; Li, Xuepu

    2014-02-15

    Identification of the resistance mechanism of insects against Bacillus thuringiensis Cry1A toxin is becoming an increasingly challenging task. This fact highlights the need for establishing new methods to further explore the molecular interactions of Cry1A toxin with insects and the receptor-binding region of Cry1A toxins for their wider application as biopesticides and a gene source for gene-modified crops. In this contribution, a quantum dot-based near-infrared fluorescence imaging method has been applied for direct dynamic tracking of the specific binding of Cry1A toxins, CrylAa and CrylAc, to the midgut tissue of silkworm. The in vitro fluorescence imaging displayed the higher binding specificity of CrylAa-QD probes compared to CrylAc-QD to the brush border membrane vesicles of midgut from silkworm. The in vivo imaging demonstrated that more CrylAa-QDs binding to silkworm midgut could be effectively and distinctly monitored in living silkworms. Furthermore, frozen section analysis clearly indicated the broader receptor-binding region of Cry1Aa compared to that of Cry1Ac in the midgut part. These observations suggest that the insecticidal activity of Cry toxins may depend on the receptor-binding sites, and this scatheless and visual near-infrared fluorescence imaging could provide a new avenue to study the resistance mechanism to maintain the insecticidal activity of B. thuringiensis toxins. PMID:24252542

  20. Seasonality and Locality Affect the Diversity of Anopheles gambiae and Anopheles coluzzii Midgut Microbiota from Ghana.

    PubMed

    Akorli, Jewelna; Gendrin, Mathilde; Pels, Nana Adjoa P; Yeboah-Manu, Dorothy; Christophides, George K; Wilson, Michael D

    2016-01-01

    Symbiotic bacteria can have important implications in the development and competence of disease vectors. In Anopheles mosquitoes, the composition of the midgut microbiota is largely influenced by the larval breeding site, but the exact factors shaping this composition are currently unknown. Here, we examined whether the proximity to urban areas and seasons have an impact on the midgut microbial community of the two major malaria vectors in Africa, An. coluzzii and An. gambiae. Larvae and pupae were collected from selected habitats in two districts of Ghana during the dry and rainy season periods. The midgut microbiota of adults that emerged from these collections was determined by 454-pyrosequencing of the 16S ribosomal DNA. We show that in both mosquito species, Shewanellaceae constituted on average of 54% and 73% of the midgut microbiota from each site in the dry and rainy season, respectively. Enterobacteriaceae was found in comparatively low abundance below 1% in 22/30 samples in the dry season, and in 25/38 samples in the rainy season. Our data indicate that seasonality and locality significantly affect both the diversity of microbiota and the relative abundance of bacterial families with a positive impact of dry season and peri-urban settings. PMID:27322614

  1. Barber Pole Sign in CT Angiography, Adult Presentation of Midgut Malrotation: A Case Report

    PubMed Central

    Garcelan-Trigo, Juan Arsenio; Tello-Moreno, Manuel; Rabaza-Espigares, Manuel Jesus; Talavera-Martinez, Ildefonso

    2015-01-01

    Adult midgut volvulus is a challenging diagnosis because of its low incidence and nonspecific symptoms. Diagnostic delay and long-term complaints are frequent in this clinical scenario. We reported a patient referred to our diagnostic imaging unit with intermittent abdominal pain, bloating and episodic vomiting for several years. He underwent barium gastrointestinal transit and abdominal ultrasound, which revealed severe gastric dilatation, food retention and slow transit until a depressed duodenojejunal flexure, with malrotation of the midgut and jejunal loops being located in the right upper quadrant. Computed tomography angiography was performed, showing rotation of the small intestine around the mesentery root, suggestive of midgut malrotation. In addition, an abnormal twisted disposition of superior mesenteric artery with corkscrew appearance was seen, shaping the pole-barber sign which was evident in volume rendering three-dimensional reconstructions. The patient underwent scheduled surgical treatment without any complication and had good outcome after hospital discharge and follow-up. Computed tomography plays an important role in evaluation of adult midgut volvulus. In addition, angiographic reconstructions can help us to assess the anatomic disposition of mesenteric vascular supply. Both of these assessments are useful in preoperative management. PMID:26557278

  2. Transcriptome of the gypsy moth (Lymantria dispar) larval midgut in response to infection by Bacillus thuringiensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptomic profiles of the lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by the biopesticide Bacillus thuringiensis kurstaki. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which...

  3. Midgut epithelium in molting silkworm: A fine balance among cell growth, differentiation, and survival.

    PubMed

    Franzetti, Eleonora; Casartelli, Morena; D'Antona, Paola; Montali, Aurora; Romanelli, Davide; Cappellozza, Silvia; Caccia, Silvia; Grimaldi, Annalisa; de Eguileor, Magda; Tettamanti, Gianluca

    2016-07-01

    The midgut of insects has attracted great attention as a system for studying intestinal stem cells (ISCs) as well as cell death-related processes, such as apoptosis and autophagy. Among insects, Lepidoptera represent a good model to analyze these cells and processes. In particular, larva-larva molting is an interesting developmental phase since the larva must deal with nutrient starvation and its organs are subjected to rearrangements due to proliferation and differentiation events. Several studies have analyzed ISCs in vitro and characterized key factors involved in their division and differentiation during molt. However, in vivo studies performed during larva-larva transition on these cells, and on the whole midgut epithelium, are fragmentary. In the present study, we analyzed the larval midgut epithelium of the silkworm, Bombyx mori, during larva-larva molting, focusing our attention on ISCs. Moreover, we investigated the metabolic changes that occur in the epithelium and evaluated the intervention of autophagy. Our data on ISCs proliferation and differentiation, autophagy activation, and metabolic and functional activities of the midgut cells shed light on the complexity of this organ during the molting phase. PMID:27349418

  4. Midgut transcriptome profiling of Anoplophora glabripennis, a lignocellulose degrading Cerambycid beetle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Wood-feeding insects often work in collaboration with microbial symbionts to degrade lignin biopolymers and release glucose and other fermentable sugars from recalcitrant plant cell wall carbohydrates, including cellulose and hemicellulose. Here, we present the midgut transcriptome of la...

  5. Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

    PubMed Central

    2010-01-01

    Background Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. Conclusions BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora. PMID:20663211

  6. Ultrastructure of the digestive system and the fate of midgut during embryonic development in Porcellio scaber (Crustacea: Isopoda).

    PubMed

    Strus, Jasna; Klepal, Waltraud; Repina, Janja; Tusek-Znidaric, Magda; Milatovic, Masa; Pipan, Ziva

    2008-07-01

    Microscopic anatomy of the digestive system in embryos and larvae of the terrestrial isopod crustacean Porcellio scaber was investigated by light bright field, fluorescence and electron microscopy. During marsupial ontogenetic development the event-dependent staging was used to discriminate the various embryonic stages. At the late embryo stage the differentiation of the ectodermal part of the gut into the complex filtering foregut and the hindgut with absorptive and transporting functions is accomplished. The gut of the marsupial manca larva is fully developed and similar to that of the adult. In early embryos the endodermal midgut gland primordia are filled with yolk and lipid globules. In late embryos the epithelium of paired midgut gland tubes is composed of two cell types; one of them exhibits orange autofluorescence. The endodermal cells located between the foregut and the midgut glands of late embryos form the prospective midgut. The cells have electron dense cytoplasm, abundant glycogen fields, endoplasmic reticulum, dictyosomes and numerous vesicles. In the adults the endodermal cells of the midgut remain only in the midgut gland ducts which connect the midgut glands and the foregut. Details of the cellular ultrastructure and morphogenesis of the ectodermal and endodermal parts of the digestive system during embryonic development of Porcellio scaber provide data for further phylogenetic and comparative studies in peracaridan crustaceans and other arthropods.

  7. Purification, characterization and sequencing of the major beta-1,3-glucanase from the midgut of Tenebrio molitor larvae.

    PubMed

    Genta, Fernando A; Bragatto, Ivan; Terra, Walter R; Ferreira, Clélia

    2009-12-01

    The major beta-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an endoglucanase that hydrolyzes beta-1,3-glucans as laminarin and yeast beta-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched beta-1,3-glucans or mixed beta-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections.

  8. A trypsin-like proteinase in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae): purification, characterization, and host plant inhibitors.

    PubMed

    Ranjbar, Mina; Zibaee, Arash; Sendi, Jalal Jalali

    2014-01-01

    A trypsin-like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G-100 and DEAE-cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 μmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh-Sabz, May-Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17-20 of Brait; fractions 18 and 21-26 of Torsh-Sabz; fractions 1-7, 11-17, and 19-21 of May-Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May-khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.

  9. Fluid absorption in the isolated midgut of adult female yellow fever mosquitoes (Aedes aegypti).

    PubMed

    Onken, Horst; Moffett, David F

    2015-07-01

    The transepithelial voltage (Vte) and the volume of isolated posterior midguts of adult female yellow fever mosquitoes (Aedes aegypti) were monitored. In all experiments, the initial Vte after filling the midgut was lumen negative, but subsequently became lumen positive at a rate of approximately 1 mV min(-1). Simultaneously, the midgut volume decreased, indicating spontaneous fluid absorption. When the midguts were filled and bathed with mosquito saline, the average rate of fluid absorption was 36.5±3.0 nl min(-1) (N=4, ±s.e.m.). In the presence of theophylline (10 mmol l(-1)), Vte reached significantly higher lumen-positive values, but the rate of fluid absorption was not affected (N=6). In the presence of NaCN (5 mmol l(-1)), Vte remained close to 0 mV (N=4) and fluid absorption was reduced (14.4±1.3 nl min(-1), N=3, ±s.e.m.). When midguts were filled with buffered NaCl (154 mmol l(-1) plus 1 mmol l(-1) HEPES) and bathed in mosquito saline with theophylline, fluid absorption was augmented (50.0±5.8 nl min(-1), N=12, ±s.e.m.). Concanamycin A (10 µmol l(-1)), ouabain (1 mmol l(-1)), and acetazolamide (1 mmol l(-1)) affected Vte in different ways, but all reduced fluid absorption by 60-70% of the value before addition of the drugs.

  10. Composition of the Spruce Budworm (Choristoneura fumiferana) Midgut Microbiota as Affected by Rearing Conditions.

    PubMed

    Landry, Mathieu; Comeau, André M; Derome, Nicolas; Cusson, Michel; Levesque, Roger C

    2015-01-01

    The eastern spruce budworm (Choristoneura fumiferana) is one of the most destructive forest insect pests in Canada. Little is known about its intestinal microbiota, which could play a role in digestion, immune protection, communication and/or development. The present study was designed to provide a first characterization of the effects of rearing conditions on the taxonomic diversity and structure of the C. fumiferana midgut microbiota, using a culture-independent approach. Three diets and insect sources were examined: larvae from a laboratory colony reared on a synthetic diet and field-collected larvae reared on balsam fir or black spruce foliage. Bacterial DNA from the larval midguts was extracted to amplify and sequence the V6-V8 region of the 16S rRNA gene, using the Roche 454 GS-FLX technology. Our results showed a dominance of Proteobacteria, mainly Pseudomonas spp., in the spruce budworm midgut, irrespective of treatment group. Taxonomic diversity of the midgut microbiota was greater for larvae reared on synthetic diet than for those collected and reared on host plants, a difference that is likely accounted for by several factors. A greater proportion of bacteria from the phylum Bacteroidetes in insects fed artificial diet constituted the main difference between this group and those reared on foliage; within the phylum Proteobacteria, the presence of the genus Bradyrhizobium was also unique to insects reared on artificial diet. Strikingly, a Bray-Curtis analysis showed important differences in microbial diversity among the treatment groups, pointing to the importance of diet and environment in defining the spruce budworm midgut microbiota. PMID:26636571

  11. Composition of the Spruce Budworm (Choristoneura fumiferana) Midgut Microbiota as Affected by Rearing Conditions

    PubMed Central

    Landry, Mathieu; Comeau, André M.; Derome, Nicolas; Cusson, Michel; Levesque, Roger C.

    2015-01-01

    The eastern spruce budworm (Choristoneura fumiferana) is one of the most destructive forest insect pests in Canada. Little is known about its intestinal microbiota, which could play a role in digestion, immune protection, communication and/or development. The present study was designed to provide a first characterization of the effects of rearing conditions on the taxonomic diversity and structure of the C. fumiferana midgut microbiota, using a culture-independent approach. Three diets and insect sources were examined: larvae from a laboratory colony reared on a synthetic diet and field-collected larvae reared on balsam fir or black spruce foliage. Bacterial DNA from the larval midguts was extracted to amplify and sequence the V6-V8 region of the 16S rRNA gene, using the Roche 454 GS-FLX technology. Our results showed a dominance of Proteobacteria, mainly Pseudomonas spp., in the spruce budworm midgut, irrespective of treatment group. Taxonomic diversity of the midgut microbiota was greater for larvae reared on synthetic diet than for those collected and reared on host plants, a difference that is likely accounted for by several factors. A greater proportion of bacteria from the phylum Bacteroidetes in insects fed artificial diet constituted the main difference between this group and those reared on foliage; within the phylum Proteobacteria, the presence of the genus Bradyrhizobium was also unique to insects reared on artificial diet. Strikingly, a Bray-Curtis analysis showed important differences in microbial diversity among the treatment groups, pointing to the importance of diet and environment in defining the spruce budworm midgut microbiota. PMID:26636571

  12. Midgut Transcriptome of the Cockroach Periplaneta americana and Its Microbiota: Digestion, Detoxification and Oxidative Stress Response

    PubMed Central

    Zhang, Jianhua; Zhang, Yixi; Li, Jingjing; Liu, Meiling; Liu, Zewen

    2016-01-01

    The cockroach, Periplaneta americana, is an obnoxious and notorious pest of the world, with a strong ability to adapt to a variety of complex environments. However, the molecular mechanism of this adaptability is mostly unknown. In this study, the genes and microbiota composition associated with the adaptation mechanism were studied by analyzing the transcriptome and 16S rDNA pyrosequencing of the P. americana midgut, respectively. Midgut transcriptome analysis identified 82,905 unigenes, among which 64 genes putatively involved in digestion (11 genes), detoxification (37 genes) and oxidative stress response (16 genes) were found. Evaluation of gene expression following treatment with cycloxaprid further revealed that the selected genes (CYP6J1, CYP4C1, CYP6K1, Delta GST, alpha-amylase, beta-glucosidase and aminopeptidase) were upregulated at least 2.0-fold at the transcriptional level, and four genes were upregulated more than 10.0-fold. An interesting finding was that three digestive enzymes positively responded to cycloxaprid application. Tissue expression profiles further showed that most of the selected genes were midgut-biased, with the exception of CYP6K1. The midgut microbiota composition was obtained via 16S rDNA pyrosequencing and was found to be mainly dominated by organisms from the Firmicutes phylum, among which Clostridiales, Lactobacillales and Burkholderiales were the main orders which might assist the host in the food digestion or detoxification of noxious compounds. The preponderant species, Clostridium cellulovorans, was previously reported to degrade lignocellulose efficiently in insects. The abundance of genes involved in digestion, detoxification and response to oxidative stress, and the diversity of microbiota in the midgut might provide P. americana high capacity to adapt to complex environments. PMID:27153200

  13. A red tide alga grown under ocean acidification up-regulates its tolerance to lower pH by increasing its photophysiological functions

    NASA Astrophysics Data System (ADS)

    Chen, S.-W.; Beardall, J.; Gao, K.-S.

    2014-05-01

    Phaeocystis globosa, a red tide alga, often forms blooms in or adjacent to coastal waters and experiences changes of pH and seawater carbonate chemistry caused by either diel/periodic fluctuation in biological activity, human activity or, in the longer term, ocean acidification due to atmospheric CO2 rise. We examined the photosynthetic physiology of this species while growing it under different pH levels induced by CO2 enrichment and investigated its acclimation to carbonate chemistry changes under different light levels. Short-term exposure to reduced pHnbs (7.70) decreased the alga's photosynthesis and light use efficiency. However, acclimation to the reduced pH level for 1-19 generations led to recovered photosynthetic activity, being equivalent to that of cells grown under pH 8.07 (control), though such acclimation required a different time span (number of generations) under different light regimes. The low-pH grown cells increased their contents of chlorophyll and carotenoids with prolonged acclimation to the acidification, with increased photosynthetic quantum yield and decreased non-photochemical quenching. The specific growth rate of the low-pH grown cells also increased to emulate that grown under the ambient pH level. This study clearly shows that Phaeocystis globosa is able to acclimate to seawater acidification by increasing its energy capture and decreasing its non-photochemcial energy loss.

  14. Distribution of tetracycline resistance genes in anaerobic treatment of waste sludge: The role of pH in regulating tetracycline resistant bacteria and horizontal gene transfer.

    PubMed

    Huang, Haining; Chen, Yinguang; Zheng, Xiong; Su, Yinglong; Wan, Rui; Yang, Shouye

    2016-10-01

    Although pH value has been widely regarded as an important factor that affects resource recovery of waste sludge, the potential influence of diverse pHs on the distribution of tetracycline resistance genes (TRGs) during sludge anaerobic treatment is largely unknown. Here we reported that in the range of pH 4-10, 0.58-1.18 log unit increase of target TRGs was observed at pH 4, compared with that at pH 7, while 0.70-1.31 log unit further removal were obtained at pH 10. Mechanism study revealed that varied pHs not only altered the community structures of tetracycline resistant bacteria (TRB), but also changed their relative abundances, benefitting the propagation (acidic pHs) or attenuation (alkaline pHs) of TRB. Further investigation indicated that the amount and gene-possessing abilities of key genetic vectors for horizontal TRGs transfer were greatly promoted at acidic pHs but restricted under alkaline conditions. PMID:27485281

  15. Sandfly (Diptera, Psychodidae, Phlebotominae) fauna of South-Western Pakistan. 1. Diagnostic morphology of Phlebotomus papatasi (Scopoli), Ph. bergeroti (Parrot) and Ph. salehi (Mesghali).

    PubMed

    Kakarsulemankhel, Juma-Khan

    2003-06-01

    A survey was conducted to study the morphology of the sandfly fauna in South-Western Pakistan (Balochistan). During the revision of different genera of sandflies the specimens of Phlebotomus papatasi (Scopoli) (N = 720), Ph. bergeroti Parrot (N = 30) and Ph. salehi Mesghali (N = 70) were encountered in various localities. These localities appear to be new records of the subgenus in the literature to date. Ph. bergeroti is reported for the first time from Pakistan and Ph. salehi from Balochistan as well. Characters of these three Pakistanese Phlebotomus are compared with the published data of these species from other countries. Keys for the identification of Pakistanese Phlebotomus are also constructed. Two female Ph. papatasi collected from indoors out of 132 female flies (1.5%) were found positive with flagellate infection in pharynx and midgut. The possible vectorial role of these flies is also discussed. Further surveys are necessary in parts of the country that have not been systematically surveyed.

  16. Regulation of [15N]urea synthesis from [5-15N]glutamine. Role of pH, hormones, and pyruvate.

    PubMed

    Nissim, I; Yudkoff, M; Brosnan, J T

    1996-12-01

    We have utilized both [5-15N]glutamine and [3-13C] pyruvate as metabolic tracers in order to: (i) examine the effect of pH, glucagon (GLU), or insulin on the precursor-product relationship between 15NH3, [15N]citrulline, and, thereby, [15N]urea synthesis and (ii) elucidate the mechanism(s) by which pyruvate stimulates [15N] urea synthesis. Hepatocytes isolated from rat were incubated at pH 6.8, 7.4, or 7.6 with 1 mM [5-15N]glutamine and 0.1 mM 14NH4Cl in the presence or the absence of [3-13C] pyruvate (2 mM). A separate series of experiments was performed at pH 7.4 in the presence of insulin or GLU. 15NH3 enrichment exceeded or was equal to that of [15N]citrulline under all conditions except for pH 7.6, when the 15N enrichment in citrulline exceeded that in ammonia. The formation of [15N]citrulline (atom % excess) was increased with higher pH. Flux through phosphate-dependent glutaminase (PDG) and [15N]urea synthesis were stimulated (p < 0.05) at pH 7.6 or with GLU and decreased (p < 0.05) at pH 6.8. Insulin had no significant effect on flux through PDG or on [15N]urea synthesis. Decreased [15N]urea production at pH 6.8 was associated with depleted aspartate and glutamate levels. Pyruvate attenuated this decrease in the aspartate and glutamate pools and stimulated [15N]urea synthesis. Production of Asp from pyruvate was increased with increasing medium pH. Approximately 80% of Asp was derived from [3-13C]pyruvate regardless of incubation pH or addition of hormone. Furthermore, approximately 20, 40, and 50% of the mitochondrial N-acetylglutamate (NAG) pool was derived from [3-13C]pyruvate at pH 6.8, 7.4, and 7.6, respectively. Both the concentration and formation of [13C]NAG from [3-13C]pyruvate were increased (p < 0.05) with glucagon and decreased (p < 0.05) with insulin or at pH 6.8. The data suggest a correlation between changes in [15N]urea synthesis and alterations in the level and synthesis of [13C]NAG from pyruvate. The current observations suggest that the

  17. S-nitrosothiols regulate cell-surface pH buffering by airway epithelial cells during the human immune response to rhinovirus.

    PubMed

    Carraro, Silvia; Doherty, Joseph; Zaman, Khalequz; Gainov, Iain; Turner, Ronald; Vaughan, John; Hunt, John F; Márquez, Javier; Gaston, Benjamin

    2006-05-01

    Human rhinovirus infection is a common trigger for asthma exacerbations. Asthma exacerbations and rhinovirus infections are both associated with markedly decreased pH and ammonium levels in exhaled breath condensates. This observation is thought to be related, in part, to decreased activity of airway epithelial glutaminase. We studied whether direct rhinovirus infection and/or the host immune response to the infection decreased airway epithelial cell surface pH in vitro. Interferon-gamma and tumor necrosis factor-alpha, but not direct rhinovirus infection, decreased pH, an effect partly associated with decreased ammonium concentrations. This effect was 1) prevented by nitric oxide synthase inhibition; 2) independent of cyclic GMP; 3) associated with an increase in endogenous airway epithelial cell S-nitrosothiol concentration; 4) mimicked by the exogenous S-nitrosothiol, S-nitroso-N-acetyl cysteine; and 5) independent of glutaminase expression and activity. We then confirmed that decreased epithelial pH inhibits human rhinovirus replication in airway epithelial cells. These data suggest that a nitric oxide synthase-dependent host response to viral infection mediated by S-nitrosothiols, rather than direct infection itself, plays a role in decreased airway surface pH during human rhinovirus infection. This host immune response may serve to protect the lower airways from direct infection in the normal host. In patients with asthma, however, this fall in pH could be associated with the increased mucus production, augmented inflammatory cell degranulation, bronchoconstriction, and cough characteristic of an asthma exacerbation. PMID:16603595

  18. Developmental Expression of Ecdysone-Related Genes Associated With Metamorphic Changes During Midgut Remodeling of Silkworm Bombyx mori (Lepidoptera:Bombycidae).

    PubMed

    Goncu, Ebru; Uranlı, Ramazan; Selek, Gozde; Parlak, Osman

    2016-01-01

    Steroid hormone 20-hydroxyecdysone is known as the systemic regulators of insect cells; however, how to impact the fate and function of mature and stem cells is unclear. For the first time, we report ecdysone regulatory cascades in both mature midgut cell and stem cell fractions related to developmental events by using histological, immunohistochemical, biochemical and gene expression analysis methods. Ecdysone receptor-B1 (EcR-B1) and ultraspiracle 1 (USP-1) mRNAs were detected mainly in mature cells during programmed cell death (PCD). Lowered E75A and probably BR-C Z4 in mature cells appear to provide a signal to the initiation of PCD. E74B, E75B and BR-C Z2 seem to be early response genes which are involved in preparatory phase of cell death. It is likely that βFTZ-F1, E74A and BR-C Z1 are probably associated with execution of death. EcR-A and USP2 mRNAs were found in stem cells during remodeling processes but EcR-B1, USP1 and E74B genes imply an important role during initial phase of metamorphic events in stem cells. BHR3 mRNAs were determined abundantly in stem cells suggesting its primary role in differentiation. All of these results showed the determination the cell fate in Bombyx mori (Linnaeus) midgut depends on type of ecdysone receptor isoforms and ecdysone-related transcription factors.

  19. Developmental Expression of Ecdysone-Related Genes Associated With Metamorphic Changes During Midgut Remodeling of Silkworm Bombyx mori (Lepidoptera:Bombycidae)

    PubMed Central

    Goncu, Ebru; Uranlı, Ramazan; Selek, Gozde; Parlak, Osman

    2016-01-01

    Steroid hormone 20-hydroxyecdysone is known as the systemic regulators of insect cells; however, how to impact the fate and function of mature and stem cells is unclear. For the first time, we report ecdysone regulatory cascades in both mature midgut cell and stem cell fractions related to developmental events by using histological, immunohistochemical, biochemical and gene expression analysis methods. Ecdysone receptor-B1 (EcR-B1) and ultraspiracle 1 (USP-1) mRNAs were detected mainly in mature cells during programmed cell death (PCD). Lowered E75A and probably BR-C Z4 in mature cells appear to provide a signal to the initiation of PCD. E74B, E75B and BR-C Z2 seem to be early response genes which are involved in preparatory phase of cell death. It is likely that βFTZ-F1, E74A and BR-C Z1 are probably associated with execution of death. EcR-A and USP2 mRNAs were found in stem cells during remodeling processes but EcR-B1, USP1 and E74B genes imply an important role during initial phase of metamorphic events in stem cells. BHR3 mRNAs were determined abundantly in stem cells suggesting its primary role in differentiation. All of these results showed the determination the cell fate in Bombyx mori (Linnaeus) midgut depends on type of ecdysone receptor isoforms and ecdysone-related transcription factors. PMID:27620558

  20. Developmental Expression of Ecdysone-Related Genes Associated With Metamorphic Changes During Midgut Remodeling of Silkworm Bombyx mori (Lepidoptera:Bombycidae)

    PubMed Central

    Goncu, Ebru; Uranlı, Ramazan; Selek, Gozde; Parlak, Osman

    2016-01-01

    Steroid hormone 20-hydroxyecdysone is known as the systemic regulators of insect cells; however, how to impact the fate and function of mature and stem cells is unclear. For the first time, we report ecdysone regulatory cascades in both mature midgut cell and stem cell fractions related to developmental events by using histological, immunohistochemical, biochemical and gene expression analysis methods. Ecdysone receptor-B1 (EcR-B1) and ultraspiracle 1 (USP-1) mRNAs were detected mainly in mature cells during programmed cell death (PCD). Lowered E75A and probably BR-C Z4 in mature cells appear to provide a signal to the initiation of PCD. E74B, E75B and BR-C Z2 seem to be early response genes which are involved in preparatory phase of cell death. It is likely that βFTZ-F1, E74A and BR-C Z1 are probably associated with execution of death. EcR-A and USP2 mRNAs were found in stem cells during remodeling processes but EcR-B1, USP1 and E74B genes imply an important role during initial phase of metamorphic events in stem cells. BHR3 mRNAs were determined abundantly in stem cells suggesting its primary role in differentiation. All of these results showed the determination the cell fate in Bombyx mori (Linnaeus) midgut depends on type of ecdysone receptor isoforms and ecdysone-related transcription factors.

  1. Developmental Expression of Ecdysone-Related Genes Associated With Metamorphic Changes During Midgut Remodeling of Silkworm Bombyx mori (Lepidoptera:Bombycidae).

    PubMed

    Goncu, Ebru; Uranlı, Ramazan; Selek, Gozde; Parlak, Osman

    2016-01-01

    Steroid hormone 20-hydroxyecdysone is known as the systemic regulators of insect cells; however, how to impact the fate and function of mature and stem cells is unclear. For the first time, we report ecdysone regulatory cascades in both mature midgut cell and stem cell fractions related to developmental events by using histological, immunohistochemical, biochemical and gene expression analysis methods. Ecdysone receptor-B1 (EcR-B1) and ultraspiracle 1 (USP-1) mRNAs were detected mainly in mature cells during programmed cell death (PCD). Lowered E75A and probably BR-C Z4 in mature cells appear to provide a signal to the initiation of PCD. E74B, E75B and BR-C Z2 seem to be early response genes which are involved in preparatory phase of cell death. It is likely that βFTZ-F1, E74A and BR-C Z1 are probably associated with execution of death. EcR-A and USP2 mRNAs were found in stem cells during remodeling processes but EcR-B1, USP1 and E74B genes imply an important role during initial phase of metamorphic events in stem cells. BHR3 mRNAs were determined abundantly in stem cells suggesting its primary role in differentiation. All of these results showed the determination the cell fate in Bombyx mori (Linnaeus) midgut depends on type of ecdysone receptor isoforms and ecdysone-related transcription factors. PMID:27620558

  2. [Estimation of the biological age in males of the taiga tick (Ixodes persulcatus: Ixodinae) by fat reserves in the midgut].

    PubMed

    Grigor'eva, L A

    2012-01-01

    Some criteria for the estimation of the biological and calendar age by the fat storage in midgut cells of Ixodes persulcatus males were established on the basis of examination of ticks from the laboratory culture.

  3. Gene expression and localization analysis of Bombyx mori bidensovirus and its putative receptor in B. mori midgut.

    PubMed

    Ito, Katsuhiko; Shimura, Sachiko; Katsuma, Susumu; Tsuda, Yasuhiro; Kobayashi, Jun; Tabunoki, Hiroko; Yokoyama, Takeshi; Shimada, Toru; Kadono-Okuda, Keiko

    2016-05-01

    Bombyx mori bidensovirus (BmBDV), which causes fatal flacherie disease in the silkworm, replicates only in midgut columnar cells. The viral resistance expressed by some silkworm strains, which is characterized as non-susceptibility irrespective of the viral dose, is determined by a single gene, nsd-2. We previously identified nsd-2 by positional cloning and found that this gene encodes a putative amino acid transporter that might function as a receptor for BmBDV. In this study, we investigated the relationship between the part of the midgut expressing nsd-2 (resistance gene), +(nsd-2) (susceptibility gene) and BmBDV propagation. Quantitative RT-PCR (qRT-PCR) analysis using total RNA isolated from the anterior, middle, and posterior parts of the midgut showed that nsd-2 and +(nsd-2) were strongly expressed in the posterior part of the midgut. The expression levels of both genes were very low in the anterior and middle parts. The qRT-PCR analysis showed that the expression levels of BmBDV-derived transcripts were correlated with the levels of +(nsd-2) expression. However, BmBDV-derived transcripts were clearly detected in all parts of the midgut. These results suggest that the infectivity of BmBDV depends mainly on the expression level of +(nsd-2) in the midgut and that viral infection is supported even by very faint expression of +(nsd-2). By contrast, the expression levels of +(nsd-2) were exceedingly low or undetectable in the middle part of the midgut, indicating that BmBDV infection might occur via another mechanism, independent of +(nsd-2), in the middle part of the midgut. PMID:26953258

  4. Transcriptome Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

    PubMed Central

    Kolliopoulou, Anna; Van Nieuwerburgh, Filip; Stravopodis, Dimitrios J.; Deforce, Dieter; Swevers, Luc; Smagghe, Guy

    2015-01-01

    Many insects can be persistently infected with viruses but do not show any obvious adverse effects with respect to physiology, development or reproduction. Here, Bombyx mori strain Daizo, persistently infected with cytoplasmic polyhedrosis virus (BmCPV), was used to study the host’s transcriptional response after pathogenic infection with the same virus in midgut tissue of larvae persistently and pathogenically infected as 2nd and 4th instars. Next generation sequencing revealed that from 13,769 expressed genes, 167 were upregulated and 141 downregulated in both larval instars following pathogenic infection. Several genes that could possibly be involved in B. mori immune response against BmCPV or that may be induced by the virus in order to increase infectivity were identified, whereas classification of differentially expressed transcripts (confirmed by qRT-PCR) resulted in gene categories related to physical barriers, immune responses, proteolytic / metabolic enzymes, heat-shock proteins, hormonal signaling and uncharacterized proteins. Comparison of our data with the available literature (pathogenic infection of persistently vs. non-persistently infected larvae) unveiled various similarities of response in both cases, which suggests that pre-existing persistent infection does not affect in a major way the transcriptome response against pathogenic infection. To investigate the possible host’s RNAi response against BmCPV challenge, the differential expression of RNAi-related genes and the accumulation of viral small RNAs (vsRNAs) were studied. During pathogenic infection, siRNA-like traces like the 2-fold up-regulation of the core RNAi genes Ago-2 and Dcr-2 as well as a peak of 20 nt small RNAs were observed. Interestingly, vsRNAs of the same size were detected at lower rates in persistently infected larvae. Collectively, our data provide an initial assessment of the relative significance of persistent infection of silkworm larvae on the host response following

  5. pH regulation in adult rat carotid body glomus cells. Importance of extracellular pH, sodium, and potassium [published erratum appears in J Gen Physiol 1993 Jan;101(1):following 144

    PubMed Central

    1992-01-01

    The course of intracellular pH (pHi) was followed in superfused (36 degrees C) single glomus (type I) cells of the freshly dissociated adult rat carotid body. The cells had been loaded with the pH-sensitive fluorescent dye 2',7'-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein. The high K(+)-nigericin method was used for calibration. The pHi of the glomus cell at pHo 7.40, without CO2, was 7.23 +/- 0.02 (n = 70); in 5% CO2/25 mM HCO3-, pHi was 7.18 +/- 0.08 (n = 9). The pHi was very sensitive to changes in pHo. Without CO2, delta pHi/delta pHo was 0.85 (pHo 6.20-8.00; 32 cells), while in CO2/HCO3- this ratio was 0.82 irrespective of whether pHo (6.80-7.40; 14 cells) was changed at constant PCO2 or at constant [HCO3-]o. The great pHi sensitivity of the glomus cell to pHo is matched only by that of the human red cell. An active Na+/H+ exchanger (apparent Km = 58 +/- 6 mM) is present in glomus cells: Na+ removal or addition of the amiloride derivative 5- (N,N-hexamethylene)-amiloride induced pHi to fall by as much as 0.9. The membrane of these cells also contains a K+/H+ exchanger. Raising [K+]o from 4.7 to 25, 50, or 140 mM reversibly raised pHi by 0.2, 0.3, and 0.6, respectively. Rb+ had no effect, but in corresponding concentrations of Tl+ alkalinization was much faster than in K+. Reducing [K+]o to 1.5 mM lowered pHi by 0.1. These pHi changes were shown not to be due to changes in membrane voltage, and were even more striking in the absence of Na+. Intrinsic buffering power (amount of strong base required to produce, in the nominal absence of CO2, a small pHi rise) increased from 3 to approximately 21 mM as pHi was lowered, but remained nearly unchanged below pHi 6.60. The fitted expression assumed the presence of one "equivalent" intracellular buffer (pK 6.41, 41 mM). The exceptional pHi sensitivity to pHo suggests that the pHi of the glomus cell is a link in the chemoreceptor's response to external acidity. PMID:1294152

  6. Regulation of hemoglobin function and whole blood oxygen affinity by carbon dioxide and pH in the loggerhead (Caretta caretta) and green sea turtle (Chelonia mydas mydas).

    PubMed

    Isaacks, R E; Harkness, D R; White, J R

    1982-01-01

    The oxygen affinity of suspensions of erythrocytes from juvenile and adult loggerhead (Caretta caretta) and green sea (Chelonia mydas mydas) turtles decreased markedly with increasing concentrations of carbon dioxide (0 to near 15%) or hydrogen ion (pH 7.6 to pH 7.2). The P50's were higher with increases in pCO2, particularly at pH near 7.4, than were the P50's with increases in hydrogen ion concentration at any given CO2 concentration. Solutions of hemoglobins from the juvenile loggerhead (8-9 mos.) and green sea (10 mos.) turtles responded to 2, 3-DPG, ATP, or inositol-P5 when added at molar ratios of phosphate to hemoglobin of 4:1 and 20:1 in 0% and 6.29% CO2 but showed no decrease in oxygen affinity at these two CO2 levels in the green turtle when the molar ratio of phosphate to hemoglobin was 0.4. These compounds had little effect on the P50 of these hemoglobins in 14.6% CO2. The P50 of the adult loggerhead turtle hemoglobin did not increase in the presence of organic phosphates beyond the effect induced by CO2 alone. The P50 of hemoglobin from the adult green sea turtle increased only slightly when the molar ratio of phosphate to hemoglobin was 20:1 and at 0 and 6% CO2 concentration; little effect was observed at 14.6% CO2. These data demonstrate that blood oxygen affinities and hemoglobin function in these two species of marine turtles are altered significantly by CO2 and to a lesser degree by pH. It is suggested that such alterations may be of significance in vivo during prolonged diving when there are dramatic rises in blood pCO2 and [H+] and profound decreases in pO2.

  7. The influence of long-term copper contaminated agricultural soil at different pH levels on microbial communities and springtail transcriptional regulation.

    PubMed

    de Boer, Tjalf E; Taş, Neslihan; Braster, Martin; Temminghoff, Erwin J M; Röling, Wilfred F M; Roelofs, Dick

    2012-01-01

    Copper has long been applied for agricultural practises. Like other metals, copper is highly persistent in the environment and biologically active long after its use has ceased. Here we present a unique study on the long-term effects (27 years) of copper and pH on soil microbial communities and on the springtail Folsomia candida an important representative of the soil macrofauna, in an experiment with a full factorial, random block design. Bacterial communities were mostly affected by pH. These effects were prominent in Acidobacteria, while Actinobacteria and Gammaroteobacteria communities were affected by original and bioavailable copper. Reproduction and survival of the collembolan F. candida was not affected by the studied copper concentrations. However, the transcriptomic responses to copper reflected a mechanism of copper transport and detoxification, while pH exerted effects on nucleotide and protein metabolism and (acute) inflammatory response. We conclude that microbial community structure reflected the history of copper contamination, while gene expression analysis of F. candida is associated with the current level of bioavailable copper. The study is a first step in the development of a molecular strategy aiming at a more comprehensive assessment of various aspects of soil quality and ecotoxicology. PMID:21882881

  8. Regulation of pH attenuates toxicity of a byproduct produced by an ethanologenic strain of Sphingomonas sp. A1 during ethanol fermentation from alginate.

    PubMed

    Fujii, Mari; Yoshida, Shiori; Murata, Kousaku; Kawai, Shigeyuki

    2014-01-01

    Marine macroalgae is a promising carbon source that contains alginate and mannitol as major carbohydrates. A bioengineered ethanologenic strain of the bacterium Sphingomonas sp. A1 can produce ethanol from alginate, but not mannitol, whereas the yeast Saccharomyces paradoxus NBRC 0259-3 can produce ethanol from mannitol, but not alginate. Thus, one practical approach for converting both alginate and mannitol into ethanol would involve two-step fermentation, in which the ethanologenic bacterium initially converts alginate into ethanol, and then the yeast produces ethanol from mannitol. In this study, we found that, during fermentation from alginate, the ethanologenic bacterium lost viability and secreted toxic byproducts into the medium. These toxic byproducts inhibited bacterial growth and killed bacterial cells and also inhibited growth of S. paradoxus NBRC 0259-3. We discovered that adjusting the pH of the culture supernatant or the culture medium containing the toxic byproducts to 6.0 attenuated the toxicity toward both bacteria and yeast, and also extended the period of viability of the bacterium. Although continuous adjustment of pH to 6.0 failed to improve the ethanol productivity of this ethanologenic bacterium, this pH adjustment worked very well in the two-step fermentation due to the attenuation of toxicity toward S. paradoxus NBRC 0259-3. These findings provide information critical for establishment of a practical system for ethanol production from brown macroalgae.

  9. HCO3- Transport through Anoctamin/Transmembrane Protein ANO1/TMEM16A in Pancreatic Acinar Cells Regulates Luminal pH.

    PubMed

    Han, Yanfeng; Shewan, Annette M; Thorn, Peter

    2016-09-23

    The identification of ANO1/TMEM16A as the likely calcium-dependent chloride channel of exocrine glands has led to a more detailed understanding of its biophysical properties. This includes a calcium-dependent change in channel selectivity and evidence that HCO3 (-) permeability can be significant. Here we use freshly isolated pancreatic acini that preserve the luminal structure to measure intraluminal pH and test the idea that ANO1/TMEM16A contributes to luminal pH balance. Our data show that, under physiologically relevant stimulation with 10 pm cholesystokinin, the luminal acid load that results from the exocytic fusion of zymogen granules is significantly blunted by HCO3 (-) buffer in comparison with HEPES, and that this is blocked by the specific TMEM16A inhibitor T16inh-A01. Furthermore, in a model of acute pancreatitis, we observed substantive luminal acidification and provide evidence that ANO1/TMEM16A acts to attenuate this pH shift. We conclude that ANO1/TMEM16A is a significant pathway in pancreatic acinar cells for HCO3 (-) secretion into the lumen.

  10. Fine-structural changes in the midgut of old Drosophila melanogaster

    NASA Technical Reports Server (NTRS)

    Anton-Erxleben, F.; Miquel, J.; Philpott, D. E.

    1983-01-01

    Senescent fine-structural changes in the midgut of Drosophila melanogaster are investigated. A large number of midgut mitochondria in old flies exhibit nodular cristae and a tubular system located perpendicular to the normal cristae orientation. Anterior intestinal cells show a senescent accumulation of age pigment, either with a surrounding two-unit membrane or without any membrane. The predominant localization of enlarged mitochondria and pigment in the luminal gut region may be related to the polarized metabolism of the intestinal cells. Findings concur with previous observations of dense-body accumulations and support the theory that mitochondria are involved in the aging of fixed post-mitotic cells. Demonstrated by statistical analyses is that mitochondrial size increase is related to mitochondrial variation increase.

  11. Two genes encoding midgut-specific maltase-like polypeptides from Anopheles gambiae.

    PubMed

    Zheng, L; Whang, L H; Kumar, V; Kafatos, F C

    1995-11-01

    Full-length cDNA clones of two genes have been isolated from the African malaria vector mosquito, Anopheles gambiae. These genes, designated Agm1 and Agm2, encode maltase-like polypeptides of 498 and 599 residues, respectively. Deduced amino acid sequences contain a putative signal peptide sequence and four potential glycosylation sites. Agm1 and Agm2 show highest similarities to the Mal1 gene from Aedes aegypti and three clustered maltase genes from Drosophila melanogaster. Both genes are located at position 46D, in the terminal division of the left arm of the third chromosome. Agm2 has very strict tissue and temporal specificity, being expressed exclusively in the adult midgut. The specificity of Agm1 is similar but appears slightly broader; transcripts of this gene are detected at a low level in the pupae, and occasionally in the adult carcass after removal of the midgut.

  12. Cytotoxic effects of neem oil in the midgut of the predator Ceraeochrysa claveri.

    PubMed

    Scudeler, Elton Luiz; Garcia, Ana Silvia Gimenes; Padovani, Carlos Roberto; Pinheiro, Patricia Fernanda Felipe; dos Santos, Daniela Carvalho

    2016-01-01

    Studies of morphological and ultrastructural alterations in target organs have been useful for evaluating the sublethal effects of biopesticides regarded as safe for non-target organisms in ecotoxicological analyses. One of the most widely used biopesticides is neem oil, and its safety and compatibility with natural enemies have been further clarified through bioassays performed to analyze the effects of indirect exposure by the intake of poisoned prey. Thus, this study examined the cellular response of midgut epithelial cells of the adult lacewing, Ceraeochrysa claveri, to neem oil exposure via intake of neem oil-contaminated prey during the larval stage. C. claveri larvae were fed Diatraea saccharalis eggs treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval stage. The adult females obtained from these treatments were used at two ages (newly emerged and at the start of oviposition) in morphological and ultrastructural analyses. Neem oil was found to cause pronounced cytotoxic effects in the adult midgut, such as cell dilation, emission of cytoplasmic protrusions, cell lysis, loss of integrity of the cell cortex, dilation of cisternae of the rough endoplasmic reticulum, swollen mitochondria, vesiculated appearance of the Golgi complex and dilated invaginations of the basal labyrinth. Epithelial cells responded to those injuries with various cytoprotective and detoxification mechanisms, including increases in cell proliferation, the number of calcium-containing cytoplasmic granules, and HSP 70 expression, autophagic processes and the development of smooth endoplasmic reticulum, but these mechanisms were insufficient for recovery from all of the cellular damage to the midgut. This study demonstrates that neem oil exposure impairs the midgut by causing sublethal effects that may affect the physiological functions of this organ, indicating the importance of studies of different life stages of this species and similar species to evaluate the

  13. Cytotoxic effects of neem oil in the midgut of the predator Ceraeochrysa claveri.

    PubMed

    Scudeler, Elton Luiz; Garcia, Ana Silvia Gimenes; Padovani, Carlos Roberto; Pinheiro, Patricia Fernanda Felipe; dos Santos, Daniela Carvalho

    2016-01-01

    Studies of morphological and ultrastructural alterations in target organs have been useful for evaluating the sublethal effects of biopesticides regarded as safe for non-target organisms in ecotoxicological analyses. One of the most widely used biopesticides is neem oil, and its safety and compatibility with natural enemies have been further clarified through bioassays performed to analyze the effects of indirect exposure by the intake of poisoned prey. Thus, this study examined the cellular response of midgut epithelial cells of the adult lacewing, Ceraeochrysa claveri, to neem oil exposure via intake of neem oil-contaminated prey during the larval stage. C. claveri larvae were fed Diatraea saccharalis eggs treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval stage. The adult females obtained from these treatments were used at two ages (newly emerged and at the start of oviposition) in morphological and ultrastructural analyses. Neem oil was found to cause pronounced cytotoxic effects in the adult midgut, such as cell dilation, emission of cytoplasmic protrusions, cell lysis, loss of integrity of the cell cortex, dilation of cisternae of the rough endoplasmic reticulum, swollen mitochondria, vesiculated appearance of the Golgi complex and dilated invaginations of the basal labyrinth. Epithelial cells responded to those injuries with various cytoprotective and detoxification mechanisms, including increases in cell proliferation, the number of calcium-containing cytoplasmic granules, and HSP 70 expression, autophagic processes and the development of smooth endoplasmic reticulum, but these mechanisms were insufficient for recovery from all of the cellular damage to the midgut. This study demonstrates that neem oil exposure impairs the midgut by causing sublethal effects that may affect the physiological functions of this organ, indicating the importance of studies of different life stages of this species and similar species to evaluate the

  14. Malrotation and Midgut Volvulus associated with Asymptomatic Duplication Cyst of Jejunum.

    PubMed

    Rahul, Sandip Kumar; Upadhyaya, Vijai Datta; Kumar, Basant

    2016-01-01

    Gastrointestinal duplications can affect any part of the alimentary tract and are notorious for their variable presentation. Their association with malrotation and midgut volvulus is rare. We describe an 8-year old boy presented with episodes of abdominal pain. Radiological workup showed whirlpool sign and abnormal relationship of mesenteric vessels. At operation, malrotation with chronic volvulus was found. Incidentally, a jejunal communicating duplication cyst was also noted. PMID:27672583

  15. Malrotation and Midgut Volvulus associated with Asymptomatic Duplication Cyst of Jejunum

    PubMed Central

    Upadhyaya, Vijai Datta; Kumar, Basant

    2016-01-01

    Gastrointestinal duplications can affect any part of the alimentary tract and are notorious for their variable presentation. Their association with malrotation and midgut volvulus is rare. We describe an 8-year old boy presented with episodes of abdominal pain. Radiological workup showed whirlpool sign and abnormal relationship of mesenteric vessels. At operation, malrotation with chronic volvulus was found. Incidentally, a jejunal communicating duplication cyst was also noted. PMID:27672583

  16. Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut.

    PubMed

    Genta, Fernando A; Blanes, Lucas; Cristofoletti, Plínio T; do Lago, Claudimir L; Terra, Walter R; Ferreira, Clélia

    2006-10-01

    Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest k(cat)/K(M) value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal Ser-Thr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane.

  17. The cytological effect of ecdysterone on the midgut cells of the flesh-fly Sarcophaga bullata.

    PubMed

    Radford, S V; Misch, D W

    1971-06-01

    Larvae of the flesh-fly, Sarcophaga bullata, were injected with the synthetic moulting hormone ecdysterone or saline at the beginning of the third and final larval instar. One group was left untreated. The ecdysterone-injected larvae showed an increase in number of secondary lysosomes in the midgut epithelial cells similar to that observed at the onset of metamorphosis, an event which would normally occur about 48 hr later in these larvae.

  18. THE CYTOLOGICAL EFFECT OF ECDYSTERONE ON THE MIDGUT CELLS OF THE FLESH-FLY SARCOPHAGA BULLATA

    PubMed Central

    Radford, Sharon V.; Misch, Donald W.

    1971-01-01

    Larvae of the flesh-fly, Sarcophaga bullata, were injected with the synthetic moulting hormone ecdysterone or saline at the beginning of the third and final larval instar. One group was left untreated. The ecdysterone-injected larvae showed an increase in number of secondary lysosomes in the midgut epithelial cells similar to that observed at the onset of metamorphosis, an event which would normally occur about 48 hr later in these larvae. PMID:5092208

  19. Incidental midgut malrotation detected during second laparotomy: Case report and literature review

    PubMed Central

    Vural, Veli; Türkoğlu, Mehmet Akif; Karatas, Gulnur

    2014-01-01

    Introduction Intestinal malrotation is defined as intestinal nonrotation or incomplete rotation around superior mesenteric artery (SMA), involving anomalies of intestinal fixation as well. The patients may be recognized incidentally during other surgical procedures or at autopsy. Here in, we present a case of midgut malrotation which was diagnosed incidentally during hepaticojejunostomy procedure for benign biliary stricture. Presentation of case A 46 years old male patient was referred to our clinic with failed surgery for biliary stricture due to extensive adhesions. Prior to our surgery, intestinal malrotation was not reported and noticed by the diagnostic tools. When the patient underwent relaparotomy, midgut malrotation was observed. Discussion Distruption in the normal embryological development of bowel is the cause of intestinal malrotation. Various anatomic configurations and anomalies resulting from rotation anomalies of midgut. Adult patients are usually asymptomatic and the anomaly is discovered only at autopsy or incidentally at surgery. The role of additional surgery especially in patients with asymptomatic disease related to malrotation is debated. Conclusion Performing loop hepaticojejunostomy with Braun enteroenterostomy is feasible and acceptable option rather than Roux-N-Y hepaticojejunostomy in case of intestinal malrotation. PMID:25533325

  20. Cytotoxic effects of thiamethoxam in the midgut and malpighian tubules of Africanized Apis mellifera (Hymenoptera: Apidae).

    PubMed

    Catae, Aline Fernanda; Roat, Thaisa Cristina; De Oliveira, Regiane Alves; Nocelli, Roberta Cornélio Ferreira; Malaspina, Osmar

    2014-04-01

    Due to its expansion, agriculture has become increasingly dependent on the use of pesticides. However, the indiscriminate use of insecticides has had additional effects on the environment. These products have a broad spectrum of action, and therefore the insecticide affects not only the pests but also non-target insects such as bees, which are important pollinators of agricultural crops and natural environments. Among the most used pesticides, the neonicotinoids are particularly harmful. One of the neonicotinoids of specific concern is thiamethoxam, which is used on a wide variety of crops and is toxic to bees. Thus, this study aimed to analyze the effects of this insecticide in the midgut and Malpighian tubule cells of Africanized Apis mellifera. Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam equal to 1/10 of LC₅₀ (0.0428 ng a.i./l L of diet). The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide.

  1. Shifts in the Midgut/Pyloric Microbiota Composition within a Honey Bee Apiary throughout a Season.

    PubMed

    Ludvigsen, Jane; Rangberg, Anbjørg; Avershina, Ekaterina; Sekelja, Monika; Kreibich, Claus; Amdam, Gro; Rudi, Knut

    2015-01-01

    Honey bees (Apis mellifera) are prominent crop pollinators and are, thus, important for effective food production. The honey bee gut microbiota is mainly host specific, with only a few species being shared with other insects. It currently remains unclear how environmental/dietary conditions affect the microbiota within a honey bee population over time. Therefore, the aim of the present study was to characterize the composition of the midgut/pyloric microbiota of a honey bee apiary throughout a season. The rationale for investigating the midgut/pyloric microbiota is its dynamic nature. Monthly sampling of a demographic homogenous population of bees was performed between May and October, with concordant recording of the honey bee diet. Mixed Sanger-and Illumina 16S rRNA gene sequencing in combination with a quantitative PCR analysis were used to determine the bacterial composition. A marked increase in α-diversity was detected between May and June. Furthermore, we found that four distinct phylotypes belonging to the Proteobacteria dominated the microbiota, and these displayed major shifts throughout the season. Gilliamella apicola dominated the composition early on, and Snodgrassella alvi began to dominate when the other bacteria declined to an absolute low in October. In vitro co-culturing revealed that G. apicola suppressed S. alvi. No shift was detected in the composition of the microbiota under stable environment/dietary conditions between November and February. Therefore, environmental/dietary changes may trigger the shifts observed in the honey bee midgut/pyloric microbiota throughout a season.

  2. Phylogenetically Diverse Burkholderia Associated with Midgut Crypts of Spurge Bugs, Dicranocephalus spp. (Heteroptera: Stenocephalidae)

    PubMed Central

    Kuechler, Stefan Martin; Matsuura, Yu; Dettner, Konrad; Kikuchi, Yoshitomo

    2016-01-01

    Diverse phytophagous heteropteran insects, commonly known as stinkbugs, are associated with specific gut symbiotic bacteria, which have been found in midgut cryptic spaces. Recent studies have revealed that members of the stinkbug families Coreidae and Alydidae of the superfamily Coreoidea are consistently associated with a specific group of the betaproteobacterial genus Burkholderia, called the “stinkbug-associated beneficial and environmental (SBE)” group, and horizontally acquire specific symbionts from the environment every generation. However, the symbiotic system of another coreoid family, Stenocephalidae remains undetermined. We herein investigated four species of the stenocephalid genus Dicranocephalus. Examinations via fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) revealed the typical arrangement and ultrastructures of midgut crypts and gut symbionts. Cloning and molecular phylogenetic analyses of bacterial genes showed that the midgut crypts of all species are colonized by Burkholderia strains, which were further assigned to different subgroups of the genus Burkholderia. In addition to the SBE-group Burkholderia, a number of stenocephalid symbionts belonged to a novel clade containing B. sordidicola and B. udeis, suggesting a specific symbiont clade for the Stenocephalidae. The symbiotic systems of stenocephalid bugs may provide a unique opportunity to study the ongoing evolution of symbiont associations in the stinkbug-Burkholderia interaction. PMID:27265344

  3. Production and characterization of monoclonal antibodies against midgut of ixodid tick, Haemaphysalis longicornis.

    PubMed

    Nakajima, Mie; Kodama, Michi; Yanase, Haruko; Iwanaga, Toshihiko; Mulenga, Albert; Ohashi, Kazuhiko; Onuma, Misao

    2003-08-14

    There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.

  4. The bacterial flora of tsetse fly midgut and its effect on trypanosome transmission.

    PubMed

    Soumana, Illiassou Hamidou; Simo, Gustave; Njiokou, Flobert; Tchicaya, Bernadette; Abd-Alla, Adly M M; Cuny, Gérard; Geiger, Anne

    2013-03-01

    The tsetse fly, Glossina palpalis is a vector of the trypanosome that causes sleeping sickness in humans and nagana in cattle along with associated human health problems and massive economic losses. The insect is also known to carry a number of symbionts such as Sodalis, Wigglesworthia, Wolbachia whose effects on the physiology of the insect have been studied in depth. However, effects of other bacterial flora on the physiology of the host and vector competence have received little attention. Epidemiological studies on tsetse fly populations from different geographic sites revealed the presence of a variety of bacteria in the midgut. The most common of the flora belong to the genera Entrobacter (most common), Enterococcus, and Acinetobacter. It was a little surprising to find such diversity in the tsetse midgut since the insect is monophagous consuming vertebrate blood only. Diversity of bacteria is normally associated with polyphagous insects. In contrast to the symbionts, the role of resident midgut bacterial flora on the physiology of the fly and vector competence remains to be elucidated. With regard, Sodalis glossinidius, our data showed that flies harbouring this symbiont have three times greater probability of being infected by trypanosomes than flies without the symbiont. The data delineated in these studies under score the need to carry out detailed investigations on the role of resident bacteria on the physiology of the fly and vector competence.

  5. Effects of gamma irradiation on the midgut ultrastructure of Glossina palpalis subspecies

    SciTech Connect

    Stiles, J.K.; Molyneux, D.H.; Wallbanks, K.R.; Van der Vloedt, A.M.

    1989-05-01

    In the sterile insect technique, insects are sterilized prior to release in areas where they are pests. The sterile males compete for and with fertile wild individuals for mates, thus reducing the population's reproductive rate. Tsetse fly (Glossina spp.) populations have been eradicated after release of laboratory-bred flies sterilized by gamma irradiation. However, no studies exist on radiation-induced damage to the midgut morphology and function of the radiation-sterilized insects. After G. palpalis palpalis and G. p. gambiensis were subjected to 130 Gy gamma radiation, their midgut damage and recovery were monitored by electron microscopy. The first sign of damage was atrophy and loss of the microvillous border from epithelial cells. The rate of cell degeneration increased, with young as well as old cells being affected and cellular debris filling the ectoperitrophic space. Muscle cells were destroyed, patches of basal lamina were left bare, intracellular virus- and rickettsia-like organisms became more frequent, and many replacement cells became unusually large. Partial recovery occurred from the 10th day postirradiation. Such changes in midgut ultrastructure and the corresponding inhibition of functions may increase the susceptibility of the fly to trypanosome infection.

  6. Discovery of midgut genes for the RNA interference control of corn rootworm.

    PubMed

    Hu, Xu; Richtman, Nina M; Zhao, Jian-Zhou; Duncan, Keith E; Niu, Xiping; Procyk, Lisa A; Oneal, Meghan A; Kernodle, Bliss M; Steimel, Joseph P; Crane, Virginia C; Sandahl, Gary; Ritland, Julie L; Howard, Richard J; Presnail, James K; Lu, Albert L; Wu, Gusui

    2016-01-01

    RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by "blebbing" of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality. PMID:27464714

  7. Midgut lysozymes of Lucilia sericata - new antimicrobials involved in maggot debridement therapy.

    PubMed

    Valachova, I; Takac, P; Majtan, J

    2014-12-01

    Larvae of Lucilia sericata are used for maggot debridement therapy (MDT) because of their ability to remove necrotic tissue and eradicate bacterial pathogens of infected wounds. So far, very few antibacterial factors have been fully characterized (eg lucifensin). Using a molecular approach, some other putative antimicrobial compounds, including three novel lysozymes, have been previously identified and predicted to be involved in MDT. Nevertheless, data on lysozymes tissue origin and their functions have never been elucidated. Therefore, the aim of this study was to investigate the expression of three lysozymes in L. sericata and confirm their antibacterial effects within MDT. Moreover, we characterized the eradication process of bacteria within the digestive system of maggots and determined the role of lysozymes in this process. We found that three lysozymes are expressed in specific sections of the L. sericata midgut. Recombinant lysozymes displayed comparable antibacterial activity against Micrococcus luteus. Furthermore, the majority of Gram-positive bacteria were destroyed in vivo within the particular section of the L. sericata midgut where lysozymes are produced. Larval ingestion and subsequent eradication of wound pathogens during their passage through the intestine of maggots are due to, at least in part, antibacterial action of three midgut lysozymes.

  8. Multiple Modes of Action of the Squamocin in the Midgut Cells of Aedes aegypti Larvae

    PubMed Central

    de Paula, Sérgio Oliveira; Martins, Gustavo Ferreira; Zanuncio, José Cola

    2016-01-01

    Annonaceous acetogenins are botanical compounds with good potential for use as insecticides. In the vector, Aedes aegypti (L.) (Diptera: Culicidae), squamocin (acetogenin) has been reported to be a larvicide and cytotoxic, but the modes of action of this molecule are still poorly understood. This study evaluated the changes in the cell morphology, and in the expression of genes, for autophagy (Atg1 and Atg8), for membrane ion transporter V-ATPase, and for water channel aquaporin-4 (Aqp4) in the midgut of A. aegypti larvae exposed to squamocin from Annona mucosa Jacq. (Annonaceae). Squamocin showed cytotoxic action with changes in the midgut epithelium and digestive cells of A. aegypti larvae, increase in the expression for autophagy gene Atg1 and Atg8, decrease in the expression of V-ATPase, decrease in the expression of Aqp4 gene in LC20 and inhibition of Apq4 genes in the midgut of this vector in LC50. These multiple modes of action for squamocin are described for the first time in insects, and they are important because different sites of action of squamocin from A. mucosa may reduce the possibility of resistance of A. aegypti to this molecule. PMID:27532504

  9. Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae.

    PubMed

    Ferreira, A H; Cristofoletti, P T; Pimenta, D C; Ribeiro, A F; Terra, W R; Ferreira, C

    2008-02-01

    A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation.

  10. Discovery of midgut genes for the RNA interference control of corn rootworm

    PubMed Central

    Hu, Xu; Richtman, Nina M.; Zhao, Jian-Zhou; Duncan, Keith E.; Niu, Xiping; Procyk, Lisa A.; Oneal, Meghan A.; Kernodle, Bliss M.; Steimel, Joseph P.; Crane, Virginia C.; Sandahl, Gary; Ritland, Julie L.; Howard, Richard J.; Presnail, James K.; Lu, Albert L.; Wu, Gusui

    2016-01-01

    RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by “blebbing” of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality. PMID:27464714

  11. Shifts in the Midgut/Pyloric Microbiota Composition within a Honey Bee Apiary throughout a Season.

    PubMed

    Ludvigsen, Jane; Rangberg, Anbjørg; Avershina, Ekaterina; Sekelja, Monika; Kreibich, Claus; Amdam, Gro; Rudi, Knut

    2015-01-01

    Honey bees (Apis mellifera) are prominent crop pollinators and are, thus, important for effective food production. The honey bee gut microbiota is mainly host specific, with only a few species being shared with other insects. It currently remains unclear how environmental/dietary conditions affect the microbiota within a honey bee population over time. Therefore, the aim of the present study was to characterize the composition of the midgut/pyloric microbiota of a honey bee apiary throughout a season. The rationale for investigating the midgut/pyloric microbiota is its dynamic nature. Monthly sampling of a demographic homogenous population of bees was performed between May and October, with concordant recording of the honey bee diet. Mixed Sanger-and Illumina 16S rRNA gene sequencing in combination with a quantitative PCR analysis were used to determine the bacterial composition. A marked increase in α-diversity was detected between May and June. Furthermore, we found that four distinct phylotypes belonging to the Proteobacteria dominated the microbiota, and these displayed major shifts throughout the season. Gilliamella apicola dominated the composition early on, and Snodgrassella alvi began to dominate when the other bacteria declined to an absolute low in October. In vitro co-culturing revealed that G. apicola suppressed S. alvi. No shift was detected in the composition of the microbiota under stable environment/dietary conditions between November and February. Therefore, environmental/dietary changes may trigger the shifts observed in the honey bee midgut/pyloric microbiota throughout a season. PMID:26330094

  12. Multiple Modes of Action of the Squamocin in the Midgut Cells of Aedes aegypti Larvae.

    PubMed

    da Silva Costa, Marilza; de Paula, Sérgio Oliveira; Martins, Gustavo Ferreira; Zanuncio, José Cola; Santana, Antônio Euzébio Goulart; Serrão, José Eduardo

    2016-01-01

    Annonaceous acetogenins are botanical compounds with good potential for use as insecticides. In the vector, Aedes aegypti (L.) (Diptera: Culicidae), squamocin (acetogenin) has been reported to be a larvicide and cytotoxic, but the modes of action of this molecule are still poorly understood. This study evaluated the changes in the cell morphology, and in the expression of genes, for autophagy (Atg1 and Atg8), for membrane ion transporter V-ATPase, and for water channel aquaporin-4 (Aqp4) in the midgut of A. aegypti larvae exposed to squamocin from Annona mucosa Jacq. (Annonaceae). Squamocin showed cytotoxic action with changes in the midgut epithelium and digestive cells of A. aegypti larvae, increase in the expression for autophagy gene Atg1 and Atg8, decrease in the expression of V-ATPase, decrease in the expression of Aqp4 gene in LC20 and inhibition of Apq4 genes in the midgut of this vector in LC50. These multiple modes of action for squamocin are described for the first time in insects, and they are important because different sites of action of squamocin from A. mucosa may reduce the possibility of resistance of A. aegypti to this molecule. PMID:27532504

  13. A specific cathepsin-L-like protease purified from an insect midgut shows antibacterial activity against gut symbiotic bacteria.

    PubMed

    Byeon, Jin Hee; Seo, Eun Sil; Lee, Jun Beom; Lee, Min Ja; Kim, Jiyeun Kate; Yoo, Jin Wook; Jung, Yunjin; Lee, Bok Luel

    2015-11-01

    Because gut symbiotic bacteria affect host biology, host insects are expected to evolve some mechanisms for regulating symbiont population. The bean bug, Riptortus pedestris, harbors the Burkholderia genus as a gut symbiont in the midgut organ, designated as the M4 region. Recently, we demonstrated that the lysate of M4B, the region adjacent to M4, harbors potent antibacterial activity against symbiotic Burkholderia but not to cultured Burkholderia. However, the bona fide substance responsible for observed antibacterial activity was not identified in the previous study. Here, we report that cathepsin-L-like protease purified from the lysate of M4B showed strong antibacterial activity against symbiotic Burkholderia but not the cultured Burkholderia. To further confirm this activity, recombinant cathepsin-L-like protease expressed in Escherichia coli also showed antibacterial activity against symbiotic Burkholderia. These results suggest that cathepsin-L-like protease purified from the M4B region plays a critical role in controlling the population of the Burkholderia gut symbiont.

  14. Cantharidin Impedes Activity of Glutathione S-Transferase in the Midgut of Helicoverpa armigera Hübner

    PubMed Central

    Khan, Rashid Ahmed; Liu, Ji Yuan; Rashid, Maryam; Wang, Dun; Zhang, Ya Lin

    2013-01-01

    Previous investigations have implicated glutathione S-transferases (GSTs) as one of the major reasons for insecticide resistance. Therefore, effectiveness of new candidate compounds depends on their ability to inhibit GSTs to prevent metabolic detoxification by insects. Cantharidin, a terpenoid compound of insect origin, has been developed as a bio-pesticide in China, and proves highly toxic to a wide range of insects, especially lepidopteran. In the present study, we test cantharidin as a model compound for its toxicity, effects on the mRNA transcription of a model Helicoverpa armigera glutathione S-transferase gene (HaGST) and also for its putative inhibitory effect on the catalytic activity of GSTs, both in vivo and in vitro in Helicoverpa armigera, employing molecular and biochemical methods. Bioassay results showed that cantharidin was highly toxic to H. armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcript ranging from 2.5 to 12.5 folds while biochemical assays showed in vivo inhibition of GSTs in midgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalized by homology and molecular docking simulations using a model GST (1PN9) as a template structure. Molecular docking simulations also confirmed accurate docking of the cantharidin molecule to the active site of HaGST impeding its catalytic activity. PMID:23528854

  15. Enteroendocrine cells are generated from stem cells through a distinct progenitor in the adult Drosophila posterior midgut

    PubMed Central

    Zeng, Xiankun; Hou, Steven X.

    2015-01-01

    Functional mature cells are continually replenished by stem cells to maintain tissue homoeostasis. In the adult Drosophila posterior midgut, both terminally differentiated enterocyte (EC) and enteroendocrine (EE) cells are generated from an intestinal stem cell (ISC). However, it is not clear how the two differentiated cells are generated from the ISC. In this study, we found that only ECs are generated through the Su(H)GBE+ immature progenitor enteroblasts (EBs), whereas EEs are generated from ISCs through a distinct progenitor pre-EE by a novel lineage-tracing system. EEs can be generated from ISCs in three ways: an ISC becoming an EE, an ISC becoming a new ISC and an EE through asymmetric division, or an ISC becoming two EEs through symmetric division. We further identified that the transcriptional factor Prospero (Pros) regulates ISC commitment to EEs. Our data provide direct evidence that different differentiated cells are generated by different modes of stem cell lineage specification within the same tissues. PMID:25670791

  16. Enzyme-regulated the changes of pH values for assembling a colorimetric and multistage interconnection logic network with multiple readouts.

    PubMed

    Huang, Yanyan; Ran, Xiang; Lin, Youhui; Ren, Jinsong; Qu, Xiaogang

    2015-04-22

    Based on enzymatic reactions-triggered changes of pH values and biocomputing, a novel and multistage interconnection biological network with multiple easy-detectable signal outputs has been developed. Compared with traditional chemical computing, the enzyme-based biological system could overcome the interference between reactions or the incompatibility of individual computing gates and offer a unique opportunity to assemble multicomponent/multifunctional logic circuitries. Our system included four enzyme inputs: β-galactosidase (β-gal), glucose oxidase (GOx), esterase (Est) and urease (Ur). With the assistance of two signal transducers (gold nanoparticles and acid-base indicators) or pH meter, the outputs of the biological network could be conveniently read by the naked eyes. In contrast to current methods, the approach present here could realize cost-effective, label-free and colorimetric logic operations without complicated instrument. By designing a series of Boolean logic operations, we could logically make judgment of the compositions of the samples on the basis of visual output signals. Our work offered a promising paradigm for future biological computing technology and might be highly useful in future intelligent diagnostics, prodrug activation, smart drug delivery, process control, and electronic applications.

  17. Enzyme-regulated the changes of pH values for assembling a colorimetric and multistage interconnection logic network with multiple readouts.

    PubMed

    Huang, Yanyan; Ran, Xiang; Lin, Youhui; Ren, Jinsong; Qu, Xiaogang

    2015-04-22

    Based on enzymatic reactions-triggered changes of pH values and biocomputing, a novel and multistage interconnection biological network with multiple easy-detectable signal outputs has been developed. Compared with traditional chemical computing, the enzyme-based biological system could overcome the interference between reactions or the incompatibility of individual computing gates and offer a unique opportunity to assemble multicomponent/multifunctional logic circuitries. Our system included four enzyme inputs: β-galactosidase (β-gal), glucose oxidase (GOx), esterase (Est) and urease (Ur). With the assistance of two signal transducers (gold nanoparticles and acid-base indicators) or pH meter, the outputs of the biological network could be conveniently read by the naked eyes. In contrast to current methods, the approach present here could realize cost-effective, label-free and colorimetric logic operations without complicated instrument. By designing a series of Boolean logic operations, we could logically make judgment of the compositions of the samples on the basis of visual output signals. Our work offered a promising paradigm for future biological computing technology and might be highly useful in future intelligent diagnostics, prodrug activation, smart drug delivery, process control, and electronic applications. PMID:25819791

  18. A lepidopteran-specific gene family encoding valine-rich midgut proteins.

    PubMed

    Odman-Naresh, Jothini; Duevel, Margret; Muthukrishnan, Subbaratnam; Merzendorfer, Hans

    2013-01-01

    Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM), an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps), which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran digestive tract facing

  19. The fine structure of the midgut epithelium in a centipede, Scolopendra cingulata (Chilopoda, Scolopendridae), with the special emphasis on epithelial regeneration.

    PubMed

    Chajec, Lukasz; Sonakowska, Lidia; Rost-Roszkowska, Magdalena M

    2014-01-01

    Scolopendra cingulata has a tube-shaped digestive system that is divided into three distinct regions: fore-, mid- and hindgut. The midgut is lined with a pseudostratified columnar epithelium which is composed of digestive, secretory and regenerative cells. Hemocytes also appear between the digestive cells of the midgut epithelium. The ultrastructure of three types of epithelial cells and hemocytes of the midgut has been described with the special emphasis on the role of regenerative cells in the protection of midgut epithelium. The process of midgut epithelium regeneration proceeds due to the ability of regenerative cells to proliferate and differentiate according to a circadian rhythm. The regenerative cells serve as unipotent stem cells that divide in an asymmetric manner. Additionally, two types of hemocytes have been distinguished among midgut epithelial cells. They enter the midgut epithelium from the body cavity. Because of the fact that numerous microorganisms occur in the cytoplasm of midgut epithelial cells, we discuss the role of hemocytes in elimination of pathogens from the midgut epithelium. The studies were conducted with the use of transmission electron microscope and immunofluorescent methods.

  20. The fine structure of the midgut epithelium in a centipede, Scolopendra cingulata (Chilopoda, Scolopendridae), with the special emphasis on epithelial regeneration.

    PubMed

    Chajec, Lukasz; Sonakowska, Lidia; Rost-Roszkowska, Magdalena M

    2014-01-01

    Scolopendra cingulata has a tube-shaped digestive system that is divided into three distinct regions: fore-, mid- and hindgut. The midgut is lined with a pseudostratified columnar epithelium which is composed of digestive, secretory and regenerative cells. Hemocytes also appear between the digestive cells of the midgut epithelium. The ultrastructure of three types of epithelial cells and hemocytes of the midgut has been described with the special emphasis on the role of regenerative cells in the protection of midgut epithelium. The process of midgut epithelium regeneration proceeds due to the ability of regenerative cells to proliferate and differentiate according to a circadian rhythm. The regenerative cells serve as unipotent stem cells that divide in an asymmetric manner. Additionally, two types of hemocytes have been distinguished among midgut epithelial cells. They enter the midgut epithelium from the body cavity. Because of the fact that numerous microorganisms occur in the cytoplasm of midgut epithelial cells, we discuss the role of hemocytes in elimination of pathogens from the midgut epithelium. The studies were conducted with the use of transmission electron microscope and immunofluorescent methods. PMID:23831526

  1. Impacts on silkworm larvae midgut proteomics by transgenic Trichoderma strain and analysis of glutathione S-transferase sigma 2 gene essential for anti-stress response of silkworm larvae.

    PubMed

    Li, Yingying; Dou, Kai; Gao, Shigang; Sun, Jianan; Wang, Meng; Fu, Kehe; Yu, Chuanjin; Wu, Qiong; Li, Yaqian; Chen, Jie

    2015-08-01

    Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain.

  2. HRG-1 enhances cancer cell invasive potential and couples glucose metabolism to cytosolic/extracellular pH gradient regulation by the vacuolar-H(+) ATPase.

    PubMed

    Fogarty, F M; O'Keeffe, J; Zhadanov, A; Papkovsky, D; Ayllon, V; O'Connor, R

    2014-09-18

    Haeme-responsive gene (HRG)-1 encodes a 16-kDa transmembrane protein that is induced by insulin-like growth factor-1 (IGF-1) and associates with the vacuolar-(H(+)) ATPase (V-ATPase). We previously reported that HRG-1 is essential for V-ATPase activity in endosomal acidification and receptor trafficking. Here, we show that in highly invasive and migratory cancer cell lines, HRG-1 and the V-ATPase are co-expressed at the plasma membrane, whereas in less invasive cell lines and non-transformed cells HRG-1 over-expression remains confined to intracellular compartments. Stable suppression of HRG-1 in invasive breast cancer MDA-MB-231 cells decreases extracellular pH, cell growth, migration and invasion. Ectopic expression of HRG-1 in non-invasive MCF-7 cells enhances V-ATPase activity, lowers the extracellular pH and increases the pH-dependent activity of MMP2 and MMP9 matrix metalloproteinases. HRG-1 enhances trafficking of the glucose transporter-1 (GLUT-1) with a concomitant increase in glucose uptake and lactate production. HRG-1 also promotes trafficking of the insulin-like growth factor I receptor (IGF-1R), β1-integrin and IGF-1 signalling. Taken together, our findings indicate that HRG-1 expression at the plasma membrane enhances V-ATPase activity, drives glycolytic flux and facilitates cancer cell growth, migration and invasion. Thus, HRG-1 may represent a novel target for selectively disrupting V-ATPase activity and the metastatic potential of cancer cells.

  3. Extracellular and Luminal pH Regulation by Vacuolar H+-ATPase Isoform Expression and Targeting to the Plasma Membrane and Endosomes*

    PubMed Central

    Smith, Gina A.; Howell, Gareth J.; Phillips, Clair; Muench, Stephen P.; Ponnambalam, Sreenivasan; Harrison, Michael A.

    2016-01-01

    Plasma membrane vacuolar H+-ATPase (V-ATPase) activity of tumor cells is a major factor in control of cytoplasmic and extracellular pH and metastatic potential, but the isoforms involved and the factors governing plasma membrane recruitment remain uncertain. Here, we examined expression, distribution, and activity of V-ATPase isoforms in invasive prostate adenocarcinoma (PC-3) cells. Isoforms 1 and 3 were the most highly expressed forms of membrane subunit a, with a1 and a3 the dominant plasma membrane isoforms. Correlation between plasma membrane V-ATPase activity and invasiveness was limited, but RNAi knockdown of either a isoform did slow cell proliferation and inhibit invasion in vitro. Isoform a1 was recruited to the cell surface from the early endosome-recycling complex pathway, its knockdown arresting transferrin receptor recycling. Isoform a3 was associated with the late endosomal/lysosomal compartment. Both a isoforms associated with accessory protein Ac45, knockdown of which stalled transit of a1 and transferrin-transferrin receptor, decreased proton efflux, and reduced cell growth and invasiveness; this latter effect was at least partly due to decreased delivery of the membrane-bound matrix metalloproteinase MMP-14 to the plasma membrane. These data indicate that in prostatic carcinoma cells, a1 and a3 isoform populations predominate in different compartments where they maintain different luminal pH. Ac45 plays a central role in navigating the V-ATPase to the plasma membrane, and hence it is an important factor in expression of the invasive phenotype. PMID:26912656

  4. NHE1, NHE2, and NHE4 contribute to regulation of cell pH in T84 colon cancer cells.

    PubMed

    Beltrán, Ana Rosa; Ramírez, Marco Antonio; Carraro-Lacroix, Luciene R; Hiraki, Yumi; Rebouças, Nancy Amaral; Malnic, Gerhard

    2008-02-01

    The isoforms of the Na+/H+ exchanger present in T84 human colon cells were identified by functional and molecular methods. Cell pH was measured by fluorescence microscopy using the probe BCECF. Based on the pH recovery after an ammonium pulse and determination of buffering capacity of these cells, the rate of H+ extrusion (JH) was 3.68 mM/min. After the use of the amiloride derivative HOE-694 at 25 microM, which inhibits the isoforms NHE1 and NHE2, there remained 43% of the above transport rate, the nature of which was investigated. Evidence of the presence of NHE1, NHE2, and NHE4 was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) (mRNA) and Western blot. There was no decrease of JH by the NHE3 inhibitor S3226 (1 microM) and no evidence of this isoform by RT-PCR was found. The following functional evidence for the presence of NHE4 was obtained: 25 microM EIPA abolished JH entirely, but NHE4 was not inhibited at 10 microM; substitution of Na by K increased the remainder, a property of NHE4; hypertonicity also increased this fraction of JH. Cl--dependent NHE was not detected: in 0 Cl- solutions JH was increased and not reduced. In 0 Cl- cell volume decreased significantly, which was abolished by the Cl- channel blocker NPPB, indicating that the 0 Cl- effect was because of reduction of cell volume. In conclusion, T84 human colon cells contain three isoforms of the Na+/H+ exchanger, NHE1, NHE2, and NHE4, but not the Cl-dependent NHE.

  5. NaRALF, a peptide signal essential for the regulation of root hair tip apoplastic pH in Nicotiana attenuata, is required for root hair development and plant growth in native soils.

    PubMed

    Wu, Jinsong; Kurten, Erin L; Monshausen, Gabriele; Hummel, Grégoire M; Gilroy, Simon; Baldwin, Ian T

    2007-12-01

    Rapid alkalinization factor (RALF) is a 49-amino-acid peptide that rapidly alkalinizes cultivated tobacco cell cultures. In the native tobacco Nicotiana attenuata, NaRALF occurs as a single-copy gene and is highly expressed in roots and petioles. Silencing the NaRALF transcript by transforming N. attenuata with an inverted-repeat construct generated plants (irRALF) with normal wild-type (WT) above-ground parts, but with roots that grew longer and produced trichoblasts that developed into abnormal root hairs. Most trichoblasts produced a localized 'bulge' without commencing root hair tip growth; fewer trichoblasts grew, but were only 10% as long as those of WT plants. The root hair phenotype was associated with slowed apoplastic pH oscillations, increased pH at the tips of trichoblasts and decreased accumulation of reactive oxygen species in the root hair initiation zone. The root hair growth phenotype was partially restored when irRALF lines were grown in a low-pH-buffered medium, and reproduced in WT plants grown in a high-pH-buffered medium. When irRALF plants were grown in pH 5.6, 6.7 and 8.1 soils together with WT plants in glasshouse experiments, they were out-competed by WT plants in basic, but not acidic, soils. When WT and irRALF lines were planted into the basic soils of the native habitat of N. attenuata in the Great Basin Desert, irRALF plants had smaller leaves, shorter stalks, and produced fewer flowers and seed capsules than did WT plants. We conclude that NaRALF is required for regulating root hair extracellular pH, the transition from root hair initiation to tip growth and plant growth in basic soils.

  6. Histopathological effects and immunolocalization of periplocoside NW from Periploca sepium Bunge on the midgut epithelium of Mythimna separata Walker larvae.

    PubMed

    Feng, Mingxing; Shi, Baojun; Zhao, Yanchao; Hu, Zhaonong; Wu, Wenjun

    2014-10-01

    Periplocoside NW (PSNW) with pregnane glycoside skeleton is a novel insecticidal compound isolated from the root bark of Periploca sepium Bunge. This compound has a potent stomach poisoning activity against several insect pests. In this study, we observed the intoxication symptoms, investigated the histopathological effects and carried out immuno-electron microscopic localization of PSNW on the midgut epithelium of oriental armyworm Mythimna separata Walker larvae for better understanding its action mechanism against insects. Ultrastructural observations showed that cell damages caused by PSNW in the midgut of M. separata larvae are related to the degeneration of brush border microvilli. The dissolution of cytoskeletal structures in the interior and on the surface of microvilli was responsible for the decrease in size and eventual disappearance of microvilli when bubbles of cytoplasmic substances protrude into the midgut lumen of M. separata, thus resulting in cell death. The immuno-electron microscopic localization research showed that gold particle appeared on the microvilli layer of the midgut of M. separate larvae firstly. The density of gold particle gradually added with the time, and finally microvilli layer was destructed severely. Meantime, the gold particles were also presented to the intracellular organelle membrane and the organelles also were destructed. Therefore, we proposed that this membrane system on insect midgut epithelium cells is the initial acting site of PSNW against insects.

  7. Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis.

    PubMed

    Cancino-Rodezno, Angeles; Lozano, Luis; Oppert, Cris; Castro, Julieta I; Lanz-Mendoza, Humberto; Encarnación, Sergio; Evans, Amy E; Gill, Sarjeet S; Soberón, Mario; Jurat-Fuentes, Juan L; Bravo, Alejandra

    2012-01-01

    Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes. PMID:22615881

  8. Midgut-enriched receptor protein tyrosine phosphatase PTP52F is required for Drosophila development during larva-pupa transition.

    PubMed

    Santhanam, Abirami; Liang, Suh-Yuen; Chen, Dong-Yuan; Chen, Guang-Chao; Meng, Tzu-Ching

    2013-01-01

    To date our understanding of Drosophila receptor protein tyrosine phosphatases (R-PTPs) in the regulation of signal transduction is limited. Of the seven R-PTPs identified in flies, six are involved in the axon guidance that occurs during embryogenesis. However, whether and how R-PTPs may control key steps of Drosophila development is not clear. In this study we investigated the potential role of Drosophila R-PTPs in developmental processes outside the neuronal system and beyond the embryogenesis stage. Through systematic data mining of available microarray databases, we found the mRNA level of PTP52F to be highly enriched in the midgut of flies at the larva-pupa transition. This finding was confirmed by gut tissue staining with a specific antibody. The unique spatiotemporal expression of PTP52F suggests that it is possibly involved in regulating metamorphosis during the transformation from larva to pupa. To test this hypothesis, we employed RNA interference to examine the defects of transgenic flies. We found that ablation of endogenous PTP52F led to high lethality characterized by the pharate adult phenotype, occurring due to post pupal eclosion failure. These results show that PTP52F plays an indispensable role during the larva-pupa transition. We also found that PTP52F could be reclassified as a member of the subtype R3 PTPs instead of as an unclassified R-PTP without a human ortholog, as suggested previously. Together, these findings suggest that Drosophila R-PTPs may control metamorphosis and other biological processes beyond our current knowledge.

  9. Orally administered H-Dmt-Tic-Lys-NH-CH2-Ph (MZ-2), a potent mu/delta-opioid receptor antagonist, regulates obese-related factors in mice.

    PubMed

    Marczak, Ewa D; Jinsmaa, Yunden; Myers, Page H; Blankenship, Terry; Wilson, Ralph; Balboni, Gianfranco; Salvadori, Severo; Lazarus, Lawrence H

    2009-08-15

    Orally active dual mu-/delta-opioid receptor antagonist, H-Dmt-Tic-Lys-NH-CH(2)-Ph (MZ-2) was applied to study body weight gain, fat content, bone mineral density, serum insulin, cholesterol and glucose levels in female ob/ob (B6.V-Lep/J homozygous) and lean wild mice with or without voluntary exercise on wheels for three weeks, and during a two week post-treatment period under the same conditions. MZ-2 (10mg/kg/day, p.o.) exhibited the following actions: (1) reduced body weight gain in sedentary obese mice that persisted beyond the treatment period without effect on lean mice; (2) stimulated voluntary running on exercise wheels of both groups of mice; (3) decreased fat content, enhanced bone mineral density (BMD), and decreased serum insulin and glucose levels in obese mice; and (4) MZ-2 (30 microM) increased BMD in human osteoblast cells (MG-63) comparable to naltrexone, while morphine inhibited mineral nodule formation. Thus, MZ-2 has potential application in the clinical management of obesity, insulin and glucose levels, and the amelioration of osteoporosis.

  10. ALL2, a Homologue of ALL1, Has a Distinct Role in Regulating pH Homeostasis in the Pathogen Cryptococcus neoformans

    PubMed Central

    Jain, Neena; Bouklas, Tejas; Gupta, Anjali; Varshney, Avanish K.; Orner, Erika P.

    2015-01-01

    Cryptococcus neoformans is a facultative intracellular fungal pathogen that has a polysaccharide capsule and causes life-threatening meningoencephalitis. Its capsule, as well as its ability to survive in the acidic environment of the phagolysosome, contributes to the pathogen's resilience in the host environment. Previously, we reported that downregulation of allergen 1 (ALL1) results in the secretion of a shorter, more viscous exopolysaccharide with less branching and structural complexity, as well as altered iron homeostasis. Now, we report on a homologous coregulated gene, allergen 2 (ALL2). ALL2's function was characterized by generating null mutants in C. neoformans. In contrast to ALL1, loss of ALL2 attenuated virulence in the pulmonary infection model. The all2Δ mutant shed a less viscous exopolysaccharide and exhibited higher sensitivity to hydrogen peroxide than the wild type, and as a result, the all2Δ mutant was more resistant to macrophage-mediated killing. Transcriptome analysis further supported the distinct function of these two genes. Unlike ALL1's involvement in iron homeostasis, we now present data on ALL2's unique function in maintaining intracellular pH in low-pH conditions. Thus, our data highlight that C. neoformans, a human-pathogenic basidiomycete, has evolved a unique set of virulence-associated genes that contributes to its resilience in the human niche. PMID:26597983

  11. Functional photoacoustic microscopy of pH

    NASA Astrophysics Data System (ADS)

    Chatni, M. Rameez; Yao, Junjie; Danielli, Amos; Favazza, Christopher P.; Maslov, Konstantin I.; Wang, Lihong V.

    2012-02-01

    pH is a tightly regulated indicator of metabolic activity. In mammalian systems, imbalance of pH regulation may result from or result in serious illness. Even though the regulation system of pH is very robust, tissue pH can be altered in many diseases such as cancer, osteoporosis and diabetes mellitus. Traditional high-resolution optical imaging techniques, such as confocal microscopy, routinely image pH in cells and tissues using pH sensitive fluorescent dyes, which change their fluorescence properties with the surrounding pH. Since strong optical scattering in biological tissue blurs images at greater depths, high-resolution pH imaging is limited to penetration depths of 1mm. Here, we report photoacoustic microscopy (PAM) of commercially available pH-sensitive fluorescent dye in tissue phantoms. Using both opticalresolution photoacoustic microscopy (OR-PAM), and acoustic resolution photoacoustic microscopy (AR-PAM), we explored the possibility of recovering the pH values in tissue phantoms. In this paper, we demonstrate that PAM was capable of recovering pH values up to a depth of 2 mm, greater than possible with other forms of optical microscopy.

  12. Restriction of Francisella novicida Genetic Diversity during Infection of the Vector Midgut

    PubMed Central

    Reif, Kathryn E.; Palmer, Guy H.; Crowder, David W.; Ueti, Massaro W.; Noh, Susan M.

    2014-01-01

    The genetic diversity of pathogens, and interactions between genotypes, can strongly influence pathogen phenotypes such as transmissibility and virulence. For vector-borne pathogens, both mammalian hosts and arthropod vectors may limit pathogen genotypic diversity (number of unique genotypes circulating in an area) by preventing infection or transmission of particular genotypes. Mammalian hosts often act as “ecological filters” for pathogen diversity, where novel variants are frequently eliminated because of stochastic events or fitness costs. However, whether vectors can serve a similar role in limiting pathogen diversity is less clear. Here we show using Francisella novicida and a natural tick vector of Francisella spp. (Dermacentor andersoni), that the tick vector acted as a stronger ecological filter for pathogen diversity compared to the mammalian host. When both mice and ticks were exposed to mixtures of F. novicida genotypes, significantly fewer genotypes co-colonized ticks compared to mice. In both ticks and mice, increased genotypic diversity negatively affected the recovery of available genotypes. Competition among genotypes contributed to the reduction of diversity during infection of the tick midgut, as genotypes not recovered from tick midguts during mixed genotype infections were recovered from tick midguts during individual genotype infection. Mediated by stochastic and selective forces, pathogen genotype diversity was markedly reduced in the tick. We incorporated our experimental results into a model to demonstrate how vector population dynamics, especially vector-to-host ratio, strongly affected pathogen genotypic diversity in a population over time. Understanding pathogen genotypic population dynamics will aid in identification of the variables that most strongly affect pathogen transmission and disease ecology. PMID:25392914

  13. Schinus terebinthifolius Leaf Extract Causes Midgut Damage, Interfering with Survival and Development of Aedes aegypti Larvae.

    PubMed

    Procópio, Thamara Figueiredo; Fernandes, Kenner Morais; Pontual, Emmanuel Viana; Ximenes, Rafael Matos; de Oliveira, Aline Rafaella Cardoso; Souza, Carolina de Santana; Melo, Ana Maria Mendonça de Albuquerque; Navarro, Daniela Maria do Amaral Ferraz; Paiva, Patrícia Maria Guedes; Martins, Gustavo Ferreira; Napoleão, Thiago Henrique

    2015-01-01

    In this study, a leaf extract from Schinus terebinthifolius was evaluated for effects on survival, development, and midgut of A. aegypti fourth instar larvae (L4), as well as for toxic effect on Artemia salina. Leaf extract was obtained using 0.15 M NaCl and evaluated for phytochemical composition and lectin activity. Early L4 larvae were incubated with the extract (0.3-1.35%, w/v) for 8 days, in presence or absence of food. Polymeric proanthocyanidins, hydrolysable tannins, heterosid and aglycone flavonoids, cinnamic acid derivatives, traces of steroids, and lectin activity were detected in the extract, which killed the larvae at an LC50 of 0.62% (unfed larvae) and 1.03% (fed larvae). Further, the larvae incubated with the extract reacted by eliminating the gut content. No larvae reached the pupal stage in treatments at concentrations between 0.5% and 1.35%, while in the control (fed larvae), 61.7% of individuals emerged as adults. The extract (1.0%) promoted intense disorganization of larval midgut epithelium, including deformation and hypertrophy of cells, disruption of microvilli, and vacuolization of cytoplasms, affecting digestive, enteroendocrine, regenerative, and proliferating cells. In addition, cells with fragmented DNA were observed. Separation of extract components by solid phase extraction revealed that cinnamic acid derivatives and flavonoids are involved in larvicidal effect of the extract, being the first most efficient in a short time after larvae treatment. The lectin present in the extract was isolated, but did not show deleterious effects on larvae. The extract and cinnamic acid derivatives were toxic to A. salina nauplii, while the flavonoids showed low toxicity. S. terebinthifolius leaf extract caused damage to the midgut of A. aegypti larvae, interfering with survival and development. The larvicidal effect of the extract can be attributed to cinnamic acid derivatives and flavonoids. The data obtained using A. salina indicates that caution

  14. Schinus terebinthifolius Leaf Extract Causes Midgut Damage, Interfering with Survival and Development of Aedes aegypti Larvae

    PubMed Central

    Procópio, Thamara Figueiredo; Fernandes, Kenner Morais; Pontual, Emmanuel Viana; Ximenes, Rafael Matos; de Oliveira, Aline Rafaella Cardoso; Souza, Carolina de Santana; Melo, Ana Maria Mendonça de Albuquerque; Navarro, Daniela Maria do Amaral Ferraz; Paiva, Patrícia Maria Guedes; Martins, Gustavo Ferreira; Napoleão, Thiago Henrique

    2015-01-01

    In this study, a leaf extract from Schinus terebinthifolius was evaluated for effects on survival, development, and midgut of A. aegypti fourth instar larvae (L4), as well as for toxic effect on Artemia salina. Leaf extract was obtained using 0.15 M NaCl and evaluated for phytochemical composition and lectin activity. Early L4 larvae were incubated with the extract (0.3–1.35%, w/v) for 8 days, in presence or absence of food. Polymeric proanthocyanidins, hydrolysable tannins, heterosid and aglycone flavonoids, cinnamic acid derivatives, traces of steroids, and lectin activity were detected in the extract, which killed the larvae at an LC50 of 0.62% (unfed larvae) and 1.03% (fed larvae). Further, the larvae incubated with the extract reacted by eliminating the gut content. No larvae reached the pupal stage in treatments at concentrations between 0.5% and 1.35%, while in the control (fed larvae), 61.7% of individuals emerged as adults. The extract (1.0%) promoted intense disorganization of larval midgut epithelium, including deformation and hypertrophy of cells, disruption of microvilli, and vacuolization of cytoplasms, affecting digestive, enteroendocrine, regenerative, and proliferating cells. In addition, cells with fragmented DNA were observed. Separation of extract components by solid phase extraction revealed that cinnamic acid derivatives and flavonoids are involved in larvicidal effect of the extract, being the first most efficient in a short time after larvae treatment. The lectin present in the extract was isolated, but did not show deleterious effects on larvae. The extract and cinnamic acid derivatives were toxic to A. salina nauplii, while the flavonoids showed low toxicity. S. terebinthifolius leaf extract caused damage to the midgut of A. aegypti larvae, interfering with survival and development. The larvicidal effect of the extract can be attributed to cinnamic acid derivatives and flavonoids. The data obtained using A. salina indicates that caution

  15. Tissue-specific transcriptome profiling of Plutella xylostella third instar larval midgut.

    PubMed

    Xie, Wen; Lei, Yanyuan; Fu, Wei; Yang, Zhongxia; Zhu, Xun; Guo, Zhaojiang; Wu, Qingjun; Wang, Shaoli; Xu, Baoyun; Zhou, Xuguo; Zhang, Youjun

    2012-01-01

    The larval midgut of diamondback moth, Plutella xylostella, is a dynamic tissue that interfaces with a diverse array of physiological and toxicological processes, including nutrient digestion and allocation, xenobiotic detoxification, innate and adaptive immune response, and pathogen defense. Despite its enormous agricultural importance, the genomic resources for P. xylostella are surprisingly scarce. In this study, a Bt resistant P. xylostella strain was subjected to the in-depth transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes in the P. xylostella larval midgut. Using Illumina deep sequencing, we obtained roughly 40 million reads containing approximately 3.6 gigabases of sequence data. De novo assembly generated 63,312 ESTs with an average read length of 416 bp, and approximately half of the P. xylostella sequences (45.4%, 28,768) showed similarity to the non-redundant database in GenBank with a cut-off E-value below 10(-5). Among them, 11,092 unigenes were assigned to one or multiple GO terms and 16,732 unigenes were assigned to 226 specific pathways. In-depth analysis identified genes putatively involved in insecticide resistance, nutrient digestion, and innate immune defense. Besides conventional detoxification enzymes and insecticide targets, novel genes, including 28 chymotrypsins and 53 ABC transporters, have been uncovered in the P. xylostella larval midgut transcriptome; which are potentially linked to the Bt toxicity and resistance. Furthermore, an unexpectedly high number of ESTs, including 46 serpins and 7 lysozymes, were predicted to be involved in the immune defense.As the first tissue-specific transcriptome analysis of P. xylostella, this study sheds light on the molecular understanding of insecticide resistance, especially Bt resistance in an agriculturally important insect pest, and lays the foundation for future functional genomics research. In addition, current

  16. Yeast Saccharomyces cerevisiae adiponectin receptor homolog Izh2 is involved in the regulation of zinc, phospholipid and pH homeostasis.

    PubMed

    Mattiazzi Ušaj, Mojca; Prelec, Metod; Brložnik, Mojca; Primo, Cecilia; Curk, Tomaž; Ščančar, Janez; Yenush, Lynne; Petrovič, Uroš

    2015-09-01

    The functional link between zinc homeostasis and membrane-related processes, including lipid metabolism regulation, extends from yeast to humans, and has a likely role in the pathogenesis of diabetes. The yeast Izh2 protein has been previously implicated in zinc ion homeostasis and in the regulation of lipid and phosphate metabolism, but its precise molecular function is not known. We performed a chemogenomics experiment to determine the genes conferring resistance or sensitivity to different environmental zinc concentrations. We then determined at normal, depleted and excess zinc concentrations, the genetic interactions of IZH2 at the genome-wide level and measured changes in the transcriptome caused by deletion of IZH2. We found evidence for an important cellular function of the Rim101 pathway in zinc homeostasis in neutral or acidic environments, and observed that phosphatidylinositol is a source of inositol when zinc availability is limited. Comparison of our experimental profiles with published gene expression and genetic interaction profiles revealed pleiotropic functions for Izh2. We propose that Izh2 acts as an integrator of intra- and extracellular signals in providing adequate cellular responses to maintain homeostasis under different external conditions, including - but not limited to - alterations in zinc concentrations.

  17. Total management of short gut secondary to midgut volvulus without prolonged total parenteral alimentation.

    PubMed

    Tepas, J J; MacLean, W C; Kolbach, S; Shermeta, D W

    1978-12-01

    Absorption studies in rats have shown that intestinal adaptation after catastrophic injury can be stimulated by early enteral feeding. Using this concept, we have devised a technique of early initiation and advancement of oral feedings that begins with Cho-Free and Polycose and gradually adds sucrose and MCT in increasing proportions. The increasing complexity and caloric density of this diet provide sufficient nutrition to allow weaning from total parenteral alimentation within 2--3 wk. Our preliminary experience in babies with midgut volvulus, necrotizing enterocolitis, and gastroschisis has been successful and uncomplicated. These patients have demonstrated consistent weight gain and have been spared the complications associated with prolonged parenteral alimentation.

  18. Dietary influences over proliferating cell nuclear antigen expression in the locust midgut.

    PubMed

    Zudaire, E; Simpson, S J; Illa, I; Montuenga, L M

    2004-06-01

    We have studied the influence of variations in dietary protein (P) and digestible carbohydrate (C), the quantity of food eaten, and insect age during the fifth instar on the expression of the proliferating cell nuclear antigen (PCNA) in the epithelial cells of the midgut (with special reference to the midgut caeca) in the African migratory locust, Locusta migratoria. Densitometric analysis of PCNA-immunostained cells was used as an indirect measure of the levels of expression of PCNA, and a PCNA cellular index (PCNA-I) was obtained. Measurements of the DNA content of the cells have also been carried out by means of microdensitometry of Feulgen-stained, thick sections of midgut. A comparison between the PCNA nuclear level and the DNA content was performed. The PCNA levels were significantly different among the cells of the five regions studied: caeca, anterior ventricle, medial ventricle, posterior ventricle and ampullae of the Malpighian tubules. We have studied in more detail the region with highest PCNA-I, i.e. the caeca. The quality and the quantity of food eaten under ad libitum conditions were highly correlated with both the PCNA and DNA levels in the caeca cells. Locusts fed a diet with a close to optimal P:C content (P 21%, C 21%) showed the highest PCNA and DNA content. In locusts fed a food that also contained a 1:1 ratio of P to C but was diluted three-fold by addition of indigestible cellulose (P 7%, C 7%), a compensatory increase in consumption was critical to maintaining PCNA levels. Our measurements also showed that the nuclear DNA content of the mature and differentiated epithelial cells was several-fold higher than the levels in the undifferentiated stem cells of the regenerative nests. These results, combined with the low number of mitotic figures found in the regenerative nests of the caeca and the marked variation in PCNA levels among groups, suggest that some type of DNA endoreduplication process may be taking place. Our data also indicate that

  19. A Small Molecule Glycosaminoglycan Mimetic Blocks Plasmodium Invasion of the Mosquito Midgut

    PubMed Central

    Ranucci, Elisabetta; Tao, Dingyin; Ferruti, Paolo; Ortega, Corrie; Staples, Gregory O.; Zaia, Joseph; Takashima, Eizo; Tsuboi, Takafumi; Borg, Natalie A.; Verotta, Luisella; Dinglasan, Rhoel R.

    2013-01-01

    Malaria transmission-blocking (T-B) interventions are essential for malaria elimination. Small molecules that inhibit the Plasmodium ookinete-to-oocyst transition in the midgut of Anopheles mosquitoes, thereby blocking sporogony, represent one approach to achieving this goal. Chondroitin sulfate glycosaminoglycans (CS-GAGs) on the Anopheles gambiae midgut surface are putative ligands for Plasmodium falciparum ookinetes. We hypothesized that our synthetic polysulfonated polymer, VS1, acting as a decoy molecular mimetic of midgut CS-GAGs confers malaria T-B activity. In our study, VS1 repeatedly reduced midgut oocyst development by as much as 99% (P<0.0001) in mosquitoes fed with P. falciparum and Plasmodium berghei. Through direct-binding assays, we observed that VS1 bound to two critical ookinete micronemal proteins, each containing at least one von Willebrand factor A (vWA) domain: (i) circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) and (ii) vWA domain-related protein (WARP). By immunofluorescence microscopy, we observed that VS1 stains permeabilized P. falciparum and P. berghei ookinetes but does not stain P. berghei CTRP knockouts or transgenic parasites lacking the vWA domains of CTRP while retaining the thrombospondin repeat region. We produced structural homology models of the first vWA domain of CTRP and identified, as expected, putative GAG-binding sites on CTRP that align closely with those predicted for the human vWA A1 domain and the Toxoplasma gondii MIC2 adhesin. Importantly, the models also identified patches of electropositive residues that may extend CTRP's GAG-binding motif and thus potentiate VS1 binding. Our molecule binds to a critical, conserved ookinete protein, CTRP, and exhibits potent malaria T-B activity. This study lays the framework for a high-throughput screen of existing libraries of safe compounds to identify those with potent T-B activity. We envision that such compounds when used as

  20. DNA Sequencing Reveals the Midgut Microbiota of Diamondback Moth, Plutella xylostella (L.) and a Possible Relationship with Insecticide Resistance

    PubMed Central

    Xia, Xiaofeng; Zheng, Dandan; Zhong, Huanzi; Qin, Bingcai; Gurr, Geoff M.; Vasseur, Liette; Lin, Hailan; Bai, Jianlin; He, Weiyi; You, Minsheng

    2013-01-01

    Background Insect midgut microbiota is important in host nutrition, development and immune response. Recent studies indicate possible links between insect gut microbiota and resistance to biological and chemical toxins. Studies of this phenomenon and symbionts in general have been hampered by difficulties in culture-based approach. In the present study, DNA sequencing was used to examine the midgut microbiota of diamondback moth (DBM), Plutella xylostella (L.), a destructive pest that attacks cruciferous crops worldwide. Its ability to develop resistance to many types of synthetic insecticide and even Bacillus thuringiensis toxins makes it an important species to study. Methodology/Principal Findings Bacteria of the DBM larval midgut in a susceptible and two insecticide (chlorpyrifos and fipronil) resistant lines were examined by Illumina sequencing sampled from an insect generation that was not exposed to insecticide. This revealed that more than 97% of the bacteria were from three orders: Enterobacteriales, Vibrionales and Lactobacillales. Both insecticide-resistant lines had more Lactobacillales and the much scarcer taxa Pseudomonadales and Xanthomonadales with fewer Enterobacteriales compared with the susceptible strain. Consistent with this, a second study observed an increase in the proportion of Lactobacillales in the midgut of DBM individuals from a generation treated with insecticides. Conclusions/Significance This is the first report of high-throughput DNA sequencing of the entire microbiota of DBM. It reveals differences related to inter- and intra-generational exposure to insecticides. Differences in the midgut microbiota among susceptible and insecticide-resistant lines are independent of insecticide exposure in the sampled generations. While this is consistent with the hypothesis that Lactobacillales or other scarcer taxa play a role in conferring DBM insecticide resistance, further studies are necessary to rule out other possibilities. Findings

  1. Imidacloprid impairs the post-embryonic development of the midgut in the yellow fever mosquito Stegomyia aegypti (=Aedes aegypti).

    PubMed

    Fernandes, K M; Gonzaga, W G; Pascini, T V; Miranda, F R; Tomé, H V V; Serrão, J E; Martins, G F

    2015-09-01

    The mosquito Stegomyia aegypti (=Aedes aegypti) (Diptera: Culicidae) is a vector for the dengue and yellow fever viruses. As blood digestion occurs in the midgut, this organ constitutes the route of entry of many pathogens. The effects of the insecticide imidacloprid on the survival of St. aegypti were investigated and the sub-lethal effects of the insecticide on midgut development were determined. Third instar larvae were exposed to different concentrations of imidacloprid (0.15, 1.5, 3.0, 6.0 and 15.0 p.p.m.) and survival was monitored every 24 h for 10 days. Midguts from imidacloprid-treated insects at different stages of development were dissected and processed for analyses by transmission electron microscopy, immunofluorescence microscopy and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays. Imidacloprid concentrations of 3.0 and 15.0 p.p.m. were found to affect midgut development similarly. Digestive cells of the fourth instar larvae (L4) midgut exposed to imidacloprid had more multilamellar bodies, abundantly found in the cell apex, and more electron-lucent vacuoles in the basal region compared with those from untreated insects. Moreover, imidacloprid interfered with the differentiation of regenerative cells, dramatically reducing the number of digestive and endocrine cells and leading to malformation of the midgut epithelium in adults. The data demonstrate that imidacloprid can reduce the survival of mosquitoes and thus indicate its potentially high efficacy in the control of St. aegypti populations.

  2. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae.

    PubMed

    Fernandez, Luisa E; Aimanova, Karlygash G; Gill, Sarjeet S; Bravo, Alejandra; Soberón, Mario

    2006-02-15

    A 65 kDa GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was characterized as a functional receptor of the Bacillus thuringiensis subsp. israelensis Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of BBMV (brush boder membrane vesicles) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein showed ALP activity. The peptide-displaying phages (P1.BBMV and P8.BBMV) that bound to the 65 kDa GPI-ALP (GPI-anchored ALP) and competed with the Cry11Aa toxin to bind to BBMV were isolated by selecting BBMV-binding peptide-phages by biopanning. GPI-ALP was shown to be preferentially distributed in Ae. aegypti in the posterior part of the midgut and in the caeca, by using P1.BBMV binding to fixed midgut tissue sections to determine the location of GPI-ALP. Cry11Aa binds to the same regions of the midgut and competed with P1.BBMV and P8.BBMV to bind to BBMV. The importance of this interaction was demonstrated by the in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that GPI-ALP is an important receptor molecule involved in Cry11Aa interaction with midgut cells and toxicity to Ae. aegypti larvae.

  3. Restriction of viral dissemination from the midgut determines incompetence of small brown planthopper as a vector of Southern rice black-streaked dwarf virus.

    PubMed

    Jia, Dongsheng; Chen, Hongyan; Mao, Qianzhuo; Liu, Qifei; Wei, Taiyun

    2012-08-01

    Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, is transmitted by the white-backed planthopper in a persistent-propagative manner. In this study, we found that another planthopper species, the small brown planthopper (SBPH), could acquire SRBSDV but not transmit it. To identify the transmission barrier for SRBSDV in SBPHs, sequential infection by SRBSDV in the organs of SBPHs was studied with immunofluorescence for viral antigens. SRBSDV initially entered the epithelial cells of the midgut, then viroplasms, the sites for viral replication, formed in the midgut of viruliferous SBPHs. Furthermore, SRBSDV spread within the midgut, but failed to disseminate from the midgut into the hemocoel or into the salivary glands. All these results indicated that the inability of SBPH to transmit SRBSDV could be due to the restriction of viral dissemination from the midgut of SBPH, which led to the failure of viral spread to the salivary glands for virus transmission.

  4. Restriction of viral dissemination from the midgut determines incompetence of small brown planthopper as a vector of Southern rice black-streaked dwarf virus.

    PubMed

    Jia, Dongsheng; Chen, Hongyan; Mao, Qianzhuo; Liu, Qifei; Wei, Taiyun

    2012-08-01

    Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, is transmitted by the white-backed planthopper in a persistent-propagative manner. In this study, we found that another planthopper species, the small brown planthopper (SBPH), could acquire SRBSDV but not transmit it. To identify the transmission barrier for SRBSDV in SBPHs, sequential infection by SRBSDV in the organs of SBPHs was studied with immunofluorescence for viral antigens. SRBSDV initially entered the epithelial cells of the midgut, then viroplasms, the sites for viral replication, formed in the midgut of viruliferous SBPHs. Furthermore, SRBSDV spread within the midgut, but failed to disseminate from the midgut into the hemocoel or into the salivary glands. All these results indicated that the inability of SBPH to transmit SRBSDV could be due to the restriction of viral dissemination from the midgut of SBPH, which led to the failure of viral spread to the salivary glands for virus transmission. PMID:22683297

  5. Morphological changes in the midgut of Aedes aegypti L. (Diptera: Culicidae) larvae following exposure to an Annona coriacea (Magnoliales: Annonaceae) extract.

    PubMed

    Costa, M S; Pinheiro, D O; Serrão, J E; Pereira, M J B

    2012-08-01

    Bioinsecticides are important in the control of disease vectors, but data regarding their physiological effects on target insects are incomplete. This study describes morphological changes that occur in the midgut of third instar Aedes aegypti L. (Diptera: Culicidae) following treatment with a methanolic extract of Annona coriacea (Magnoliales: Annonaceae). Dissected midguts were subdivided into anterior and posterior regions and analyzed by light and scanning electron microscopy. Insects exposed to the extract displayed intense, destructive cytoplasmic vacuolization in columnar and regenerative midgut cells. The apical surfaces of columnar cells exhibited cytoplasmic protrusions oriented toward the lumen, suggesting that these cells could be involved in apocrine secretory processes and/or apoptosis. We report that A. coriacea extracts induced morphological alterations in the midgut of A. aegypti midgut larvae, supporting the use of plant extracts for control of the dengue vector.

  6. Glutathione S-transferase in the midgut tissue of gypsy moth (Lymantria dispar) caterpillars exposed to dietary cadmium.

    PubMed

    Vlahović, Milena; Ilijin, Larisa; Mrdaković, Marija; Todorović, Dajana; Matić, Dragana; Lazarević, Jelica; Mataruga, Vesna Perić

    2016-06-01

    Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 and 30μg Cd/g dry food was examined. Based on the enzyme reaction through conjugation with glutathione, overall activity remained unaltered after acute and chronic treatment. No-observed-effect-concentration (10μg Cd/g dry food) significantly increased activity only after 3-day recovery following cadmium administration. Almost all comparisons of the indices of phenotypic plasticity revealed statistically significant differences. Despite the facts that GST has important role in xenobiotic biotransformation, our results indicate that this enzyme in insect midgut does not represent the key factor in cadmium detoxification.

  7. Impact on bacterial community in midguts of the Asian corn borer larvae by transgenic Trichoderma strain overexpressing a heterologous chit42 gene with chitin-binding domain.

    PubMed

    Li, Yingying; Fu, Kehe; Gao, Shigang; Wu, Qiong; Fan, Lili; Li, Yaqian; Chen, Jie

    2013-01-01

    This paper is the first report of the impact on the bacterial community in the midgut of the Asian corn borer (Ostrinia furnacalis) by the chitinase from the transgenic Trichoderma strain. In this study, we detected a change of the bacterial community in the midgut of the fourth instar larvae by using a culture-independent method. Results suggested that Proteobacteria and Firmicutes were the most highly represented phyla, being present in all the midgut bacterial communities. The observed species richness was simple, ranging from four to five of all the 16S rRNA clone libraries. When using Trichoderma fermentation liquids as additives, the percentages of the dominant flora in the total bacterial community in larval midgut changed significantly. The community of the genus Ochrobactrum in the midgut decreased significantly when the larvae were fed with the fermentation liquids of the transgenic Trichoderma strain Mc4. However, the Enterococcus community increased and then occupied the vacated niche of the Ochrobactrum members. Furthermore, the Shannon-Wiener (H) and the Simpson (1-D) indexes of the larval midgut bacterial library treated by feeding fermentation liquids of the transgenic Trichoderma strain Mc4 was the lowest compared with the culture medium, fermentation liquids of the wild type strain T30, and the sterile artificial diet. The Enterococcus sp. strain was isolated and characterized from the healthy larvae midgut of the Asian corn borer. An infection study of the Asian corn borer larvae using Enterococcus sp. ACB-1 revealed that a correlation existed between the increased Enterococcus community in the larval midgut and larval mortality. These results demonstrated that the transgenic Trichoderma strain could affect the composition of the midgut bacterial community. The change of the midgut bacterial community might be viewed as one of the factors resulting in the increased mortality of the Asian corn borer larvae.

  8. Cadmium-binding proteins in midgut gland of freshwater crayfish Procambarus clarkii

    SciTech Connect

    Del Ramo, J.; Pastor, A.; Torreblanca, A.; Medina, J.; Diza-Mayans, J.

    1989-02-01

    Metallothioneins, metal binding proteins, were originally isolated and characterized by Margoshes and Vallee. These proteins have a high affinity for various heavy metals, particularly cadmium and mercury and have extensively been studied in mammals. Metal binding proteins have been observed in a variety of marine invertebrates; however, there is very little information available on metal binding proteins in freshwater invertebrates, and particularly in freshwater crustaceans. Cadmium is an ubiquitous non essential element which possesses high toxicity to aquatic organisms. Cadmium binding proteins observed in invertebrates have similar characteristics to mammalian metallothioneins. In 1978, the American red crayfish appeared in Albufera Lake and the surrounding rice fields (Valencia, Spain). Albufera Lake and the surrounding rice fields waters are subjected to very heavy loads of sewage and toxic industrial residues (including heavy metals) from the many urban and wastewaters in this area. In previous reports the authors studied the toxicity and accumulation of cadmium on Procambarus clarkii of Albufera Lake. This crayfish shows a high resistance to cadmium and a great accumulation rate of this metal in several tissues, including midgut gland. Since Procambarus clarkii shows a high resistance to cadmium, the presence of cadmium binding proteins (Cd-BP) in midgut gland of these crayfish would be expected. This report describes results on the characterization of Cd-BPs obtained from cadmium exposed crayfish Procambarus clarkii, demonstrating their presence in this freshwater crayfish.

  9. Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut.

    PubMed

    Rupp, Ingrid; Sologub, Ludmilla; Williamson, Kim C; Scheuermayer, Matthias; Reininger, Luc; Doerig, Christian; Eksi, Saliha; Kombila, Davy U; Frank, Matthias; Pradel, Gabriele

    2011-04-01

    Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 μm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive "nanotubes" in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.

  10. Culture-independent analysis of midgut microbiota in the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae).

    PubMed

    Campbell, Corey L; Mummey, Daniel L; Schmidtmann, Edward T; Wilson, William C

    2004-05-01

    Differences in midgut microbial communities inhabiting Culicoides spp., insect vectors of virus pathogens, may affect the variation observed in the ability of these biting midges to propagate arthropod-borne viruses. As a first step toward addressing this hypothesis, midgut bacterial communities were compared between Culicoides species expected to be efficient and inefficient vectors of virus pathogens. We used 16S rDNA sequence and restriction fragment information to provisionally identify 36 bacterial genera from guts of wild adult female biting midges, Culicoides sonorensis Wirth and Jones and Culicoides variipennis (Coquillet), from two geographical locations. Bacterial identification was made by sequence analysis of 16S rDNA fragments and by terminal restriction fragment length polymorphism analysis of polymerase chain reaction-amplified 16S rDNA fragments from adult guts. Of 36 bacterial genera identified, 12 had been previously identified in other insects: Comomonas, Enterobacter, Klebsiella, Acinetobacter, Pseudomonas, Stenotrophomonas, Staphylococcus, Chryseobacterium, Moraxella, Acholeplasma, Flavobacterium, and Rickettsia, Significant differences in bacterial community composition were found between all three groups of wild adult females analyzed: live-trapped C. sonorensis, laboratory-emerged C. sonorensis, and laboratory-emerged C. variipennis.

  11. High-frequency conjugative transfer of antibiotic resistance genes to Yersinia pestis in the flea midgut.

    PubMed

    Hinnebusch, B Joseph; Rosso, Marie-Laure; Schwan, Tom G; Carniel, Elisabeth

    2002-10-01

    The acquisition of foreign DNA by horizontal transfer from unrelated organisms is a major source of variation leading to new strains of bacterial pathogens. The extent to which this occurs varies widely, due in part to lifestyle factors that determine exposure to potential donors. Yersinia pestis, the plague bacillus, infects normally sterile sites in its mammalian host, but forms dense aggregates in the non-sterile digestive tract of its flea vector to produce a transmissible infection. Here we show that unrelated co-infecting bacteria in the flea midgut are readily incorporated into these aggregates, and that this close physical contact leads to high-frequency conjugative genetic exchange. Transfer of an antibiotic resistance plasmid from an Escherichia coli donor to Y. pestis occurred in the flea midgut at a frequency of 10-3 after only 3 days of co-infection, and after 4 weeks 95% of co-infected fleas contained an average of 103 antibiotic-resistant Y. pestis transconjugants. Thus, transit in its arthropod vector exposes Y. pestis to favourable conditions for efficient genetic exchange with microbial flora of the flea gut. Horizontal gene transfer in the flea may be the source of antibiotic-resistant Y. pestis strains recently isolated from plague patients in Madagascar. PMID:12406213

  12. Posterior midgut epithelial cells differ in their organization of the membrane skeleton from other drosophila epithelia.

    PubMed

    Baumann, O

    2001-11-01

    In epithelial cells, the various components of the membrane skeleton are segregated within specialized subregions of the plasma membrane, thus contributing to the development and stabilization of cell surface polarity. It has previously been shown that, in various Drosophila epithelia, the membrane skeleton components ankyrin and alphabeta-spectrin reside at the lateral surface, whereas alphabeta(H)-spectrin is restricted to the apical domain. By use of confocal immunofluorescence microscopy, the present study characterizes the membrane skeleton of epithelial cells in the posterior midgut, leading to a number of unexpected results. First, ankyrin and alphabeta-spectrin are not detected on the entire lateral surface but appear to be restricted to the apicolateral area, codistributing with fasciclin III at smooth septate junctions. The presumptive ankyrin-binding proteins neuroglian and Na(+),K(+)-ATPase, however, do not colocalize with ankyrin. Second, alphabeta(H)-spectrin is enriched at the apical domain but is also present in lower amounts on the entire lateral surface, colocalizing apicolaterally with ankyrin/alphabeta-spectrin. Finally, despite the absence of zonulae adherentes, F-actin, beta(H)-spectrin, and nonmuscle myosin-II are enriched in the midlateral region. Thus, the model established for the organization of the membrane skeleton in Drosophila epithelia does not hold for the posterior midgut, and there is quite some variability between the different epithelia with respect to the organization of the membrane skeleton.

  13. Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos.

    PubMed

    Games, P D; Alves, S N; Katz, B B; Tomich, J M; Serrão, J E

    2016-09-01

    Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide-induced responses in the mosquito.

  14. Internalization of Sambucus nigra agglutinins I and II in insect midgut CF-203 cells.

    PubMed

    Shahidi-Noghabi, Shahnaz; Van Damme, Els J M; De Vos, Winnok H; Smagghe, Guy

    2011-04-01

    In this project, the uptake mechanisms and localization of two lectins from Sambucus nigra, further referred to as S. nigra agglutinin (SNA)-I and SNA-II, into insect midgut CF-203 cells were studied. SNA-I is a chimeric lectin belonging to the class of ribosome-inactivating proteins, whereas SNA-II is a hololectin devoid of enzymatic activity. Internalization of the fluorescein isothiocyanate-labeled lectin was investigated using confocal microscopy. Both lectins were internalized into the cytoplasm of CF-203 cells at similar rates. Preexposure of the insect midgut cells to specific inhibitors of clathrin- and caveolae-mediated endocytosis resulted in an inhibition of lectin uptake in CF-203 cells and caspase-induced cytotoxicity caused by SNA-I and SNA-II, confirming the involvement of both endocytosis pathways. Further studies demonstrated that the uptake mechanism(s) for both lectins required phosphoinositide 3-kinases, but did not depend on the actin cytoskeleton. Since the hololectin SNA-II apparently uses a similar endocytosis pathway as the chimerolectin SNA-I, it can be concluded that the endocytosis process mainly relies on the carbohydrate-binding activity of the lectins under investigation. © 2011 Wiley Periodicals, Inc. PMID:21254203

  15. The Anopheles-midgut APN1 structure reveals a new malaria transmission-blocking vaccine epitope.

    PubMed

    Atkinson, Sarah C; Armistead, Jennifer S; Mathias, Derrick K; Sandeu, Maurice M; Tao, Dingyin; Borhani-Dizaji, Nahid; Tarimo, Brian B; Morlais, Isabelle; Dinglasan, Rhoel R; Borg, Natalie A

    2015-07-01

    Mosquito-based malaria transmission-blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however, AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission have remained elusive. Here we present the 2.65-Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profiles of three monoclonal antibodies (mAbs) to AnAPN1, including mAb 4H5B7, which effectively blocks transmission of natural strains of Plasmodium falciparum. Using the AnAPN1 structure, we map the conformation-dependent 4H5B7 neoepitope to a previously uncharacterized region on domain 1 and further demonstrate that nonhuman-primate neoepitope-specific IgG also blocks parasite transmission. We discuss the prospect of a new biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV. PMID:26075520

  16. Metabolism of cydiastatin 4 and analogues by enzymes associated with the midgut and haemolymph of Manduca sexta larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The degradation of synthetic cydiastatin 4 (ARPYSFGL-amide) and cydiastatin 4 analogues cydiastatin 4a (PPPPPARPYSFGL-amide) and cydiastatin 4b (PPPPPARPYSF[Acpc]L-amide) by enzymes associated with the midgut and/or haemolymph of the tobacco hawkmoth moth, Manduca sexta were investigated using rever...

  17. Amount and metal composition of midgut gland metallothionein in shore crabs (Carcinus maenas) after exposure to cadmium in the food.

    PubMed

    Pedersen, Knud Ladegaard; Bach, Louise Thornhøj; Bjerregaard, Poul

    2014-05-01

    Accumulation of cadmium in aquatic invertebrates may compromise human food safety and anthropogenic additions of cadmium to coastal areas cause concern. Induction of crustacean metallothionein has been suggested as a useful biomarker for contamination of the aquatic environment with cadmium. We investigated how exposure to low concentrations of cadmium in the food affects the subcellular binding of cadmium with the shore crab Carcinus maenas as model organism. Approximately 80% of the assimilated cadmium was bound in the soluble fraction of the midgut gland and of this, 82% was found in the metallothionein fraction. Metallothionein synthesis was only induced at the highest exposure level. However, the number of cadmium atoms bound per molecule of metallothionein increased linearly with exposure, from approximately 0.18 in the control group to 1.4 in a group administered food containing 5.1 μg Cd g(-1). We noted a marked interaction between the presence of copper and zinc in the midgut gland and the binding of cadmium. The usefulness of crustacean midgut gland metallothionein as a biomarker for cadmium exposure at modest levels was questioned since exposures at levels producing significant increases in the tissue contents of the metal did not result in elevated concentrations of metallothionein in the midgut gland. PMID:24685622

  18. Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus

    PubMed Central

    Ghanim, Murad; Fattah-Hosseini, Somayeh; Levy, Amit; Cilia, Michelle

    2016-01-01

    Candidatus Liberibacter asiaticus (CLas) is a phloem-limited, gram-negative, fastidious bacterium that is associated with the development of citrus greening disease, also known as Huanglongbing (HLB). CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri, in a circulative manner. Two major barriers to transmission within the insect are the midgut and the salivary glands. We performed a thorough microscopic analysis within the insect midgut following exposure to CLas-infected citrus trees. We observed changes in nuclear architecture, including pyknosis and karyorrhexis as well as changes to the actin cytoskeleton in CLas-exposed midgut cells. Further analyses showed that the changes are likely due to the activation of programmed cell death as assessed by Annexin V staining and DNA fragmentation assays. These results suggest that exposure to CLas-infected trees induces apoptotic responses in the psyllid midgut that should be further investigated. Understanding the adaptive significance of the apoptotic response has the potential to create new approaches for controlling HLB. PMID:27630042

  19. Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus.

    PubMed

    Ghanim, Murad; Fattah-Hosseini, Somayeh; Levy, Amit; Cilia, Michelle

    2016-01-01

    Candidatus Liberibacter asiaticus (CLas) is a phloem-limited, gram-negative, fastidious bacterium that is associated with the development of citrus greening disease, also known as Huanglongbing (HLB). CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri, in a circulative manner. Two major barriers to transmission within the insect are the midgut and the salivary glands. We performed a thorough microscopic analysis within the insect midgut following exposure to CLas-infected citrus trees. We observed changes in nuclear architecture, including pyknosis and karyorrhexis as well as changes to the actin cytoskeleton in CLas-exposed midgut cells. Further analyses showed that the changes are likely due to the activation of programmed cell death as assessed by Annexin V staining and DNA fragmentation assays. These results suggest that exposure to CLas-infected trees induces apoptotic responses in the psyllid midgut that should be further investigated. Understanding the adaptive significance of the apoptotic response has the potential to create new approaches for controlling HLB.

  20. The effects of Bt Cry1Ie toxin on bacterial diversity in the midgut of Apis mellifera ligustica (Hymenoptera: Apidae).

    PubMed

    Jia, Hui-Ru; Geng, Li-Li; Li, Yun-He; Wang, Qiang; Diao, Qing-Yun; Zhou, Ting; Dai, Ping-Li

    2016-01-01

    The honey bee has been regarded as a key species in the environmental risk assessment of biotech crops. Here, the potential adverse effects of Cry1Ie toxin on the midgut bacteria of the worker bees (Apis mellifera ligustica) were investigated under laboratory conditions. Newly emerged bees were fed with different concentrations of Cry1Ie toxin syrups (20 ng/mL, 200 ng/mL, and 20 μg/mL), pure sugar syrup, and 48 ppb of imidacloprid syrups, then sampled after 15 and 30 d. We characterized the dominant midgut bacteria and compared the composition and structure of the midgut bacterial community in all samples using the Illumina MiSeq platform targeting the V3-V4 regions of 16S rDNA. No significant differences in the diversity of the midgut bacteria were observed between the five treatments. This work was the first to show the effects of Cry1Ie toxin on honey bees, and our study provided a theoretical basis for the biosafety assessment of transgenic Cry1Ie maize. PMID:27090812

  1. Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus.

    PubMed

    Ghanim, Murad; Fattah-Hosseini, Somayeh; Levy, Amit; Cilia, Michelle

    2016-01-01

    Candidatus Liberibacter asiaticus (CLas) is a phloem-limited, gram-negative, fastidious bacterium that is associated with the development of citrus greening disease, also known as Huanglongbing (HLB). CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri, in a circulative manner. Two major barriers to transmission within the insect are the midgut and the salivary glands. We performed a thorough microscopic analysis within the insect midgut following exposure to CLas-infected citrus trees. We observed changes in nuclear architecture, including pyknosis and karyorrhexis as well as changes to the actin cytoskeleton in CLas-exposed midgut cells. Further analyses showed that the changes are likely due to the activation of programmed cell death as assessed by Annexin V staining and DNA fragmentation assays. These results suggest that exposure to CLas-infected trees induces apoptotic responses in the psyllid midgut that should be further investigated. Understanding the adaptive significance of the apoptotic response has the potential to create new approaches for controlling HLB. PMID:27630042

  2. Transepithelial flux of an allotostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transepithelial transport of cydiastatin 4 and analogues across flat sheet preparations of the anterior midgut of larvae of the tobacco hawkmoth moth, Manduca sexta, was investigated using a combination of reversed-phase high performance liquid chromatography (RP-HPLC), enzyme linked immunosorbe...

  3. The effects of Bt Cry1Ie toxin on bacterial diversity in the midgut of Apis mellifera ligustica (Hymenoptera: Apidae)

    PubMed Central

    Jia, Hui-Ru; Geng, Li-Li; Li, Yun-He; Wang, Qiang; Diao, Qing-Yun; Zhou, Ting; Dai, Ping-Li

    2016-01-01

    The honey bee has been regarded as a key species in the environmental risk assessment of biotech crops. Here, the potential adverse effects of Cry1Ie toxin on the midgut bacteria of the worker bees (Apis mellifera ligustica) were investigated under laboratory conditions. Newly emerged bees were fed with different concentrations of Cry1Ie toxin syrups (20 ng/mL, 200 ng/mL, and 20 μg/mL), pure sugar syrup, and 48 ppb of imidacloprid syrups, then sampled after 15 and 30 d. We characterized the dominant midgut bacteria and compared the composition and structure of the midgut bacterial community in all samples using the Illumina MiSeq platform targeting the V3–V4 regions of 16S rDNA. No significant differences in the diversity of the midgut bacteria were observed between the five treatments. This work was the first to show the effects of Cry1Ie toxin on honey bees, and our study provided a theoretical basis for the biosafety assessment of transgenic Cry1Ie maize. PMID:27090812

  4. Live imaging of baculovirus infection of midgut epithelium cells: a functional assay of per os infectivity factors.

    PubMed

    Mu, Jingfang; van Lent, Jan W M; Smagghe, Guy; Wang, Yun; Chen, Xinwen; Vlak, Just M; van Oers, Monique M

    2014-11-01

    The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host-range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with EGFP fused to the nucleocapsid to monitor virus entry into primary midgut epithelium cells ex vivo using confocal fluorescence microscopy. The EGFP-labelled virus showed similar BV virulence and ODV infectivity as WT virus. The ability to bind and enter host cells was then visualized for WT AcMNPV and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, whilst PIF1 and PIF2 play important roles in the entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process. PMID:25006078

  5. Transcriptome of the lymantria dispar (gypsy moth) larval midgut and its response to infection by bacillus thuringiensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control in nature. RNA-Seq approaches were used to define a set of ...

  6. The receptor of Bacillus sphaericus binary toxin in Culex pipiens (Diptera: Culicidae) midgut: molecular cloning and expression.

    PubMed

    Darboux, I; Nielsen-LeRoux, C; Charles, J F; Pauron, D

    2001-09-01

    Culex pipiens larval midgut is the primary target of the binary toxin (Bin) present in parasporal inclusions of Bacillus sphaericus. Cpm1, a 60-kDa protein purified from brush border membranes, has been proposed as the receptor of the Bin toxin in the midgut epithelial cells of mosquitoes. We have cloned and characterized the corresponding cDNA from midgut of Culex pipiens larvae. The open reading frame predicted a 580 amino-acid protein with a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus. The amino acid sequence of the cloned Cpm1 exhibited 39-43% identities with insect maltases (alpha-glucosidases and alpha-amylases). Recombinant Cpm1 expressed in E. coli specifically bound to the Bin toxin and had a significant alpha-glucosidase activity but no alpha-amylase activity. These results support the view that Cpm1 is an alpha-glucosidase expressed in Culex midgut where it constitutes the receptor for the Bin toxin. To date, this is the first component involved in the mosquitocidal activity of the Bacillus sphaericus Bin toxin to be characterized. Its identification provides a key step to elucidate the mode of action of the Bin toxin and the mechanisms of resistance developed against it by some mosquito strains. PMID:11483434

  7. Limited effect of reactive oxygen species on the composition of susceptible essential amino acids in the midguts of Lymantria dispar caterpillars.

    PubMed

    Barbehenn, Raymond V; Niewiadomski, Julie; Kochmanski, Joseph; Constabel, C Peter

    2012-11-01

    The essential amino acids (EAAs) arginine, histidine, lysine, and methionine, as well as cysteine (semiessential), are believed to be susceptible to reactions with reactive oxygen species (ROS) in biological systems. The decreased availability of these EAAs could harm insect nutrition, since several of them can also be limiting for protein synthesis. However, no in vivo studies have quantified the effect of ROS in the midguts of insect herbivores on EAA composition. This study examined the association between elevated levels of ROS in the midgut fluid of Lymantria dispar caterpillars and the compositions of EAAs (protein-bound + protein-free) in their midgut fluid and frass. Contrary to expectation, the compositions of EAAs were not significantly decreased by ROS in midgut fluid ex vivo when incubated with phenolic compounds. Two in vivo comparisons of low- and high-ROS-producing leaves also showed similar results: there were no significant decreases in the compositions of EAAs in the midgut fluids and/or frass of larvae with elevated levels of ROS in their midguts. In addition, waste nitrogen excretion was not significantly increased from larvae on high-ROS treatments, as would be expected if ROS produced unbalanced EAA compositions. These results suggest that L. dispar larvae are able to tolerate elevated levels of ROS in their midguts without nutritionally significant changes in the compositions of susceptible EAAs in their food.

  8. Fluorescence Localization and Comparative Ultrastructural Study of Periplocoside NW from Periploca sepium Bunge in the Midgut of the Oriental Amyworm, Mythimna separata Walker (Lepidoptera: Noctuidae)

    PubMed Central

    Feng, Mingxing; Zhao, Juan; Zhang, Jiwen; Hu, Zhaonong; Wu, Wenjun

    2014-01-01

    Periplocoside NW (PSNW) is a novel insecticidal compound isolated from the root bark of Periploca sepium Bunge and has potent stomach toxicity against some insect pests. Previous studies showed that the Mythimna separata larva is sensitive to PSNW, but the Agrotis ispilon larva is insensitive. In this study, preliminary target localization on the midgut of M. separata larvae was conducted via a fluorescence labeling technique. A comparative ultrastructural study on the effects of PSNW on the midguts of M. separata and A. ispilon larvae was performed. Symptom observation results showed that typical stomach toxicity was induced by PSNW in M. separata larvae. Fluorescence localization results showed that PSNW binds to the midgut cells of M. separata larvae. Ultrastructure observations showed destruction of the microvilli, organelle, and cytomembrane in the midgut cells of M. separata larvae, whereas no obvious changes were observed in midgut cells of A. ispilon larvae. These results were consistent with the insecticidal activity of PSNW. Therefore, PSNW might act on the midgut tissues of the insects, and one or more binding sites of PSNW may exist in M. separata larvae midgut cell cytomembranes. PMID:24831268

  9. Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis.

    PubMed

    Suzuki, Ken; Sakamoto, Hironori; Shinozaki, Yukiko; Tabata, Jun; Watanabe, Takashi; Mochizuki, Atsushi; Koitabashi, Motoo; Fujii, Takeshi; Tsushima, Seiya; Kitamoto, Hiroko K

    2013-09-01

    Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca(2+) or Mg(2+) at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).

  10. Lectin-carbohydrate recognition mechanism of Plasmodium berghei in the midgut of malaria vector Anopheles stephensi using quantum dot as a new approach.

    PubMed

    Basseri, Hamid R; Javazm, Mahdi Salari; Farivar, Leila; Abai, Mohammad R

    2016-04-01

    Potential targets of Plasmodium ookinetes at the mosquito midgut walls were investigated in relation to interfering malarial transmission. In this study, the essential application of Quantum Dots (QDs) was used to examine the interaction between Plasmodium berghei ookinetes and the Anopheles stephensi midgut, based on lectin-carbohydrate recognition. Two significant lectins were utilized to determine this interaction. Two QDs, cadmium telluride (CdTe)/CdS and cadmium selenide (CdSe)/CdS, were employed in staining Plasmodium ookinete to study its interaction in the midgut of the mosquito vector in vivo. Concurrently, two lectins, wheat germ agglutinin (WGA) and concanavalin A (Con A), were inadvertently exploited to mask lectin binding sites between ookinetes and mosquito midgut cells. The numbers of ookinetes in both lumen and epithelial cells were eventually counted, following adequate preparation of wax sections extracted from whole midgut, and subsequent examination using a differential interference contrast a fluorescence microscopic technique. Interestingly, we detected that neither of the QDs mutated ookinete invasion into the midgut cells of the investigated mosquitoes. QD staining of ookinetes remained permanent despite the effective embedding procedure. The massive binding potency of ookinetes to midgut cells of the cross-examined mosquitoes undoubtedly revealed that Con A did not interrupt ookinete penetration into the midgut wall. In contrast, WGA inhibited ookinete invasion into the midgut cells. The results proved that QD nanoparticles are biocompatible, non-toxic to P. berghei and stable to photobleaching. The QDs staining, which was successfully implemented for ookinete labelling, is a simple and effective tool which plays a crucial role in bioimaging including the study of parasite-vector interactions.

  11. The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

    PubMed

    Beton, Daniela; Guzzo, Cristiane R; Ribeiro, Alberto F; Farah, Chuck S; Terra, Walter R

    2012-09-01

    Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents.

  12. A Kazal-type inhibitor is modulated by Trypanosoma cruzi to control microbiota inside the anterior midgut of Rhodnius prolixus.

    PubMed

    Soares, Tatiane S; Buarque, Diego S; Queiroz, Bruna R; Gomes, Cícera M; Braz, Glória R C; Araújo, Ricardo N; Pereira, Marcos H; Guarneri, Alessandra A; Tanaka, Aparecida S

    2015-05-01

    The triatomine insect, Rhodnius prolixus, is a vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. The parasite must overcome immune response and microbiota to develop inside the midgut of triatomines. In this study, we expressed, purified and characterized a Kazal-type inhibitor from the midgut of R. prolixus, named RpTI, which may be involved in microbiota - T. cruzi interactions. The qPCR showed that the RpTI transcript was primarily expressed in tissues from the intestinal tract and that it was upregulated in the anterior midgut after T. cruzi infection. A 315-bp cDNA fragment encoding the mature protein was cloned into the pPIC9 vector and expressed in Pichia pastoris system. Recombinant RpTI (rRpTI) was purified on a trypsin-Sepharose column and had a molecular mass of 11.5 kDa as determined by SDS-PAGE analysis. This protein inhibited trypsin (Ki = 0.42 nM), whereas serine proteases from the coagulation cascade were not inhibited. Moreover, trypanocidal assays revealed that rRpTI did not interfere in the viability of T. cruzi trypomastigotes. The RpTI transcript was also knocked down by RNA interference prior to infection of R. prolixus with T. cruzi. The amount of T. cruzi in the anterior midgut was significantly lower in RpTI knockdown insects compared to the non-silenced groups. We also verified that the bacterial load is higher in the anterior midgut of silenced and infected R. prolixus compared to non-silenced and infected insects. Our results suggest that T. cruzi infection increases the expression of RpTI to mediate microbiota modulation and is important for parasite immediately after infection with R. prolixus.

  13. Trilogy of foregut, midgut and hindgut atresias presenting in reverse order

    PubMed Central

    Patel, Ramnik V; Jackson, Paul; De Coppi, Paolo; Pierro, Agostino

    2014-01-01

    We present a case of triple gut atresias (foregut, midgut and hindgut) with multiple congenital anomalies presenting as imperforate anus. Abdominal radiography showed the double bubble sign. Upper gastrointestinal study through a nasogastric tube confirmed duodenal obstruction. Exploratory laparotomy, duodeno-duodenostomy for duodenal atresia and a left descending colostomy for anorectal malformation were performed. During extubation, the nasogastric tube came out and could not be reinserted by the anaesthetic team under laryngoscopic guidance. A chest radiograph showed the tube curled in the upper pouch. Bronchoscopy and oesophagoscopy confirmed oesophageal atresia (OA) with a distal tracheoesophageal fistula (TOF). The patient underwent right-sided extrapleural thoracotomy and division of the fistula with primary repair of OA uneventfully. Triple gut atresias presenting in reverse order with multiple anomalies is rare and passage of a nasogastric tube into the stomach in the presence of OA+TOF is exceptional. Alimentary tract obstruction should be corrected in proximal to distal direction. PMID:24835810

  14. Temporal differences in blood meal detection from the midguts of Triatoma infestans.

    PubMed

    Pinto, Jesus; Roellig, Dawn M; Gilman, Robert H; Calderón, Maritza; Bartra, Carlos; Salazar, Renzo; Bern, Caryn; Ancca-Juárez, Jenny; Levy, Michael; Náquira, Cesar; Cama, Vitaliano

    2012-01-01

    We used genus/species specific PCRs to determine the temporal persistence of host DNA in Triatoma infestans experimentally fed on blood from six common vertebrate species: humans, domestic dogs, guinea pigs, chickens, mice, and pigs. Twenty third or fourth instar nymphs per animal group were allowed to feed to engorgement, followed by fasting-maintenance in the insectary. At 7, 14, 21, or 28 days post-feeding, the midgut contents from five triatomines per group were tested with the respective PCR assay. DNA from all vertebrate species was detected in at least four of five study nymphs at seven and 14 days post-feeding. DNA of humans, domestic dogs, guinea pigs, pigs, and chickens were more successfully detected (80-100%) through day 21, and less successfully (20-100%) at day 28. Findings demonstrate that species-specific PCRs can consistently identify feeding sources of T. infestans within two weeks, a biologically relevant time interval.

  15. Resident microbiota of the gypsy moth midgut harbors antibiotic resistance determinants.

    PubMed

    Allen, Heather K; Cloud-Hansen, Karen A; Wolinski, Joseph M; Guan, Changhui; Greene, Serena; Lu, Shyue; Boeyink, Mallory; Broderick, Nichole A; Raffa, Kenneth F; Handelsman, Jo

    2009-03-01

    Little is known about the significance of insects as environmental reservoirs of antibiotic-resistant bacteria. We characterized the antibiotic resistome of the microbial community in gypsy moth larval midguts by applying functional metagenomics to cultured isolates. The minimum inhibitory concentrations of 12 antibiotics were determined for 44 cultured isolates, and antibiotic resistance genes were selected from metagenomic libraries derived from DNA extracted from a pool of the isolates. Six unique clones were identified. Two were highly resistant to penicillin-type beta-lactams, two were moderately resistant to erythromycin, and two were moderately resistant to a range of antibiotics, including erythromycin, carbenicillin, and chloramphenicol. Sequence analysis predicted that the active genes encoded efflux pumps, a transcriptional activator of efflux pump protein expression, and an extended-spectrum class A beta-lactamase. Insect guts are a reservoir of antibiotic resistance genes with the potential for dissemination.

  16. Changes in Drosophila melanogaster midgut proteins in response to dietary Bowman-Birk inhibitor.

    PubMed

    Li, H-M; Margam, V; Muir, W M; Murdock, L L; Pittendrigh, B R

    2007-10-01

    The midgut proteome of Drosophila melanogaster was compared in larvae fed dietary Bowman-Birk inhibitor (BBI) vs. larvae fed a control diet. By using two-dimensional gel electrophoresis, nine differentially expressed proteins were observed, which were associated with enzymes or transport functions such as sterol carrier protein X (SCPX), ubiquitin-conjugating enzyme, endopeptidase, receptor signalling protein kinase, ATP-dependent RNA helicase and alpha-tocopherol transport. Quantitative real-time PCR verified differential expression of transcripts coding for six of the proteins observed from the proteomic analysis. BBI evidently affects expression of proteins associated with protein degradation, transport and fatty acid catabolism. We then tested the hypothesis that SCPX was critical for the Drosophila third instars' response to BBI treatment. Inhibition of SCPX caused the third instars to become more susceptible to dietary BBI. PMID:17725801

  17. Interaction between Host Complement and Mosquito-Midgut-Stage Plasmodium berghei

    PubMed Central

    Margos, Gabriele; Navarette, Sandra; Butcher, Geoff; Davies, Alex; Willers, Christine; Sinden, Robert E.; Lachmann, Peter J.

    2001-01-01

    After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement on Plasmodium developmental stages in the mosquito blood meal using the rodent malaria parasite Plasmodium berghei and rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion of P. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages of P. berghei survived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules. PMID:11447187

  18. Intracellular pH in human arterial smooth muscle. Regulation by Na+/H+ exchange and a novel 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na(+)- and HCO3(-)-dependent mechanism

    SciTech Connect

    Neylon, C.B.; Little, P.J.; Cragoe, E.J. Jr.; Bobik, A. )

    1990-10-01

    We investigated in a physiological salt solution (PSS) containing HCO3- the intracellular pH (pHi) regulating mechanisms in smooth muscle cells cultured from human internal mammary arteries, using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and 22Na+ influx rates. The recovery of pHi from an equivalent intracellular acidosis was more rapid when the cells were incubated in CO2/HCO3(-)-buffered PSS than in HEPES-buffered PSS. Recovery of pHi was dependent on extracellular Na+ (Km, 13.1 mM); however, it was not attenuated by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), indicating the absence of SITS-sensitive HCO3(-)-dependent mechanisms. Recovery instead appeared mostly dependent on processes sensitive to 5-(N-ethyl-N-isopropyl)amiloride (EIPA), indicating the involvement of Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism. Differentiation between this HCO3(-)-dependent mechanism and Na+/H+ exchange was achieved after depletion of cellular ATP. Under these conditions, the NH4Cl-induced 22Na+ influx rate stimulated by intracellular acidosis was markedly attenuated in HEPES-buffered PSS but not in CO2/HCO3(-)-buffered PSS. EIPA also appeared to inhibit the two mechanisms differentially. In HEPES-buffered PSS containing 20 mM Na+, the EIPA inhibition curve for the intracellular acidosis-induced 22Na+ influx was monophasic (IC50, 39 nM), whereas in an identical CO2/HCO3(-)-buffered PSS, the inhibition curve exhibited biphasic characteristics (IC50, 37.3 nM and 312 microM). Taken together, the results indicate that Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism play an important role in regulating the pHi of human vascular smooth muscle.

  19. Genotoxic effects of starvation and dimethoate in haemocytes and midgut gland cells of wolf spider Xerolycosa nemoralis (Lycosidae).

    PubMed

    Wilczek, Grażyna; Mędrzak, Monika; Augustyniak, Maria; Wilczek, Piotr; Stalmach, Monika

    2016-06-01

    The aim of this study was to assess the genotoxic effects of starvation and dimethoate (organophosphate insecticide) in female and male wolf spiders Xerolycosa nemoralis (Lycosidae) exposed to the stressors under laboratory conditions. DNA damage was measured in haemocytes and midgut gland cells using the comet assay. In response to the two stressing factors, both cell types showed %TDNA, tail length (TL) and OTM values higher in males than in females. Level of DNA damage in haemocytes was greater than in midgut gland cells. In both sexes, the strongest genotoxicity was recorded at single application of dimethoate. After five-time exposure to the pesticide, genotoxic effects of a single dose were sustained in males and reduced to the control level in females. Starvation stress was well tolerated by the females, in which neither cell type was affected by DNA damage. However, in male haemocytes food deprivation induced severe DNA damage, what suggests suppression of the defence potential at prolonged starvation periods.

  20. Glutathione S-transferase in the midgut tissue of gypsy moth (Lymantria dispar) caterpillars exposed to dietary cadmium.

    PubMed

    Vlahović, Milena; Ilijin, Larisa; Mrdaković, Marija; Todorović, Dajana; Matić, Dragana; Lazarević, Jelica; Mataruga, Vesna Perić

    2016-06-01

    Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 and 30μg Cd/g dry food was examined. Based on the enzyme reaction through conjugation with glutathione, overall activity remained unaltered after acute and chronic treatment. No-observed-effect-concentration (10μg Cd/g dry food) significantly increased activity only after 3-day recovery following cadmium administration. Almost all comparisons of the indices of phenotypic plasticity revealed statistically significant differences. Despite the facts that GST has important role in xenobiotic biotransformation, our results indicate that this enzyme in insect midgut does not represent the key factor in cadmium detoxification. PMID:27084993

  1. Vaccinia mature virus fusion regulator A26 protein binds to A16 and G9 proteins of the viral entry fusion complex and dissociates from mature virions at low pH.

    PubMed

    Chang, Shu-Jung; Shih, Ao-Chun; Tang, Yin-Liang; Chang, Wen

    2012-04-01

    Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422-8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive.

  2. Association of Cry1Ac Toxin Resistance in Helicoverpa zea (Boddie) with Increased Alkaline Phosphatase Levels in the Midgut Lumen

    PubMed Central

    Caccia, Silvia; Moar, William J.; Chandrashekhar, Jayadevi; Oppert, Cris; Anilkumar, Konasale J.; Jurat-Fuentes, Juan Luis

    2012-01-01

    Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae. PMID:22685140

  3. Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen.

    PubMed

    Caccia, Silvia; Moar, William J; Chandrashekhar, Jayadevi; Oppert, Cris; Anilkumar, Konasale J; Jurat-Fuentes, Juan Luis; Ferré, Juan

    2012-08-01

    Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae. PMID:22685140

  4. Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches

    PubMed Central

    2012-01-01

    Background Bacillus thuringiensis var. israelensis (Bti) is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis). Results Several Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti-receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut. Conclusions By combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti-resistance mechanisms in mosquitoes. PMID:22703117

  5. Phoxim-induced damages of Bombyx mori larval midgut and titanium dioxide nanoparticles protective role under phoxim-induced toxicity.

    PubMed

    Su, Junju; Li, Bing; Cheng, Shen; Zhu, Zhou; Sang, Xuezi; Gui, Suxin; Xie, Yi; Sun, Qingqing; Cheng, Zhe; Cheng, Jie; Hu, Rengping; Shen, Weide; Xia, Qingyou; Zhao, Ping; Hong, Fashui

    2014-12-01

    Phoxim (O,O-diethyl O-(alpha-cyanobenzylideneamino) phosphorothioate) is a powerful organophosphorus pesticide with high potential for Bombyx mori larvae of silkworm exposure. However, it is possible that during the phoxim metabolism, there is generation of reactive oxygen species (ROS) and phoxim may produce oxidative stress and neurotoxicity in an intoxicated silkworm. Titanium dioxide nanoparticles (TiO2 NPs) pretreatment has been demonstrated to increase antioxidant capacity and acetylcholinesterase (AChE) activity in organisms. This study was, therefore, undertaken to determine phoxim-induced oxidative stress and neurotoxicity to determine whether phoxim intoxication alters the antioxidant system and AChE activity in the B. mori larval midgut, and to determine whether TiO2 NPs pretreatment attenuates phoxim-induced toxicity. The findings suggested that phoxim exposure decreased survival of B. mori larvae, increased malondialdehyde (MDA), carbonyl and 8-OHdG levels, and ROS accumulation in the midgut. Furthermore, phoxim significantly decreased the activities of AChE, superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione-S-transferase (GST), and levels of ascorbic acid (AsA), reduced glutathione (GSH), and thiol in the midgut. TiO2 pretreatment, however, could increase AChE activity, and remove ROS via activating SOD, CAT, APX, GR, and GST, and accelerating AsA-GSH cycle, thus attenuated lipid, protein, and DNA peroxidation and improve B. mori larval survival under phoxim-induced toxicity. Moreover, this experimental system would help nanomaterials to be applied in the sericulture.

  6. Novel Histopathological and Molecular Effects of Natural Compound Pellitorine on Larval Midgut Epithelium and Anal Gills of Aedes aegypti

    PubMed Central

    Perumalsamy, Haribalan; Kim, Jun-Ran; Oh, Sang Mi; Jung, Je Won; Ahn, Young-Joon; Kwon, Hyung Wook

    2013-01-01

    The yellow fever mosquito, Aedes aegypti, is a vector for transmitting dengue fever and yellow fever. In this study, we assessed the histopathological and molecular effects of pellitorine, an isobutylamide alkaloid, on the third instar of Ae. aegypti larvae. At 5 mg/l concentration of pellitorine, the whole body of the treated larvae became dark in color, particularly damaged thorax and abdominal regions. Pellitorine was targeted mainly on midgut epithelium and anal gills, indicating variably dramatic degenerative responses of the midgut through a sequential epithelial disorganization. The anterior and posterior midgut was entirely necrosed, bearing only gut lumen residues inside the peritrophic membranes. Pellitorine caused comprehensive damage of anal gill cells and branches of tracheole and debris was found in hemolymph of the anal gills. RT-PCR analysis indicates that the compound inhibited gene expression encoding V-type H+-ATPase and aquaporine 4 after treatment with 2.21 mg/l pellitorine. These results verify that pellitorine merits further study as a potential larvicide with a specific target site and a lead molecule for the control of mosquito populations. PMID:24260359

  7. Ferritin acts as the most abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers (Nilaparvata lugens).

    PubMed

    Du, J; Foissac, X; Carss, A; Gatehouse, A M; Gatehouse, J A

    2000-04-01

    The mannose-specific snowdrop lectin [Galanthus nivalis agglutinin (GNA)] displays toxicity to the rice brown planthopper Nilaparvata lugens. A 26kDa GNA-binding polypeptide from N. lugens midgut was identified by lectin blotting and affinity chromatography, and characterized by N-terminal sequencing. This polypeptide is the most abundant binding protein for GNA in the N. lugens midgut. A cDNA (fersub2) encoding this protein was isolated from an N. lugens cDNA library. The deduced amino acid sequence shows significant homology to ferritin subunits from Manduca sexta and other arthropods, plants and vertebrates, and contains a putative N-glycosylation site. Native ferritin was purified from whole insects as a protein of more than 400kDa in size and characterized biochemically. Three subunits of 20, 26 and 27kDa were released from the native complex. The 26kDa subunit binds GNA, and its N-terminal sequence was identical to that of fersub2. A second cDNA (fersub1), exhibiting strong homology with dipteran ferritin, was identified as an abundant cDNA in an N. lugens midgut-specific cDNA library, and could encode the larger ferritin subunit. The fersub1 cDNA carries a stem-loop structure (iron-responsive element) upstream from the start codon, similar to structures that have been shown to play a role in the control of ferritin synthesis in other insects.

  8. Differential Midgut Attachment of Leishmania (Viannia) braziliensis in the Sand Flies Lutzomyia (Nyssomyia) whitmani and Lutzomyia (Nyssomyia) intermedia

    PubMed Central

    Soares, Rodrigo P.; Margonari, Carina; Secundino, Nágila C.; Macêdo, Maria E.; da Costa, Simone M.; Rangel, Elizabeth F.; Pimenta, Paulo F.; Turco, Salvatore J.

    2010-01-01

    The interaction between Leishmania and sand flies has been demonstrated in many Old and New World species. Besides the morphological differentiation from procyclic to infective metacyclic promastigotes, the parasite undergoes biochemical transformations in its major surface lipophosphoglycan (LPG). An upregulation of β-glucose residues was previously shown in the LPG repeat units from procyclic to metacyclic phase in Leishmania (Viannia) braziliensis, which has not been reported in any Leishmania species. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the Subgenus Leishmania. These adaptations were explored for the first time in a species from the Subgenus Viannia, L. (V.) braziliensis with its natural vectors Lutzomyia (Nyssomyia) intermedia and Lutzomyia (Nyssomyia) whitmani. Using two in vitro binding techniques, phosphoglycans (PGs) derived from procyclic and metacyclic parasites were able to bind to the insect midgut and inhibit L. braziliensis attachment. Interestingly, L. braziliensis procyclic parasite attachment was ∼11-fold greater in the midgut of L. whitmani than in L. intermedia. The epidemiological relevance of L. whitmani as a vector of American Cutaneous Leishmaniasis (ACL) in Brazil is discussed. PMID:20011070

  9. Tissue- and time-dependent transcription in Ixodes ricinus salivary glands and midguts when blood feeding on the vertebrate host

    PubMed Central

    Kotsyfakis, Michalis; Schwarz, Alexandra; Erhart, Jan; Ribeiro, José M. C.

    2015-01-01

    Ixodes ricinus is a tick that transmits the pathogens of Lyme and several arboviral diseases. Pathogens invade the tick midgut, disseminate through the hemolymph, and are transmitted to the vertebrate host via the salivary glands; subverting these processes could be used to interrupt pathogen transfer. Here, we use massive de novo sequencing to characterize the transcriptional dynamics of the salivary and midgut tissues of nymphal and adult I. ricinus at various time points after attachment on the vertebrate host. Members of a number of gene families show stage- and time-specific expression. We hypothesize that gene expression switching may be under epigenetic control and, in support of this, identify 34 candidate proteins that modify histones. I. ricinus-secreted proteins are encoded by genes that have a non-synonymous to synonymous mutation rate even greater than immune-related genes. Midgut transcriptome (mialome) analysis reveals several enzymes associated with protein, carbohydrate, and lipid digestion, transporters and channels that might be associated with nutrient uptake, and immune-related transcripts including antimicrobial peptides. This publicly available dataset supports the identification of protein and gene targets for biochemical and physiological studies that exploit the transmission lifecycle of this disease vector for preventative and therapeutic purposes. PMID:25765539

  10. Genetic transformation of midgut bacteria from the red imported fire ant (Solenopsis invicta).

    PubMed

    Medina, Freder; Li, Haiwen; Vinson, S Bradleigh; Coates, Craig J

    2009-05-01

    In our previous study we isolated 10 bacterial species from fourth-instar larval midguts of the red imported fire ant, Solenopsis invicta. Here we report the genetic transformation and reintroduction of three species (Kluyvera cryocrescens, Serratia marcescens, and isolate 38) into the fire ant host. All three species were transformed with the plasmid vector, pZeoDsRed. High expression levels of DsRed were observed and the plasmid is maintained in these bacteria at 37 degrees C in the absence of antibiotic selection for at least 9 days of subculturing. The transformed bacteria were successfully reintroduced into fire ant larvae and survived in the fire ant gut for at least 7 days. Upon pupal emergence, 7 days after reintroduction, transformed bacteria can still be isolated, however, most were passed out in the meconium. We further demonstrated that the engineered bacteria could be spread within the colony by feeding this meconium to naive larvae with the aid of worker fire ants.

  11. Transcriptome of the Lymantria dispar (Gypsy Moth) Larval Midgut in Response to Infection by Bacillus thuringiensis

    PubMed Central

    Sparks, Michael E.; Blackburn, Michael B.; Kuhar, Daniel; Gundersen-Rindal, Dawn E.

    2013-01-01

    Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which 838, 1,248 and 3,305 were respectively partitioned into high-, mid- and low-quality tiers on the basis of homology information. Digital gene expression profiles suggested genes differentially expressed at 24 hours post infection, and qRT-PCR analyses were performed for verification. The differentially expressed genes primarily associated with digestive function, including α-amylase, lipase and carboxypeptidase; immune response, including C-type lectin 4; developmental genes such as arylphorin; as well as a variety of binding proteins: cellular retinoic acid binding protein (lipid-binding), insulin-related peptide binding protein (protein-binding) and ovary C/EBPg transcription factor (nucleic acid-binding). This is the first study conducted to specifically investigate gypsy moth response to a bacterial infection challenge using large-scale sequencing technologies, and the results highlight important genes that could be involved in biopesticide resistance development or could serve as targets for biologically-based control mechanisms of this insect pest. PMID:23658687

  12. Ookinete-induced midgut peroxidases detonate the time bomb in anopheline mosquitoes.

    PubMed

    Kumar, Sanjeev; Barillas-Mury, Carolina

    2005-07-01

    Previous analysis of the temporal-spatial relationship between ookinete migration and the cellular localization of genes mediating midgut immune defense responses suggested that, in order to survive, parasites must complete invasion before toxic chemicals ("a bomb") are generated by the invaded cell. Recent studies indicate that ookinete invasion induces tyrosine nitration as a two-step reaction, in which NOS induction is followed by a localized increase in peroxidase activity. Peroxidases utilize nitrite and hydrogen peroxide as substrates, and detonate the time bomb by generating reactive nitrogen intermediates, such as nitrogen dioxide, which mediate nitration. There is evidence that peroxidases also mediate antimicrobial responses to bacteria, fungi and parasites in a broad range of biological systems including humans and plants. Defense reactions that generate toxic chemicals are also potentially harmful to the host mounting the response and often results in apoptosis. The two-step nitration pathway is probably an ancient response, as it has also been described in vertebrate leukocytes and probably evolved as a mechanism to circumscribe the toxic products generated during defense responses involving protein nitration. PMID:15894189

  13. Rhynchophorus ferrugineus midgut cell line to evaluate insecticidal potency of different plant essential oils.

    PubMed

    Rizwan-ul-Haq, Muhammad; Aljabr, Ahmed Mohammed

    2015-03-01

    Cell cultures can be a potent and strong tool to evaluate the insecticidal efficiency of natural products. Plant essential oils have long been used as the fragrance or curative products around the world which means that they are safer to be used in close proximity of humans and mammals. In this study, a midgut cell line, developed from Rhynchophorus ferrugineus (RPW-1), was used for screening essential oils from nine different plants. Assays revealed that higher cell mortality was observed at 500 ppm which reached to 86, 65, 60, 59, 56, 54, 54, 53, and 53%, whereas lowest cell mortality at 1 ppm remained at 41, 23, 20, 17, 16, 15, 14, 13, and 10%, for Azadirachta indica, Piper nigrum, Mentha spicata, Cammiphora myrrha, Elettaria cardamomum, Zingiber officinale, Curcuma longa, Schinus molle, and Rosm