Azadirachtin Affects the Growth of Spodoptera litura Fabricius by Inducing Apoptosis in Larval Midgut.

PubMed

Shu, Benshui; Zhang, Jingjing; Cui, Gaofeng; Sun, Ranran; Yi, Xin; Zhong, Guohua

2018-01-01

Azadirachtin, the environmentally friendly botanical pesticide, has been used as an antifeedant and pest growth regulator in integrated pest management for decades. It has shown strong biological activity against Spodoptera litura , but the mechanism of toxicity remains unclear. The present study showed that azadirachtin inhibited the growth of S. litura larvae, which was resulted by structure destroy and size inhibition of the midgut. Digital gene expression (DGE) analysis of midgut suggested that azadirachtin regulated the transcriptional level of multiple unigenes involved in mitogen-activated protein kinase (MAPK) and calcium apoptotic signaling pathways. Simultaneously, the expression patterns of some differentially expressed unigenes were verified by quantitative real time-PCR (qRT-PCR). In addition, the enhanced terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, the increased expression of caspase family members and apoptosis-binding motif 1 (IBM1) on both gene and protein level and the release of cytochrome c from mitochondria to cytoplasm were induced in midgut after azadirachtin treatment. These results demonstrated that azadirachtin induced structural alteration in S. litura larval midgut by apoptosis activation. These alterations may affect the digestion and absorption of nutrients and eventually lead to the growth inhibition of larvae.

Azadirachtin Affects the Growth of Spodoptera litura Fabricius by Inducing Apoptosis in Larval Midgut

PubMed Central

Shu, Benshui; Zhang, Jingjing; Cui, Gaofeng; Sun, Ranran; Yi, Xin; Zhong, Guohua

2018-01-01

Azadirachtin, the environmentally friendly botanical pesticide, has been used as an antifeedant and pest growth regulator in integrated pest management for decades. It has shown strong biological activity against Spodoptera litura, but the mechanism of toxicity remains unclear. The present study showed that azadirachtin inhibited the growth of S. litura larvae, which was resulted by structure destroy and size inhibition of the midgut. Digital gene expression (DGE) analysis of midgut suggested that azadirachtin regulated the transcriptional level of multiple unigenes involved in mitogen-activated protein kinase (MAPK) and calcium apoptotic signaling pathways. Simultaneously, the expression patterns of some differentially expressed unigenes were verified by quantitative real time-PCR (qRT-PCR). In addition, the enhanced terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, the increased expression of caspase family members and apoptosis-binding motif 1 (IBM1) on both gene and protein level and the release of cytochrome c from mitochondria to cytoplasm were induced in midgut after azadirachtin treatment. These results demonstrated that azadirachtin induced structural alteration in S. litura larval midgut by apoptosis activation. These alterations may affect the digestion and absorption of nutrients and eventually lead to the growth inhibition of larvae. PMID:29535638

iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

PubMed

Gao, Kun; Deng, Xiang-Yuan; Shang, Meng-Ke; Qin, Guang-Xing; Hou, Cheng-Xiang; Guo, Xi-Jie

2017-01-30

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis

Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

PubMed Central

Khattab, Ayman; Barroso, Marta; Miettinen, Tiera; Meri, Seppo

2015-01-01

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood. PMID:25679788

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.

Midgut Protease Activity During Larval Development of Anastrepha obliqua (Diptera: Tephritidae) Fed With Natural and Artificial Diet

PubMed Central

Rivera-Ciprian, José Pedro; Aceituno-Medina, Marysol; Guillen, Karina

2017-01-01

Abstract In this study, we examined the activity of two serine proteases (chymotrypsin and trypsin) and two metalloproteases (carboxypeptidases A and B) during larval development in Anastrepha obliqua fed natural (mango fruit) and artificial (formulation used in mass-rearing) diets. Proteolytic activity of chymotrypsin, trypsin, carboxypeptidase A, and carboxypeptidase B was detected in the midgut of different instars of A. obliqua and was strongly affected by the pH and diet type. The protein content of the natural and artificial diets was similar. Enzymatic activity was higher in the midgut of the larvae fed the natural diet than in larvae fed the artificial diet. The activity of the endopeptidases (chymotrypsin and trypsin) was lower than those of the exopeptidases (carboxypeptidases A and B). The pH of the midgut varied from acidic to neutral. The results indicate that in the midgut of the larvae reared on both types of diet, the level of carboxypeptidase activity was approximately 100-fold greater than the level of chymotrypsin activity and 10,000-fold greater than the level of trypsin. In conclusion, carboxypeptidase A and B are the main proteases involved in the digestion of proteins in the larvae of A. obliqua. The natural diet showed a high bioaccessibility. A clear tendency to express high activities of chymotrypsin and trypsin was observed by the third instar. Our research contributes to the planning and development of novel bioaccessibility assays to understand the nutrition processing of A. obliqua larvae under mass-rearing conditions for sterile insect technique.

PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris.

PubMed

Jang, Seong Han; Jang, Ho Am; Lee, Junbeom; Kim, Jong Uk; Lee, Seung Ah; Park, Kyoung-Eun; Kim, Byung Hyun; Jo, Yong Hun; Lee, Bok Luel

2017-06-01

Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (Δ phaP1 , Δ phaP2 , Δ phaP3 , and Δ phaP4 mutants), one phaR gene-depleted mutant, and a phaR -complemented mutant (Δ phaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second-instar nymphs, and colonization ability and fitness parameters were examined. In vitro , the Δ phaP3 and Δ phaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the Δ phaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia -infected insects. However, other phaP family gene-depleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the Δ phaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut. IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA

PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris

PubMed Central

Jang, Seong Han; Jang, Ho Am; Lee, Junbeom; Kim, Jong Uk; Lee, Seung Ah; Park, Kyoung-Eun; Kim, Byung Hyun; Jo, Yong Hun

2017-01-01

ABSTRACT Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (ΔphaP1, ΔphaP2, ΔphaP3, and ΔphaP4 mutants), one phaR gene-depleted mutant, and a phaR-complemented mutant (ΔphaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second-instar nymphs, and colonization ability and fitness parameters were examined. In vitro, the ΔphaP3 and ΔphaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the ΔphaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia-infected insects. However, other phaP family gene-depleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the ΔphaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut. IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA granules

Intestinal Rotation Abnormalities and Midgut Volvulus.

PubMed

Langer, Jacob C

2017-02-01

Rotation abnormalities may be asymptomatic or may be associated with obstruction caused by bands, midgut volvulus, or associated atresia or web. The most important goal of clinicians is to determine whether the patient has midgut volvulus with intestinal ischemia, in which case an emergency laparotomy should be done. If the patient is not acutely ill, the next goal is to determine whether the patient has a narrow-based small bowel mesentery. In general, the outcomes for children with a rotation abnormality are excellent, unless there has been midgut volvulus with significant intestinal ischemia. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Regulation of pH During Amelogenesis.

PubMed

Lacruz, Rodrigo S; Nanci, Antonio; Kurtz, Ira; Wright, J Timothy; Paine, Michael L

2010-02-01

During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation.

The midgut transcriptome of Aedes aegypti fed with saline or protein meals containing chikungunya virus reveals genes potentially involved in viral midgut escape.

PubMed

Dong, Shengzhang; Behura, Susanta K; Franz, Alexander W E

2017-05-15

The mosquito Aedes aegypti is the primary vector for medically important arthropod-borne viruses, including chikungunya virus (CHIKV). Following oral acquisition, an arbovirus has to persistently infect several organs in the mosquito before becoming transmissible to another vertebrate host. A major obstacle an arbovirus has to overcome during its infection cycle inside the mosquito is the midgut escape barrier, representing the exit mechanism arboviruses utilize when disseminating from the midgut. To understand the transcriptomic basis of midgut escape and to reveal genes involved in the process, we conducted a comparative transcriptomic analysis of midgut samples from mosquitoes which had received a saline meal (SM) or a protein meal (PM) (not) containing CHIKV. CHIKV which was orally acquired by a mosquito along with a SM or PM productively infected the midgut epithelium and disseminated to secondary tissues. A total of 27 RNA-Seq libraries from midguts of mosquitoes that had received PM or SM (not) containing CHIKV at 1 and 2 days post-feeding were generated and sequenced. Fewer than 80 genes responded differentially to the presence of CHIKV in midguts of mosquitoes that had acquired the virus along with SM or PM. SM feeding induced differential expression (DE) of 479 genes at day 1 and 314 genes at day 2 when compared to midguts of sugarfed mosquitoes. By comparison, PM feeding induced 6029 DE genes at day 1 and 7368 genes at day 2. Twenty-three DE genes encoding trypsins, metalloproteinases, and serine-type endopeptidases were significantly upregulated in midguts of mosquitoes at day 1 following SM or PM ingestion. Two of these genes were Ae. aegypti late trypsin (AeLT) and serine collagenase 1 precursor (AeSP1). In vitro, recombinant AeLT showed strong matrix metalloproteinase activity whereas recombinant AeSP1 did not. By substituting a bloodmeal for SM, we identified midgut-expressed genes not involved in blood or protein digestion. These included genes

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Shin-Hae; Park, Joung-Sun; Kim, Young-Shin

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 inmore » 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.« less

Org-1 is required for the diversification of circular visceral muscle founder cells and normal midgut morphogenesis

PubMed Central

Schaub, Christoph; Frasch, Manfred

2013-01-01

The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. In this study we report that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. We demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells we genetically dissected known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. We conclude that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut. PMID:23380635

Org-1 is required for the diversification of circular visceral muscle founder cells and normal midgut morphogenesis.

PubMed

Schaub, Christoph; Frasch, Manfred

2013-04-15

The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. In this study we report that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. We demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells we genetically dissected known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. We conclude that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular Genetic Analysis of Midgut Serine Proteases in Aedes aegypti Mosquitoes

PubMed Central

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

2009-01-01

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

Differential protein modulation in midguts of Aedes aegypti infected with chikungunya and dengue 2 viruses.

PubMed

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

2010-10-05

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. 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. 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 virus survival, replication and transmission, suggesting a subversion of

pH regulation in glycosomes of procyclic form Trypanosoma brucei.

PubMed

Lin, Sheng; Voyton, Charles; Morris, Meredith T; Ackroyd, P Christine; Morris, James C; Christensen, Kenneth A

2017-05-12

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na + ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na + /H + exchangers are required for glycosomal pH regulation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues

PubMed Central

Wang, Yanhai; Jin, Binbin; Liu, Peiwen; Li, Jing; Chen, Xiaoguang; Gu, Jinbao

2018-01-01

. albopictus; however, only AalPiwi5–7 and AalAgo3(1–2) were readily detected in the midgut. The characteristics of piRNAs in DENV-2-infected Ae. albopictus adult females were similar to those previously described for flavivirus infections but were not observed in the midgut. The reduced levels of vepiRNAs and incomplete expression of PIWI pathway genes in midgut samples from DENV-2-infected Ae. albopictus suggests that viral regulation of host piRNAs may not be an important factor in the midgut. PMID:29690553

The development of malaria parasites in the mosquito midgut

PubMed Central

Bennink, Sandra; Kiesow, Meike J.

2016-01-01

Summary The mosquito midgut stages of malaria parasites are crucial for establishing an infection in the insect vector and to thus ensure further spread of the pathogen. Parasite development in the midgut starts with the activation of the intraerythrocytic gametocytes immediately after take‐up and ends with traversal of the midgut epithelium by the invasive ookinetes less than 24 h later. During this time period, the plasmodia undergo two processes of stage conversion, from gametocytes to gametes and from zygotes to ookinetes, both accompanied by dramatic morphological changes. Further, gamete formation requires parasite egress from the enveloping erythrocytes, rendering them vulnerable to the aggressive factors of the insect gut, like components of the human blood meal. The mosquito midgut stages of malaria parasites are unprecedented objects to study a variety of cell biological aspects, including signal perception, cell conversion, parasite/host co‐adaptation and immune evasion. This review highlights recent insights into the molecules involved in gametocyte activation and gamete formation as well as in zygote‐to‐ookinete conversion and ookinete midgut exit; it further discusses factors that can harm the extracellular midgut stages as well as the measures of the parasites to protect themselves from any damage. PMID:27111866

Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae).

PubMed

Fialho, Maria do Carmo Q; Terra, Walter R; Moreira, Nathália R; Zanuncio, José C; Serrão, Jose Eduardo

2013-07-01

The predatory stinkbug Podisus nigrispinus has been utilized in biological control programs. Its midgut is anatomically divided into anterior, middle and posterior regions, which play different roles in the digestive process. We describe the midgut ultrastructure and the secretion of digestive enzymes in the midgut of P. nigrispinus. Midguts were analyzed with transmission electron microscopy and the digestive enzymes amylase, cathepsin L, aminopeptidase and α-glucosidase were immunolocalized. The ultrastructural features of the digestive cells in the anterior, middle and posterior midgut regions suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, storage and excretion. The digestive enzymes have different distribution along the midgut regions of the predator P. nigrispinus. Amylase, aminopeptidase and α-glucosidase occur in three midgut regions, whereas cathepsin L occurs in the middle and posterior midgut regions. The anterior midgut region of P. nigrispinus seems to play a role in water absorption, the middle midgut may be involved in nutrient absorption and the posterior midgut region is responsible for water transport to the midgut lumen. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Brain-midgut short neuropeptide F mechanism that inhibits digestive activity of the American cockroach, Periplaneta americana upon starvation.

PubMed

Mikani, Azam; Wang, Qiu-Shi; Takeda, Makio

2012-03-01

Immunohistochemical reactivity against short neuropeptide F (sNPF) was observed in the brain-corpus cardiacum and midgut paraneurons of the American cockroach, Periplaneta americana. Four weeks of starvation increased the number of sNPF-ir cells in the midgut epithelium but the refeeding decreased the number in 3h. Dramatic rises in sNPF contents in the midgut epithelium and hemolymph of roaches starved for 4 weeks were confirmed by ELISA. Starvation for 4 weeks reduced α-amylase, protease and lipase activities in the midgut of P. americana but refeeding restored these to high levels. Co-incubation of dissected midgut with sNPF at physiological concentrations inhibited α-amylase, protease and lipase activities. sNPF injection into the hemocoel led to a decrease in α-amylase, protease and lipase activities, whereas PBS injection had no effects. The injection of d-(+)-trehalose and l-proline into the hemocoel of decapitated adult male cockroaches that had been starved for 4 weeks had no effect on these digestive enzymes. However, injection into the hemocoel of head-intact starved cockroaches stimulated digestive activity. Injection of d-(+)-trehalose and l-proline into the lumen of decapitated cockroaches that had been starved for 4 weeks increased enzymes activities and suppressed sNPF in the midgut. Our data indicate that sNPF from the midgut paraneurons suppresses α-amylase, protease and lipase activities during starvation. Injection of d-(+)-trehalose/l-proline into the hemocoel of head-intact starved cockroach decreased the hemolymph sNPF content, which suggests that sNPF could be one of the brain factors, demonstrating brain-midgut interplay in the regulation of digestive activities and possibly nutrition-associated behavioral modifications. Copyright © 2011 Elsevier Inc. All rights reserved.

Histone deacetylase-mediated regulation of endolysosomal pH.

PubMed

Prasad, Hari; Rao, Rajini

2018-05-04

The pH of the endolysosomal system is tightly regulated by a balance of proton pump and leak mechanisms that are critical for storage, recycling, turnover, and signaling functions in the cell. Dysregulation of endolysosomal pH has been linked to aging, amyloidogenesis, synaptic dysfunction, and various neurodegenerative disorders, including Alzheimer's disease. Therefore, understanding the mechanisms that regulate luminal pH may be key to identifying new targets for managing these disorders. Meta-analysis of yeast microarray databases revealed that nutrient-limiting conditions inhibited the histone deacetylase (HDAC) Rpd3 and thereby up-regulated transcription of the endosomal Na + /H + exchanger Nhx1, resulting in vacuolar alkalinization. Consistent with these findings, Rpd3 inhibition by the HDAC inhibitor and antifungal drug trichostatin A induced Nhx1 expression and vacuolar alkalinization. Bioinformatics analysis of Drosophila and mouse databases revealed that caloric control of the Nhx1 orthologs DmNHE3 and NHE6, respectively, is also mediated by HDACs. We show that NHE6 is a target of the transcription factor cAMP-response element-binding protein (CREB), a known regulator of cellular responses to low-nutrient conditions, providing a molecular mechanism for nutrient- and HDAC-dependent regulation of endosomal pH. Of note, pharmacological targeting of the CREB pathway to increase NHE6 expression helped regulate endosomal pH and correct defective clearance of amyloid Aβ in an apoE4 astrocyte model of Alzheimer's disease. These observations from yeast, fly, mouse, and cell culture models point to an evolutionarily conserved mechanism for HDAC-mediated regulation of endosomal NHE expression. Our insights offer new therapeutic strategies for modulation of endolysosomal pH in fungal infection and human disease. © 2018 Prasad and Rao.

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

pH sensing and regulation in cancer.

PubMed

Damaghi, Mehdi; Wojtkowiak, Jonathan W; Gillies, Robert J

2013-12-17

Cells maintain intracellular pH (pHi) within a narrow range (7.1-7.2) by controlling membrane proton pumps and transporters whose activity is set by intra-cytoplasmic pH sensors. These sensors have the ability to recognize and induce cellular responses to maintain the pHi, often at the expense of acidifying the extracellular pH. In turn, extracellular acidification impacts cells via specific acid-sensing ion channels (ASICs) and proton-sensing G-protein coupled receptors (GPCRs). In this review, we will discuss some of the major players in proton sensing at the plasma membrane and their downstream consequences in cancer cells and how these pH-mediated changes affect processes such as migration and metastasis. The complex mechanisms by which they transduce acid pH signals to the cytoplasm and nucleus are not well understood. However, there is evidence that expression of proton-sensing GPCRs such as GPR4, TDAG8, and OGR1 can regulate aspects of tumorigenesis and invasion, including cofilin and talin regulated actin (de-)polymerization. Major mechanisms for maintenance of pHi homeostasis include monocarboxylate, bicarbonate, and proton transporters. Notably, there is little evidence suggesting a link between their activities and those of the extracellular H(+)-sensors, suggesting a mechanistic disconnect between intra- and extracellular pH. Understanding the mechanisms of pH sensing and regulation may lead to novel and informed therapeutic strategies that can target acidosis, a common physical hallmark of solid tumors.

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.

MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Donly, B. Cameron, E-mail: Cam.Donly@agr.gc.ca

Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, asmore » well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues. -- Highlights: •The transcriptome of MacoNPV ODV in larval midgut was measured by RNA-seq and digital PCR. •The earliest genes expressed included fusion protein, hoar, and me53. •p6.9 was highly expressed late but polH and p10 were less so. •These patterns are unique from BV of other baculoviruses in tissue culture cells.« less

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.

Constant pH Accelerated Molecular Dynamics Investigation of the pH Regulation Mechanism of Dinoflagellate Luciferase.

PubMed

Donnan, Patrick H; Ngo, Phong D; Mansoorabadi, Steven O

2018-01-23

The bioluminescence reaction in dinoflagellates involves the oxidation of an open-chain tetrapyrrole by the enzyme dinoflagellate luciferase (LCF). The activity of LCF is tightly regulated by pH, where the enzyme is essentially inactive at pH ∼8 and optimally active at pH ∼6. Little is known about the mechanism of LCF or the structure of the active form of the enzyme, although it has been proposed that several intramolecularly conserved histidine residues in the N-terminal region are important for the pH regulation mechanism. Here, constant pH accelerated molecular dynamics was employed to gain insight into the conformational activation of LCF induced by acidification.

Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy.

PubMed

Izumi, Hiroto; Torigoe, Takayuki; Ishiguchi, Hiroshi; Uramoto, Hidetaka; Yoshida, Yoichiro; Tanabe, Mizuho; Ise, Tomoko; Murakami, Tadashi; Yoshida, Takeshi; Nomoto, Minoru; Kohno, Kimitoshi

2003-12-01

One of the major obstacles to the successful treatment of cancer is the complex biology of solid tumour development. Although regulation of intracellular pH has been shown to be critically important for many cellular functions, pH regulation has not been fully investigated in the field of cancer. It has, however, been shown that cellular pH is crucial for biological functions such as cell proliferation, invasion and metastasis, drug resistance and apoptosis. Hypoxic conditions are often observed during the development of solid tumours and lead to intracellular and extracellular acidosis. Cellular acidosis has been shown to be a trigger in the early phase of apoptosis and leads to activation of endonucleases inducing DNA fragmentation. To avoid intracellular acidification under such conditions, pH regulators are thought to be up-regulated in tumour cells. Four major types of pH regulator have been identified: the proton pump, the sodium-proton exchanger family (NHE), the bicarbonate transporter family (BCT) and the monocarboxylate transporter family (MCT). Here, we describe the structure and function of pH regulators expressed in tumour tissue. Understanding pH regulation in tumour cells may provide new ways of inducing tumour-specific apoptosis, thus aiding cancer chemotherapy.

The discovery of a novel antagonist - Manduca sexta allatotropin analogue - as an insect midgut active ion transport inhibitor.

PubMed

Deng, Xi-le; Kai, Zhen-Peng; Chamberlin, Mary E; Horodyski, Frank M; Yang, Xin-Ling

2016-11-01

The midgut is an important site for both nutrient absorption and ionic regulation in lepidopteran larvae, major pests in agriculture. The larval lepidopteran midgut has become a potent insecticide target over the past few decades. Recent studies have shown that an insect neuropeptide, Manduca sexta allatotropin (Manse-AT), exhibits inhibition of active ion transport (AIT) across the larval midgut epithelium. The full characteristic of the AIT inhibition capacity of Manse-AT is essential to assay. In this study, AIT inhibition across the M. sexta midgut by Manse-AT and its analogues in a range of concentrations was assayed. The structure-activity relationship of Manse-AT was also studied by truncated and alanine-replacement strategies. Our results identified three residues, Thr4, Arg6 and Phe8, as the most important components for activity on the midgut. Replacement of Glu1, Met2 and Met3 reduced the potency of the analogues. The conservative substitution of Gly7 with alanine had little effect on the potency of the analogues. We demonstrated for the first time that Manse-AT (10-13) behaves as a potent antagonist in vitro on active ion transport across the epithelium of the posterior midgut in M. sexta. Structure-activity studies of Manse-AT are useful in developing lead compounds for the design and testing of synthetic antagonists, ultimately to develop potent and specific pest control strategies. Manse-AT (10-13) has been discovered as the first Manse-AT antagonist, with a significant effect and a short sequence compared with other insect neuropeptides. It may be a new potential pest control agent in the future. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Targeting pH regulating proteins for cancer therapy-Progress and limitations.

PubMed

Parks, Scott K; Pouysségur, Jacques

2017-04-01

Tumour acidity induced by metabolic alterations and incomplete vascularisation sets cancer cells apart from normal cellular physiology. This distinguishing tumour characteristic has been an area of intense study, as cellular pH (pH i ) disturbances disrupt protein function and therefore multiple cellular processes. Tumour cells effectively utilise pH i regulating machinery present in normal cells with enhancements provided by additional oncogenic or hypoxia induced protein modifications. This overall improvement of pH regulation enables maintenance of an alkaline pH i in the continued presence of external acidification (pH e ). Considerable experimentation has revealed targets that successfully disrupt tumour pH i regulation in efforts to develop novel means to weaken or kill tumour cells. However, redundancy in these pH-regulating proteins, which include Na + /H + exchangers (NHEs), carbonic anhydrases (CAs), Na + /HCO 3 - co-transporters (NBCs) and monocarboxylate transporters (MCTs) has prevented effective disruption of tumour pH i when individual protein targeting is performed. Here we synthesise recent advances in understanding both normoxic and hypoxic pH regulating mechanisms in tumour cells with an ultimate focus on the disruption of tumour growth, survival and metastasis. Interactions between tumour acidity and other cell types are also proving to be important in understanding therapeutic applications such as immune therapy. Promising therapeutic developments regarding pH manipulation along with current limitations are highlighted to provide a framework for future research directives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Morphometry of the midgut of Melipona quadrifasciata anthidioides (Lepeletier) (Hymenoptera: Apidae) during metamorphosis.

PubMed

Cruz, L C; Araújo, V A; Dolder, H; Araújo, A P A; Serrão, J E; Neves, C A

2011-01-01

In Hymenoptera, midgut changes begin in the last instar. At this stage, the larval epithelial digestive cells degenerate, leaving only the basal membrane and the regenerative cells which will develop into a new epithelium during the pupal stage and in the adult. Epithelium renewal is followed by changes in volume and shape of the midgut. Morphometric analysis of digestive cells and total midgut volume of Melipona quadrifasciata anthidioides (Lepeletier) were conducted to verify whether cell volume increase are sufficient to account for the total midgut volume increase that occurs during metamorphosis. An increase in midgut volume was verified in spite of the scarcity of cell proliferation found during metamorphosis. At the end of metamorphosis, the increase in cell volume was not sufficient to explain the increase in volume of the midgut, indicating that an increase in the number of digestive cells is apparently necessary. Nevertheless, the mechanism by which regenerative cells reconstitute the epithelium during metamorphosis remains unknown.

Primary midgut, salivary gland, and ovary cultures from Boophilus microplus.

PubMed

Mosqueda, Juan; Cossío-Bayugar, Raquel; Rodríguez, Elba; Falcón, Alfonso; Ramos, Alberto; Figueroa, Julio V; Alvarez, Antonio

2008-12-01

Primary cell cultures from different tick organs are a valuable tool for host parasite research in the study of the protozoan Babesia sp., which infects different organs of the tick. In this work we describe the generation of midgut, salivary gland, and ovary primary cell cultures from dissections of Boophilus microplus. Midguts, salivary glands, and ovaries were dissected from B. microplus ticks on different days after bovine infestation; different enzymatic disaggregating protocols were tested in the presence of proteolytic enzymes, such as trypsin and collagenase type I and II, for tissue disaggregation and primary cell culture generation. The dissected tick organs obtained 18-20 days after bovine infestation showed a major cellular differentiation and were easier to identify by cellular morphology. The enzymatic disaggregation results showed that each tissue required a different proteolytic enzyme for optimal disaggregation; collagenase type I produced the most complete disaggregation for ovaries but not for midgut or salivary glands. Collagenase type II was effective for salivary glands but performed poorly on ovaries and midgets, and typsin was effective for midguts only. The midgut and ovary primary cell cultures were maintained for 4 weeks in optimal conditions after the cells were no longer viable. The salivary gland cell cultures were viable for 8 months.

Differential expression of chemosensory-protein genes in midguts in response to diet of Spodoptera litura.

PubMed

Yi, Xin; Qi, Jiangwei; Zhou, Xiaofan; Hu, Mei Ying; Zhong, Guo Hua

2017-03-22

While it has been well characterized that chemosensory receptors in guts of mammals have great influence on food preference, much remains elusive in insects. Insect chemosensory proteins (CSPs) are soluble proteins that could deliver chemicals to olfactory and gustatory receptors. Recent studies have identified a number of CSPs expressed in midgut in Lepidoptera insects, which started to reveal their roles in chemical recognition and stimulating appetite in midgut. In this study, we examined expression patterns in midgut of 21 Spodoptera litura CSPs (SlitCSPs) characterized from a previously reported transcriptome, and three CSPs were identified to be expressed highly in midgut. The orthologous relationships between midgut expressed CSPs in S. litura and those in Bombyx mori and Plutella xylostella also suggest a conserved pattern of CSP expression in midgut. We further demonstrated that the expression of midgut-CSPs may change in response to different host plants, and SlitCSPs could bind typical chemicals from host plant in vitro. Overall, our results suggested midgut expressed SlitCSPs may have functional roles, likely contributing to specialization and adaption to different ecosystems. Better knowledge of this critical component of the chemsensation signaling pathways in midguts may improve our understanding of food preference processes in a new perspective.

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis.

PubMed

Khan, Meraj A; Philip, Lijy M; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pH e ) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H + ions reducing the intracellular pH (pH i ). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pH e (ranging from 6.6 to 7.8; every 0.2 units) increased pH i of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H + ions, pH i is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pH e promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pH e -mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H + ions, whereas each bicarbonate HCO3 - ion binds 1H + ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

Lufenuron impact upon Anthonomus grandis Boheman (Coleoptera: Curculionidae) midgut and its reflection in gametogenesis.

PubMed

Costa, Hilton Nobre; da Cunha, Franklin Magliano; Cruz, Glaucilane Santos; D'assunção, Carolline Guimarães; Rolim, Guilherme Gomes; Barros, Maria Edna Gomes; Breda, Mariana Oliveira; Teixeira, Alvaro Aguiar Coelho; Teixeira, Valéria Wanderley

2017-04-01

The insecticide Match® (lufenuron), one of the main insect growth regulators used in pest control, has been presented as a viable alternative against the boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), by inhibiting chitin synthesis. Thus, this study aimed to examine whether Match® interferes in the synthesis of the peritrophic matrix, leading to changes in the midgut epithelium, resulting in nutritional deficiency and reflecting, thereby, in the gametogenesis process of A. grandis. Floral cotton buds were immersed in the insecticide solution (800μL of Match®+200mL of distilled water) and offered to the adult insects. The midguts of the insects were evaluated after 24 and 120h after feeding. The gonads were evaluated after 120h. The results showed that Match®, in both evaluation periods, induced histopathological alterations such as disorganization, vacuolization and desquamation of the midgut epithelium; histochemical modifications in the distribution patterns of carbohydrates, although without quantitative changes; and a strong decrease in protein levels. No apoptosis were observed, however, there was an increase in the number of regenerative cell nests. In the testicles, a reduction in the amount of spermatozoids and reduced carbohydrate levels were observed, but no difference in protein levels. The ovarioles presented structural disorganization of follicular cells, yolk reduction and decrease in protein levels, however, no change in carbohydrates levels was noted. Therefore, it is concluded that Match® performs histopathologic and histochemical alterations in the midgut epithelium and the gonads of A. grandis adults, reflecting in the gametogenesis process, presenting itself as a promising tool in the management of this pest on cotton crops. Copyright © 2016 Elsevier Inc. All rights reserved.

Brevibacillus laterosporus pathogenesis and local immune response regulation in the house fly midgut.

PubMed

Mura, Maria Elena; Ruiu, Luca

2017-05-01

The insect midgut represents the primary site of action of the entompathogenic bacterium Brevibacillus laterosporus. While most studies on this microorganism focus on the identification and characterization of possible virulence factors and toxins, little is known about the insect immune defense mechanisms that are activated against this pathogen. In this study we have investigated the local immune response of different house fly stages to B. laterosporus at the transcriptional level, and we tested the hypothesis that an improvement in entomopathogenicity can be achieved by impairing host innate immunity. Gene expression analyses showed that immediately after spore ingestion (6-12h) both larvae and adults increased the transcription rate of immune related genes in the midgut tissues, with special regard to those encoding for the main house fly antimicrobial peptides (AMPs) (i.e., attacin, cecropin, defensin, diptericin, domesticin, muscin) and for prophenoloxydase that is normally involved in the cascade of events leading to the generation of reactive oxygen species (ROS) and other factors with antibacterial properties. In experiments evaluating the use of an immunosuppressive agent to enhance the virulence of B. laterosporus against adult house flies, a significant downregulation of the same genes was observed 12-24h after the administration of sub-lethal doses of the botanical compound azadirachtin. Consequently, a significant increase in B. laterosporus entomopathogenic action was observed when flies were preliminarily or simultaneously exposed to a sub-lethal dose of azadirachtin. These results provide an important contribution to the prospect of employing immune-impairing tools to implement pest management strategies. Copyright © 2017 Elsevier Inc. All rights reserved.

Gene expression profiling provides insights into the immune mechanism of Plutella xylostella midgut to microbial infection.

PubMed

Lin, Junhan; Xia, Xiaofeng; Yu, Xiao-Qiang; Shen, Jinhong; Li, Yong; Lin, Hailan; Tang, Shanshan; Vasseur, Liette; You, Minsheng

2018-03-20

Insect gut immunity plays a key role in defense against microorganism infection. The knowledge of insect gut immunity has been obtained mostly from Drosophila melanogaster. Little is known about gut immunity in the diamondback moth, Plutella xylostella (L.), a pest destroying cruciferous crops worldwide. In this study, expressions of the immune-related genes in the midgut of P. xylostella orally infected with Staphylococcus aureus, Escherichia coli and Pichia pastoris were profiled by RNA-seq and qRT-PCR approaches. The results revealed that the Toll, IMD, JNK and JAK-STAT pathways and possibly the prophenoloxidase activation system in P. xylostella could be activated by oral infections, and moricins, gloverins and lysozyme2 might act as important effectors against microorganisms. Subsequent knock-down of IMD showed that this gene was involved in regulating the expression of down-stream genes in the IMD pathway. Our work indicates that the Toll, IMD, JNK and JAK-STAT pathways may synergistically modulate immune responses in the P. xylostella midgut, implying a complex and diverse immune system in the midgut of insects. Copyright © 2018 Elsevier B.V. All rights reserved.

The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies.

PubMed

Dostálová, Anna; Votýpka, Jan; Favreau, Amanda J; Barbian, Kent D; Volf, Petr; Valenzuela, Jesus G; Jochim, Ryan C

2011-05-10

Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles. A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal. This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative

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

PubMed

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

2016-01-15

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 × 10(9) 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. 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 midgut epithelium of an

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buarque, Diego S.; Spindola, Leticia M.N.; Martins, Rafael M.

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 Tigutcystatinmore » 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.« less

pCO2 and pH regulation of cerebral blood flow

PubMed Central

Yoon, SeongHun; Zuccarello, Mario; Rapoport, Robert M.

2012-01-01

CO2 serves as one of the fundamental regulators of cerebral blood flow (CBF). It is widely considered that this regulation occurs through pCO2-driven changes in pH of the cerebral spinal fluid (CSF), with elevated and lowered pH causing direct relaxation and contraction of the smooth muscle, respectively. However, some findings also suggest that pCO2 acts independently of and/or in conjunction with altered pH. This action may be due to a direct effect of CSF pCO2 on the smooth muscle as well as on the endothelium, nerves, and astrocytes. Findings may also point to an action of arterial pCO2 on the endothelium to regulate smooth muscle contractility. Thus, the effects of pH and pCO2 may be influenced by the absence/presence of different cell types in the various experimental preparations. Results may also be influenced by experimental parameters including myogenic tone as well as solutions containing significantly altered HCO3− concentrations, i.e., solutions routinely employed to differentiate the effects of pH from pCO2. In sum, it appears that pCO2, independently and in conjunction with pH, may regulate CBF. PMID:23049512

Transepithelial SCFA fluxes link intracellular and extracellular pH regulation of mouse colonocytes.

PubMed

Chu, S; Montrose, M H

1997-10-01

We have studied pH regulation in both intracellular and extracellular compartments of mouse colonic crypts, using distal colonic mucosa with intact epithelial architecture. In this work, we question how transepithelial SCFA gradients affect intracellular pH (pHi) and examine interactions between extracellular pH (pHo) and pHi regulation in crypts of distal colonic epithelium from mouse. We studied pH regulation in three adjacent compartments of distal colonic epithelium (crypt lumen, crypt epithelial cell cytosol, and lamina propria) with SNARF-1 (a pH sensitive fluorescent dye), digital imaging microscopy (for pHi), and confocal microscopy (for pHo). Combining results from the three compartments allows us to find how pHi and pHo are regulated and related under the influence of physiological transepithelial SCFA gradients, and develop a better understanding of pH regulation mechanisms in colonic crypts. Results suggest a complex interdependency between SCFA fluxes and pHo values, which can directly affect how strongly SCFAs acidify colonocytes.

Chitinases of the avian malaria parasite Plasmodium gallinaceum, a class of enzymes necessary for parasite invasion of the mosquito midgut.

PubMed

Vinetz, J M; Valenzuela, J G; Specht, C A; Aravind, L; Langer, R C; Ribeiro, J M; Kaslow, D C

2000-04-07

The Plasmodium ookinete produces chitinolytic activity that allows the parasite to penetrate the chitin-containing peritrophic matrix surrounding the blood meal in the mosquito midgut. Since the peritrophic matrix is a physical barrier that the parasite must cross to invade the mosquito, and the presence of allosamidin, a chitinase inhibitor, in a blood meal prevents the parasite from invading the midgut epithelium, chitinases (3.2.1.14) are potential targets of malaria parasite transmission-blocking interventions. We have purified a chitinase of the avian malaria parasite Plasmodium gallinaceum and cloned the gene, PgCHT1, encoding it. PgCHT1 encodes catalytic and substrate-binding sites characteristic of family 18 glycohydrolases. Expressed in Escherichia coli strain AD494 (DE3), recombinant PgCHT1 was found to hydrolyze polymeric chitin, native chitin oligosaccharides, and 4-methylumbelliferone derivatives of chitin oligosaccharides. Allosamidin inhibited recombinant PgCHT1 with an IC(50) of 7 microM and differentially inhibited two chromatographically separable P. gallinaceum ookinete-produced chitinase activities with IC(50) values of 7 and 12 microM, respectively. These two chitinase activities also had different pH activity profiles. These data suggest that the P. gallinaceum ookinete uses products of more than one chitinase gene to initiate mosquito midgut invasion.

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

PubMed

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; Silva-Neto, Mário Alberto Cardoso

2016-10-01

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. 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. 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.

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.

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

PubMed Central

Khan, Meraj A.; Philip, Lijy M.; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity

PubMed Central

Bando, Hironori; Okado, Kiyoshi; Guelbeogo, Wamdaogo M.; Badolo, Athanase; Aonuma, Hiroka; Nelson, Bryce; Fukumoto, Shinya; Xuan, Xuenan; Sagnon, N'Fale; Kanuka, Hirotaka

2013-01-01

A critical stage in malaria transmission occurs in the Anopheles mosquito midgut, when the malaria parasite, Plasmodium, ingested with blood, first makes contact with the gut epithelial surface. To understand the response mechanisms within the midgut environment, including those influenced by resident microbiota against Plasmodium, we focus on a midgut bacteria species' intra-specific variation that confers diversity to the mosquito's competency for malaria transmission. Serratia marcescens isolated from either laboratory-reared mosquitoes or wild populations in Burkina Faso shows great phenotypic variation in its cellular and structural features. Importantly, this variation is directly correlated with its ability to inhibit Plasmodium development within the mosquito midgut. Furthermore, this anti-Plasmodium function conferred by Serratia marcescens requires increased expression of the flagellum biosynthetic pathway that is modulated by the motility master regulatory operon, flhDC. These findings point to new strategies for controlling malaria through genetic manipulation of midgut bacteria within the mosquito. PMID:23571408

Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

PubMed Central

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; Pennacchio, Francesco

2016-01-01

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

Intrauterine midgut volvulus without malrotation: Diagnosis from the ‘coffee bean sign’

PubMed Central

Park, Jun Seok; Cha, Seong Jae; Kim, Beom Gyu; Kim, Yong Seok; Choi, Yoo Shin; Chang, In Taik; Kim, Gwang Jun; Lee, Woo Seok; Kim, Gi Hyeon

2008-01-01

Fetal midgut volvulus is quite rare, and most cases are associated with abnormalities of intestinal rotation or fixation. We report a case of midgut volvulus without malrotation, associated with a meconium pellet, during the gestation period. This 2.79 kg, 33-wk infant was born via a spontaneous vaginal delivery caused by preterm labor. Prenatal ultrasound showed dilated bowel loops with the appearance of a ‘coffee bean sign’. This patient had an unusual presentation with a distended abdomen showing skin discoloration. An emergency laparotomy revealed a midgut volvulus and a twisted small bowel, caused by complicated meconium ileus. Such nonspecific prenatal radiological signs and a low index of suspicion of a volvulus during gestation might delay appropriate surgical management and result in ischemic necrosis of the bowel. Preterm labor, specific prenatal sonographic findings (for example, the coffee bean sign) and bluish discoloration of the abdominal wall could suggest intrauterine midgut volvulus requiring prompt surgical intervention. PMID:18322966

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

Proteomic analysis of Aedes aegypti midgut during post-embryonic development and of the female mosquitoes fed different diets.

PubMed

Fernandes, Kenner Morais; de Magalhães-Júnior, Marcos Jorge; Baracat-Pereira, Maria Cristina; Martins, Gustavo Ferreira

2016-12-01

In this work we analyzed protein expression in the Aedes aegypti midgut during the larval (fourth instar, L4), pupal, and adult stages [including newly emerged (NE), sugar-fed (SF) and blood-fed (BF) females]. Two-dimensional electrophoresis showed 13 spots in the midgut of larvae, 95 in the midgut of pupae, 90 in the midgut of NE, and 76 in the midgut of SF or BF females. In the larval midguts, high serpin expression was noted, while in the pupae, protein abundance was lower than in the NE, SF, and BF females. The spots related to proteins linked to energy production, protein metabolism, signaling, and transport were highly expressed in the NE stage, while spots related proteins involved in translation were abundant in SF and BF females. The differential abundance of proteins in the midgut of A. aegypti at different developmental stages supports the necessity for midgut development during immature stage followed by the necessity of proteins related to digestion in adults. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion

PubMed Central

Dong, Shengzhang; Balaraman, Velmurugan; Kantor, Asher M.; Lin, Jingyi; Grant, DeAna G.; Held, Nicole L.

2017-01-01

In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus) that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB), is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV) as a model in Aedes aegypti, we demonstrate that the basal lamina (BL) of the extracellular matrix (ECM) surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24–32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs) are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (Ae)MMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase) activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP) in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in vitro

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

Catalase protects Aedes aegypti from oxidative stress and increases midgut infection prevalence of Dengue but not Zika.

PubMed

Oliveira, José Henrique M; Talyuli, Octávio A C; Goncalves, Renata L S; Paiva-Silva, Gabriela Oliveira; Sorgine, Marcos Henrique F; Alvarenga, Patricia Hessab; Oliveira, Pedro L

2017-04-01

Digestion of blood in the midgut of Aedes aegypti results in the release of pro-oxidant molecules that can be toxic to the mosquito. We hypothesized that after a blood meal, the antioxidant capacity of the midgut is increased to protect cells against oxidative stress. Concomitantly, pathogens present in the blood ingested by mosquitoes, such as the arboviruses Dengue and Zika, also have to overcome the same oxidative challenge, and the antioxidant program induced by the insect is likely to influence infection status of the mosquito and its vectorial competence. We found that blood-induced catalase mRNA and activity in the midgut peaked 24 h after feeding and returned to basal levels after the completion of digestion. RNAi-mediated silencing of catalase (AAEL013407-RB) reduced enzyme activity in the midgut epithelia, increased H2O2 leakage and decreased fecundity and lifespan when mosquitoes were fed H2O2. When infected with Dengue 4 and Zika virus, catalase-silenced mosquitoes showed no alteration in infection intensity (number of plaque forming units/midgut) 7 days after the infectious meal. However, catalase knockdown reduced Dengue 4, but not Zika, infection prevalence (percent of infected midguts). Here, we showed that blood ingestion triggers an antioxidant response in the midgut through the induction of catalase. This protection facilitates the establishment of Dengue virus in the midgut. Importantly, this mechanism appears to be specific for Dengue because catalase silencing did not change Zika virus prevalence. In summary, our data suggest that redox balance in the midgut modulates mosquito vectorial competence to arboviral infections.

Value of pH regulators in the diagnosis, prognosis and treatment of cancer.

PubMed

Granja, Sara; Tavares-Valente, Diana; Queirós, Odília; Baltazar, Fátima

2017-04-01

Altered metabolism, associated with acidification of the extracellular milieu, is one of the major features of cancer. As pH regulation is crucial for the maintenance of all biological functions, cancer cells rely on the activity of lactate exporters and proton transporters to regulate their intracellular pH. The major players in cancer pH regulation are proton pump ATPases, sodium-proton exchangers (NHEs), monocarboxylate transporters (MCTs), carbonic anhydrases (CAs) and anion exchangers (AEs), which have been shown to be upregulated in several human malignancies. Thanks to the activity of the proton pumps and transporters, tumours acidify their microenvironment, becoming more aggressive and resistant to therapy. Thus, targeting tumour pH may contribute to more effective anticancer strategies for controlling tumour progression and therapeutic resistance. In the present study, we review the role of the main pH regulators expressed in human cancer cells, including their diagnostic and prognostic value, as well as their usefulness as therapeutic targets. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation of Vacuolar pH in Citrus limon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lincoln Taiz

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 lemonmore » 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.« less

An aposymbiotic primary coral polyp counteracts acidification by active pH regulation

NASA Astrophysics Data System (ADS)

Ohno, Yoshikazu; Iguchi, Akira; Shinzato, Chuya; Inoue, Mayuri; Suzuki, Atsushi; Sakai, Kazuhiko; Nakamura, Takashi

2017-01-01

Corals build their skeletons using extracellular calcifying fluid located in the tissue-skeleton interface. However, the mechanism by which corals control the transport of calcium and other ions from seawater and the mechanism of constant alkalization of calcifying fluid are largely unknown. To address these questions, we performed direct pH imaging at calcification sites (subcalicoblastic medium, SCM) to visualize active pH upregulation in live aposymbiotic primary coral polyps treated with HCl-acidified seawater. Active alkalization was observed in all individuals using vital staining method while the movement of HPTS and Alexa Fluor to SCM suggests that certain ions such as H+ could diffuse via a paracellular pathway to SCM. Among them, we discovered acid-induced oscillations in the pH of SCM (pHSCM), observed in 24% of polyps examined. In addition, we discovered acid-induced pH up-regulation waves in 21% of polyps examined, which propagated among SCMs after exposure to acidified seawater. Our results showed that corals can regulate pHSCM more dynamically than was previously believed. These observations will have important implications for determining how corals regulate pHSCM during calcification. We propose that corals can sense ambient seawater pH via their innate pH-sensitive systems and regulate pHSCM using several unknown pH-regulating ion transporters that coordinate with multicellular signaling occurring in coral tissue.

Passage of ingested Mansonella ozzardi (Spirurida: Onchocercidae) microfilariae through the midgut of Aedes aegypti (Diptera: Culicidae).

PubMed

Vaughan, Jefferson A; Bell, Jeffrey A; Turell, Michael J; Chadee, Dave D

2007-01-01

When virus and microfilariae are ingested concurrently by a mosquito, microfilariae (mf) may penetrate the mosquito midgut and introduce virus directly into the mosquito hemocoel, allowing mosquitoes to become infectious much sooner than normal and enhancing transmission of viruses by mosquitoes. Mansonella ozzardi (Manson) is a benign filarial nematode parasite of humans in Latin America and is transmitted by black flies (Diptera: Simuliidae) and biting midges (Diptera: Ceratopogonidae). Because M. ozzardi and dengue are sympatric, we wanted to know whether M. ozzardi mf had the ability to penetrate the midgut of Aedes aegypti (L.) (Diptera: Culicidae) and thus play a potential role in the enhancement of dengue transmission. To test this, the F1 progeny from locally collected Ae. aegypti were fed on M. ozzardi-infected human males in an endemic village in northern Trinidad. Mosquitoes were dissected at various times after feeding and examined for mf in the midguts and thoraces. Microfilariae penetrated the midguts of 43% of 63 mosquitoes that ingested mf. Overall, 11% of mf penetrated the midgut by 17 h after being ingested. The intensity of midgut penetration was positively correlated to the numbers of mf ingested. Because midgut penetration is a key requirement for mf enhancement to occur, the potential exists that M. ozzardi could be involved in the enhancement of dengue virus transmission.

Intracellular pH regulation in rat round spermatids.

PubMed

Osses, N; Pancetti, F; Benos, D J; Reyes, J G

1997-07-01

Intracellular pH has been shown to be an important physiological parameter in cell cycle control and differentiation, aspects that are central to the spermatogenic process. However, the pH regulatory mechanisms in spermatogenic cells have not been systematically explored. In this work, measuring intracellular pH (pHi) with a fluorescent probe (BCECF), membrane potential with a fluorescent lipophilic anion (bisoxonol), and net movement of acid using a pH-stat system, we have found that rat round spermatids regulate pHi by means of a V-type H(+)-ATPase, a HCO3- entry pathway, a Na+/HCO3- dependent transport system, and a putative proton conductive pathway. Rat spermatids do not have functional base extruder transport systems. These pH regulatory characteristics seem specially designed to withstand acid challenges, and can generate sustained alkalinization upon acid exit stimulation.

A model of lysosomal pH regulation

PubMed Central

Ishida, Yoichi; Nayak, Smita

2013-01-01

Lysosomes must maintain an acidic luminal pH to activate hydrolytic enzymes and degrade internalized macromolecules. Acidification requires the vacuolar-type H+-ATPase to pump protons into the lumen and a counterion flux to neutralize the membrane potential created by proton accumulation. Early experiments suggested that the counterion was chloride, and more recently a pathway consistent with the ClC-7 Cl–/H+ antiporter was identified. However, reports that the steady-state luminal pH is unaffected in ClC-7 knockout mice raise questions regarding the identity of the carrier and the counterion. Here, we measure the current–voltage characteristics of a mammalian ClC-7 antiporter, and we use its transport properties, together with other key ion regulating elements, to construct a mathematical model of lysosomal pH regulation. We show that results of in vitro lysosome experiments can only be explained by the presence of ClC-7, and that ClC-7 promotes greater acidification than Cl–, K+, or Na+ channels. Our models predict strikingly different lysosomal K+ dynamics depending on the major counterion pathways. However, given the lack of experimental data concerning acidification in vivo, the model cannot definitively rule out any given mechanism, but the model does provide concrete predictions for additional experiments that would clarify the identity of the counterion and its carrier. PMID:23712550

Catalase protects Aedes aegypti from oxidative stress and increases midgut infection prevalence of Dengue but not Zika

PubMed Central

Goncalves, Renata L. S.; Paiva-Silva, Gabriela Oliveira; Sorgine, Marcos Henrique F.; Alvarenga, Patricia Hessab; Oliveira, Pedro L.

2017-01-01

Background Digestion of blood in the midgut of Aedes aegypti results in the release of pro-oxidant molecules that can be toxic to the mosquito. We hypothesized that after a blood meal, the antioxidant capacity of the midgut is increased to protect cells against oxidative stress. Concomitantly, pathogens present in the blood ingested by mosquitoes, such as the arboviruses Dengue and Zika, also have to overcome the same oxidative challenge, and the antioxidant program induced by the insect is likely to influence infection status of the mosquito and its vectorial competence. Methodology/Principal findings We found that blood-induced catalase mRNA and activity in the midgut peaked 24 h after feeding and returned to basal levels after the completion of digestion. RNAi-mediated silencing of catalase (AAEL013407-RB) reduced enzyme activity in the midgut epithelia, increased H2O2 leakage and decreased fecundity and lifespan when mosquitoes were fed H2O2. When infected with Dengue 4 and Zika virus, catalase-silenced mosquitoes showed no alteration in infection intensity (number of plaque forming units/midgut) 7 days after the infectious meal. However, catalase knockdown reduced Dengue 4, but not Zika, infection prevalence (percent of infected midguts). Conclusion/Significance Here, we showed that blood ingestion triggers an antioxidant response in the midgut through the induction of catalase. This protection facilitates the establishment of Dengue virus in the midgut. Importantly, this mechanism appears to be specific for Dengue because catalase silencing did not change Zika virus prevalence. In summary, our data suggest that redox balance in the midgut modulates mosquito vectorial competence to arboviral infections. PMID:28379952

PhERF6, interacting with EOBI, negatively regulates fragrance biosynthesis in petunia flowers.

PubMed

Liu, Fei; Xiao, Zhina; Yang, Li; Chen, Qian; Shao, Lu; Liu, Juanxu; Yu, Yixun

2017-09-01

In petunia, the production of volatile benzenoids/phenylpropanoids determines floral aroma, highly regulated by development, rhythm and ethylene. Previous studies identified several R2R3-type MYB trans-factors as positive regulators of scent biosynthesis in petunia flowers. Ethylene response factors (ERFs) have been shown to take part in the signal transduction of hormones, and regulation of metabolism and development processes in various plant species. Using virus-induced gene silencing technology, a negative regulator of volatile benzenoid biosynthesis, PhERF6, was identified by a screen for regulators of the expression of genes related to scent production. PhERF6 expression was temporally and spatially connected with scent production and was upregulated by exogenous ethylene. Up-/downregulation of the mRNA level of PhERF6 affected the expression of ODO1 and several floral scent-related genes. PhERF6 silencing led to a significant increase in the concentrations of volatiles emitted by flowers. Yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays indicated that PhERF6 interacted with the N-terminus of EOBI, which includes two DNA binding domains. Our results show that PhERF6 negatively regulates volatile production in petunia flowers by competing for the binding of the c-myb domains of the EOBI protein with the promoters of genes related to floral scent. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Phosphorus Imaging as a Tool for Studying the pH Metabolism in Living Insects

NASA Astrophysics Data System (ADS)

Skibbe, U.; Christeller, J. T.; Eccles, C. D.; Laing, W. A.; Callaghan, P. T.

1995-09-01

Comparative 31P NMR and 1H NMR imaging experiments at submillimeter pixel resolution were carried out, using a specially constructed solenoidal RF coil. Chemical-shift imaging is used to provide pH maps from the midgut of a Lepidopteran larvae and to demonstrate physiological dependence in the resulting images, The titration curve of pH versus chemical shift for inorganic phosphate is extended beyond the "normal" biological range to the strong alkaline limit.

Active site characterization and molecular cloning of Tenebrio molitor midgut trehalase and comments on their insect homologs.

PubMed

Gomez, Ana; Cardoso, Christiane; Genta, Fernando A; Terra, Walter R; Ferreira, Clélia

2013-08-01

The soluble midgut trehalase from Tenebrio molitor (TmTre1) was purified after several chromatographic steps, resulting in an enzyme with 58 kDa and pH optimum 5.3 (ionizing active groups in the free enzyme: pK(e1) = 3.8 ± 0.2 pK(e2) = 7.4 ± 0.2). The purified enzyme corresponds to the deduced amino acid sequence of a cloned cDNA (TmTre1-cDNA), because a single cDNA coding a soluble trehalase was found in the T. molitor midgut transcriptome. Furthermore, the mass of the protein predicted to be coded by TmTre1-cDNA agrees with that of the purified enzyme. TmTre1 has the essential catalytic groups Asp 315 and Glu 513 and the essential Arg residues R164, R217, R282. Carbodiimide inactivation of the purified enzyme at different pH values reveals an essential carboxyl group with pKa = 3.5 ± 0.3. Phenylglyoxal modified a single Arg residue with pKa = 7.5 ± 0.2, as observed in the soluble trehalase from Spodoptera frugiperda (SfTre1). Diethylpyrocarbonate modified a His residue that resulted in a less active enzyme with pK(e1) changed to 4.8 ± 0.2. In TmTre1 the modified His residue (putatively His 336) is more exposed than the His modified in SfTre1 (putatively His 210) and that affects the ionization of an Arg residue. The architecture of the active site of TmTre1 and SfTre1 is different, as shown by multiple inhibition analysis, the meaning of which demands further research. Trehalase sequences obtained from midgut transcriptomes (pyrosequencing and Illumina data) from 8 insects pertaining to 5 different orders were used in a cladogram, together with other representative sequences. The data suggest that the trehalase gene went duplication and divergence prior to the separation of the paraneopteran and holometabolan orders and that the soluble trehalase derived from the membrane-bound one by losing the C-terminal transmembrane loop. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

A Lepidopteran-Specific Gene Family Encoding Valine-Rich Midgut Proteins

PubMed Central

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

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

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.

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

Larval Mid-Gut Responses to Sub-Lethal Dose of Cry Toxin in Lepidopteran Pest Achaea janata.

PubMed

Chauhan, Vinod K; Dhania, Narender K; Chaitanya, R K; Senthilkumaran, Balasubramanian; Dutta-Gupta, Aparna

2017-01-01

The lack of homogeneity in field application of Bacillus thuringiensis formulation often results in ingestion of sub-lethal doses of the biopesticide by a fraction of pest population and there by promotes the toxin tolerance and resistance in long term. Gut regeneration seems to be one of the possible mechanism by which this is accomplished. However, the existing information is primarily derived from in vitro studies using mid-gut cell cultures. Present study illustrates cellular and molecular changes in mid-gut epithelium of a Bt -susceptible polyphagous insect pest castor semilooper, Achaea janata in response to a Cry toxin formulation. The present report showed that prolonged exposure to sub-lethal doses of Cry toxin formulation has deleterious effect on larval growth and development. Histological analysis of mid-gut tissue exhibits epithelial cell degeneration, which is due to necrotic form of cell death followed by regeneration through enhanced proliferation of mid-gut stem cells. Cell death is demonstrated by confocal microscopy, flow-cytometry, and DNA fragmentation analysis. Cell proliferation in control vs. toxin-exposed larvae is evaluated by bromodeoxyuridine (BrdU) labeling and toluidine blue staining. Intriguingly, in situ mRNA analysis detected the presence of arylphorin transcripts in larval mid-gut epithelial cells. Quantitative PCR analysis further demonstrates altered expression of arylphorin gene in toxin-exposed larvae when compared with the control. The coincidence of enhanced mid-gut cell proliferation coincides with the elevated arylphorin expression upon Cry intoxication suggests that it might play a role in the regeneration of mid-gut epithelial cells.

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.

Analysis of gene expression in the midgut of Bombyx mori during the larval molting stage.

PubMed

Yang, Bing; Huang, Wuren; Zhang, Jie; Xu, Qiuyun; Zhu, Shoulin; Zhang, Qiaoli; Beerntsen, Brenda T; Song, Hongsheng; Ling, Erjun

2016-11-03

Insects can be models for understanding human intestinal infection and pathology. Molting, a special period during which the old insect cuticle is shed and a new one is produced, is crucial for insect development. Holometabolous insects may experience several larva-to-larva moltings to become larger, a pupal molt and adult eclosion to become adults. During the larval molts, they stop feeding and become quiescent. Although the molting larvae become quiescent, it is not known if changes in microbiome, physiology, development and immunity of midguts occur. Transcriptome analysis indicated that functions such as metabolism, digestion, and transport may become reduced due to the downregulated expression of many associated genes. During the molting stage, midguts harbor less microflora and DNA synthesis decreases. Both ecdysone and juvenile hormone in the larval midgut likely degrade after entering the larva-to-larva molting stage. However, at 12 h after ecdysis, the feeding larvae of 5th instars that were injected with 20-hydroxyecdysone entered a molting-like stage, during which changes in midgut morphology, DNA synthesis, gene expression, and microflora exhibited the same patterns as observed in the actual molting state. This study is important for understanding insect midgut physiology, development and immunity during a special development stage when no food is ingested. Although the molting larva becomes immobile and quiescent, we demonstrate that numerous changes occur in midgut morphology, physiology, metabolism and microbiome during this period.

Lack of Connection Between Midgut Cell Autophagy Gene Expression and BmCPV Infection in the Midgut of Bombyx mori

PubMed Central

Yang, Xiaobing; Wu, Suli; Wu, Yongpeng; Liu, Yang; Qian, Yonghua; Jiao, Feng

2015-01-01

Autophagy is associated with multiple biological processes and has protective and defensive functions with respect to immunity, inflammation, and resistance to microbial infection. In this experiment, we wished to investigate whether autophagy is a factor in the midgut cell response of Bombyx mori to infection by the B. mori cytoplasmic polyhedrosis virus (BmCPV). Our results indicated that the expression of three autophagy-related genes (BmAtg8, BmAtg5, and BmAtg7) in the midgut did not change greatly after BmCPV infection in B. mori. Basal ATG8/ATG8PE protein expression was detected in different B. mori tissues by using western blot analysis. Immunohistochemistry showed that the ATG8/ATG8PE proteins were located mainly in the cytoplasm. ATG8/ATG8PE protein levels decreased at 12 and 16 h after BmCPV infection. Our results indicate that autophagy responded slightly to BmCPV infection, but could not prevent the invasion and replication of the virus. PMID:26163666

A novel quick transendoscopic enteral tubing in mid-gut: technique and training with video.

PubMed

Long, Chuyan; Yu, Yan; Cui, Bota; Jagessar, Sabreen Abdul Rahman; Zhang, Jie; Ji, Guozhong; Huang, Guangming; Zhang, Faming

2018-03-13

This study aimed to evaluate the feasibility, safety, and value of a quick technique for transendoscopic enteral tubing (TET) through mid-gut. A prospective interventional study was performed in a single center. A TET tube was inserted into mid-gut through the nasal orifice and fixed on the pylorus wall by one tiny titanium endoscopic clip under anesthesia. The feasibility, safety, success rate, and satisfaction with TET placement were evaluated for enteral nutrition or fecal microbiota transplantation. A total of 86 patients underwent mid-gut TET. The success rate of the TET procedure was 98.8% (85/86). Mean tubing time of the TET procedure was 4.2 ± 1.9 min. 10 cases of procedure was enough for training of general endoscopist to shorten the procedure time (7.0 min vs 4.0 min, p < 0.05). 97.7% (84/86) of patients were satisfied with the TET placement. Procedure-related and tube-related adverse events were observed in 8.1% (7/86) and 7.0% (6/86) of patients respectively. There were no moderate to severe adverse events during tube extubation. TET through mid-gut is a novel, convenient, reliable and safe procedure for mid-gut administration with a high degree of patient satisfaction. This research was retrospectively registered with clinicaltrials.gov. Trial registration date: 29th November 2017. NCT03335982 .

Differential ammonia metabolism in Aedes aegypti fat body and midgut tissues

PubMed Central

Scaraffia, Patricia Y.; Zhang, Quigfen; Thorson, Kelsey; Wysocki, Vicki H.; Miesfeld, Roger L.

2010-01-01

In order to understand at the tissue level how Aedes aegypti copes with toxic ammonia concentrations that result from the rapid metabolism of blood meal proteins, we investigated the incorporation of 15N from 15NH4Cl into amino acids using an in vitro tissue culture system. Fat body or midgut tissues from female mosquitoes were incubated in an Aedes saline solution supplemented with glucose and 15NH4Cl for 10–40 minutes. The media was then mixed with deuterium-labeled amino acids, dried and derivatized. The 15N-labeled and unlabeled amino acids in each sample were quantified by mass spectrometry techniques. The results demonstrate that both tissues efficiently incorporate ammonia into amino acids, however, the specific metabolic pathways are distinct. In the fat body, the 15N from 15NH4Cl is first incorporated into the amide side chain of Gln and then into the amino group of Gln, Glu, Ala and Pro. This process mainly occurs via the glutamine synthetase (GS) and glutamate synthase (GltS) pathway. In contrast, 15N in midgut is first incorporated into the amino group of Glu and Ala, and then into the amide side chain of Gln. Interestingly, our data show that the GS/GltS pathway is not functional in the midgut. Instead, midgut cells detoxify ammonia by glutamate dehydrogenase, alanine aminotransferase and GS. These data provide new insights into ammonia metabolism in A. aegypti mosquitoes. PMID:20206632

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

Investigation of the midgut structure and ultrastructure in Cimex lectularius and Cimex pipistrelli (Hemiptera: Cimicidae).

PubMed

Rost-Roszkowska, M M; Vilimova, J; Włodarczyk, A; Sonakowska, L; Kamińska, K; Kaszuba, F; Marchewka, A; Sadílek, D

2017-02-01

Cimicidae are temporary ectoparasites, which means that they cannot obtain food continuously. Both Cimex species examined here, Cimex lectularius (Linnaeus 1758) and Cimex pipistrelli (Jenyns 1839), can feed on a non-natal host, C. lectularius from humans on bats, C. pipistrelli on humans, but never naturally. The midgut of C. lectularius and C. pipistrelli is composed of three distinct regions-the anterior midgut (AMG), which has a sack-like shape, the long tube-shaped middle midgut (MMG), and the posterior midgut (PMG). The different ultrastructures of the AMG, MMG, and PMG in both of the species examined suggest that these regions must fulfill different functions in the digestive system. Ultrastructural analysis showed that the AMG fulfills the role of storing food and synthesizing and secreting enzymes, while the MMG is the main organ for the synthesis of enzymes, secretion, and the storage of the reserve material. Additionally, both regions, the AMG and MMG, are involved in water absorption in the digestive system of both Cimex species. The PMG is the part of the midgut in which spherites accumulate. The results of our studies confirm the suggestion of former authors that the structure of the digestive tract of insects is not attributed solely to diet but to the basic adaptation of an ancestor.

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transcriptional Signatures in Response to Wheat Germ Agglutinin and Starvation in Drosophila melanogaster Larval Midgut

USDA-ARS?s Scientific Manuscript database

One function of plant lectins such as wheat germ agglutinin (WGA) is to serve as defenses against herbivorous insects. The midgut is one critical site affected by dietary lectins. We observed marked cellular, structural, and gene expression changes in the midguts of Drosophila melanogaster third-i...

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

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.

Recurrent intestinal volvulus in midgut malrotation causing acute bowel obstruction: A case report

PubMed Central

Sheikh, Fayed; Balarajah, Vickna; Ayantunde, Abraham Abiodun

2013-01-01

Intestinal malrotation occurs when there is a disruption in the normal embryological development of the bowel. The majority of patients present with clinical features in childhood, though rarely a first presentation can take place in adulthood. Recurrent bowel obstruction in patients with previous abdominal operation for midgut malrotation is mostly due to adhesions but very few reported cases have been due to recurrent volvulus. We present the case of a 22-year-old gentleman who had laparotomy in childhood for small bowel volvulus and then presented with acute bowel obstruction. Preoperative computerised tomography scan showed small bowel obstruction and features in keeping with midgut malrotation. Emergency laparotomy findings confirmed midgut malrotation with absent appendix, abnormal location of caecum, ascending colon and small bowel. In addition, there were small bowel volvulus and a segment of terminal ileal stricture. Limited right hemicolectomy was performed with excellent postoperative recovery. This case is presented to illustrate a rare occurrence and raise an awareness of the possibility of dreadful recurrent volvulus even several years following an initial Ladd’s procedure for midgut malrotation. Therefore, one will need to exercise a high index of suspicion and this becomes very crucial in order to ensure prompt surgical intervention and thereby preventing an attendant bowel ischaemia with its associated high fatality. PMID:23556060

Recurrent intestinal volvulus in midgut malrotation causing acute bowel obstruction: A case report.

PubMed

Sheikh, Fayed; Balarajah, Vickna; Ayantunde, Abraham Abiodun

2013-03-27

Intestinal malrotation occurs when there is a disruption in the normal embryological development of the bowel. The majority of patients present with clinical features in childhood, though rarely a first presentation can take place in adulthood. Recurrent bowel obstruction in patients with previous abdominal operation for midgut malrotation is mostly due to adhesions but very few reported cases have been due to recurrent volvulus. We present the case of a 22-year-old gentleman who had laparotomy in childhood for small bowel volvulus and then presented with acute bowel obstruction. Preoperative computerised tomography scan showed small bowel obstruction and features in keeping with midgut malrotation. Emergency laparotomy findings confirmed midgut malrotation with absent appendix, abnormal location of caecum, ascending colon and small bowel. In addition, there were small bowel volvulus and a segment of terminal ileal stricture. Limited right hemicolectomy was performed with excellent postoperative recovery. This case is presented to illustrate a rare occurrence and raise an awareness of the possibility of dreadful recurrent volvulus even several years following an initial Ladd's procedure for midgut malrotation. Therefore, one will need to exercise a high index of suspicion and this becomes very crucial in order to ensure prompt surgical intervention and thereby preventing an attendant bowel ischaemia with its associated high fatality.

Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment.

PubMed

Xia, J. H.; Roberts, JKM.

1996-05-01

We tested the hypothesis that H+ extrusion contributes to cytoplasmic pH regulation and tolerance of anoxia in maize (Zea mays) root tips. We studied root tips of whole seedlings that were acclimated to a low-oxygen environment by pretreatment in 3% (v/v) O2. Acclimated root tips characteristically regulate cytoplasmic pH near neutrality and survive prolonged anoxia, whereas nonacclimated tips undergo severe cytoplasmic acidosis and die much more quickly. We show that the plasma membrane H+-ATPase can operate under anoxia and that net H+ extrusion increases when cytoplasmic pH falls. However, at an external pH near 6.0, H+ extrusion contributes little to cytoplasmic pH regulation. At more acidic external pH values, net H+ flux into root tips increases dramatically, leading to a decrease in cytoplasmic pH and reduced tolerance of anoxia. We present evidence that, under these conditions, H+ pumps are activated to partly offset acidosis due to H+ influx and, thereby, contribute to cytoplasmic pH regulation and tolerance of anoxia. The regulation of H+ extrusion under anoxia is discussed with respect to the acclimation response and mechanisms of intracellular pH regulation in aerobic plant cells.

Lack of Connection Between Midgut Cell Autophagy Gene Expression and BmCPV Infection in the Midgut of Bombyx mori.

PubMed

Yang, Xiaobing; Wu, Suli; Wu, Yongpeng; Liu, Yang; Qian, Yonghua; Jiao, Feng

2015-01-01

Autophagy is associated with multiple biological processes and has protective and defensive functions with respect to immunity, inflammation, and resistance to microbial infection. In this experiment, we wished to investigate whether autophagy is a factor in the midgut cell response of Bombyx mori to infection by the B. mori cytoplasmic polyhedrosis virus (BmCPV). Our results indicated that the expression of three autophagy-related genes (BmAtg8, BmAtg5, and BmAtg7) in the midgut did not change greatly after BmCPV infection in B. mori. Basal ATG8/ATG8PE protein expression was detected in different B. mori tissues by using western blot analysis. Immunohistochemistry showed that the ATG8/ATG8PE proteins were located mainly in the cytoplasm. ATG8/ATG8PE protein levels decreased at 12 and 16 h after BmCPV infection. Our results indicate that autophagy responded slightly to BmCPV infection, but could not prevent the invasion and replication of the virus. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

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

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fine structure of the midgut epithelium in the millipede Telodeinopus aoutii (Myriapoda, Diplopoda) with special emphasis on epithelial regeneration.

PubMed

Rost-Roszkowska, M M; Kszuk-Jendrysik, M; Marchewka, A; Poprawa, I

2018-01-01

The midgut of millipedes is composed of a simple epithelium that rests on a basal lamina, which is surrounded by visceral muscles and hepatic cells. As the material for our studies, we chose Telodeinopus aoutii (Demange, 1971) (Kenyan millipede) (Diplopoda, Spirostreptida), which lives in the rain forests of Central Africa. This commonly reared species is easy to obtain from local breeders and easy to culture in the laboratory. During our studies, we used transmission and scanning electron microscopes and light and fluorescent microscopes. The midgut epithelium of the species examined here shares similarities to the structure of the millipedes analyzed to date. The midgut epithelium is composed of three types of cells-digestive, secretory, and regenerative cells. Evidence of three types of secretion have been observed in the midgut epithelium: merocrine, apocrine, and microapocrine secretion. The regenerative cells of the midgut epithelium in millipedes fulfill the role of midgut stem cells because of their main functions: self-renewal (the ability to divide mitotically and to maintain in an undifferentiated state) and potency (ability to differentiate into digestive cells). We also confirmed that spot desmosomes are common intercellular junctions between the regenerative and digestive cells in millipedes.

A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae.

PubMed

Kumar, Sanjeev; Molina-Cruz, Alvaro; Gupta, Lalita; Rodrigues, Janneth; Barillas-Mury, Carolina

2010-03-26

Extracellular matrices in diverse biological systems are cross-linked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that a peroxidase, secreted by the Anopheles gambiae midgut, and dual oxidase form a dityrosine network that decreases gut permeability to immune elicitors. This network protects the microbiota by preventing activation of epithelial immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses.

An unexpected cause of small bowel obstruction in an adult patient: midgut volvulus

PubMed Central

Söker, Gökhan; Yılmaz, Cengiz; Karateke, Faruk; Gülek, Bozkurt

2014-01-01

The most important complication of intestinal malrotation is midgut volvulus because it may lead to intestinal ischaemia and necrosis. A 29-year-old male patient was admitted to the emergency department with abdominal pain. Ultrasonography (US), colour Doppler ultrasonography (CDUS), CT and barium studies were carried out. On US and CDUS, twisting of intestinal segments around the superior mesenteric artery (SMA) and superior mesenteric vein (SMV) and alteration of the SMA–SMV relationship were detected. CT demonstrated that the small intestine was making a rotation around the SMA and SMV, which amounted to more than 360°. The upper gastrointestinal barium series revealed a corkscrew appearance of the duodenum and proximal jejunum, which is a pathognomonic finding of midgut volvulus. Prior knowledge of characteristic imaging findings of midgut volvulus is essential in order to reach proper diagnosis and establish proper treatment before the development of intestinal ischaemia and necrosis. PMID:24811563

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.

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus.

PubMed

Costa, Inês A; Samuels, Richard I; Bifano, Thaís D; Terra, Walter R; Silva, Carlos P

2011-03-01

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106kDa (gel filtration) and 55kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, AβNA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3, Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. Copyright © 2010 Elsevier Inc. All rights reserved.

RNA-seq analyses of the midgut from blood- and serum-fed Ixodes ricinus ticks

PubMed Central

Perner, Jan; Provazník, Jan; Schrenková, Jana; Urbanová, Veronika; Ribeiro, José M. C.; Kopáček, Petr

2016-01-01

Adult females of the genus Ixodes imbibe blood meals exceeding about 100 times their own weight within 7‒9 days. During this period, ticks internalise components of host blood by endocytic digest cells that line the tick midgut epithelium. Using RNA-seq, we aimed to characterise the midgut transcriptome composition in adult Ixodes ricinus females during early and late phase of engorgement. To address specific adaptations to the haemoglobin-rich diet, we compared the midgut transcriptomes of genetically homogenous female siblings fed either bovine blood or haemoglobin-depleted serum. We noted that tick gut transcriptomes are subject to substantial temporal-dependent expression changes between day 3 and day 8 of feeding. In contrast, the number of transcripts significantly affected by the presence or absence of host red blood cells was low. Transcripts relevant to the processes associated with blood-meal digestion were analysed and involvement of selected encoded proteins in the tick midgut physiology discussed. A total of 7215 novel sequences from I. ricinus were deposited in public databases as an additional outcome of this study. Our results broaden the current knowledge of tick digestive system and may lead to the discovery of potential molecular targets for efficient tick control. PMID:27824139

Diaphorina citri Nymphs Are Resistant to Morphological Changes Induced by "Candidatus Liberibacter asiaticus" in Midgut Epithelial Cells.

PubMed

Mann, Marina; Fattah-Hosseini, Somayeh; Ammar, El-Desouky; Stange, Richard; Warrick, EricaRose; Sturgeon, Kasie; Shatters, Robert; Heck, Michelle

2018-04-01

" Candidatus Liberibacter asiaticus" is the causative bacterium associated with citrus greening disease. " Ca Liberibacter asiaticus" is transmitted by Diaphorina citri more efficiently when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared midguts of D. citri insects reared on healthy or " Ca Liberibacter asiaticus"-infected citrus trees using quantitative PCR, confocal microscopy, and mitochondrial superoxide staining for evidence of oxidative stress. Consistent with its classification as propagative, " Ca Liberibacter asiaticus" titers were higher in adults than in nymphs. Our previous work showed that adult D. citri insects have basal levels of karyorrhexis (fragmentation of the nucleus) in midgut epithelial cells, which is increased in severity and frequency in response to " Ca Liberibacter asiaticus." Here, we show that nymphs exhibit lower levels of early-stage karyorrhexis than adults and are refractory to the induction of advanced karyorrhexis by " Ca Liberibacter asiaticus" in the midgut epithelium. MitoSox Red staining showed that guts of infected adults, particularly males, experienced oxidative stress in response to " Ca Liberibacter asiaticus." A positive correlation between the titers of " Ca Liberibacter asiaticus" and the Wolbachia endosymbiont was observed in adult and nymph midguts, suggesting an interplay between these bacteria during development. We hypothesize that the resistance of the nymph midgut to late-stage karyorrhexis through as yet unknown molecular mechanisms benefits " Ca Liberibacter asiaticus" for efficient invasion of midgut epithelial cells, which may be a factor explaining the developmental dependency of " Ca Liberibacter asiaticus" acquisition by the vector.

A midgut lysate of the Riptortus pedestris has antibacterial activity against LPS O-antigen-deficient Burkholderia mutants.

PubMed

Jang, Ho Am; Seo, Eun Sil; Seong, Min Young; Lee, Bok Luel

2017-02-01

Riptortus pedestris, a common pest in soybean fields, harbors a symbiont Burkholderia in a specialized posterior midgut region of insects. Every generation of second nymphs acquires new Burkholderia cells from the environment. We compared in vitro cultured Burkholderia with newly in vivo colonized Burkholderia in the host midgut using biochemical approaches. The bacterial cell envelope of in vitro cultured and in vivo Burkholderia differed in structure, as in vivo bacteria lacked lipopolysaccharide (LPS) O-antigen. The LPS O-antigen deficient bacteria had a reduced colonization rate in the host midgut compared with that of the wild-type Burkholderia. To determine why LPS O-antigen-deficient bacteria are less able to colonize the host midgut, we examined in vitro survival rates of three LPS O-antigen-deficient Burkholderia mutants and lysates of five different midgut regions. The LPS O-antigen-deficient mutants were highly susceptible when cultured with the lysate of a specific first midgut region (M1), indicating that the M1 lysate contains unidentified substance(s) capable of killing LPS O-antigen-deficient mutants. We identified a 17 kDa protein from the M1 lysate, which was enriched in the active fractions. The N-terminal sequence of the protein was determined to be a soybean Kunitz-type trypsin inhibitor. These data suggest that the 17 kDa protein, which was originated from a main soybean source of the R. pedestris host, has antibacterial activity against the LPS O-antigen deficient (rough-type) Burkholderia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Andrographolide powder treatment as antifeedant decreased digestive enzyme activity from Plutella xylostella (L.) larvae midgut

NASA Astrophysics Data System (ADS)

Madihah, Malini, Desak Made; Roviani, Hana; Rani, Nessa Vidya; Hermawan, Wawan

2018-02-01

Andrographolide, an active compound of Andrographis paniculata, has shown antifeedant activity against Plutella xylostella larvae by disrupting the midgut histological structures. This study aims to determine the activity of andrographolide in crystallized powder form against several digestive enzymes from the midgut of 4th instar P. xylostella larvae. The concentrations used were 0 (control), 1000, 1600, 2500, 4000 and 6500 ppm with four replications each. No-choice antifeedant test with leaf disc method is used in a bioassay for 24 hours. The midgut was dissected from 2nd until 6th segment of 4th instar larvae and was homogenized in iced-buffer solution. Furthermore, larvae's midgut samples were centrifuged at 10,000 rpm, 4°C for 20 min and the supernatant is used as enzyme source. The results showed that andrographolide significantly reduces the amylase, invertase, protease and trypsin activity, as well as total protein concentration compared with control (p<0.05) in a dose-dependent manner. This study provides information about the mode of action of andrographolide in inhibiting feed activity by the reduced digestive enzyme activity of 4th instar P. xylostella larvae.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+-ATPase.

PubMed

Onken, Horst; Patel, Malay; Javoroncov, Margarita; Izeirovski, Sejmir; Moffett, Stacia B; Moffett, David F

2009-03-01

Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.

Regulation of internal pH by the coldwater coral Desmophyllum dianthus

NASA Astrophysics Data System (ADS)

Wall, M.; Schmidt, G. M.; Richter, C.; de Beer, D.

2016-02-01

In the Patagonian fjords of Chile, large aggregations of the coldwater coral Desmophyllum dianthus build the structural and functional basis for a highly diverse benthic ecosystem. Interestingly, D. dianthus growths in both, high-pH (aragonite-supersaturated) and low-pH (aragonite-undersaturated) waters in near-surface and deep waters, respectively. This indicates a high adaptability of these corals to regulate and control calcification. Measurements of the skeletal boron isotopic composition (d11B) in D. dianthus indicate an upregulation of the internal calcifying pH (pHcf) in response to external pH (pHsw) in culturing experiments simulating ocean acidification. A physiological underpinning of pHcf upregulation in corals under different pHsw is, however, so far lacking. Direct measurements at the site of calcification in corals are limited to a few studies on tropical corals. Comparable studies for coldwater corals are wanting. We used microsensors for pH, calcium and oxygen to assess pHcf in D. dianthus in relation to calcium dynamics and respiration along the coral polyp under different pHsw. We found pHcf to be linked to pHsw but no upregulation of pHcf compared to pHsw as well as a strong spatial heterogeneity in pHcf. This suggests a highly complex pH regulation inconsistent with the hitherto upregulation models and suggests that rather the internal carbon pool and not pH is upregulated to enable calcification in D. dianthus.

Effects of gamma irradiation on the midgut ultrastructure of Glossina palpalis subspecies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stiles, J.K.; Molyneux, D.H.; Wallbanks, K.R.

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 wasmore » 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.« less

Possible Insecticidal Mechanisms Mediated by Immune-Response-Related Cry-Binding Proteins in the Midgut Juice of Plutella xylostella and Spodoptera exigua.

PubMed

Lu, Keyu; Gu, Yuqing; Liu, Xiaoping; Lin, Yi; Yu, Xiao-Qiang

2017-03-15

Cry toxins are insecticidal toxin proteins produced by a spore-forming Gram-positive bacterium Bacillus thuringiensis. Interactions between the Cry toxins and the receptors from midgut brush border membrane vesicles (BBMVs), such as cadherin, alkaline phosphatase, and aminopeptidase, are key steps for the specificity and insecticidal activity of Cry proteins. However, little is known about the midgut juice proteins that may interfere with Cry binding to the receptors. To validate the hypothesis that there exist Cry-binding proteins that can interfere with the insecticidal process of Cry toxins, we applied Cry1Ab1-coupled Sepharose beads to isolate Cry-binding proteins form midgut juice of Plutella xylostella and Spodoptera exigua. Trypsin-like serine proteases and Dorsal were found to be Cry1Ab1-binding proteins in the midgut juice of P. xylostella. Peroxidase-C (POX-C) was found to be the Cry1Ab1-binding protein in the midgut juice of S. exigua. We proposed possible insecticidal mechanisms of Cry1Ab1 mediated by the two immune-related proteins: Dorsal and POX-C. Our results suggested that there exist, in the midgut juice, Cry-binding proteins, which are different from BBMV-specific receptors.

Termiticidal lectins from Myracrodruon urundeuva (Anacardiaceae) cause midgut damage when ingested by Nasutitermes corniger (Isoptera: Termitidae) workers.

PubMed

Lima, Thâmarah A; Fernandes, Kenner M; Oliveira, Ana Patrícia S; Dornelles, Leonardo P; Martins, Gustavo F; Napoleão, Thiago H; Paiva, Patrícia Mg

2017-05-01

Myracrodruon urundeuva is a hardwood tree, and its bark, heartwood and leaf contain lectins (MuBL, MuHL and MuLL respectively) with termiticidal activity against Nasutitermes corniger. In this work, the effects of these lectins on the midgut of N. corniger workers were evaluated. The insects were supplied with an artificial diet containing the lectins at their respective LC 50 (previously determined). At 48 h after treatment, the midguts were dissected and fixed for histopathology analyses. Toluidine-blue-stained midguts from lectin-treated workers showed disorganisation, with the presence of debris in the lumen and the absence of brush border. Fluorescence microscopy revealed that the numbers of digestive and proliferating cells were lower in lectin-treated individuals than in the control, and caspase-3 staining confirmed the occurrence of cell apoptosis. Enteroendocrine cells were not seen in the treated individuals. The midguts from treated insects showed greater staining for peroxidase than the control, suggesting that the lectins caused oxidative stress. Staining with wheat germ agglutinin conjugated to FITC revealed that the lectins interfered with the integrity of the peritrophic matrix. This study showed that termiticidal lectins from M. urundeuva cause severe injuries, oxidative stress and cell death in the midgut of N. corniger workers. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

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. © 2016. Published by The Company of Biologists Ltd.

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. © 2015 The Royal Entomological Society.

Diaphorina citri Nymphs Are Resistant to Morphological Changes Induced by “Candidatus Liberibacter asiaticus” in Midgut Epithelial Cells

PubMed Central

2018-01-01

ABSTRACT “Candidatus Liberibacter asiaticus” is the causative bacterium associated with citrus greening disease. “Ca. Liberibacter asiaticus” is transmitted by Diaphorina citri more efficiently when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared midguts of D. citri insects reared on healthy or “Ca. Liberibacter asiaticus”-infected citrus trees using quantitative PCR, confocal microscopy, and mitochondrial superoxide staining for evidence of oxidative stress. Consistent with its classification as propagative, “Ca. Liberibacter asiaticus” titers were higher in adults than in nymphs. Our previous work showed that adult D. citri insects have basal levels of karyorrhexis (fragmentation of the nucleus) in midgut epithelial cells, which is increased in severity and frequency in response to “Ca. Liberibacter asiaticus.” Here, we show that nymphs exhibit lower levels of early-stage karyorrhexis than adults and are refractory to the induction of advanced karyorrhexis by “Ca. Liberibacter asiaticus” in the midgut epithelium. MitoSox Red staining showed that guts of infected adults, particularly males, experienced oxidative stress in response to “Ca. Liberibacter asiaticus.” A positive correlation between the titers of “Ca. Liberibacter asiaticus” and the Wolbachia endosymbiont was observed in adult and nymph midguts, suggesting an interplay between these bacteria during development. We hypothesize that the resistance of the nymph midgut to late-stage karyorrhexis through as yet unknown molecular mechanisms benefits “Ca. Liberibacter asiaticus” for efficient invasion of midgut epithelial cells, which may be a factor explaining the developmental dependency of “Ca. Liberibacter asiaticus” acquisition by the vector. PMID:29311247

The risk of midgut volvulus in patients with abdominal wall defects: A multi-institutional study.

PubMed

Fawley, Jason A; Abdelhafeez, Abdelhafeez H; Schultz, Jessica A; Ertl, Allison; Cassidy, Laura D; Peter, Shawn St; Wagner, Amy J

2017-01-01

The management of malrotation in patients with congenital abdominal wall defects has varied among surgeons. We were interested in investigating the risk of midgut volvulus in patients with gastroschisis and omphalocele to help determine if these patients may benefit from undergoing a Ladd procedure. A retrospective chart review was performed for all patients managed at three institutions born between 1/1/2000 and 12/31/2008 with a diagnosis of gastroschisis or omphalocele. Patient charts were reviewed through 12/31/2012 for occurrence of midgut volvulus or need for second laparotomy. Of the 414 patients identified with abdominal wall defects, 299 patients (72%) had gastroschisis, and 115 patients (28%) had omphalocele. The mean gestational age at birth was 36.1±2.3weeks, and the mean birth weight was 2.57±0.7kg. There were a total of 8 (1.9%) cases of midgut volvulus: 3 (1.0%) patients with gastroschisis compared to 5 patients (4.4%) with omphalocele (p=0.04). Patients with omphalocele have a greater risk of developing midgut volvulus, and a Ladd procedure should be considered during definitive repair to mitigate these risks. III; retrospective comparative study. Copyright © 2017 Elsevier Inc. All rights reserved.

A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

PubMed Central

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. PMID:25502928

Functional and molecular characterization of transmembrane intracellular pH regulators in human dental pulp stem cells.

PubMed

Chen, Gunng-Shinng; Lee, Shiao-Pieng; Huang, Shu-Fu; Chao, Shih-Chi; Chang, Chung-Yi; Wu, Gwo-Jang; Li, Chung-Hsing; Loh, Shih-Hurng

2018-06-01

Homeostasis of intracellular pH (pH i ) plays vital roles in many cell functions, such as proliferation, apoptosis, differentiation and metastasis. Thus far, Na + -H + exchanger (NHE), Na + -HCO 3 - co-transporter (NBC), Cl - /HCO 3 - exchanger (AE) and Cl - /OH - exchanger (CHE) have been identified to co-regulate pH i homeostasis. However, functional and biological pH i -regulators in human dental pulp stem cells (hDPSCs) have yet to be identified. Microspectrofluorimetry technique with pH-sensitive fluorescent dye, BCECF, was used to detect pH i changes. NH 4 Cl and Na + -acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pH i -regulators were detected by Western blot technique. The resting pH i was no significant difference between that in HEPES-buffered (nominal HCO 3 - -free) solution or CO 2 /HCO 3 -buffered system (7.42 and 7.46, respectively). The pH i recovery following the induced-intracellular acidosis was blocked completely by removing [Na + ] o , while only slowed (-63%) by adding HOE694 (a NHE1 specific inhibitor) in HEPES-buffered solution. The pH i recovery was inhibited entirely by removing [Na + ] o , while adding HOE 694 pulse DIDS (an anion-transporter inhibitor) only slowed (-55%) the acid extrusion. Both in HEPES-buffered and CO 2 /HCO 3 -buffered system solution, the pH i recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl - ] o . Western blot analysis showed the isoforms of pH i regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs. We demonstrate for the first time that resting pH i is significantly higher than 7.2 and meditates functionally by two Na + -dependent acid extruders (NHE and NBC), two Cl - -dependent acid loaders (CHE and AE) and one Na + -independent acid extruder(s) in hDPSCs. These findings provide novel insight for basic and clinical treatment of dentistry. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

2016-01-04

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.

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

Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment.

PubMed

Myšková, Jitka; Dostálová, Anna; Pěničková, Lucie; Halada, Petr; Bates, Paul A; Volf, Petr

2016-07-25

Leishmania parasites are transmitted by phlebotomine sand flies and a crucial step in their life-cycle is the binding to the sand fly midgut. Laboratory studies on sand fly competence to Leishmania parasites suggest that the sand flies fall into two groups: several species are termed "specific/restricted" vectors that support the development of one Leishmania species only, while the others belong to so-called "permissive" vectors susceptible to a wide range of Leishmania species. In a previous study we revealed a correlation between specificity vs permissivity of the vector and glycosylation of its midgut proteins. Lutzomyia longipalpis and other four permissive species tested possessed O-linked glycoproteins whereas none were detected in three specific vectors examined. We used a combination of biochemical, molecular and parasitological approaches to characterize biochemical and biological properties of O-linked glycoprotein of Lu. longipalpis. Lectin blotting and mass spectrometry revealed that this molecule with an apparent molecular weight about 45-50 kDa corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis. We produced a recombinant glycoprotein rLuloG with molecular weight around 45 kDa. Anti-rLuloG antibodies localize the native glycoprotein on epithelial midgut surface of Lu. longipalpis. Although we could not prove involvement of LuloG in Leishmania attachment by blocking the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of Leishmania promastigotes. We characterized a novel O-glycoprotein from sand fly Lutzomyia longipalpis. It has mucin-like properties and is localized on the luminal side of the midgut epithelium. Recombinant form of the protein binds to Leishmania parasites in vitro. We propose a role of this molecule in Leishmania attachment to sand fly midgut.

The mechanism by which a propeptide-encoded pH sensor regulates spatiotemporal activation of furin.

PubMed

Williamson, Danielle M; Elferich, Johannes; Ramakrishnan, Parvathy; Thomas, Gary; Shinde, Ujwal

2013-06-28

The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation.

The Mechanism by Which a Propeptide-encoded pH Sensor Regulates Spatiotemporal Activation of Furin*

PubMed Central

Williamson, Danielle M.; Elferich, Johannes; Ramakrishnan, Parvathy; Thomas, Gary; Shinde, Ujwal

2013-01-01

The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation. PMID:23653353

Infection dynamics of Nosema ceranae in honey bee midgut and host cell apoptosis.

PubMed

Kurze, Christoph; Le Conte, Yves; Kryger, Per; Lewkowski, Oleg; Müller, Thomas; Moritz, Robin F A

2018-05-01

Nosema ceranae is an intracellular microsporidian parasite that infects epithelial cells of the honey bee (Apis mellifera) midgut. Previous studies have shown that Nosema may alter cell renewal and apoptosis in honey bees. We found that the amount of apoptotic cells progressively declines from the anterior towards posterior regions of the midgut in Nosema-infected sensitive bees. There was no such pattern in the infected Nosema tolerant honey bees and controls. These data provide additional evidence that N. ceranae appears to alter apoptosis in its host cells for its own advantage. Copyright © 2018 Elsevier Inc. All rights reserved.

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

2016-09-15

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.

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1.

PubMed

Galeone, Antonio; Han, Seung Yeop; Huang, Chengcheng; Hosomi, Akira; Suzuki, Tadashi; Jafar-Nejad, Hamed

2017-08-04

Mutations in the human N- glycanase 1 ( NGLY1 ) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. Here we show that Drosophila Pngl encodes an N -glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, our studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N -glycanase enzyme.

Coordinated regulation of intracellular pH by two glucose-sensing pathways in yeast.

PubMed

Isom, Daniel G; Page, Stephani C; Collins, Leonard B; Kapolka, Nicholas J; Taghon, Geoffrey J; Dohlman, Henrik G

2018-02-16

The yeast Saccharomyces cerevisiae employs multiple pathways to coordinate sugar availability and metabolism. Glucose and other sugars are detected by a G protein-coupled receptor, Gpr1, as well as a pair of transporter-like proteins, Rgt2 and Snf3. When glucose is limiting, however, an ATP-driven proton pump (Pma1) is inactivated, leading to a marked decrease in cytoplasmic pH. Here we determine the relative contribution of the two sugar-sensing pathways to pH regulation. Whereas cytoplasmic pH is strongly dependent on glucose abundance and is regulated by both glucose-sensing pathways, ATP is largely unaffected and therefore cannot account for the changes in Pma1 activity. These data suggest that the pH is a second messenger of the glucose-sensing pathways. We show further that different sugars differ in their ability to control cellular acidification, in the manner of inverse agonists. We conclude that the sugar-sensing pathways act via Pma1 to invoke coordinated changes in cellular pH and metabolism. More broadly, our findings support the emerging view that cellular systems have evolved the use of pH signals as a means of adapting to environmental stresses such as those caused by hypoxia, ischemia, and diabetes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcriptomic Survey of the Midgut of Anthonomus grandis (Coleoptera: Curculionidae)

PubMed Central

Salvador, Ricardo; Príncipi, Darío; Berretta, Marcelo; Fernández, Paula; Paniego, Norma; Sciocco-Cap, Alicia; Hopp, Esteban

2014-01-01

Abstract Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase , and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information. PMID:25473064

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.

Calcium-calmodulin and pH regulate protein tyrosine phosphorylation in stallion sperm.

PubMed

González-Fernández, L; Macías-García, B; Velez, I C; Varner, D D; Hinrichs, K

2012-10-01

The mechanisms leading to capacitation in stallion sperm are poorly understood. The objective of our study was to define factors associated with regulation of protein tyrosine phosphorylation in stallion sperm. Stallion sperm were incubated for 4 h in modified Whitten's media with or without bicarbonate, calcium, or BSA. When sperm were incubated in air at 30×10⁶/ml at initial pH 7.25, protein tyrosine phosphorylation was detected only in medium containing 25 mM bicarbonate alone; calcium and BSA inhibited phosphorylation. Surprisingly, this inhibition did not occur when sperm were incubated at 10×10⁶/ml. The final pH values after incubation at 30×10⁶ and 10×10⁶ sperm/ml were 7.43 ± 0.04 and 7.83 ± 0.07 (mean ± s.e.m.) respectively. Sperm were then incubated at initial pH values of 7.25, 7.90, or 8.50 in either air or 5% CO₂. Protein tyrosine phosphorylation increased with increasing final medium pH, regardless of the addition of bicarbonate or BSA. An increase in environmental pH was observed when raw semen was instilled into the uteri of estrous mares and retrieved after 30 min (from 7.47 ± 0.10 to 7.85 ± 0.08), demonstrating a potential physiological role for pH regulation of capacitation. Sperm incubated in the presence of the calmodulin (CaM) inhibitor W-7 exhibited a dose-dependent increase in protein tyrosine phosphorylation, suggesting that the inhibitory effect of calcium was CaM mediated. These results show for the first time a major regulatory role of external pH, calcium, and CaM in stallion sperm protein tyrosine phosphorylation.

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

Treesearch

Algimantas P. Valaitis

2008-01-01

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...

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

2015-08-03

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. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Extracellular pH regulation in microdomains of colonic crypts: effects of short-chain fatty acids.

PubMed Central

Chu, S; Montrose, M H

1995-01-01

It has been suggested that transepithelial gradients of short-chain fatty acids (SCFAs; the major anions in the colonic lumen) generate pH gradients across the colonic epithelium. Quantitative confocal microscopy was used to study extracellular pH in mouse distal colon with intact epithelial architecture, by superfusing tissue with carboxy SNARF-1 (a pH-sensitive fluorescent dye). Results demonstrate extracellular pH regulation in two separate microdomains surrounding colonic crypts: the crypt lumen and the subepithelial tissue adjacent to crypt colonocytes. Apical superfusion with (i) a poorly metabolized SCFA (isobutyrate), (ii) an avidly metabolized SCFA (n-butyrate), or (iii) a physiologic mixture of acetate/propionate/n-butyrate produced similar results: alkalinization of the crypt lumen and acidification of subepithelial tissue. Effects were (i) dependent on the presence and orientation of a transepithelial SCFA gradient, (ii) not observed with gluconate substitution, and (iii) required activation of sustained vectorial acid/base transport by SCFAs. Results suggest that the crypt lumen functions as a pH microdomain due to slow mixing with bulk superfusates and that crypts contribute significant buffering capacity to the lumen. In conclusion, physiologic SCFA gradients cause polarized extracellular pH regulation because epithelial architecture and vectorial transport synergize to establish regulated microenvironments. Images Fig. 1 Fig. 3 PMID:7724557

Midgut volvulus following laparoscopic gastric banding--a rare and dangerous situation.

PubMed

Arbell, Dan; Koplewitz, Benjamin; Zamir, Gideon; Bala, Miklosh

2007-06-01

Intestinal malrotation is usually encountered in infants. Its main complication is midgut volvulus, a situation that presents itself with bilious vomiting. This symptom allows for early surgical treatment. A delay in diagnosis and treatment may lead to catastrophic sequelae, such as extensive bowel necrosis and death. This situation is rare but well known in adults. Laparoscopic gastric banding is a popular option for treating morbid obesity. One of the consequences of this procedure may be impaired vomiting when there is an obstruction below the band. In this paper, we present a case in which a patient suffered from midgut volvulus 4 years after a laparoscopic gastric banding. Owing to impaired vomiting, the diagnosis was delayed, therefore, severely endangering the patient. This case prompted us to suggest that malrotation should be actively sought after before or during any bariatric procedure.

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. Copyright © 2012 Elsevier Ltd. All rights reserved.

De novo transcriptome sequencing and comparative analysis of midgut tissues of four non-model insects pertaining to Hemiptera, Coleoptera, Diptera and Lepidoptera.

PubMed

Gazara, Rajesh K; Cardoso, Christiane; Bellieny-Rabelo, Daniel; Ferreira, Clélia; Terra, Walter R; Venancio, Thiago M

2017-09-05

Despite the great morphological diversity of insects, there is a regularity in their digestive functions, which is apparently related to their physiology. In the present work we report the de novo midgut transcriptomes of four non-model insects from four distinct orders: Spodoptera frugiperda (Lepidoptera), Musca domestica (Diptera), Tenebrio molitor (Coleoptera) and Dysdercus peruvianus (Hemiptera). We employed a computational strategy to merge assemblies obtained with two different algorithms, which substantially increased the quality of the final transcriptomes. Unigenes were annotated and analyzed using the eggNOG database, which allowed us to assign some level of functional and evolutionary information to 79.7% to 93.1% of the transcriptomes. We found interesting transcriptional patterns, such as: i) the intense use of lysozymes in digestive functions of M. domestica larvae, which are streamlined and adapted to feed on bacteria; ii) the up-regulation of orthologous UDP-glycosyl transferase and cytochrome P450 genes in the whole midguts different species, supporting the existence of an ancient defense frontline to counter xenobiotics; iii) evidence supporting roles for juvenile hormone binding proteins in the midgut physiology, probably as a way to activate genes that help fight anti-nutritional substances (e.g. protease inhibitors). The results presented here shed light on the digestive and structural properties of the digestive systems of these distantly related species. Furthermore, the produced datasets will also be useful for scientists studying these insects. Copyright © 2017. Published by Elsevier B.V.

Transcriptomic survey of the midgut of Anthonomus grandis (Coleoptera: Curculionidae).

PubMed

Salvador, Ricardo; Príncipi, Darío; Berretta, Marcelo; Fernández, Paula; Paniego, Norma; Sciocco-Cap, Alicia; Hopp, Esteban

2014-01-01

Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase, and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

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

PubMed Central

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

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

Purification and characterization of midgut α-amylase in a predatory bug, Andralus spinidens

PubMed Central

Sorkhabi-Abdolmaleki, Sahar; Zibaee, Arash; Hoda, Hassan; FazeliDinan, Mahmoud

2014-01-01

Abstract α-Amylases are widespread enzymes that catalyze endohydrolysis of long α-1,4-glucan chains such as starch and glycogen. The highest amylolytic activity was found in 5th instar nymphs and midgut of the predatory bug, Andrallus spinidens F. (Hemiptera: Pentatomidae). The α-amylase was purified following a three-step procedure. The purified α-amylase had a specific activity of 13.46 U/mg protein, recovery of 4.21, purification fold of 13.87, and molecular weight of 21.3 kDa. The enzyme had optimal pH and temperature of 7 and 45°C, respectively. Na+, Mn+, Mg2+, and Zn2+ significantly decreased activity of the purified α-amylase, but some concentrations of K+, Ca2+, and Cu2+ had the opposite effect. EDTA, EGTA, and DTC significantly decreased enzymatic activity, showing the presence of metal ions in the catalytic site of the enzyme. Kinetic parameters of the purified α-amylase showed a Km of 3.71% in starch and 4.96% for glycogen, suggesting that the enzyme had a higher affinity for starch. PMID:25373212

Ecdysone has an effect on the regeneration of midgut epithelial cells that is distinct from 20-hydroxyecdysone in the silkworm Bombyx mori.

PubMed

Tanaka, Y; Yukuhiro, F

1999-12-01

We investigated the effects of two ecdysteroids, ecdysone (E) and 20-hydroxyecdysone (20E), on silkworm larval development. Silkworm larvae, Bombyx mori, were fed an artificial diet supplemented with 20E during the fourth instar to promote premature molting. At the onset of the fifth instar, these precocious fifth-instar larvae were fed diets supplemented with either E or 20E to determine the effects of the two ecdysteroids on the morphology of midgut epithelial cells. Regeneration of midgut epithelial cells normally occurs only during the molting period. However, in larvae fed E, complete replacement of midgut epithelial cells was observed 24 h before the larvae entered apolysis. In larvae fed 20E, the morphology of midgut epithelial cells was disrupted, leading to death of the larvae during the fifth instar. We also observed similar differences in the effects of the two ecdysteroids in an in vitro experiment. These results suggest that E has a specific effect on the morphological change of midgut epithelial cells in precocious fifth-instar larvae that is distinct from 20E. Copyright 1999 Academic Press.

Isolation of midgut escape mutants of two American genotype dengue 2 viruses from Aedes aegypti

PubMed Central

2013-01-01

Background Several studies have shown that American genotype dengue 2 viruses (DENV2) have reduced viral fitness in the mosquito vector, Aedes aegypti, compared to other DENV2 genotypes. Diminished replication efficiency or inability to efficiently traverse membrane barriers encompassing organs such as the midgut or salivary glands are considered major factors negatively impacting viral fitness in the mosquito. Results We analyzed the vector competence of Ae. aegypti for two American DENV2 strains, QR94 and PR159 originating from Mexico and Puerto-Rico, respectively. Both strains infected mosquito midguts following acquisition of infectious bloodmeals. However, DENV2-QR94 and DENV2-PR159 poorly disseminated from the midgut at 7 or 14 days post-bloodmeal (pbm). We detected one virus isolate, EM33, among 31 DENV2-QR94 infected mosquitoes, and one isolate, EM41, among 121 DENV2-PR159 infected mosquitoes, generating high virus titers in mosquito carcasses at 7 days pbm. In oral challenge experiments, EM33 and EM41 showed midgut dissemination rates of 40-50%. Replication efficiency of EM41 in secondary mosquito tissue was similar to that of a dissemination-competent control strain, whereas the replication efficiency of EM33 was significantly lower than that of the control virus. The genome sequence of DENV2-QR94 encoded seven unique amino acids (aa), which were not found in 100 of the most closely related DENV2 strains. EM33 had one additional aa change, E202K, in the E protein. DENV2-PR159 encoded four unique aa residues, one of them E202K, whereas EM41 had two additional aa substitutions, Q77E in the E protein and E93D in NS3. Conclusions Our results indicate that the midgut of Ae. aegypti acts as a selective sieve for DENV2 in which genetically distinct, dissemination-competent virus variants are rapidly selected from the viral quasispecies to be transmitted to vertebrates. PMID:23937713

Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness.

PubMed

Vijay, Sonam; Rawal, Ritu; Kadian, Kavita; Singh, Jagbir; Adak, Tridibesh; Sharma, Arun

2018-05-08

Midgut invasion, a major bottleneck for malaria parasites transmission is considered as a potential target for vector-parasite interaction studies. New intervention strategies are required to explore the midgut proteins and their potential role in refractoriness for malaria control in Anopheles mosquitoes. To better understand the midgut functional proteins of An. culicifacies susceptible and refractory species, proteomic approaches coupled with bioinformatics analysis is an effective means in order to understand the mechanism of refractoriness. In the present study, an integrated in solution- in gel trypsin digestion approach, along with Isobaric tag for relative and absolute quantitation (iTRAQ)-Liquid chromatography/Mass spectrometry (LC/MS/MS) and data mining were performed to identify the proteomic profile and differentially expressed proteins in Anopheles culicifacies susceptible species A and refractory species B. Shot gun proteomics approaches led to the identification of 80 proteins in An. culicifacies susceptible species A and 92 in refractory species B and catalogue was prepared. iTRAQ based proteomic analysis identified 48 differentially expressed proteins from total 130 proteins. Of these, 41 were downregulated and 7 were upregulated in refractory species B in comparison to susceptible species A. We report that the altered midgut proteins identified in naturally refractory mosquitoes are involved in oxidative phosphorylation, antioxidant and proteolysis process that may suggest their role in parasite growth inhibition. Furthermore, real time polymerase chain reaction (PCR) analysis of few proteins indicated higher expression of iTRAQ upregulated protein in refractory species than susceptible species. This study elucidates the first proteome of the midguts of An. culicifacies sibling species that attempts to analyze unique proteogenomic interactions to provide insights for better understanding of the mechanism of refractoriness. Functional implications

Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence.

PubMed

Pauchet, Y; Wilkinson, P; Vogel, H; Nelson, D R; Reynolds, S E; Heckel, D G; ffrench-Constant, R H

2010-02-01

The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.

Cytological lesions in the midgut of Tribolium confusum larvae exposed to gamma radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jafri, R.H.; Ismail, M.

1977-01-01

The major cytological lesions in Tribolium confusum after irradiation were displayed by the midgut epithelium. At 24 hr following exposure to 5.3 kR, the regenerative cells called nidi appeared numerous. They gradually disappeared with increases in dosage and time in accordance with Arndt-Schulze's Law. The columnar epithelial cells and their nuclei appeared swollen and vacuolated on the fifth and twelfth day following exposure to 5.3 kR. They appeared disorganized and shed into the lumen of the midgut on the twelfth and fifth day following 50- and 70-kR irradiation, respectively. The basement membrane and the muscularis appeared loose on the fifthmore » and twelfth day dollowing 70-kR irradiation. It was observed that once the catabolic activity, i.e., histolysis, was initiated in the midgut, it continued to accelerate with increasing dose and time. Thus, the last effects at low doses, 5.3 and 10 kR, appeared as immediate effects at high doses, 50 and 70 kR. The differentiated cells, i.e., columnar epithelial cells, appeared radioresistant as compared to undifferentiated cells, i.e., regenerative cells, which appeared radiosensitive in accordance with the principle of Bergonie and Tribondeau.« less

Endogenous cyclo-oxygenase activity regulates mouse gastric surface pH

PubMed Central

Baumgartner, Heidi K; Kirbiyik, Uzay; Coskun, Tamer; Chu, Shaoyou; Montrose, Marshall H

2002-01-01

In the stomach, production of prostaglandins by cyclo-oxygenase (COX) is believed to be important in mucosal defence. We tested the hypothesis that endogenous COX activity is required for protective gastric surface pH control. Intact stomachs of anaesthetized mice were perfused with a weakly buffered solution (150 mmNaCl + 4 mm Homopipes) at pH values from 2.5 to 7.0. Gastric effluents were collected to measure pH and estimate amounts of acid or alkali secretion in nanomoles secreted per minute. A switch from net acid to net alkali secretion was seen in response to acidifying luminal pH with an apparent ‘set point’ between pH 4 and 5. At luminal pH 3, the net alkali secretion (12.7 ± 2.8 nmol OH− equivalents min−1) was abolished (2.2 ± 1.7 nmol OH− min−1) by the non-specific COX inhibitor indomethacin (5 mg kg−1 I.P.). Similar inhibition was observed using a COX-1 inhibitor (SC-560; 10 mg kg−1 I.P.), but not a COX-2 inhibitor (NS-398; 10 mg kg−1 I.P.). Subsequent treatment with 16,16-dimethyl prostaglandin E2 (dm-PGE2; 1 mg kg−1 I.P.) rescued the alkali secretion (21.8 ± 2.7 nmol OH− min−1). In either the absence or presence of the H+,K+-ATPase inhibitor omeprazole (60 mg kg−1 I.P.), indomethacin blocked similar amounts of net alkali secretion (10.5 ± 2.7 and 16.4 ± 3.4 nmol OH− min−1, respectively). We also used in vivo confocal microscopy to examine pH near the mucosal surface. The gastric mucosal surface of anaesthetized mice was exposed and mucosal surface pH was imaged using the fluorescence intensity ratio of Cl-NERF as a pH indicator. Results showed a switch from a continuous net acid to net alkali secretion by the stomach in response to changing superfusate pH from 5 to 3. At luminal pH 3, the relatively alkaline surface pH (4.3 ± 0.1) was acidified (3.6 ± 0.2) by indomethacin, and subsequent dm-PGE2 restored surface pH (4.2 ± 0.2). We conclude that the pre-epithelial alkaline layer is regulated by endogenous COX

Effects of periplocoside X on midgut cells and digestive enzymes activity of the soldiers of red imported fire ant.

PubMed

Li, Yan; Zeng, Xin-Nian

2013-07-01

The pathological effects of ingested periplocoside X, an insecticidal component isolated from the root of Periploca sepium Bunge, on the midgut epithelial cells of the soldiers of red imported fire ant were studied and the symptom was described. The results showed that periplocoside X could induce a severe, time-dependent cytotoxicity in the midgut epithelial cells. An optical microscopy showed that epithelial cells swelled firstly and then lysed. Transmission electron microscopy (TEM) showed that numerous swollen lysosomes were appeared, microvilli were disrupted and sloughed off, and the numbers of the rough endoplasmic reticulum and the mitochondria decreased sharply in earlier stage. Numerous vacuoles were observed in the later stage. Finally, periplocoside X resulted in cell death by cytolysis. Assay of main three digestive enzymes activity indicated that amylase activity was significantly inhibited, but no significant changes were seen for lipase activity and total protease activity. So it is suggested that periplocoside X induced mainly to organic damage of midgut epithelium cells of insect. In all, insect midgut is one of targets for periplocoside X. Copyright © 2013 Elsevier Inc. All rights reserved.

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Formation and dissociation of proteasome storage granules are regulated by cytosolic pH.

PubMed

Peters, Lee Zeev; Hazan, Rotem; Breker, Michal; Schuldiner, Maya; Ben-Aroya, Shay

2013-05-27

The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae).

PubMed

Fruttero, Leonardo L; Demartini, Diogo R; Rubiolo, Edilberto R; Carlini, Célia R; Canavoso, Lilián E

2014-09-01

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [(3)H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-(3)H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus. Copyright © 2014 Elsevier Ltd. All rights reserved.

TPC2 controls pigmentation by regulating melanosome pH and size

PubMed Central

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

2016-01-01

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 Ca2+ release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca2+ sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size. PMID:27140606

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.

Regulation of neuronal pH by the metabotropic Zn(2+)-sensing Gq-coupled receptor, mZnR/GPR39.

PubMed

Ganay, Thibault; Asraf, Hila; Aizenman, Elias; Bogdanovic, Milos; Sekler, Israel; Hershfinkel, Michal

2015-12-01

Synaptically released Zn(2+) acts as a neurotransmitter, in part, by activating the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39). In previous work using epithelial cells, we described crosstalk between Zn(2+) signaling and changes in intracellular pH and/or extracellular pH (pHe). As pH changes accompany neuronal activity under physiological and pathological conditions, we tested whether Zn(2+) signaling is involved in regulation of neuronal pH. Here, we report that up-regulation of a major H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), is induced by mZnR/GPR39 activation in an extracellular-regulated kinase 1/2-dependent manner in hippocampal neurons in vitro. We also observed that changes in pHe can modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. Similarly, Zn(2+)-dependent extracellular-regulated kinase 1/2 phosphorylation and up-regulation of NHE activity were absent at acidic pHe. Thus, our results suggest that when pHe is maintained within the physiological range, mZnR/GPR39 activation can up-regulate NHE-dependent recovery from intracellular acidification. During acidosis, as pHe drops, mZnR/GPR39-dependent NHE activation is inhibited, thereby attenuating further H(+) extrusion. This mechanism may serve to protect neurons from excessive decreases in pHe. Thus, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. We show that the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39) activation induces up-regulation of a major neuronal H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), thereby enhancing neuronal recovery from intracellular acidification. Changes in extracellular pH (pHe), however, modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. This mechanism may serve to protect neurons from excessive

Extracellular pH Regulates Zinc Signaling via an Asp Residue of the Zinc-sensing Receptor (ZnR/GPR39)*

PubMed Central

Cohen, Limor; Asraf, Hila; Sekler, Israel; Hershfinkel, Michal

2012-01-01

Zinc activates a specific Zn2+-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca2+ responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na+/H+ exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn2+ binding site, His17 or His19, or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp313 with alanine resulted in similar Ca2+ responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na+/H+ exchange at pH 7.4 and pH 6.5. Substitution of Asp313 to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp313, which was shown to modulate Zn2+ binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity. PMID:22879599

Extracellular pH regulates zinc signaling via an Asp residue of the zinc-sensing receptor (ZnR/GPR39).

PubMed

Cohen, Limor; Asraf, Hila; Sekler, Israel; Hershfinkel, Michal

2012-09-28

Zinc activates a specific Zn(2+)-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca(2+) responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na(+)/H(+) exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn(2+) binding site, His(17) or His(19), or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp(313) with alanine resulted in similar Ca(2+) responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na(+)/H(+) exchange at pH 7.4 and pH 6.5. Substitution of Asp(313) to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp(313), which was shown to modulate Zn(2+) binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity.

Phase 3 Trial of 177Lu-Dotatate for Midgut Neuroendocrine Tumors.

PubMed

Strosberg, Jonathan; El-Haddad, Ghassan; Wolin, Edward; Hendifar, Andrew; Yao, James; Chasen, Beth; Mittra, Erik; Kunz, Pamela L; Kulke, Matthew H; Jacene, Heather; Bushnell, David; O'Dorisio, Thomas M; Baum, Richard P; Kulkarni, Harshad R; Caplin, Martyn; Lebtahi, Rachida; Hobday, Timothy; Delpassand, Ebrahim; Van Cutsem, Eric; Benson, Al; Srirajaskanthan, Rajaventhan; Pavel, Marianne; Mora, Jaime; Berlin, Jordan; Grande, Enrique; Reed, Nicholas; Seregni, Ettore; Öberg, Kjell; Lopera Sierra, Maribel; Santoro, Paola; Thevenet, Thomas; Erion, Jack L; Ruszniewski, Philippe; Kwekkeboom, Dik; Krenning, Eric

2017-01-12

Patients with advanced midgut neuroendocrine tumors who have had disease progression during first-line somatostatin analogue therapy have limited therapeutic options. This randomized, controlled trial evaluated the efficacy and safety of lutetium-177 ( 177 Lu)-Dotatate in patients with advanced, progressive, somatostatin-receptor-positive midgut neuroendocrine tumors. We randomly assigned 229 patients who had well-differentiated, metastatic midgut neuroendocrine tumors to receive either 177 Lu-Dotatate (116 patients) at a dose of 7.4 GBq every 8 weeks (four intravenous infusions, plus best supportive care including octreotide long-acting repeatable [LAR] administered intramuscularly at a dose of 30 mg) ( 177 Lu-Dotatate group) or octreotide LAR alone (113 patients) administered intramuscularly at a dose of 60 mg every 4 weeks (control group). The primary end point was progression-free survival. Secondary end points included the objective response rate, overall survival, safety, and the side-effect profile. The final analysis of overall survival will be conducted in the future as specified in the protocol; a prespecified interim analysis of overall survival was conducted and is reported here. At the data-cutoff date for the primary analysis, the estimated rate of progression-free survival at month 20 was 65.2% (95% confidence interval [CI], 50.0 to 76.8) in the 177 Lu-Dotatate group and 10.8% (95% CI, 3.5 to 23.0) in the control group. The response rate was 18% in the 177 Lu-Dotatate group versus 3% in the control group (P<0.001). In the planned interim analysis of overall survival, 14 deaths occurred in the 177 Lu-Dotatate group and 26 in the control group (P=0.004). Grade 3 or 4 neutropenia, thrombocytopenia, and lymphopenia occurred in 1%, 2%, and 9%, respectively, of patients in the 177 Lu-Dotatate group as compared with no patients in the control group, with no evidence of renal toxic effects during the observed time frame. Treatment with 177 Lu

Phylogenetically Diverse Burkholderia Associated with Midgut Crypts of Spurge Bugs, Dicranocephalus spp. (Heteroptera: Stenocephalidae).

PubMed

Kuechler, Stefan Martin; Matsuura, Yu; Dettner, Konrad; Kikuchi, Yoshitomo

2016-06-25

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.

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

Female-Specific Specialization of a Posterior End Region of the Midgut Symbiotic Organ in Plautia splendens and Allied Stinkbugs

PubMed Central

Hayashi, Toshinari; Hosokawa, Takahiro; Meng, Xian-Ying; Koga, Ryuichi

2015-01-01

Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens. A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris. These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae. PMID:25636847

Bacillus thuringiensis toxins trigger receptor shedding from gypsy moth midgut cells

Treesearch

Algimantas P. Valaitis

2007-01-01

The mechanism of action of the Cry1 insecticidal proteins produced by Bacillus thuringiensis (Bt) begins with the processing of these proteins in the larval gut. After proteolytic activation, the Bt toxins bind to specific midgut receptors and insert into the membrane of the gut epithelial cells, causing insect death.

PINK1 is required for timely cell-type specific mitochondrial clearance during Drosophila midgut metamorphosis.

PubMed

Liu, Yan; Lin, Jingjing; Zhang, Minjie; Chen, Kai; Yang, Shengxi; Wang, Qun; Yang, Hongqin; Xie, Shusen; Zhou, Yongjian; Zhang, Xi; Chen, Fei; Yang, Yufeng

2016-11-15

Mitophagy is the selective degradation of mitochondria by autophagy, which is an important mitochondrial quality and quantity control process. During Drosophila metamorphosis, the degradation of midgut involves a large change in length and organization, which is mediated by autophagy. Here we noticed a cell-type specific mitochondrial clearance process that occurs in enterocytes (ECs), while most mitochondria remain in intestinal stem cells (ISCs) during metamorphosis. Although PINK1/PARKIN represent the canonical pathway for the elimination of impaired mitochondria in varied pathological conditions, their roles in developmental processes or normal physiological conditions have been less studied. To examine the potential contribution of PINK1 in developmental processes, we monitored the dynamic expression pattern of PINK1 in the midgut development by taking advantage of a newly CRISPR/Cas9 generated knock-in fly strain expressing PINK1-mCherry fusion protein that presumably recapitulates the endogenous expression pattern of PINK1. We disclosed a spatiotemporal correlation between the expression pattern of PINK1 and the mitochondrial clearance or persistence in ECs or ISCs respectively. By mosaic genetic analysis, we then demonstrated that PINK1 and PARKIN function epistatically to mediate the specific timely removal of mitochondria, and are involved in global autophagy in ECs during Drosophila midgut metamorphosis, with kinase-dead PINK1 exerting dominant negative effects. Taken together, our studies concluded that the PINK1/PARKIN is crucial for timely cell-type specific mitophagy under physiological conditions and demonstrated again that Drosophila midgut metamorphosis might serve as an elegant in vivo model to study autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

Acute exposure of mercury chloride stimulates the tissue regeneration program and reactive oxygen species production in the Drosophila midgut.

PubMed

Chen, Zhi; Wu, Xiaochun; Luo, Hongjie; Zhao, Lingling; Ji, Xin; Qiao, Xianfeng; Jin, Yaping; Liu, Wei

2016-01-01

We used Drosophila as an animal model to study the digestive tract in response to the exposure of inorganic mercury (HgCl2). We found that after oral administration, mercury was mainly sequestered within the midgut. This resulted in increased cell death, which in turn stimulated the tissue regeneration program, including accelerated proliferation and differentiation of the intestinal stem cells (ISCs). We further demonstrated that these injuries correlate closely with the excessive production of the reactive oxygen species (ROS), as vitamin E, an antioxidant reagent, efficiently suppressed the HgCl2-induced phenotypes of midgut and improved the viability. We propose that the Drosophila midgut could serve as a suitable model to study the treatment of acute hydrargyrism on the digestive systems. Copyright © 2015 Elsevier B.V. All rights reserved.

The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies.

PubMed

Jochim, Ryan C; Teixeira, Clarissa R; Laughinghouse, Andre; Mu, Jianbing; Oliveira, Fabiano; Gomes, Regis B; Elnaiem, Dia-Eldin; Valenzuela, Jesus G

2008-01-14

In the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi. Comparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5), two peritrophin like proteins, a trypsin like protein (Lltryp1), two chymotrypsin like proteins (LuloChym1A and 2) and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3), a trypsin like protein (Lltryp2) and an astacin-like metalloprotease (LuloAstacin). Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5), a Chymotrypsin (LuloChym1A) and a carboxypeptidase (LuloCpepA1), among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1), a trypsin-like protein (Lltryp2) and an unknown protein. This transcriptome analysis represents the largest set of sequence data reported from a specific sand

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.

Intracellular pH in mammalian stages of Trypanosoma cruzi is K+-dependent and regulated by H+-ATPases.

PubMed

Van Der Heyden, N; Docampo, R

2000-02-05

Regulation of intracellular pH (pHi) was investigated in Trypanosoma cruzi amastigotes and trypomastigotes using 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF). pHi was determined to be 7.33 +/- 0.08 and 7.35 +/- 0.07 in amastigotes and trypomastigotes, respectively, and there were no significant differences in the regulation of pH, between the two stages. Steady-state pHi, recovery of pHi from acidification, and H+-efflux were all decreased markedly by the H+-ATPase inhibitors N,N'-dicyclohexylcarbodi-imide (DCCD), diethylstilbestrol (DES) and N-ethylmaleimide (NEM) supporting a significant role for a plasma membrane H+-ATPase in the regulation of pHi. pHi was maintained at neutrality over a range of external pH (pHe) from 5-8 in parasites suspended in a buffer containing Na+ and K+ (standard buffer) but was acidified at low pHe in the absence of these cations (choline buffer). The pHi of trypomastigotes decreased significantly when they transformed into amastigotes. The rate of recovery of pHi by acidified parasites was similar in Na+-free buffer and standard buffer but was slower in the absence of K+ (K+-free or choline buffer) and parasites suspended in choline buffer were acidic by 0.25 pH units as compared with controls. Ba2+ and Cs+ decreased the pHi of parasites suspended in standard but not choline buffer suggesting the presence of an inward directed K+ channel. The pHi of amastigotes and trypomastigotes suspended in Cl(-)-free buffer was decreased by 0.13 and 0.2 pH units, respectively, supporting the presence of a chloride conductive channel. No evidence of pH regulation via a Na+/H+ or Cl-/HCO3- exchanger was found. These results are consistent with the presence of a plasma membrane H+-ATPase that regulates pHi and is supported by K+ and Cl- channels.

Salinity alters snakeskin and mesh transcript abundance and permeability in midgut and Malpighian tubules of larval mosquito, Aedes aegypti.

PubMed

Jonusaite, Sima; Donini, Andrew; Kelly, Scott P

2017-03-01

This study examined the distribution and localization of the septate junction (SJ) proteins snakeskin (Ssk) and mesh in osmoregulatory organs of larval mosquito (Aedes aegypti), as well as their response to altered environmental salt levels. Ssk and mesh transcripts and immunoreactivity were detected in tissues of endodermal origin such as the midgut and Malpighian tubules of A. aegypti larvae, but not in ectodermally derived hindgut and anal papillae. Immunolocalization of Ssk and mesh in the midgut and Malpighian tubules indicated that both proteins are concentrated at regions of cell-cell contact between epithelial cells. Transcript abundance of ssk and mesh was higher in the midgut and Malpighian tubules of brackish water (BW, 30% SW) reared A. aegypti larvae when compared with freshwater (FW) reared animals. Therefore, [ 3 H]polyethylene glycol (MW 400Da, PEG-400) flux was examined across isolated midgut and Malpighian tubule preparations as a measure of their paracellular permeability. It was found that PEG-400 flux was greater across the midgut of BW versus FW larvae while the Malpighian tubules of BW-reared larvae had reduced PEG-400 permeability in conjunction with increased Cl - secretion compared to FW animals. Taken together, data suggest that Ssk and mesh are found in smooth SJs (sSJs) of larval A. aegypti and that their abundance alters in association with changes in epithelial permeability when larvae reside in water of differing salt content. This latter observation suggests that Ssk and mesh play a role in the homeostatic control of salt and water balance in larval A. aegypti. Copyright © 2016 Elsevier Inc. All rights reserved.

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…

Midgut pseudotumors and the maintenance of tissue homeostasis: studies on aging and manipulated stick insects.

PubMed

Holtmann, Matthias; Dorn, August

2009-02-01

Stick insects (Carausius morosus) develop pseudotumors in aging adults. Pseudotumor formation starts at the M2 midgut region where an accumulation of stomatogastric nerve terminals is observed. Pseudotumors arise from dying columnar cells whose basal parts form an "amorphous substance" at the basement membrane whereas the apical parts, including the nucleus, are expelled into the gut lumen. The "amorphous substance" is ensheathed by hemocytes. These nodules, which do not melanize, characterize the phenotype of the pseudotumors. With age, cell death and pseudotumor infestation increases. It is shown that the maintenance of midgut tissue homoeostasis is disturbed and becomes more serious with growing pseudotumor incidence. The increased death rate of differentiated columnar cells is no longer compensated by the proliferation of regenerative cells, i.e., intestinal stem cells, in the midgut nidi. The appearance of "holes" in the intestinal wall is evidently a causative factor of premature death. Extirpation of the hypocerebral ganglion in young adults of the stick insect (before the onset of spontaneous pseudotumor formation) provokes the apoptosis of a large number of columnar cells within 24 h and the formation of pseudotumors that are histologically identical with spontaneous ones. We conclude that the stomatogastric nervous system plays a decisive role in the regulatory mechanism maintaining midgut tissue homeostasis. The possibility of experimentally manipulating the regulatory system provides a valuable tool for the exploration of extrinsic factors involved into the feedback circuitry of tissue homeostasis. The fact that comparable pseudotumors were observed in a number of orthopteromorphan species, where they could also be induced by the interruption of the stomatogastric nervous system, indicates that its role in tissue homoeostasis may be widespread in insects and possibly represent a general principle. (c) 2008 Wiley-Liss, Inc.

Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases

PubMed Central

Dixon, Daniel P.; Van Ekeris, Leslie; Linser, Paul J.

2017-01-01

In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control strategies into the

The effect of diet on the expression of lipase genes in the midgut of the lightbrown apple moth (Epiphyas postvittana Walker; Tortricidae).

PubMed

Christeller, J T; Poulton, J; Markwick, N M; Simpson, R M

2010-02-01

We have identified lipase-like genes from an Epiphyas postvittana larval midgut EST library. Of the 10 pancreatic lipase family genes, six appear to encode active lipases and four encode inactive lipases, based on the presence/absence of essential catalytic residues. The four gastric lipase family genes appear to encode active proteins. Phylogenetic analysis of 54 lepidopteran pancreatic lipase proteins resolved the clade into five groups of midgut origin and a sixth of non-midgut lipases. The inactive proteins formed two separate groups with highly conserved mutations. The lepidopteran midgut lipases formed a ninth subfamily of pancreatic lipases. Eighteen insect and human gastric lipases were analysed phylogenetically with only very weak support for any groupings. Gene expression was measured in the larval midgut following feeding on five artificial diets and on apple leaves. The artificial diets contained different levels of triacylglycerol, linoleic acid and cholesterol. Significant changes in gene expression (more than 100-fold for active pancreatic lipases) were observed. All the inactive lipases were also highly expressed. The gastric lipase genes were expressed at lower levels and suppressed in larvae feeding on leaves. Together, protein motif analysis and the gene expression data suggest that, in phytophagous lepidopteran larvae, the pancreatic lipases may function in vivo as galactolipases and phospholipases whereas the gastric lipases may function as triacylglycerol hydrolases.

In Vitro Inhibition of Leishmania Attachment to Sandfly Midguts and LL-5 Cells by Divalent Metal Chelators, Anti-gp63 and Phosphoglycans.

PubMed

Soares, Rodrigo Pedro; Altoé, Ellen Cristina Félix; Ennes-Vidal, Vítor; da Costa, Simone M; Rangel, Elizabeth Ferreira; de Souza, Nataly Araújo; da Silva, Vanderlei Campos; Volf, Petr; d'Avila-Levy, Claudia Masini

2017-07-01

Leishmania braziliensis and Leishmania infantum are the causative agents of cutaneous and visceral leishmaniasis, respectively. Several aspects of the vector-parasite interaction involving gp63 and phosphoglycans have been individually assayed in different studies. However, their role under the same experimental conditions was not studied yet. Here, the roles of divalent metal chelators, anti-gp63 antibodies and purified type I phosphoglycans (PGs) were evaluated during in vitro parasite attachment to the midgut of the vector. Parasites were treated with divalent metal chelators or anti-gp63 antibodies prior to the interaction with Lutzomyia longipalpis/Lutzomyia intermedia midguts or sand fly LL-5 cells. In vitro binding system was used to examine the role of PG and gp63 in parallel. Treatment with divalent metal chelators reduced Le. infantum adhesion to the Lu. longipalpis midguts. The most effective compound (Phen) inhibited the binding in both vectors. Similar results were observed in the interaction between both Leishmania species and the cell line LL-5. Finally, parallel experiments using anti-gp63-treated parasites and PG-incubated midguts demonstrated that both approaches substantially inhibited attachment in the natural parasite-vector pairs Le. infantum/Lu. longipalpis and Le. braziliensis/Lu. intermedia. Our results suggest that gp63 and/or PG are involved in parasite attachment to the midgut of these important vectors. Copyright © 2017 Elsevier GmbH. All rights reserved.

The Phosphorolytic Exoribonucleases Polynucleotide Phosphorylase and RNase PH Stabilize sRNAs and Facilitate Regulation of Their mRNA Targets

PubMed Central

2016-01-01

ABSTRACT Gene regulation by base pairing between small noncoding RNAs (sRNAs) and their mRNA targets is an important mechanism that allows bacteria to maintain homeostasis and respond to dynamic environments. In Gram-negative bacteria, sRNA pairing and regulation are mediated by several RNA-binding proteins, including the sRNA chaperone Hfq and polynucleotide phosphorylase (PNPase). PNPase and its homolog RNase PH together represent the two 3′ to 5′ phosphorolytic exoribonucleases found in Escherichia coli; however, the role of RNase PH in sRNA regulation has not yet been explored and reported. Here, we have examined in detail how PNPase and RNase PH interact to support sRNA stability, activity, and base pairing in exponential and stationary growth conditions. Our results indicate that these proteins facilitate the stability and regulatory function of the sRNAs RyhB, CyaR, and MicA during exponential growth. PNPase further appears to contribute to pairing between RyhB and its mRNA targets. During stationary growth, each sRNA responded differently to the absence or presence of PNPase and RNase PH. Finally, our results suggest that PNPase and RNase PH stabilize only Hfq-bound sRNAs. Taken together, these results confirm and extend previous findings that PNPase participates in sRNA regulation and reveal that RNase PH serves a similar, albeit more limited, role as well. These proteins may, therefore, act to protect sRNAs from spurious degradation while also facilitating regulatory pairing with their targets. IMPORTANCE In many bacteria, Hfq-dependent base-pairing sRNAs facilitate rapid changes in gene expression that are critical for maintaining homeostasis and responding to stress and environmental changes. While a role for Hfq in this process was identified more than 2 decades ago, the identity and function of the other proteins required for Hfq-dependent regulation by sRNAs have not been resolved. Here, we demonstrate that PNPase and RNase PH, the two

Host-derived transferrin is maintained and transferred from midgut to ovary in Haemaphysalis longicornis ticks.

PubMed

Mori, Hiroyuki; Galay, Remil Linggatong; Maeda, Hiroki; Matsuo, Tomohide; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

2014-03-01

Transferrin is known to be an iron transporter in vertebrates and several arthropods. Iron from host blood is essential for ovarian development in blood-sucking arthropods. However, tick transferrin has been identified in only a few species, and its function has yet to be elucidated, resulting in incomplete understanding of iron metabolism in ticks. Here, we investigated the transfer of host-derived transferrin in the hard tick Haemaphysalis longicornis using immunological methods. Western blot showed that host-derived transferrin was maintained in all developmental stages of ticks up to 28 days after engorgement and was detected in the midgut and the ovary of adult females following blood feeding. However, no host-derived transferrin was detected in eggs after laying or in larvae after hatching, indicating that host-derived transferrin is not transferred to offspring transovarially. Indirect immunofluorescent antibody testing showed the localization of host-derived transferrin in digestive cells of the midgut and oocytes of the ovary from engorged adult females. These results suggest that host-derived transferrin is transferred to the ovary through the midgut and the hemolymph, and raise the possibility of the function of host-derived transferrin as an iron source in the ovary, providing additional insight on iron metabolism in ticks. Copyright © 2013 Elsevier GmbH. All rights reserved.

Abscisic acid induces a transient shift in signaling that enhances NF-κB-mediated parasite killing in the midgut of Anopheles stephensi without reducing lifespan or fecundity.

PubMed

Glennon, Elizabeth K K; Torrevillas, Brandi K; Morrissey, Shannon F; Ejercito, Jadrian M; Luckhart, Shirley

2017-07-13

Abscisic acid (ABA) is naturally present in mammalian blood and circulating levels can be increased by oral supplementation. We showed previously that oral ABA supplementation in a mouse model of Plasmodium yoelii 17XNL infection reduced parasitemia and gametocytemia, spleen and liver pathology, and parasite transmission to the mosquito Anopheles stephensi fed on these mice. Treatment of cultured Plasmodium falciparum with ABA at levels detected in our model had no effects on asexual growth or gametocyte formation in vitro. However, ABA treatment of cultured P. falciparum immediately prior to mosquito feeding significantly reduced oocyst development in A. stephensi via ABA-dependent synthesis of nitric oxide (NO) in the mosquito midgut. Here we describe the mechanisms of effects of ABA on mosquito physiology, which are dependent on phosphorylation of TGF-β-activated kinase 1 (TAK1) and associated with changes in homeostatic gene expression and activity of kinases that are central to metabolic regulation in the midgut epithelium. Collectively, the timing of these effects suggests a transient physiological shift that enhances NF-κB-dependent innate immunity without significantly altering mosquito lifespan or fecundity. ABA is a highly conserved regulator of immune and metabolic homeostasis within the malaria vector A. stephensi with potential as a transmission-blocking supplemental treatment.

Encapsidated Host Factors in Alphavirus Particles Influence Midgut Infection of Aedes aegypti.

PubMed

Mackenzie-Liu, David; Sokoloski, Kevin J; Purdy, Sarah; Hardy, Richard W

2018-05-16

Transmission of mosquito-borne viruses requires the efficient infection of both a permissive vertebrate host and a competent mosquito vector. The infectivity of Sindbis virus (SINV), the type species of the Alphavirus genus, is influenced by both the original and new host cell. We have shown that infection of vertebrate cells by SINV, chikungunya virus (CHIKV), and Ross River virus (RRV) produces two subpopulations of virus particles separable based on density. In contrast, a single population of viral particles is produced by mosquito cells. Previous studies demonstrated that the denser vertebrate-derived particles and the mosquito-derived particles contain components of the small subunit of the host cell ribosome, whereas the less dense vertebrate-derived particles do not. Infection of mice with RRV showed that both particle subpopulations are produced in an infected vertebrate, but in a tissue specific manner with serum containing only the less dense version of the virus particles. Previous infectivity studies using SINV particles have shown that the denser particles (SINV Heavy ) and mosquito derived particles SINV C6/36 are significantly more infectious in vertebrate cells than the less dense vertebrate derived particles (SINV Light ). The current study shows that SINV Light particles, initiate the infection of the mosquito midgut more efficiently than SINV Heavy particles and that this enhanced infectivity is associated with an exacerbated immune response to SINV Light infection in midgut tissues. The enhanced infection of SINV Light is specific to the midgut as intrathoracically injected virus do not exhibit the same fitness advantage. Together, our data indicate a biologically significant role for the SINV Light subpopulation in the efficient transmission from infected vertebrates to the mosquito vector.

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Different rate-limiting activities of intracellular pH regulators for HCO3- secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts.

PubMed

Ueno, Kaori; Hirono, Chikara; Kitagawa, Michinori; Shiba, Yoshiki; Sugita, Makoto

2016-11-01

Intracellular pH (pH i ) regulation fundamentally participates in maintaining HCO 3 - release from HCO 3 - -secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na + -H + exchanger (NHE) to pH i regulation for HCO 3 - secretion by cAMP and Ca 2+ signals. Resting pH i was dispersed between 7.4 and 7.9. Forskolin consistently decreased pH i showing the dominance of pH i -lowering activities, but carbachol gathered pH i around 7.6. CA inhibition suppressed the forskolin-induced decrease in pH i , while it allowed carbachol to consistently increase pH i by revealing that carbachol prominently activated NHE via Ca 2+ -calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pH i , which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pH i -lowering CA and pH i -raising NHE, respectively, to regulate pH i for HCO 3 - secretion.

Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways.

PubMed

Virgilio, Stela; Cupertino, Fernanda Barbosa; Ambrosio, Daniela Luz; Bertolini, Maria Célia

2017-06-09

Glycogen and trehalose are storage carbohydrates and their levels in microorganisms vary according to environmental conditions. In Neurospora crassa, alkaline pH stress highly influences glycogen levels, and in Saccharomyces cerevisiae, the response to pH stress also involves the calcineurin signaling pathway mediated by the Crz1 transcription factor. Recently, in yeast, pH stress response genes were identified as targets of Crz1 including genes involved in glycogen and trehalose metabolism. In this work, we present evidence that in N. crassa the glycogen and trehalose metabolism is modulated by alkaline pH and calcium stresses. We demonstrated that the pH signaling pathway in N. crassa controls the accumulation of the reserve carbohydrates glycogen and trehalose via the PAC-3 transcription factor, which is the central regulator of the signaling pathway. The protein binds to the promoters of most of the genes encoding enzymes of glycogen and trehalose metabolism and regulates their expression. We also demonstrated that the reserve carbohydrate levels and gene expression are both modulated under calcium stress and that the response to calcium stress may involve the concerted action of PAC-3. Calcium activates growth of the Δpac-3 strain and influences its glycogen and trehalose accumulation. In addition, calcium stress differently regulates glycogen and trehalose metabolism in the mutant strain compared to the wild-type strain. While glycogen levels are decreased in both strains, the trehalose levels are significantly increased in the wild-type strain and not affected by calcium in the mutant strain when compared to mycelium not exposed to calcium. We previously reported the role of PAC-3 as a transcription factor involved in glycogen metabolism regulation by controlling the expression of the gsn gene, which encodes an enzyme of glycogen synthesis. In this work, we extended the investigation by studying in greater detail the effects of pH on the metabolism of the

Variability in larval gut pH regulation defines sensitivity to ocean acidification in six species of the Ambulacraria superphylum.

PubMed

Hu, Marian; Tseng, Yung-Che; Su, Yi-Hsien; Lein, Etienne; Lee, Hae-Gyeong; Lee, Jay-Ron; Dupont, Sam; Stumpp, Meike

2017-10-11

The unusual rate and extent of environmental changes due to human activities may exceed the capacity of marine organisms to deal with this phenomenon. The identification of physiological systems that set the tolerance limits and their potential for phenotypic buffering in the most vulnerable ontogenetic stages become increasingly important to make large-scale projections. Here, we demonstrate that the differential sensitivity of non-calcifying Ambulacraria (echinoderms and hemichordates) larvae towards simulated ocean acidification is dictated by the physiology of their digestive systems. Gastric pH regulation upon experimental ocean acidification was compared in six species of the superphylum Ambulacraria. We observed a strong correlation between sensitivity to ocean acidification and the ability to regulate gut pH. Surprisingly, species with tightly regulated gastric pH were more sensitive to ocean acidification. This study provides evidence that strict maintenance of highly alkaline conditions in the larval gut of Ambulacraria early life stages may dictate their sensitivity to decreases in seawater pH. These findings highlight the importance of identifying and understanding pH regulatory systems in marine larval stages that may contribute to substantial energetic challenges under near-future ocean acidification scenarios. © 2017 The Author(s).

Requirements for Ion and Solute Transport, and pH Regulation During Enamel Maturation

PubMed Central

LACRUZ, RODRIGO S.; SMITH, CHARLES E.; MOFFATT, PIERRE; CHANG, EUGENE H.; BROMAGE, TIMOTHY G.; BRINGAS, PABLO; NANCI, ANTONIO; BANIWAL, SANJEEV K.; ZABNER, JOSEPH; WELSH, MICHAEL J.; KURTZ, IRA; PAINE, MICHAEL L.

2012-01-01

Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, amutant porcine(CFTR-ΔF508) and the NBCe1-null mouse.Our data show that two SLCs(AE2 and NBCe1),Cftr,and Car2, Car3,Car6,and Car12 are all significantly up-regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of Cftr-ΔF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1-null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild-type. These data demonstrate the importance of many non-matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth. PMID:21732355

Recognition and Binding of the PF2 Lectin to α-Amylase From Zabrotes subfasciatus (Coleoptera:Bruchidae) Larval Midgut

PubMed Central

Lagarda-Diaz, I.; Geiser, D.; Guzman-Partida, A.M.; Winzerling, J.; Vazquez-Moreno, L.

2014-01-01

Abstract Amylases are an important family of enzymes involved in insect carbohydrate metabolism that are required for the survival of insect larvae. For this reason, enzymes from starch-dependent insects are targets for insecticidal control. PF2 ( Olneya tesota ) is a lectin that is toxic to Zabrotes subfasciatus (Coleoptera: Bruchidae) larvae. In this study, we evaluated recognition of the PF2 lectin to α-amylases from Z. subfasciatus midgut and the effect of PF2 on α-amylase activity. PF2 caused a decrease of total amylase activity in vitro. Subsequently, several α-amylase isoforms were isolated from insect midgut tissues using ion exchange chromatography. Three enzyme isoforms were verified by an in-gel assay for amylase activity; however, only one isoform was recognized by antiamylase serum and PF2. The identity of this Z. subfasciatus α-amylase was confirmed by liquid chromatography−tandem mass spectrometry. The findings strongly suggest that a glycosylated α-amylase isoform from larval Z. subfasciatus midgut interacts with PF2, which interferes with starch digestion. PMID:25528751

Tissue-Specific Transcriptome Profiling of Plutella Xylostella Third Instar Larval Midgut

PubMed Central

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 416bp, 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 indentified 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

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

Purification and characterization of midgut α-amylase in a predatory bug, Andralus spinidens.

PubMed

Sorkhabi-Abdolmaleki, Sahar; Zibaee, Arash; Hoda, Hassan; Fazeli-Dinan, Mahmoud

2014-05-13

α-Amylases are widespread enzymes that catalyze endohydrolysis of long α-1,4-glucan chains such as starch and glycogen. The highest amylolytic activity was found in 5th instar nymphs and midgut of the predatory bug, Andrallus spinidens F. (Hemiptera: Pentatomidae). The α-amylase was purified following a three-step procedure. The purified α-amylase had a specific activity of 13.46 U/mg protein, recovery of 4.21, purification fold of 13.87, and molecular weight of 21.3 kDa. The enzyme had optimal pH and temperature of 7 and 45°C, respectively. Na+, Mn+, Mg2+, and Zn2+ significantly decreased activity of the purified α-amylase, but some concentrations of K+, Ca2+, and Cu2+ had the opposite effect. EDTA, EGTA, and DTC significantly decreased enzymatic activity, showing the presence of metal ions in the catalytic site of the enzyme. Kinetic parameters of the purified α-amylase showed a Km of 3.71% in starch and 4.96% for glycogen, suggesting that the enzyme had a higher affinity for starch. This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.

Spatial distribution of digestive proteinases in the midgut of the Pacific white shrimp (Litopenaeus vannamei) indicates the existence of endo-ectoperitrophic circulation in Crustacea.

PubMed

Alexandre, Daniel; Ozório, Renata A; Derner, Roberto B; Fracalossi, Débora M; Oliveira, Gabriel B; Samuels, Richard I; Terra, Walter R; Silva, Carlos P

2014-01-01

The effect of dietary protein concentration on the spatial distribution of digestive proteinases in the shrimp Litopenaeus vannamei indicates the existence of endo-ectoperitrophic enzyme circulation in this species. Samples recovered from the midgut gland tissues, stomach contents, three different portions of the midgut and feces were used for quantitative and qualitative analyses of the composition and distribution of the digestive proteinases. Animals were divided into three different groups: (1) animals (controls) fed with a commercial 35% protein diet, (2) animals fed with a commercial diet supplemented with ovalbumin to a final protein concentration of 60%; (3) animals fed with an 80% protein diet. Quantitative determinations using different substrates and zymograms showed that increasing protein concentration in the diet alters the distribution of proteinases along the digestive tract. Composition of proteinases in the midgut gland, stomach contents, midgut sections and feces were similar, but not identical. Chymotrypsin and trypsin paralogues were identified in all enzyme sources in a concentration gradient along the midgut in the control shrimp, the expected distribution supporting the existence of a recycling mechanism. The occurrence of a peritrophic membrane in other Decapoda suggests that endo-ectoperitrophic circulation of digestive enzymes and nutrients may also occur in other crustaceans and also extends beyond the Insecta. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut.

PubMed

Verma, Sudha; Das, Sushmita; Mandal, Abhishek; Ansari, Md Yousuf; Kumari, Sujata; Mansuri, Rani; Kumar, Ajay; Singh, Ruby; Saini, Savita; Abhishek, Kumar; Kumar, Vijay; Sahoo, Ganesh Chandra; Das, Pradeep

2017-06-23

In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment. In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays. We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to

Systemic Activin signaling independently regulates sugar homeostasis, cellular metabolism, and pH balance in Drosophila melanogaster

PubMed Central

Ghosh, Arpan C.; O’Connor, Michael B.

2014-01-01

The ability to maintain cellular and physiological metabolic homeostasis is key for the survival of multicellular organisms in changing environmental conditions. However, our understanding of extracellular signaling pathways that modulate metabolic processes remains limited. In this study we show that the Activin-like ligand Dawdle (Daw) is a major regulator of systemic metabolic homeostasis and cellular metabolism in Drosophila. We find that loss of canonical Smad signaling downstream of Daw leads to defects in sugar and systemic pH homeostasis. Although Daw regulates sugar homeostasis by positively influencing insulin release, we find that the effect of Daw on pH balance is independent of its role in insulin signaling and is caused by accumulation of organic acids that are primarily tricarboxylic acid (TCA) cycle intermediates. RNA sequencing reveals that a number of TCA cycle enzymes and nuclear-encoded mitochondrial genes including genes involved in oxidative phosphorylation and β-oxidation are up-regulated in the daw mutants, indicating either a direct or indirect role of Daw in regulating these genes. These findings establish Activin signaling as a major metabolic regulator and uncover a functional link between TGF-β signaling, insulin signaling, and metabolism in Drosophila. PMID:24706779

A MIDGUT DIGESTIVE PHOSPHOLIPASE A2 IN LARVAL MOSQUITOES, AEDES ALBOPICTUS AND CULEX QUINQUEFASCIATUS

USDA-ARS?s Scientific Manuscript database

Phospholipase A2 (PLA2) is a secretory digestive enzyme that hydrolyzes ester bond at sn-2 position of dietary phospholipids, creating free fatty acid and lysophopholipid. The free fatty acids (arachidonic acid) are absorbed into midgut cells. Aedes albopictus and Culex quinquefasciatus digestive PL...

Alteration of carbohydrates metabolism and midgut glucose absorption in Gromphadorhina portentosa after subchronic exposure to imidacloprid and fenitrothion.

PubMed

Sawczyn, Tomasz; Dolezych, Bogdan; Klosok, Marcin; Augustyniak, Maria; Stygar, Dominika; Buldak, Rafal J; Kukla, Michal; Michalczyk, Katarzyna; Karcz-Socha, Iwona; Zwirska-Korczala, Krystyna

2012-01-01

This study was undertaken to test the hypothesis that following exposure to insecticides, changes take place in the metabolism of carbohydrates and absorption in the midgut of insects. The Madagascar hissing cockroach (Gromphadorhina portentosa) was chosen for the experiment as a model organism, due to it being easy to breed and its relatively large alimentary tract, which was important when preparing the microperfusion midgut bioassay. In each group of cockroaches treated with imidacloprid and fenitrothion, absorption of glucose, expressed as the area under the curve (AUC), was elevated compared to the control group. Glucose in the hemolymph of the examined insects was present in a vestigial amount, often below the threshold of determination, so the determinable carbohydrate indices were: hemolymph trehalose concentration and fat body glycogen content. The level of trehalose found in the hemolymph of insects when exposed to fenitrothion, and irrespective of the level of concentration mixed into food, were significantly lower when comparing to the control samples. Imidacloprid acted analogically with one exception at the concentration of 10 mg·kg(-1) dry food where trehalose concentration did not differ from the control values. Coupling with fat body glycogen concentration was less visible and appeared only at the concentrations of 5 and 10 mg imidacloprid·kg(-1) dry food. As described in this study changes in the sugar distribution and midgut glucose absorption indicate that insects cover the increased energy needs induced by insecticides; also at the gastrointestinal tract level. The result indicates that the midgut glucose absorption parameters could be considered as a non-specific biomarker of insecticide toxicity.

Recognition and binding of the PF2 lectin to α-amylase from Zabrotes subfasciatus (Coleoptera:Bruchidae) larval midgut.

PubMed

Lagarda-Diaz, I; Geiser, D; Guzman-Partida, A M; Winzerling, J; Vazquez-Moreno, L

2014-01-01

Amylases are an important family of enzymes involved in insect carbohydrate metabolism that are required for the survival of insect larvae. For this reason, enzymes from starch-dependent insects are targets for insecticidal control. PF2 (Olneya tesota) is a lectin that is toxic to Zabrotes subfasciatus (Coleoptera: Bruchidae) larvae. In this study, we evaluated recognition of the PF2 lectin to α-amylases from Z. subfasciatus midgut and the effect of PF2 on α-amylase activity. PF2 caused a decrease of total amylase activity in vitro. Subsequently, several α-amylase isoforms were isolated from insect midgut tissues using ion exchange chromatography. Three enzyme isoforms were verified by an in-gel assay for amylase activity; however, only one isoform was recognized by antiamylase serum and PF2. The identity of this Z. subfasciatus α-amylase was confirmed by liquid chromatography-tandem mass spectrometry. The findings strongly suggest that a glycosylated α-amylase isoform from larval Z. subfasciatus midgut interacts with PF2, which interferes with starch digestion. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

Baculoviral mid-gut gland necrosis (BMN) of kuruma shrimp (Penaeus japonicus) larvae in Japanese intensive culture systems

NASA Astrophysics Data System (ADS)

Sano, T.; Nishimura, T.; Fukuda, H.; Hayashida, T.; Momoyama, K.

1984-03-01

In many shrimp farms in the Kyushu and Chugoku areas of Japan, the so-called mid-gut gland cloudy disease of kuruma shrimp larvae (Penaeus japonicus) has occurred since 1971. The pathological changes associated with this baculoviral mid-gut gland necrosis (BMN) are extensive cellular necrosis, collapse of mid-gut gland cells, nuclear hypertrophy and finally karyorrhexis. Electron microscopic examination revealed the presence of virions and virogenic stages in the affected nuclei. Average length and diameter of the virions detected was 310 and 72 nm, respectively; nucleocapsids were 250 nm in size. Virions enclosing 2 nucleocapsids within a single envelope were rarely found. The spirally arranged capsomeres were at an angle of 37 to 38° to a horizontal line meeting at right angles with the long axis of the virion. Infectivity trials resulted in high mortality of healthy mysis and juveniles (2nd post-larval stage). Juveniles at the 9th post-larval stage showed no mortality, although they could be infected easily by the agent. Hypertrophied nuclei in squashed and stained preparations of the affected gland cells can be considered to be of reliable presumptive diagnostic character, and fluorescent antibody staining can be employed to confirm the diagnosis of BMN.

Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil.

PubMed

de Oliveira, Davi Marcos Souza; da Silva, Bruno José Martins; de Sena, Chubert Bernardo Castro; Lima, José Aprígio Nunes; Vasconcelos Dos Santos, Thiago; Silveira, Fernando Tobias; Silva, Edilene Oliveira

2016-09-01

Leishmaniasis are worldwide diseases that occur in 98 countries including Brazil, transmitted by the bite of female phlebotomines during blood feeding. In Brazil it is known that some species of sand flies as Lutzomyia longipalpis sensun latum (vector of Leishmania infantum chagasi), Lutzomyia flaviscutellata (vector of Leishmania (Leishmania) amazonensis) and Lutzomyia antunesi [suspected vector of Leishmania (Viannia) lindenbergi] are incriminated of transmitting the parasite Leishmania for the vertebrate host. The phlebotomine-parasite is mediated by the attachment of the promastigote lipophosphoglycan (LPG) to the midgut epithelium. However, another mechanism that is LPG-independent and mediated by N-acetyl-galactosamine (GalNAc) seems to occur in some species of phlebotomines that are classified as permissive. The aim of this study was to characterize the carbohydrate residues that, probably, play a role in parasite attachment to the midgut of phlebotomine from colony and field populations from the Brazilian Amazonian region. We observed the presence of GalNAc, mannose, galactose and GlcNAc in all phlebotomine species. A binding assay between L. (L.) amazonensis and L. i.chagasi to the midguts of different species of phlebotomines was performed. The attachment of both Leishmania and vector species suggests the presence of GalNAc on the midgut surfaces. Thus, these results suggested that GalNAc is a possible binding sites of Leishmania in sand flies from the Brazilian Amazonian region. Copyright © 2016 Elsevier Inc. All rights reserved.

Bacteria in midguts of field-collected Anopheles albimanus block Plasmodium vivax sporogonic development.

PubMed

Gonzalez-Ceron, Lilia; Santillan, Frida; Rodriguez, Mario H; Mendez, Domingo; Hernandez-Avila, Juan E

2003-05-01

Bacterial infections were investigated in midguts of insectary and field-collected Anopheles albimanus Weidemann from southern Mexico. Serratia marcescens, Enterobacter cloacae and Enterobacter amnigenus 2, Enterobacter sp., and Serratia sp. were isolated in field samples obtained in 1998, but only Enterobacter sp. was recovered in field samples of 1997 and no bacteria were isolated from insectary specimens. These bacteria were offered along with Plasmodium vivax infected blood to aseptic insectary An. albimanus, and the number of infected mosquitoes as well as the oocyst densities assessed after 7d. Plasmodium vivax infections in mosquitoes co-infected with En. amnigenus 2, En. cloacae, and S. marcensces were 53, 17, and 210 times, respectively, lower than in control mosquitoes, and the mean oocyst density in mosquitoes co-infected with En. cloacae was 2.5 times lower than in controls. Mortality was 13 times higher in S. marcensces-infected mosquitoes compared with controls. The overall midgut bacterial infection in mosquito field populations may influence P. vivax transmission, and could contribute to explain the annual variations in malaria incidence observed in the area.

Sensors and regulators of intracellular pH.

PubMed

Casey, Joseph R; Grinstein, Sergio; Orlowski, John

2010-01-01

Protons dictate the charge and structure of macromolecules and are used as energy currency by eukaryotic cells. The unique function of individual organelles therefore depends on the establishment and stringent maintenance of a distinct pH. This, in turn, requires a means to sense the prevailing pH and to respond to deviations from the norm with effective mechanisms to transport, produce or consume proton equivalents. A dynamic, finely tuned balance between proton-extruding and proton-importing processes underlies pH homeostasis not only in the cytosol, but in other cellular compartments as well.

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

Intestinal alkaline phosphatase regulates protective surface microclimate pH in rat duodenum.

PubMed

Mizumori, Misa; Ham, Maggie; Guth, Paul H; Engel, Eli; Kaunitz, Jonathan D; Akiba, Yasutada

2009-07-15

Regulation of localized extracellular pH (pH(o)) maintains normal organ function. An alkaline microclimate overlying the duodenal enterocyte brush border protects the mucosa from luminal acid. We hypothesized that intestinal alkaline phosphatase (IAP) regulates pH(o) due to pH-sensitive ATP hydrolysis as part of an ecto-purinergic pH regulatory system, comprised of cell-surface P2Y receptors and ATP-stimulated duodenal bicarbonate secretion (DBS). To test this hypothesis, we measured DBS in a perfused rat duodenal loop, examining the effect of the competitive alkaline phosphatase inhibitor glycerol phosphate (GP), the ecto-nucleoside triphosphate diphosphohydrolase inhibitor ARL67156, and exogenous nucleotides or P2 receptor agonists on DBS. Furthermore, we measured perfusate ATP concentration with a luciferin-luciferase bioassay. IAP inhibition increased DBS and luminal ATP output. Increased luminal ATP output was partially CFTR dependent, but was not due to cellular injury. Immunofluorescence localized the P2Y(1) receptor to the brush border membrane of duodenal villi. The P2Y(1) agonist 2-methylthio-ADP increased DBS, whereas the P2Y(1) antagonist MRS2179 reduced ATP- or GP-induced DBS. Acid perfusion augmented DBS and ATP release, further enhanced by the IAP inhibitor l-cysteine, and reduced by the exogenous ATPase apyrase. Furthermore, MRS2179 or the highly selective P2Y(1) antagonist MRS2500 co-perfused with acid induced epithelial injury, suggesting that IAP/ATP/P2Y signalling protects the mucosa from acid injury. Increased DBS augments IAP activity presumably by raising pH(o), increasing the rate of ATP degradation, decreasing ATP-mediated DBS, forming a negative feedback loop. The duodenal epithelial brush border IAP-P2Y-HCO(3-) surface microclimate pH regulatory system effectively protects the mucosa from acid injury.

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange.

PubMed

Montrose, M H; Murer, H

1986-01-01

Suspensions of LLC-PK1 cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na+ and K+ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05 +/- 0.01, n = 5) of cells which have recovered in (pH 7.4) Na+-containing medium is not affected over several minutes by addition of 100 microM amiloride or removal of extracellular Na+ (Na+o less than 1 mM). In contrast, when the cells recover from an acid load (caused by NH4 preincubation and removal), the recovery is largely Na+ dependent and is sensitive to 100 microM amiloride. These results suggest that with resting pH near neutrality, both Na+o/H+i and Na+i/H+o exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na+o/H+i exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H+ efflux or net Na+ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a "set point" of Na+/H+ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.

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.

pH Regulation of Pectate Lyase Secretion Modulates the Attack of Colletotrichum gloeosporioides on Avocado Fruits†

PubMed Central

Yakoby, Nir; Kobiler, Ilana; Dinoor, Amos; Prusky, Dov

2000-01-01

Growth of Colletotrichum gloeosporioides in pectolytic enzyme-inducing medium (PEIM) increased the pH of the medium from 3.8 to 6.5. Pectate lyase (PL) secretion was detected when the pH reached 5.8, and the level of secretion increased up to pH 6.5. PL gene (pel) transcript production began at pH 5.0 and increased up to pH 5.7. PL secretion was never detected when the pH of the inducing medium was lower than 5.8 or when C. gloeosporioides hyphae were transferred from PL-secreting conditions at pH 6.5 to pH 3.8. This behavior differed from that of polygalacturonase (PG), where pg transcripts and protein secretion were detected at pH 5.0 and continued up to 5.7. Under in vivo conditions, the pH of unripe pericarp of freshly harvested avocado (Persea americana cv. Fuerte) fruits, resistant to C. gloeosporioides attack, was 5.2, whereas in ripe fruits, when decay symptoms were expressed, the pericarp pH had increased to 6.3. Two avocado cultivars, Ardit and Ettinger, which are resistant to C. gloeosporioides attack, had pericarp pHs of less than 5.5, which did not increase during ripening. The present results suggest that host pH regulates the secretion of PL and may affect C. gloeosporioides pathogenicity. The mechanism found in avocado may have equivalents in other postharvest pathosystems and suggests new approaches for breeding against and controlling postharvest diseases. PMID:10698767

Functional characterization of PhGR and PhGRL1 during flower senescence in the petunia.

PubMed

Yang, Weiyuan; Liu, Juanxu; Tan, Yinyan; Zhong, Shan; Tang, Na; Chen, Guoju; Yu, Yixun

2015-09-01

Petunia PhGRL1 suppression accelerated flower senescence and increased the expression of the genes downstream of ethylene signaling, whereas PhGR suppression did not. Ethylene plays an important role in flowers senescence. Homologous proteins Green-Ripe and Reversion to Ethylene sensitivity1 are positive regulators of ethylene responses in tomato and Arabidopsis, respectively. The petunia flower has served as a model for the study of ethylene response during senescence. In this study, petunia PhGR and PhGRL1 expression was analyzed in different organs, throughout floral senescence, and after exogenous ethylene treatment; and the roles of PhGR and PhGRL1 during petunia flower senescence were investigated. PhGRL1 suppression mediated by virus-induced gene silencing accelerated flower senescence and increased ethylene production; however, the suppression of PhGR did not. Taken together, these data suggest that PhGRL1 is involved in negative regulation of flower senescence, possibly via ethylene production inhibition and consequently reduced ethylene signaling activation.

A Systems Level Analysis Reveals Transcriptomic and Proteomic Complexity in Ixodes Ricinus Midgut and Salivary Glands During Early Attachment and Feeding*

PubMed Central

Schwarz, Alexandra; Tenzer, Stefan; Hackenberg, Michael; Erhart, Jan; Gerhold-Ay, Aslihan; Mazur, Johanna; Kuharev, Jörg; Ribeiro, José M. C.; Kotsyfakis, Michail

2014-01-01

Although pathogens are usually transmitted within the first 24–48 h of attachment of the castor bean tick Ixodes ricinus, little is known about the tick's biological responses at these earliest phases of attachment. Tick midgut and salivary glands are the main tissues involved in tick blood feeding and pathogen transmission but the limited genomic information for I. ricinus delays the application of high-throughput methods to study their physiology. We took advantage of the latest advances in the fields of Next Generation RNA-Sequencing and Label-free Quantitative Proteomics to deliver an unprecedented, quantitative description of the gene expression dynamics in the midgut and salivary glands of this disease vector upon attachment to the vertebrate host. A total of 373 of 1510 identified proteins had higher expression in the salivary glands, but only 110 had correspondingly high transcript levels in the same tissue. Furthermore, there was midgut-specific expression of 217 genes at both the transcriptome and proteome level. Tissue-dependent transcript, but not protein, accumulation was revealed for 552 of 885 genes. Moreover, we discovered the enrichment of tick salivary glands in proteins involved in gene transcription and translation, which agrees with the secretory role of this tissue; this finding also agrees with our finding of lower tick t-RNA representation in the salivary glands when compared with the midgut. The midgut, in turn, is enriched in metabolic components and proteins that support its mechanical integrity in order to accommodate and metabolize the ingested blood. Beyond understanding the physiological events that support hematophagy by arthropod ectoparasites, we discovered more than 1500 proteins located at the interface between ticks, the vertebrate host, and the tick-borne pathogens. Thus, our work significantly improves the knowledge of the genetics underlying the transmission lifecycle of this tick species, which is an essential step for

Determination of nitric oxide metabolites, nitrate and nitrite, in Anopheles culicifacies mosquito midgut and haemolymph by anion exchange high-performance liquid chromatography: plausible mechanism of refractoriness

PubMed Central

Sharma, Arun; Raghavendra, Kamaraju; Adak, Tridibesh; Dash, Aditya P

2008-01-01

Background The diverse physiological and pathological role of nitric oxide in innate immune defenses against many intra and extracellular pathogens, have led to the development of various methods for determining nitric oxide (NO) synthesis. NO metabolites, nitrite (NO2-) and nitrate (NO3-) are produced by the action of an inducible Anopheles culicifacies NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes. Method While exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of An. culicifacies, a sensitive, specific and cost effective high performance liquid chromatography (HPLC) method was developed, which is not influenced by the presence of biogenic amines, for the determination of NO2- and NO3- from mosquito mid-guts and haemolymph. Results This method is based on extraction, efficiency, assay reproducibility and contaminant minimization. It entails de-proteinization by centrifugal ultra filtration through ultracel 3 K filter and analysis by high performance anion exchange liquid chromatography (Sphereclone, 5 μ SAX column) with UV detection at 214 nm. The lower detection limit of the assay procedure is 50 pmoles in all midgut and haemolymph samples. Retention times for NO2- and NO3- in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 nM and 1 mM. Recoveries of NO2- and NO3- from spiked samples (1–100 μM) and from the extracted standards (1–100 μM) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO2- and NO3- in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO2- and NO3- in midguts and haemolymph of An. culicifacies sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology. Conclusion HPLC is a sensitive and accurate technique

Intracellular pH regulation in hepatocytes isolated from three teleost species.

PubMed

Furimsky, M; Moon, T W; Perry, S F

1999-09-01

The mechanisms of intracellular pH (pH(i)) regulation were studied in hepatocytes isolated from three species of teleost: rainbow trout (Oncorhynchus mykiss), black bullhead (Ameiurus melas) and American eel (Anguilla rostrata). Intracellular pH was monitored over time using the pH-sensitive fluorescent dye BCECF in response to acid loading under control conditions and in different experimental media containing either low Na(+) or Cl(-) concentrations, the Na(+)-H(+) exchanger blocker amiloride or the blocker of the V-type H(+)-ATPase, bafilomycin A(1). In trout and bullhead hepatocytes, recovery to an intracellular acid load occurred principally by way of a Na(+)-dependent amiloride-sensitive Na(+)-H(+) exchanger. In eel hepatocytes, the Na(+)-H(+) exchanger did not contribute to recovery to an acid load though evidence suggests that it is present on the cell membrane and participates in the maintenance of steady-state pH(i). The V-type H(+)-ATPase did not participate in recovery to an acid load in any species. A Cl(-)-HCO(3)(-) exchanger may play a role in recovery to an acid load in eel hepatocytes by switching off and retaining base that would normally be tonically extruded. Thus, it is clear that hepatocytes isolated from the three species are capable of regulating pH(i), principally by way of a Na(+)-H(+) exchanger and a Cl(-)-HCO(3)(-) exchanger, but do not exploit identical mechanisms for pH(i) recovery. J. Exp. Zool. 284:361-367, 1999. Copyright 1999 Wiley-Liss, Inc.

Molecular and biochemical responses in the midgut of the silkworm, Bombyx mori, infected with Nosema bombycis.

PubMed

Li, Zhi; Wang, Yu; Wang, Linling; Zhou, Zeyang

2018-03-06

Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. However, there is little information available of how microsporidia obtain nutrients and energy from host cells. The purpose of this study was to investigate the energy and material requirements of Nosema bombycis for the invasion procedure through analyzing the global variation of the gene expression, protein abundance, fatty acids level and ATP flux induced by the microsporidia N. bombycis infection in the midgut of the silkworm Bombyx mori. A suppression subtractive hybridization (SSH) and quantitative real-time PCR (qPCR) analysis were performed to identify the genes upregulated in the midgut of B. mori 48 h following N. bombycis infection. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to annotate and summarize the differentially expressed genes, according to the categories 'molecular function', 'cellular component' and 'biological process'. To evaluate the nutrition material and energy costs in B.mori infected by N. bombycis, biochemical analysis was performed to determine the variation of protein abundance, fatty acid levels and ATP flux with or without the microsporidia N. bombycis infection in the midgut of the silkworm B. mori. A total of 744 clones were obtained, 288 clones were randomly selected for sequencing, and 110 unigenes were generated. Amongst these, 49.21%, 30.16% and 14.29% genes were involved in 19 molecular functions, 19 biological processes and nine cellular components, respectively. A total of 11 oxidative phosphorylation- and eight proton-coupled ATP synthesis-related genes were upregulated. Seven protein degradation-, three fat degradation-related genes were upregulated, and no genes related to the de novo synthesis of amino acids and fatty acids were significantly upregulated. The data from the biochemical analysis showed the contents of total protein and ATP of B. mori

Midgut glycosidases activities in monophagous larvae of Apollo butterfly, Parnassius apollo ssp. frankenbergeri.

PubMed

Nakonieczny, Mirosław; Michalczyk, Katarzyna; Kedziorski, Andrzej

2006-10-01

Parnassius apollo (Lepidoptera, Papilionidae) declines on numerous localities all over Europe. Its local subspecies frankenbergeri, inhabiting the Pieniny Mts (southern Poland) and successfully recovered from extinction, is monophagous in larval stage. In natural conditions, it completes development on the orpine Sedum telephium ssp. maximum. Since proper quality and quantity of necessary nutritional compounds of the food plant ensure developmental success, the digestive processes in the insect midgut should reflect adaptation to a specific food source. The paper presents, for the first time, the activity of detected glycolytic enzymes in midgut tissue and liquid gut contents of the L4 and L5 instars of P. apollo larvae. alpha-Amylase plays the main role in utilization of carbohydrates, contrary to cellulase activity. Saccharase seems to be the main disaccharidase, and high activity of beta-glycosidase enables hydrolysis of the plant glycosides. Trehalase activity was unexpectedly low and comparable to those of cellobiase and lactase. alpha-Amylolytic and other glycolytic activities indicate that larvae utilize starch and other carbohydrate compounds as energy sources. Possible use of some plant allelochemicals as energy sources by Apollo larvae is discussed.

Ethylene regulates phosphorus remobilization and expression of a phosphate transporter (PhPT1) during petunia corolla senescence

PubMed Central

Chapin, Laura J.; Jones, Michelle L.

2009-01-01

The programmed degradation of macromolecules during petal senescence allows the plant to remobilize nutrients from dying to developing tissues. Ethylene is involved in regulating the timing of nucleic acid degradation in petunia, but it is not clear if ethylene has a role in the remobilization of phosphorus during petal senescence. To investigate ethylene's role in nutrient remobilization, the P content of petals (collectively called the corolla) during early development and senescence was compared in ethylene-sensitive wild type Petunia×hybrida ‘Mitchell Diploid’ (MD) and transgenic petunias with reduced sensitivity to ethylene (35S::etr1-1). When compared to the total P content of corollas on the day of flower opening (the early non-senescing stage), P in MD corollas had decreased 74% by the late stage of senescence (advanced wilting). By contrast, P levels were only reduced by an average of 32% during etr1-1 corolla (lines 44568 and Z00-35-10) senescence. A high-affinity phosphate transporter, PhPT1 (PhPht1;1), was cloned from senescing petunia corollas by RT-PCR. PhPT1 expression was up-regulated during MD corolla senescence and a much smaller increase was detected during the senescence of etr1-1 petunia corollas. PhPT1 mRNA levels showed a rapid increase in detached corollas (treated at 1 d after flower opening) following treatment with low levels of ethylene (0.1 μl l-1). Transcripts accumulated in the presence of the protein synthesis inhibitor, cycloheximide, indicating that PhPT1 is a primary ethylene response gene. PhPT1 is a putative phosphate transporter that may function in Pi translocation during senescence. PMID:19380421

Effects of neem oil (Azadirachta indica A. Juss) on the replacement of the midgut epithelium in the lacewing Ceraeochrysa claveri during larval-pupal metamorphosis.

PubMed

Scudeler, Elton Luiz; Padovani, Carlos Roberto; Santos, Daniela Carvalho Dos

2014-06-01

Larvae of the lacewing Ceraeochrysa claveri were fed on eggs of Diatraeasaccharalis treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval period. Pupae obtained from treated larvae were used in the study at five days after the completion of cocoon spinning to investigate the effects of neem oil on the replacement of the midgut epithelium during the larval-pupal transition. We observed that the old larval epithelium was shed into the midgut lumen and transformed into the yellow body. Old cells from the yellow body were destroyed by apoptosis and autophagy and were not affected by neem oil. However, neem oil did affect the new pupal epithelium. Cells from treated pupae showed cellular injuries such as a loss of microvilli, cytoplasmic vacuolization, an increase of glycogen stores, deformation of the rough endoplasmic reticulum and dilation of the perinuclear space. Additionally, the neem oil treatment resulted in the release of cytoplasmic protrusions, rupture of the plasma membrane and leakage of cellular debris into the midgut lumen, characteristics of cell death by necrosis. The results indicate that neem oil ingestion affects the replacement of midgut epithelium, causing cytotoxic effects that can alter the organism's physiology due to extensive cellular injuries. Copyright © 2014 Elsevier GmbH. All rights reserved.

pH modulation of glial glutamate transporters regulates synaptic transmission in the nucleus of the solitary tract

PubMed Central

McCrimmon, Donald R.; Martina, Marco

2013-01-01

The nucleus of the solitary tract (NTS) is the major site for termination of visceral sensory afferents contributing to homeostatic regulation of, for example, arterial pressure, gastric motility, and breathing. Whereas much is known about how different neuronal populations influence these functions, information about the role of glia remains scant. In this article, we propose that glia may contribute to NTS functions by modulating excitatory neurotransmission. We found that acidification (pH 7.0) depolarizes NTS glia by inhibiting K+-selective membrane currents. NTS glia also showed functional expression of voltage-sensitive glutamate transporters, suggesting that extracellular acidification regulates synaptic transmission by compromising glial glutamate uptake. To test this hypothesis, we evoked glutamatergic slow excitatory potentials (SEPs) in NTS neurons with repetitive stimulation (20 pulses at 10 Hz) of the solitary tract. This SEP depends on accumulation of glutamate following repetitive stimulation, since it was potentiated by blocking glutamate uptake with dl-threo-β-benzyloxyaspartic acid (TBOA) or a glia-specific glutamate transport blocker, dihydrokainate (DHK). Importantly, extracellular acidification (pH 7.0) also potentiated the SEP. This effect appeared to be mediated through a depolarization-induced inhibition of glial transporter activity, because it was occluded by TBOA and DHK. In agreement, pH 7.0 did not directly alter d-aspartate-induced responses in NTS glia or properties of presynaptic glutamate release. Thus acidification-dependent regulation of glial function affects synaptic transmission within the NTS. These results suggest that glia play a modulatory role in the NTS by integrating local tissue signals (such as pH) with synaptic inputs from peripheral afferents. PMID:23615553

pH and external Ca(2+) regulation of a small conductance Cl(-) channel in kidney distal tubule.

PubMed

Sauvé, R; Cai, S; Garneau, L; Klein, H; Parent, L

2000-12-20

A single channel characterization of the Cl(-) channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl(-) channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca(2+) regulation of a small conductance Cl(-) channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability P(O). Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel P(O) with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca(2+) concentration, with a dose-dependent increase in channel activity as a function of the cis Ca(2+) concentration. It is concluded on the basis of these results that the small conductance Cl(-) channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca(2+).

Onset of vitellogenin production and vitellogenesis, and their relationship to changes in the midgut epithelium and oocytes in the tick Dermacentor variabilis.

PubMed

Coons, L B; Lamoreaux, W J; Rosell-Davis, R; Tarnowski, B I

1989-05-01

In Dermacentor variabilis (Say), the onset of vitellogenin production and vitellogenesis (uptake of vitellogenin into oocytes) began during the rapid-engorgement feeding period. Mating was required for both vitellogenin production and vitellogenesis to complete the tick's life cycle. Complete immunological identity, as measured by Ouchterlony's double diffusion test, existed between vitellogenin from the fat body, midgut and hemolymph, and vitellin from the ovaries and eggs. Antivitellin antibody did not react with host hemoglobin nor with fat body, midgut, and ovary extracts from feeding females prior to rapid engorgement, feeding unmated females, or unfed or fed males. Some unmated females fed for 13 days and then hand-detached from the host eventually began oviposition after going through a preoviposition period. In these ticks, organ extracts from the midgut, fat body and ovary reacted with antivitellin antibody. The presence or absence of presumed vitellogenic cells in the midgut and yolk bodies in oocytes corresponded with the presence or absence of vitellogenin and vitellogenesis as measured by Ouchterlony's test. Presumed vitellogenic cells increased in size during the preoviposition period. These cells reached their greatest size during the time when the most eggs were being produced, and then declined in size toward the end of oviposition. Vitellogenin was deposited directly into developing yolk bodies in oocytes and was not processed through lysosomes. Feeding was the process that initiated the formation of eggshell cuticle. Detachment from the host was required for the initiation of oviposition.

Side Effects of Neem Oil on the Midgut Endocrine Cells of the Green Lacewing Ceraeochrysa claveri (Navás) (Neuroptera: Chrysopidae).

PubMed

Scudeler, E L; Santos, D C

2014-04-01

We described the ultrastructure of Ceraeochrysa claveri (Navás) midgut endocrine cells in larva, pupa, and adult, and evaluated the side effects of ingested neem oil, a botanical insecticide obtained from the seeds of the neem tree (Azadirachta indica), on these cells. During the larval period, C. claveri were fed (ad libitum) Diatraea saccharalis (F.) eggs treated with neem oil at concentrations of 0.5%, 1%, or 2%. Transmission electron microscopy showed that two subtypes of endocrine cells, namely granular and vesicular, occurred in the midgut epithelium during the three stages of the life cycle. Both cell types did not reach the midgut lumen and were positioned basally in the epithelium. The endocrine cells did not show extensive infoldings of the basal plasma membrane, and there were numerous secretory granules in the basal region of the cytoplasm. In the granular endocrine cells, the granules were completely filled with a dense matrix. In the vesicular endocrine cells, the main secretory products consisted of haloed vesicles. Ultrastructural examination indicated that only the granular endocrine cells exhibited signs of morphologic changes of cell injury present in all life cycle stages after the larvae were chronically exposed to neem oil by ingestion. The major cellular damage consisted of dilatation and vesiculation of the rough endoplasmic reticulum and the development of smooth endoplasmic reticulum and mitochondrial swelling. Our data suggest that cytotoxic effects on midgut endocrine cells can contribute to a generalized disruption of the physiological processes in this organ due to a general alteration of endocrine function.

Transport of H(+), Na(+) and K(+) across the posterior midgut of blood-fed mosquitoes (Aedes aegypti).

PubMed

Pacey, Evan K; O'Donnell, Michael J

2014-02-01

Following ingestion of a blood meal, the adult female mosquito undergoes a massive diuresis during which Na(+), Cl(-) and water are secreted at high rates by the Malpighian tubules. In the hours following completion of diuresis, digestion of the K(+)-rich blood cells provides a source of energy as well as amino acids for proteins in the developing eggs. Although the transport of inorganic ions by the Malpighian tubules of blood-fed mosquitoes has been extensively characterized, relatively little is known of the epithelial transport mechanisms responsible for movement of Na(+), H(+), and K(+) across the posterior midgut. In this paper we have used the Scanning Ion-selective Electrode Technique (SIET) to measure the basal (unstimulated) rates of transport of K(+), Na(+) and H(+) across the isolated posterior midgut at intervals after the blood meal. We have also measured luminal concentrations of Na(+) and K(+) and the transepithelial electrical potential at the same time points and have calculated the electrochemical potentials for Na(+), K(+) and H(+) across the midgut. SIET measurements reveal absorption (lumen to bath) of Na(+) and H(+) and secretion of K(+) for the first 2h after blood-feeding. By 24h after the meal, absorption of Na(+) and H(+) remains active while there is an electrochemical gradient favouring absorption of K(+). Inhibition by ouabain and Ba(2+) suggest a role for the Na(+)/K(+)-ATPase and K(+) channels in absorption of Na(+) and K(+), respectively. Inhibition of H(+) absorption by acetazolamide implicates carbonic anhydrase in transepithelial H(+) transport. Copyright © 2014 Elsevier Ltd. All rights reserved.

Histochemical study of lectin binding sites in fourth and fifth instar gypsy moth larval midgut epithelium

Treesearch

Algimantas P. Valaitis

2011-01-01

There is evidence that the gypsy moth, Lymantria dispar, midgut epithelial brush border membrane has membrane-bound glycoconjugates, such as BTR-270 and aminopeptidase N (APN), which function as high affinity binding sites (receptors) for the insecticidal proteins produced by Bacillus thuringiensis (Bt). As gypsy...

Coral calcifying fluid pH is modulated by seawater carbonate chemistry not solely seawater pH.

PubMed

Comeau, S; Tambutté, E; Carpenter, R C; Edmunds, P J; Evensen, N R; Allemand, D; Ferrier-Pagès, C; Tambutté, S; Venn, A A

2017-01-25

Reef coral calcification depends on regulation of pH in the internal calcifying fluid (CF) in which the coral skeleton forms. However, little is known about calcifying fluid pH (pH CF ) regulation, despite its importance in determining the response of corals to ocean acidification. Here, we investigate pH CF in the coral Stylophora pistillata in seawater maintained at constant pH with manipulated carbonate chemistry to alter dissolved inorganic carbon (DIC) concentration, and therefore total alkalinity (A T ). We also investigate the intracellular pH of calcifying cells, photosynthesis, respiration and calcification rates under the same conditions. Our results show that despite constant pH in the surrounding seawater, pH CF is sensitive to shifts in carbonate chemistry associated with changes in [DIC] and [A T ], revealing that seawater pH is not the sole driver of pH CF Notably, when we synthesize our results with published data, we identify linear relationships of pH CF with the seawater [DIC]/[H + ] ratio, [A T ]/ [H + ] ratio and [[Formula: see text

TOR complex 1 regulates the yeast plasma membrane proton pump and pH and potassium homeostasis.

PubMed

Mahmoud, Shima; Planes, María Dolores; Cabedo, Marc; Trujillo, Cristina; Rienzo, Alessandro; Caballero-Molada, Marcos; Sharma, Sukesh C; Montesinos, Consuelo; Mulet, José Miguel; Serrano, Ramón

2017-07-01

We have identified in yeast a connection between two master regulators of cell growth: a biochemical connection involving the TORC1 protein kinase (which activates protein synthesis, nutrient uptake, and anabolism) and a biophysical connection involving the plasma membrane proton-pumping H + -ATPase Pma1 (which drives nutrient and K + uptake and regulates pH homeostasis). Raising the temperature to nonpermissive values in a TOR thermosensitive mutant decreases Pma1 activity. Rapamycin, a TORC1 inhibitor, inhibits Pma1 dependent on its receptor Fpr1 and on the protein phosphatase Sit4, a TORC1 effector. Mutation of either Sit4 or Tco89, a nonessential subunit of TORC1, decreases proton efflux, K + uptake, intracellular pH, cell growth, and tolerance to weak organic acids. Tco89 does not affect Pma1 activity but activates K + transport. © 2017 Federation of European Biochemical Societies.

Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel.

PubMed

He, Li; Si, Guangwei; Huang, Jiuhong; Samuel, Aravinthan D T; Perrimon, Norbert

2018-03-01

Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Ca 2+ levels, and increases in cytosolic Ca 2+ resemble the Piezo overexpression phenotype, suggesting that Piezo functions through Ca 2+ signalling. Further studies suggest that Ca 2+ signalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Ca 2+ in response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.

Ultrastructural Effects of Bacillus thuringiensis var. san diego on Midgut Cells of the Cottonwood Leaf Beetle1

Treesearch

Leah S. Bauer; Stuart H. Pankratz

1992-01-01

Sequential observations of the ultrastructural effects of Bacillus thuringiensis var. san diego were made on midgut epithelial cells of the cottonwood leaf beetle, Chrysomela scripta F. Larvae imbibed a droplet of B. thuringiensis var. san diego containing endotoxin and live...

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

PubMed

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.

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.

The effect of starvation and re-feeding on mitochondrial potential in the midgut of Neocaridina davidi (Crustacea, Malacostraca)

PubMed Central

Włodarczyk, Agnieszka; Sonakowska, Lidia; Kamińska, Karolina; Marchewka, Angelika; Wilczek, Grażyna; Wilczek, Piotr; Student, Sebastian; Rost-Roszkowska, Magdalena

2017-01-01

The midgut in the freshwater shrimp Neocaridina davidi (previously named N. heteropoda) (Crustacea, Malacostraca) is composed of a tube-shaped intestine and a large hepatopancreas that is formed by numerous blind-ended tubules. The precise structure and ultrastructure of these regions were presented in our previous papers, while here we focused on the ultrastructural changes that occurred in the midgut epithelial cells (D-cells in the intestine, B- and F- cells in the hepatopancreas) after long-term starvation and re-feeding. We used transmission electron microscopy, light and confocal microscopes and flow cytometry to describe all of the changes that occurred due to the stressor with special emphasis on mitochondrial alterations. A quantitative assessment of cells with depolarized mitochondria helped us to establish whether there is a relationship between starvation, re-feeding and the inactivation/activation of mitochondria. The results of our studies showed that in the freshwater shrimp N. davidi that were analyzed, long-term starvation activates the degeneration of epithelial cells at the ultrastructural level and causes an increase of cells with depolarized (non-active) mitochondria. The process of re-feeding leads to the gradual regeneration of the cytoplasm of the midgut epithelial cells; however, these changes were observed at the ultrastructural level. Additionally, re-feeding causes the regeneration of mitochondrial ultrastructure. Therefore, we can state that the increase in the number of cells with polarized mitochondria occurs slowly and does not depend on ultrastructural alterations. PMID:28282457

The effect of starvation and re-feeding on mitochondrial potential in the midgut of Neocaridina davidi (Crustacea, Malacostraca).

PubMed

Włodarczyk, Agnieszka; Sonakowska, Lidia; Kamińska, Karolina; Marchewka, Angelika; Wilczek, Grażyna; Wilczek, Piotr; Student, Sebastian; Rost-Roszkowska, Magdalena

2017-01-01

The midgut in the freshwater shrimp Neocaridina davidi (previously named N. heteropoda) (Crustacea, Malacostraca) is composed of a tube-shaped intestine and a large hepatopancreas that is formed by numerous blind-ended tubules. The precise structure and ultrastructure of these regions were presented in our previous papers, while here we focused on the ultrastructural changes that occurred in the midgut epithelial cells (D-cells in the intestine, B- and F- cells in the hepatopancreas) after long-term starvation and re-feeding. We used transmission electron microscopy, light and confocal microscopes and flow cytometry to describe all of the changes that occurred due to the stressor with special emphasis on mitochondrial alterations. A quantitative assessment of cells with depolarized mitochondria helped us to establish whether there is a relationship between starvation, re-feeding and the inactivation/activation of mitochondria. The results of our studies showed that in the freshwater shrimp N. davidi that were analyzed, long-term starvation activates the degeneration of epithelial cells at the ultrastructural level and causes an increase of cells with depolarized (non-active) mitochondria. The process of re-feeding leads to the gradual regeneration of the cytoplasm of the midgut epithelial cells; however, these changes were observed at the ultrastructural level. Additionally, re-feeding causes the regeneration of mitochondrial ultrastructure. Therefore, we can state that the increase in the number of cells with polarized mitochondria occurs slowly and does not depend on ultrastructural alterations.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

pH regulators in invadosomal functioning: proton delivery for matrix tasting.

PubMed

Brisson, Lucie; Reshkin, Stephan J; Goré, Jacques; Roger, Sébastien

2012-01-01

Invadosomes are actin-rich finger-like cellular structures sensing and interacting with the surrounding extracellular matrix (ECM) and involved in its proteolytic remodeling. Invadosomes are structures distinct from other adhesion complexes, and have been identified in normal cells that have to cross tissue barriers to fulfill their function such as leukocytes, osteoclasts and endothelial cells. They also represent features of highly aggressive cancer cells, allowing them to escape from the primary tumor, to invade surrounding tissues and to reach systemic circulation. They are localized to the ventral membrane of cells grown under 2-dimensional conditions and are supposed to be present all around cells grown in 3-dimensional matrices. Indeed invadosomes are key structures in physiological processes such as inflammation and the immune response, bone remodeling, tissue repair, but also in pathological conditions such as osteopetrosis and the development of metastases. Invadosomes are subdivided into podosomes, found in normal cells, and into invadopodia specific for cancer cells. While these two structures exhibit differences in organization, size, number and half-life, they share similarities in molecular composition, participation in cell-matrix adhesion and promoting matrix degradation. A key determinant in invadosomal function is the recruitment and release of proteases, such as matrix metalloproteinases (MMPs), serine proteases and cysteine cathepsins, together with their activation in a tightly controlled and highly acidic microenvironment. Therefore numerous pH regulators such as V-ATPases and Na(+)/H(+) exchangers, are found in invadosomes and are directly involved in their constitution as well as their functioning. This review focuses on the participation of pH regulators in invadosome function in physiological and pathological conditions, with a particular emphasis on ECM remodeling by osteoclasts during bone resorption and by cancer cells. Copyright �

Functional photoacoustic microscopy of pH.

PubMed

Chatni, Muhammad Rameez; Yao, Junjie; Danielli, Amos; Favazza, Christopher P; Maslov, Konstantin I; Wang, Lihong V

2011-10-01

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, an imbalance of pH regulation may result from or result in serious illness. In this paper, we report photoacoustic microscopy (PAM) of a commercially available pH-sensitive fluorescent dye (SNARF-5F carboxylic acid) in tissue phantoms. We demonstrated that PAM is capable of pH imaging in absolute values at tissue depths of up to 2.0 mm, greater than possible with other forms of optical microscopy.

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. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of novel serine proteinase gene transcripts in the midguts of two tropical insect pests, Scirpophaga incertulas (Wk.) and Helicoverpa armigera (Hb.).

PubMed

Mazumdar-Leighton, S; Babu, C R; Bennett, J

2000-01-01

We have used RT PCR and 3'RACE to identify diverse serine proteinase genes expressed in the midguts of the rice yellow stem borer (Scirpophaga incertulas) and Asian corn borer (Helicoverpa armigera). The RT-PCR primers encoded the conserved regions around the active site histidine57 and serine195 of Drosophila melanogaster alpha trypsin, including aspartate189 of the specificity pocket. These primers amplified three transcripts (SiP1-3) from midguts of S. incertulas, and two transcripts (HaP1-2) from midguts of H. armigera. The five RT PCR products were sequenced to permit design of gene-specific forward primers for use with anchored oligo dT primers in 3'RACE. Sequencing of the 3'RACE products indicated that SiP1, SiP2 and HaP1 encoded trypsin-like serine proteinases, while HaP2 encoded a chymotrypsin-like serine proteinases. The SiP3 transcript proved to be an abundant 960 nt mRNA encoding a trypsin-like protein in which the active site serine195 was replaced by aspartate. The possible functions of this unusual protein are discussed.

Successive Onset of Molecular, Cellular and Tissue-Specific Responses in Midgut Gland of Littorina littorea Exposed to Sub-Lethal Cadmium Concentrations

PubMed Central

Benito, Denis; Izagirre, Urtzi; Dallinger, Reinhard; Soto, Manu

2017-01-01

Cadmium (Cd) is one of the most harmful metals, being toxic to most animal species, including marine invertebrates. Among marine gastropods, the periwinkle (Littorina littorea) in particular can accumulate high amounts of Cd in its midgut gland. In this organ, the metal can elicit extensive cytological and tissue-specific alterations that may reach, depending on the intensity of Cd exposure, from reversible lesions to pathological cellular disruptions. At the same time, Littorina littorea expresses a Cd-specific metallothionein (MT) that, due to its molecular features, expectedly exerts a protective function against the adverse intracellular effects of this metal. The aim of the present study was, therefore, to assess the time course of MT induction in the periwinkle’s midgut gland on the one hand, and cellular and tissue-specific alterations in the digestive organ complex (midgut gland and digestive tract) on the other, upon exposure to sub-lethal Cd concentrations (0.25 and 1 mg Cd/L) over 21 days. Depending on the Cd concentrations applied, the beginning of alterations of the assessed parameters followed distinct concentration-dependent and time-dependent patterns, where the timeframe for the onset of the different response reactions became narrower at higher Cd concentrations compared to lower exposure concentrations. PMID:28829377

Distinct pH regulation of slow and rapid anion channels at the plasma membrane of Arabidopsis thaliana hypocotyl cells.

PubMed

Colcombet, Jean; Lelièvre, Françoise; Thomine, Sébastien; Barbier-Brygoo, Hélène; Frachisse, Jean-Marie

2005-07-01

Variations in both intracellular and extracellular pH are known to be involved in a wealth of physiological responses. Using the patch-clamp technique on Arabidopsis hypocotyl cells, it is shown that rapid-type and slow-type anion channels at the plasma membrane are both regulated by pH via distinct mechanisms. Modifications of pH modulate the voltage-dependent gating of the rapid channel. While intracellular alkalinization facilitates channel activation by shifting the voltage gate towards negative potentials, extracellular alkalinization shifts the activation threshold to more positive potentials, away from physiological resting membrane potentials. By contrast, pH modulates slow anion channel activity in a voltage-independent manner. Intracellular acidification and extracellular alkalinization increase slow anion channel currents. The possible role of these distinct modulations in physiological processes involving anion efflux and modulation of extracellular and/or intracellular pH, such as elicitor and ABA signalling, are discussed.

The role of mitochondrial reactive oxygen species in pH regulation in articular chondrocytes.

PubMed

Milner, P I; Wilkins, R J; Gibson, J S

2007-07-01

To examine the effect of O(2) and the role, and source, of reactive oxygen species (ROS) on pH regulation in articular chondrocytes. Cartilage from equine metacarpo/tarsophalangeal joints was digested (collagenase) to isolate chondrocytes and loaded with 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein, a pH-sensitive fluorophore. O(2) tension was maintained using Eschweiler tonometers and a Wosthoff gas mixer. Cells were exposed to agents which alter ROS levels, mitochondrial inhibitors and/or inhibitors of protein phosphorylation. ROS levels were determined by dichlorofluorescein and mitochondrial membrane potential measured using JC-1. pH homeostasis was dependent on ROS. Na(+)/H(+) exchanger (NHE) activity was inhibited at low O(2) tension (acid efflux reducing from 2.30+/-0.05 to 1.27+/-0.11mMmin(-1) at 1%). NHE activity correlated with ROS levels (r(2)=0.65). ROS levels were increased by antimycin A (with levels at 1% O(2) tension increasing from 59+/-9% of the value at 20% to 87+/-7%), but reduced by rotenone, myxothiazol and diphenyleneiodonium. Hypoxia induced depolarisation of the mitochondrial membrane potential (with JC-1 red-green fluorescence ratio at 1% O(2) tension decreasing to 40+/-10% of the value at 20%). The response to changes in O(2) and to antimycin A was inhibited by staurosporine, wortmanin and calyculin A. The fall in ROS levels in hypoxia reduces the ability of articular chondrocytes to regulate pH, inhibiting NHE activity via changes in protein phosphorylation. The site of ROS generation is likely to be mitochondrial electron transport chain complex III. These effects are important to understanding normal chondrocyte function and response to altered O(2) tension.

Refinements in the short-circuit technique and its application to active potassium transport across the cecropia midgut.

PubMed

Wood, J L; Moreton, R B

1978-12-01

1. The conventional, two-electrode method for measuring potential difference across an epithelium is subject to error due to potential gradients caused by current flow in the bathing medium. Mathematical analysis shows that the error in measuring short-circuit current is proportional to the resistivity of the bathing medium and to the separation of the two recording electrodes. It is particularly serious for the insect larval midgut, where the resistivity of the medium is high, and that of the tissue is low. 2. A system has been devised, which uses a third recording electrode to monitor directly the potential gradient in the bathing medium. By suitable electrical connexions, the gradient can be automatically compensated, leaving a residual error which depends on the thickness of the tissue, but not on the electrode separation. Because the thicknesses of most epithelia are smaller than the smallest practical electrode spacing, this error is smaller than that inherent in a two-electrode system. 3. Since voltage-gradients are automatically compensated, it is possible to obtain continuous readings of potential and current. A 'voltage-clamp' circuit is described, which allows the time-course of the short-circuit current to be studied. 4.The three-electrode system has been used to study the larval midgut of Hyalophora cecropia. The average results from five experiments were: initial potential difference (open-circuit): 98+/-11 mV (S.E.M.); short-circuit current at time 60 min: 498+/-160 microA cm=2; 'steady-state' resistance at 60 min: 150+/-26 omega cm2. The current is equivalent to a net potassium transport of 18.6 mu-equiv cm-2 h-1. 5. The electrical parameters of the midgut change rapidly with time. The potential difference decays with a half-time of about 158 min, the resistance increases with a half-time of about 16 min, and the short-circuit current decays as the sum of two exponential terms, with half-times of about 16 and 158 min respectively. In addition

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.

[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.

Proton Transport and pH Control in Fungi.

PubMed

Kane, Patricia M

2016-01-01

Despite diverse and changing extracellular environments, fungi maintain a relatively constant cytosolic pH and numerous organelles of distinct lumenal pH. Key players in fungal pH control are V-ATPases and the P-type proton pump Pma1. These two proton pumps act in concert with a large array of other transporters and are highly regulated. The activities of Pma1 and the V-ATPase are coordinated under some conditions, suggesting that pH in the cytosol and organelles is not controlled independently. Genomic studies, particularly in the highly tractable S. cerevisiae, are beginning to provide a systems-level view of pH control, including transcriptional responses to acid or alkaline ambient pH and definition of the full set of regulators required to maintain pH homeostasis. Genetically encoded pH sensors have provided new insights into localized mechanisms of pH control, as well as highlighting the dynamic nature of pH responses to the extracellular environment. Recent studies indicate that cellular pH plays a genuine signaling role that connects nutrient availability and growth rate through a number of mechanisms. Many of the pH control mechanisms found in S. cerevisiae are shared with other fungi, with adaptations for their individual physiological contexts. Fungi deploy certain proton transport and pH control mechanisms not shared with other eukaryotes; these regulators of cellular pH are potential antifungal targets. This review describes current and emerging knowledge proton transport and pH control mechanisms in S. cerevisiae and briefly discusses how these mechanisms vary among fungi.

Proton Transport and pH Control in Fungi

PubMed Central

Kane, Patricia M.

2018-01-01

Despite diverse and changing extracellular environments, fungi maintain a relatively constant cytosolic pH and numerous organelles of distinct lumenal pH. Key players in fungal pH control are V-ATPases and the P-type proton pump Pma1. These two proton pumps act in concert with a large array of other transporters and are highly regulated. The activities of Pma1 and the V-ATPaseare coordinated under some conditions, suggesting that pH in the cytosol and organelles is not controlled independently. Genomic studies, particularly in the highly tractable S. cerevisiae, are beginning to provide a systems-level view of pH control, including transcriptional responses to acid or alkaline ambient pH and definition of the full set of regulators required to maintain pH homeostasis. Genetically encoded pH sensors have provided new insights into localized mechanisms of pH control, as well as highlighting the dynamic nature of pH responses to the extracellular environment. Recent studies indicate that cellular pH plays a genuine signaling role that connects nutrient availability and growth rate through a number of mechanisms. Many of the pH control mechanisms found in S. cerevisiae are shared with other fungi, with adaptations for their individual physiological contexts. Fungi deploy certain proton transport and pH control mechanisms not shared with other eukaryotes; these regulators of cellular pH are potential antifungal targets. This re view describes current and emerging knowledge proton transport and pH control mechanisms in S. cerevisiae and briefly discusses how these mechanisms vary among fungi. PMID:26721270

Robustness of the bacterial community in the cabbage white butterfly larval midgut.

PubMed

Robinson, Courtney J; Schloss, Patrick; Ramos, Yolied; Raffa, Kenneth; Handelsman, Jo

2010-02-01

Microbial communities typically vary in composition and structure over space and time. Little is known about the inherent characteristics of communities that govern various drivers of these changes, such as random variation, changes in response to perturbation, or susceptibility to invasion. In this study, we use 16S ribosomal RNA gene sequences to describe variation among bacterial communities in the midguts of cabbage white butterfly (Pieris rapae) larvae and examine the influence of community structure on susceptibility to invasion. We compared communities in larvae experiencing the same conditions at different times (temporal variation) or fed different diets (perturbation). The most highly represented phylum was Proteobacteria, which was present in all midgut communities. The observed species richness ranged from six to 15, and the most abundant members affiliated with the genera Methylobacteria, Asaia, Acinetobacter, Enterobacter, and Pantoea. Individual larvae subjected to the same conditions at the same time harbored communities that were highly similar in structure and membership, whereas the communities observed within larval populations changed with diet and over time. In addition, structural changes due to perturbation coincided with enhanced susceptibility to invasion by Enterobacter sp. NAB3R and Pantoea stewartii CWB600, suggesting that resistance to invasion is in part governed by community structure. These findings along with the observed conservation of membership at the phylum level, variation in structure and membership at lower taxonomic levels, and its relative simplicity make the cabbage white butterfly larval community an attractive model for studying community dynamics and robustness.

Regulation of intracellular pH in the rabbit cortical collecting tubule.

PubMed Central

Weiner, I D; Hamm, L L

1990-01-01

The cortical collecting tubule (CCT) is an important nephron segment for Na+, K+, water and acid-base transport. Differential loading characteristics of the pH sensitive dye 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) and basolateral Cl- removal were used to identify and study intracellular pH (pHi) regulation in each of three cell types involved in this transport. Both principal cells and beta-intercalated cells were found to have a basolateral Na+/H+ exchanger based on the Na+ and amiloride sensitivity of pHi recovery from acid loads. Intercalated cells demonstrated abrupt pHi changes with basolateral Cl- removal. alpha-intercalated cells alkalinized; beta-intercalated cells acidified. In the beta-intercalated cells, luminal Cl- removal blocked changes in pHi in response to changes in luminal HCO3- or peritubular Cl-, providing direct evidence for a luminal Cl-/HCO3- exchanger. In principal cells, brief removal of either peritubular or luminal Cl- resulted in no change in pHi; however, return of peritubular Cl- after prolonged removal resulted in a rapid fall in pHi consistent with a basolateral Cl-/HCO3- exchanger, which may be relatively inactive under baseline conditions. Therefore, Cl-/HCO3- exchange is present in all three cell types but varies in location and activity. PMID:2153152

Changes in pH and NADPH regulate the DNA binding activity of neuronal PAS domain protein 2, a mammalian circadian transcription factor.

PubMed

Yoshii, Katsuhiro; Tajima, Fumihisa; Ishijima, Sumio; Sagami, Ikuko

2015-01-20

Neuronal PAS domain protein 2 (NPAS2) is a core clock transcription factor that forms a heterodimer with BMAL1 to bind the E-box in the promoter of clock genes and is regulated by various environmental stimuli such as heme, carbon monoxide, and NAD(P)H. In this study, we investigated the effects of pH and NADPH on the DNA binding activity of NPAS2. In an electrophoretic mobility shift (EMS) assay, the pH of the reaction mixture affected the DNA binding activity of the NPAS2/BMAL1 heterodimer but not that of the BMAL1/BMAL1 homodimer. A change in pH from 7.0 to 7.5 resulted in a 1.7-fold increase in activity in the absence of NADPH, and NADPH additively enhanced the activity up to 2.7-fold at pH 7.5. The experiments using truncated mutants revealed that N-terminal amino acids 1-61 of NPAS2 were sufficient to sense the change in both pH and NADPH. We further analyzed the kinetics of formation and DNA binding of the NPAS2/BMAL1 heterodimer at various pH values. In the absence of NADPH, a change in pH from 6.5 to 8.0 decreased the KD(app) value of the E-box from 125 to 22 nM, with an 8-fold increase in the maximal level of DNA binding for the NPAS2/BMAL1 heterodimer. The addition of NADPH resulted in a further decrease in KD(app) to 9 nM at pH 8.0. Furthermore, NPAS2-dependent transcriptional activity in a luciferase assay using NIH3T3 cells also increased with the pH of the culture medium. These results suggest that NPAS2 has a role as a pH and metabolite sensor in regulating circadian rhythms.

Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome

PubMed Central

Jiang, Jian; Huang, Ying; Shu, Changlong; Soberón, Mario; Bravo, Alejandra; Liu, Chunqing; Song, Fuping; Lai, Jinsheng

2017-01-01

ABSTRACT The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), containing two cry genes (cry8-like and cry8Ga), is toxic to Holotrichia oblita larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMVs) in H. oblita larvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. The results of saturation binding assays demonstrated that Cry8-like bound specifically to binding sites on BBMVs from H. oblita, and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in the midgut from H. oblita larvae were identified by pulldown assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the H. oblita midgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like-binding proteins. Eight Cry8-like-binding proteins were obtained from pulldown assays conducted with BBMVs. The LC-MS/MS data for these proteins were successfully matched with the H. oblita transcriptome, and BLASTX results identified five proteins as serine protease, transferrin-like, uncharacterized protein LOC658236 of Tribolium castaneum, ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase, alkaline phosphatase, and cadherin. IMPORTANCE Holotrichia oblita is one of the main soil-dwelling pests in China. The larvae damage the roots of crops, resulting in significant yield reductions and economic losses. H. oblita is difficult to control, principally due to its soil-dwelling habits. In recent years, some Cry8 toxins from Bacillus thuringiensis were shown to be active against this pest. Study of the mechanism

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.

Diaphorina citri nymphs are resistant to morphological changes induced by “Candidatus Liberibacter asiaticus” in midgut epithelial cells

USDA-ARS?s Scientific Manuscript database

“Candidatus Liberibacter asiaticus” is the causative bacterium associated with citrus greening disease. “Ca. L. asiaticus” is transmitted by Diaphorina citri more efficiently when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared midguts of D. citri reared on hea...

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus

PubMed Central

Bevensee, Mark O.; Weed, Regina A.; Boron, Walter F.

1997-01-01

We studied the regulation of intracellular pH (pHi) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pHi. Intrinsic buffering power (βI) was 10.5 mM (pH unit)−1 at pHi 7.0, and decreased linearly with pHi; the best-fit line to the data had a slope of −10.0 mM (pH unit)−2. In the absence of HCO3 −, pHi recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pHi 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pHi. Acid extrusion was inhibited 68% (pHi 6.23) by substituting Li+ for Na+ in the bath solution. Switching from a CO2/HCO3 −-free to a CO2/HCO3 −-containing bath solution caused mean steady state pHi to increase from 6.82 to 6.90, due to a Na+-driven HCO3 − transporter. The HCO3 −-induced pHi increase was unaffected by amiloride, but was inhibited 75% (pHi 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pHi 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO2/HCO3 −-induced pHi increase was blocked when external Na+ was replaced with N-methyl-d-glucammonium (NMDG+). In the presence of HCO3 −, the Na+-driven HCO3 − transporter contributed to the pHi recovery from an acid load. For example, HCO3 − shifted the plot of acid-extrusion rate vs. pHi by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO3 − increased acid extrusion 3.8-fold (pHi 6.20). When astrocytes were acid loaded in amiloride, with Li+ as the major cation, HCO3 − failed to elicit a substantial increase in pHi. Thus, Li+ does not appear to substitute well for Na+ on the HCO3 − transporter. We conclude that an amiloride

Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture-independent methods.

PubMed

Broderick, Nichole A; Raffa, Kenneth F; Goodman, Robert M; Handelsman, Jo

2004-01-01

Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the gypsy moth (Lymantria dispar L.), whose gut is highly alkaline, by using traditional culturing and culture-independent methods. We also examined the effects of diet on microbial composition. Analysis of individual third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the gamma-Proteobacteria and low G+C gram-positive divisions and that the cultured members represented more than half of the phylotypes identified. Less frequently detected taxa included members of the alpha-Proteobacterium, Actinobacterium, and Cytophaga/Flexibacter/Bacteroides divisions. The 16S rRNA gene sequences from 7 of the 15 cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that an Enterococcus species and culture-independent analysis indicated that an Entbacter sp. were both present in all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among individual insects. These results provide the first comprehensive description of the microbial diversity of a lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut bacterial community.

Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis.

PubMed

Martins, Erica Soares; Monnerat, Rose Gomes; Queiroz, Paulo Roberto; Dumas, Vinicius Fiuza; Braz, Shélida Vasconcelos; de Souza Aguiar, Raimundo Wagner; Gomes, Ana Cristina Menezes Mendes; Sánchez, Jorge; Bravo, Alejandra; Ribeiro, Bergmann Morais

2010-02-01

Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis. 2009. Published by Elsevier Ltd.

Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Minghui; Zhang, Wei K.; Benvin, Nicole M.

The activities of organellar ion channels are often regulated by Ca2+ and H+, which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca2+/pH regulation of TRPML1, a Ca2+-release channel crucial for endolysosomal function. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained crystal structures of the 213-residue luminal domain of human TRPML1 containing three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore loop. Cysteine cross-linking and cryo-EMmore » analyses confirmed that this architecture occurs in the full-length channel. Structure–function studies demonstrated that Ca2+ and H+ interact with the luminal pore and exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal-domain structure and cause TRPML1 mislocalization. Our study reveals the structural underpinnings of TRPML1's regulation, assembly and pathogenesis.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh

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 weremore » 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.« less

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

2016-07-05

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.

Pseudoxanthomonas icgebensis sp. nov., isolated from the midgut of Anopheles stephensi field-collected larvae.

PubMed

Rani, Asha; Sharma, Anil; Adak, Tridibes; Bhatnagar, Raj K

2010-10-01

A Gram-negative, aerobic, golden yellow, rod-shaped bacterium, a strain designated ICGEB-L15(T), was isolated from the larval midgut of Anopheles stephensi captured in District Jhajjar, Haryana, India. The strain ICGEB-L15(T) grows at 30-50°C (optimum 30-37°C), pH 6.5-8.5 (optimum 7.0-8.0) and in the presence of 2% NaCl. The major fatty acids were iso-C(15:0) (22.5% of total fatty acid), anteiso-C(15:0) (16.5%), iso-C(17:1) 9c (10.3%), iso-C(16:0) (7.3%), C(16:0) (6.1%), and iso-C(11:0) (5.3%). The strain showed the highest 16S rRNA gene sequence similarities with the type strains Pseudoxanthomonas daejeonensis KCTC 12207(T) (97.4%), Pseudoxanthomonas kaohsiungensis J36(T) (97.17%), and Pseudoxanthomonas mexicana AMX 26B(T) (97.11%). The DNA relatedness between ICGEB-L15(T) and Pseudoxanthomonas daejeonensis KCTC 12207(T), Pseudoxanthomonas kaohsiungensis J36(T) and Pseudoxanthomonas mexicana AMX 26B(T) was 24.5%, 28.2%, and 33.6%, respectively. The G+C content of genomic DNA was 69.9 mol%. The major isoprenoid quinone of strain ICGEB-L15(T) was Q-8. The strain ICGEB-L15(T) represents a novel species of the genus Pseudoxanthomonas based on physiological, biochemical and phylogenetic properties; therefore, the name Pseudoxanthomonas icgebensis sp. nov. is proposed. The type strain is ICGEB-L15(T) (=KACC 14090(T) =DSM 22536(T)).

A SLC4 family bicarbonate transporter is critical for intracellular pH regulation and biomineralization in sea urchin embryos.

PubMed

Hu, Marian Y; Yan, Jia-Jiun; Petersen, Inga; Himmerkus, Nina; Bleich, Markus; Stumpp, Meike

2018-05-01

Efficient pH regulation is a fundamental requisite of all calcifying systems in animals and plants but with the underlying pH regulatory mechanisms remaining largely unknown. Using the sea urchin larva, this work identified the SLC4 HCO 3 - transporter family member SpSlc4a10 to be critically involved in the formation of an elaborate calcitic endoskeleton. SpSlc4a10 is specifically expressed by calcifying primary mesenchyme cells with peak expression during de novo formation of the skeleton. Knock-down of SpSlc4a10 led to pH regulatory defects accompanied by decreased calcification rates and skeleton deformations. Reductions in seawater pH, resembling ocean acidification scenarios, led to an increase in SpSlc4a10 expression suggesting a compensatory mechanism in place to maintain calcification rates. We propose a first pH regulatory and HCO 3 - concentrating mechanism that is fundamentally linked to the biological precipitation of CaCO 3 . This knowledge will help understanding biomineralization strategies in animals and their interaction with a changing environment. © 2018, Hu et al.

A SLC4 family bicarbonate transporter is critical for intracellular pH regulation and biomineralization in sea urchin embryos

PubMed Central

Yan, Jia-Jiun; Petersen, Inga; Himmerkus, Nina; Bleich, Markus; Stumpp, Meike

2018-01-01

Efficient pH regulation is a fundamental requisite of all calcifying systems in animals and plants but with the underlying pH regulatory mechanisms remaining largely unknown. Using the sea urchin larva, this work identified the SLC4 HCO3- transporter family member SpSlc4a10 to be critically involved in the formation of an elaborate calcitic endoskeleton. SpSlc4a10 is specifically expressed by calcifying primary mesenchyme cells with peak expression during de novo formation of the skeleton. Knock-down of SpSlc4a10 led to pH regulatory defects accompanied by decreased calcification rates and skeleton deformations. Reductions in seawater pH, resembling ocean acidification scenarios, led to an increase in SpSlc4a10 expression suggesting a compensatory mechanism in place to maintain calcification rates. We propose a first pH regulatory and HCO3- concentrating mechanism that is fundamentally linked to the biological precipitation of CaCO3. This knowledge will help understanding biomineralization strategies in animals and their interaction with a changing environment. PMID:29714685

Afforestation neutralizes soil pH.

PubMed

Hong, Songbai; Piao, Shilong; Chen, Anping; Liu, Yongwen; Liu, Lingli; Peng, Shushi; Sardans, Jordi; Sun, Yan; Peñuelas, Josep; Zeng, Hui

2018-02-06

Soil pH regulates soil biogeochemical processes and has cascading effects on terrestrial ecosystem structure and functions. Afforestation has been widely adopted to increase terrestrial carbon sequestration and enhance water and soil preservation. However, the effect of afforestation on soil pH is still poorly understood and inconclusive. Here we investigate the afforestation-caused soil pH changes with pairwise samplings from 549 afforested and 148 control plots in northern China. We find significant soil pH neutralization by afforestation-afforestation lowers pH in relatively alkaline soil but raises pH in relatively acid soil. The soil pH thresholds (T pH ), the point when afforestation changes from increasing to decreasing soil pH, are species-specific, ranging from 5.5 (Pinus koraiensis) to 7.3 (Populus spp.) with a mean of 6.3. These findings indicate that afforestation can modify soil pH if tree species and initial pH are properly matched, which may potentially improve soil fertility and promote ecosystem productivity.

pH regulation of mitochondrial branch chain alpha-keto acid transport and oxidation in rat heart mitochondria.

PubMed

Hutson, S M

1987-07-15

.1. Changes in the rate of branched chain alpha-keto acid oxidation, particularly when ATP was added (increase delta pH), were found to parallel the pH effects observed on branched chain alpha-keto acid uptake. Therefore, transport, and by implication oxidation, can be regulated by pH changes within the physiological range. Furthermore, intracellular pH may affect the degree of compartmentation between the cytosolic and mitochondrial branched chain alpha-keto acid pools.

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

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

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

Fetal midgut volvulus: report of eight cases.

PubMed

Sciarrone, A; Teruzzi, E; Pertusio, A; Bastonero, S; Errante, G; Todros, T; Viora, E

2016-01-01

To evaluate whether prenatal diagnosis of intestinal midgut volvulus (a rare condition due to the small bowel loops twisting) can improve the prognosis of the newborns. In our Prenatal Diagnosis Center, eight cases of intestinal volvulus observed between 2007 and 2014 were retrospectively considered. Ultrasonographic signs can be direct and specific (whirlpool sign, coffee bean sign) or indirect and non-specific (abdominal mass, dilated bowel loops, pseudocysts, ascites, polyhydramnios). Prenatal diagnosis was performed at 20-34 weeks of gestation. All newborns were exposed to an emergency surgery: the major complication was due to cystic fibrosis. An early suspicion of intestinal volvulus allows the clinician to refer the patient to a tertiary center so to confirm the diagnosis and perform an appropriate follow-up in order to identify the proper time of delivery. The prognosis of the babies with prenatal intestinal volvulus depends on the length of the segment involved, on the level of intestinal obstruction, on the presence of meconium peritonitis and on the gestational age at birth. Our experience, according with the literature, suggests that ascites and absence of abdominal peristalsis are ultrasonographic signs that, in the third trimester of pregnancy, correctly lead to an immediate delivery intervention.

The digestive system of the "stick bug" Cladomorphus phyllinus (Phasmida, Phasmatidae): a morphological, physiological and biochemical analysis.

PubMed

Monteiro, Emiliano C; Tamaki, Fábio K; Terra, Walter R; Ribeiro, Alberto F

2014-03-01

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data. Copyright © 2013 Elsevier Ltd. All rights reserved.

Antagonistic regulation, yet synergistic defense: effect of bergapten and protease inhibitor on development of cowpea bruchid Callosobruchus maculatus.

PubMed

Guo, Fengguang; Lei, Jiaxin; Sun, Yucheng; Chi, Yong Hun; Ge, Feng; Patil, Bhimanagouda S; Koiwa, Hisashi; Zeng, Rensen; Zhu-Salzman, Keyan

2012-01-01

The furanocoumarin compound bergapten is a plant secondary metabolite that has anti-insect function. When incorporated into artificial diet, it retarded cowpea bruchid development, decreased fecundity, and caused mortality at a sufficient dose. cDNA microarray analysis indicated that cowpea bruchid altered expression of 543 midgut genes in response to dietary bergapten. Among these bergapten-regulated genes, 225 have known functions; for instance, those encoding proteins related to nutrient transport and metabolism, development, detoxification, defense and various cellular functions. Such differential gene regulation presumably facilitates the bruchids' countering the negative effect of dietary bergapten. Many genes did not have homology (E-value cutoff 10(-6)) with known genes in a BlastX search (206), or had homology only with genes of unknown function (112). Interestingly, when compared with the transcriptomic profile of cowpea bruchids treated with dietary soybean cysteine protease inhibitor N (scN), 195 out of 200 coregulated midgut genes are oppositely regulated by the two compounds. Simultaneous administration of bergapten and scN attenuated magnitude of change in selected oppositely-regulated genes, as well as led to synergistic delay in insect development. Therefore, targeting insect vulnerable sites that may compromise each other's counter-defensive response has the potential to increase the efficacy of the anti-insect molecules.

pH regulation of recombinant glucoamylase production in Fusarium venenatum JeRS 325, a transformant with a Fusarium oxysporum alkaline (trypsin-like) protease promoter.

PubMed

Wiebe, M G; Robson, G D; Shuster, J R; Trinci, A P

1999-08-05

Fusarium venenatum (formerly Fusarium graminearum) JeRS 325 produces heterologous glucoamylase (GAM) under the regulation of a Fusarium oxysporum alkaline (trypsin-like) protease promoter. The glucoamylase gene was used as a reporter gene to study the effects of ammonium and pH on GAM production under the control of the alkaline protease promoter. Between pH 4.0 and 5.8, GAM production in glucose-limited chemostat cultures of JeRS 325 grown at a dilution rate of 0.10 h-1 (doubling time, 6.9 h) on (NH4)2SO4 medium increased in a linear manner with increase in pH. However, at pH 4.0 and below GAM production was almost completely repressed in glucose-limited chemostat cultures grown on (NH4)2SO4 or NaNO3 medium. Thus GAM production in JeRS 325 is regulated by culture pH, not by the nature of the nitrogen source in the medium. The difficulty of using unbuffered medium when investigating putative ammonium repression is also shown. The study demonstrates the potential for use of the alkaline protease promoter in F. graminearum for the production of recombinant proteins in a pH dependent man ner. Copyright 1999 John Wiley & Sons, Inc.

Identification of a molecular pH sensor in coral.

PubMed

Barott, Katie L; Barron, Megan E; Tresguerres, Martin

2017-11-15

Maintaining stable intracellular pH (pHi) is essential for homeostasis, and requires the ability to both sense pH changes that may result from internal and external sources, and to regulate downstream compensatory pH pathways. Here we identified the cAMP-producing enzyme soluble adenylyl cyclase (sAC) as the first molecular pH sensor in corals. sAC protein was detected throughout coral tissues, including those involved in symbiosis and calcification. Application of a sAC-specific inhibitor caused significant and reversible pHi acidosis in isolated coral cells under both dark and light conditions, indicating sAC is essential for sensing and regulating pHi perturbations caused by respiration and photosynthesis. Furthermore, pHi regulation during external acidification was also dependent on sAC activity. Thus, sAC is a sensor and regulator of pH disturbances from both metabolic and external origin in corals. Since sAC is present in all coral cell types, and the cAMP pathway can regulate virtually every aspect of cell physiology through post-translational modifications of proteins, sAC is likely to trigger multiple homeostatic mechanisms in response to pH disturbances. This is also the first evidence that sAC modulates pHi in any non-mammalian animal. Since corals are basal metazoans, our results indicate this function is evolutionarily conserved across animals. © 2017 The Author(s).

Dinotefuran-induced morphophysiological changes in the ovaries and midgut of semi-engorged females Rhipicephalus sanguineus Latreille, 1806 (Acari: Ixodidae) ticks.

PubMed

de Oliveira, Patrícia Rosa; Remédio, Rafael Neodini; Bechara, Gervásio Henrique; Anholeto, Luis Adriano; Mathias, Maria Izabel Camargo

2016-02-01

The present study demonstrated the effects of dinotefuran (active compound of the Protetor Pet® acaricide) in germ cells and the digestive processes of semi-engorged females of R. sanguineus exposed to different concentrations of the chemical. For this purpose, 120 semi-engorged females were divided into four treatment groups with 30 individuals each: group I or control (distilled water), group II (5000 ppm), group III (6250 ppm), and group IV (8334 ppm of dinotefuran). All ticks were immersed in different concentrations of dinotefuran or in distilled water for 5 min and then were dried and stored in biological oxygen demand (BOD) incubator for 7 days. The results show the action of this compound, exhibiting morphohistologic and histochemical changes in the oocytes and the midgut cells of individuals of different groups, which were compared with those of group I (control). The alterations occurred mainly in relation to the size of the germ cells and yolk granules; presence, quantity, size, and location of vacuoles found in the cytoplasm of these germ cells; the damage occurred in the generative cells of the midgut; the size of the digestive cells; the quantity of blood elements captured, accumulated digestive wastes and digestive vacuoles found in the cytoplasm of the digestive cells of the midgut, as well as the amount and distribution of proteins, polysaccharides, lipids of all cells in both organs. So, it has demonstrated the effectiveness of dinotefuran in the reduction of fertility and digestive processes of semi-engorged females of R. sanguineus, data that points the possibility of employing this chemical to control these ectoparasites.

Regulation of net bicarbonate transport in rabbit cortical collecting tubule by peritubular pH, carbon dioxide tension, and bicarbonate concentration.

PubMed Central

Breyer, M D; Kokko, J P; Jacobson, H R

1986-01-01

The effects of changes in peritubular pH, carbon dioxide tension (PCO2), and HCO3- concentration on net HCO3- transport was examined in in vitro perfused cortical collecting tubules (CCTs) from unpretreated New Zealand white rabbits. Lowering peritubular HCO3- concentration and pH by reciprocal replacement of HCO3- with Cl-, significantly stimulated net HCO3- absorption. Lowering peritubular HCO3- concentration and pH, by substitution of HCO3- with gluconate, while keeping Cl- concentration constant, also stimulated net HCO3- absorption. Raising peritubular HCO3- concentration and pH, by reciprocal replacement of Cl- with HCO3-, inhibited net HCO3- absorption (or stimulated net HCO3- secretion). When the tubule was cooled, raising peritubular HCO3- concentration had no effect on net HCO3- transport, suggesting these results are not due to the passive flux of HCO3- down its concentration gradient. The effect of changes in ambient PCO2 on net HCO3- transport were also studied. Increasing the ambient PCO2 from 40 mmHg to either 80 or 120 mmHg, allowing pH to fall, had no effect on net HCO3- transport. Similarly, lowering ambient PCO2 to 14 mmHg had no effect on net HCO3- transport. Simultaneously increasing peritubular HCO3- concentration and PCO2, without accompanying changes in peritubular pH, i.e., isohydric changes, stimulated net HCO3- secretion to the same degree as nonisohydric increases in peritubular HCO3- concentration. Likewise, isohydric lowering of peritubular HCO3- concentration and PCO2 stimulated net HCO3- absorption. We conclude that: acute changes in peritubular HCO3- concentration regulate acidification in the CCT and these effects are mediated by a transcellular process; acute changes in ambient PCO2 within the physiologic range have no effect on HCO3- transport in the in vitro perfused CCT; and acute in vitro regulation of CCT acidification is independent of peritubular pH. PMID:3084564

The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix.

PubMed

Arakane, Y; Muthukrishnan, S; Kramer, K J; Specht, C A; Tomoyasu, Y; Lorenzen, M D; Kanost, M; Beeman, R W

2005-10-01

Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1-specific RNA interference (RNAi) disrupted all three types of moult (larval-larval, larval-pupal and pupal-adult) and greatly reduced whole-body chitin content. Exon-specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval-pupal and pupal-adult moults, whereas splice variant 8b was required only for the latter. TcCHS2-specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi-mediated down-regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut.

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

K-targeted strategy for isolation of phenolic alkaloids of Nelumbo nucifera Gaertn by counter-current chromatography using lysine as a pH regulator.

PubMed

Wang, Yanyan; Zhang, Lihong; Zhou, Hui; Guo, Xiuyun; Wu, Shihua

2017-03-24

Counter-current chromatography (CCC) is an efficient liquid-liquid partition chromatography technique without support matrix. Despite there are many significant advancements in the CCC separation of natural products especially for non-ionic neutral compounds, CCC isolation of ionic compounds including alkaloids is still a challenging process guide by classical partition coefficients (K) or distribution ratio (K C ) because their partition coefficient could not be equal to distribution ratio in common ionic conditions. Here, taking the extract of embryo of the seed of Nelumbo nucifera Gaertn as sample, we introduced a modified K-targeted strategy for isolation of phenolic alkaloids by use of lysine as a pH regulator. The results indicated that if the mass of basic regulators such as aqueous ammonia and lysine added into the solvent system were high enough to inhibit the ionization of the targeted alkaloids, the distribution ratio of targets with ionic and non-ionic molecular forms got stable and might not been changed as the concentration of the pH regulator. In this case, the distribution ratio of target was almost equal to the partition coefficient. Thus, the targets could be isolated by K-targeted CCC separation through adding a certain amount pH regulators into the solvent system. Further experiments also showed that the sample concentration was an important factor on the distribution ratio of targets. Meanwhile, CCC experiments indicated that lysine was more suitable than aqueous ammonia for the separation of phenolic alkaloids because the chemical property of lysine-target complex in the CCC fractions was more stable. Therefore, the preparative CCC separation was performed using 20mM lysine as a pH regulator with more than 800mg injection mass. After simple back-extraction with dichloromethane, the lysine in the CCC fraction was removed completely and pure isoliensinine and neferine were obtained. In summary, the whole results indicated that the modified K

Spinosad-mediated effects on the walking ability, midgut, and Malpighian tubules of Africanized honey bee workers.

PubMed

Lopes, Marcos Pereira; Fernandes, Kenner Morais; Tomé, Hudson Vaner Ventura; Gonçalves, Wagner Gonzaga; Miranda, Franciane Rosa; Serrão, José Eduardo; Martins, Gustavo Ferreira

2018-06-01

The global decline in Apis mellifera colonies is attributed to multiple factors, including pesticides. The bioinsecticide spinosad was initially recognized as safe for non-target organisms; however, its toxicity has been changing this view. Here, we investigated the survival, behavioral changes, and structural changes in the midgut and Malpighian tubules of A. mellifera treated orally with a spinosad formulation. The field-recommended concentration of spinosad killed 100% of the bees. The 5% and 50% lethal concentrations (LC 5 and LC 50 , respectively) of spinosad altered the behavioral activity, reducing the walking distance and velocity, and increased the resting time in comparison to the control. The LC 50 caused disorganization of the epithelia of tested organs and induced oxidative stress and cell death. The present work provides new insights into the debate about the role of bioinsecticides in the mortality of Africanized honey bees. Even at very low concentrations, the spinosad formulation was toxic to the vital organs midgut and Malpighian tubules and adversely affected walking behavior. This detailed evaluation of the impact of the bioinsecticide on A. mellifera will contribute to the clarification of disturbances probably caused by spinosad formulations, which can be used to develop more sustainable protocols in agriculture. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

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

2016-08-01

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.

Coral calcifying fluid pH is modulated by seawater carbonate chemistry not solely seawater pH

PubMed Central

Tambutté, E.; Carpenter, R. C.; Edmunds, P. J.; Evensen, N. R.; Allemand, D.; Ferrier-Pagès, C.; Tambutté, S.; Venn, A. A.

2017-01-01

Reef coral calcification depends on regulation of pH in the internal calcifying fluid (CF) in which the coral skeleton forms. However, little is known about calcifying fluid pH (pHCF) regulation, despite its importance in determining the response of corals to ocean acidification. Here, we investigate pHCF in the coral Stylophora pistillata in seawater maintained at constant pH with manipulated carbonate chemistry to alter dissolved inorganic carbon (DIC) concentration, and therefore total alkalinity (AT). We also investigate the intracellular pH of calcifying cells, photosynthesis, respiration and calcification rates under the same conditions. Our results show that despite constant pH in the surrounding seawater, pHCF is sensitive to shifts in carbonate chemistry associated with changes in [DIC] and [AT], revealing that seawater pH is not the sole driver of pHCF. Notably, when we synthesize our results with published data, we identify linear relationships of pHCF with the seawater [DIC]/[H+] ratio, [AT]/ [H+] ratio and []. Our findings contribute new insights into the mechanisms determining the sensitivity of coral calcification to changes in seawater carbonate chemistry, which are needed for predicting effects of environmental change on coral reefs and for robust interpretations of isotopic palaeoenvironmental records in coral skeletons. PMID:28100813

Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

PubMed

Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

2012-04-15

Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

Serratia glossinae sp. nov., isolated from the midgut of the tsetse fly Glossina palpalis gambiensis.

PubMed

Geiger, A; Fardeau, M-L; Falsen, E; Ollivier, B; Cuny, G

2010-06-01

We report the isolation of a novel bacterium, strain C1(T), from the midgut of the tsetse fly Glossina palpalis gambiensis, one of the vector insects responsible for transmission of the trypanosomes that cause sleeping sickness in sub-Saharan African countries. Strain C1(T) is a motile, facultatively anaerobic, rod-like bacterium (0.8-1.0 microm in diameter; 2-6 microm long) that grows as single cells or in chains. Optimum growth occurred at 25-35 degrees C, at pH 6.7-8.4 and in medium containing 5-20 g NaCl l(-1). The bacterium hydrolysed urea and used L-lysine, L-ornithine, citrate, pyruvate, D-glucose, D-mannitol, inositol, D-sorbitol, melibiose, amygdalin, L-arabinose, arbutin, aesculin, D-fructose, D-galactose, glycerol, maltose, D-mannose, raffinose, trehalose and d-xylose; it produced acetoin, reduced nitrate to nitrite and was positive for beta-galactosidase and catalase. The DNA G+C content was 53.6 mol%. It was related phylogenetically to members of the genus Serratia, family Enterobacteriaceae, the type strain of Serratia fonticola being its closest relative (99 % similarity between 16S rRNA gene sequences). However, DNA-DNA relatedness between strain C1(T) and S. fonticola DSM 4576(T) was only 37.15 %. Therefore, on the basis of morphological, nutritional, physiological and fatty acid analysis and genetic criteria, strain C1(T) is proposed to be assigned to a novel Serratia species, Serratia glossinae sp. nov. (type strain C1(T) =DSM 22080(T) =CCUG 57457(T)).

Assessing the genotoxic effects of two lipid peroxidation products (4-oxo-2-nonenal and 4-hydroxy-hexenal) in haemocytes and midgut cells of Drosophila melanogaster larvae.

PubMed

Demir, Eşref; Marcos, Ricard

2017-07-01

Lipid peroxidation products can induce tissue damage and are implicated in diverse pathological conditions, including aging, atherosclerosis, brain disorders, cancer, lung and various liver disorders. Since in vivo studies produce relevant information, we have selected Drosophila melanogaster as a suitable in vivo model to characterise the potential risks associated to two lipid peroxidation products namely 4-oxo-2-nonenal (4-ONE) and 4-hydroxy-hexenal (4-HHE). Toxicity, intracellular reactive oxygen species production, and genotoxicity were the end-points evaluated. Haemocytes and midgut cells were the evaluated targets. Results showed that both compounds penetrate the intestine of the larvae, affecting midgut cells, and reaching haemocytes. Significant genotoxic effects, as determined by the comet assay, were observed in both selected cell targets in a concentration/time dependent manner. This study highlights the importance of D. melanogaster as a model organism in the study of the different biological effects caused by lipid peroxidation products entering via ingestion. This is the first study reporting genotoxicity data in haemocytes and midgut cells of D. melanogaster larvae for the two selected compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Low pH, aluminum, and phosphorus coordinately regulate malate exudation through GmALMT1 to improve soybean adaptation to acid soils.

PubMed

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

2013-03-01

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.

Low Medium pH Value Enhances Anthocyanin Accumulation in Malus Crabapple Leaves

PubMed Central

Tian, Ji; Jin, Kaina; Yao, Yuncong

2014-01-01

Anthocyanin is a critical factor involved in coloration of plant tissues, but the mechanism how medium pH values affect anthocyanin accumulation in woody plants is unknown. We analyzed anthocyanin composition and the expression of elements encoding anthocyanin and flavonols biosynthesis underlying different medium pH values by using three different leave color type cultivars. HPLC analysis demonstrated that high medium pH values treatment induced a dramatic decrease in the concentration of cyaniding in crabapple leaves. Conversely, the high medium pH values induced up-regulation of the content of flavones and flavonols, suggesting that low pH treatment-induced anthocyanin accumulation. Quantitative real time PCR experiment showed the expression level of anthocyanidin synthase (McANS) and uridine diphosphate glucose flavonoid 3-O-glucosyltransferase (McUFGT) was up-regulated by low pH values treatment, and high medium pH value treatment up-regulate the transcription level of flavonol synthase (McFLS). Meanwhile, several MYB TFs have been suggested in the regulation of pH responses. These results strongly indicate that the low pH treatment-induced anthocyanin accumulation is mediated by the variation of mRNA transcription of the anthocyanin biosynthetic genes. PMID:24914811

The PH Domain of PDK1 Exhibits a Novel, Phospho-Regulated Monomer-Dimer Equilibrium With Important Implications for Kinase Domain Activation: Single Molecule and Ensemble Studies†

PubMed Central

Ziemba, Brian P.; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J.

2013-01-01

Phosphoinositide-Dependent Kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4-5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric state(s) of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. The present study investigates the binding of purified WT and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single molecule and ensemble measurements. Single molecule analysis of the brightness of fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric, while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single molecule analysis of 2-D diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little protein penetration into the bilayer as observed for other PH domains. The 2-D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that enables greater protein insertion into

Long photoperiods sustain high pH in Arctic kelp forests.

PubMed

Krause-Jensen, Dorte; Marbà, Núria; Sanz-Martin, Marina; Hendriks, Iris E; Thyrring, Jakob; Carstensen, Jacob; Sejr, Mikael Kristian; Duarte, Carlos M

2016-12-01

Concern on the impacts of ocean acidification on calcifiers, such as bivalves, sea urchins, and foraminifers, has led to efforts to understand the controls on pH in their habitats, which include kelp forests and seagrass meadows. The metabolism of these habitats can lead to diel fluctuation in pH with increases during the day and declines at night, suggesting no net effect on pH at time scales longer than daily. We examined the capacity of subarctic and Arctic kelps to up-regulate pH in situ and experimentally tested the role of photoperiod in determining the capacity of Arctic macrophytes to up-regulate pH. Field observations at photoperiods of 15 and 24 hours in Greenland combined with experimental manipulations of photoperiod show that photoperiods longer than 21 hours, characteristic of Arctic summers, are conducive to sustained up-regulation of pH by kelp photosynthesis. We report a gradual increase in pH of 0.15 units and a parallel decline in pCO 2 of 100 parts per million over a 10-day period in an Arctic kelp forest over midsummer, with ample scope for continued pH increase during the months of continuous daylight. Experimental increase in CO 2 concentration further stimulated the capacity of macrophytes to deplete CO 2 and increase pH. We conclude that long photoperiods in Arctic summers support sustained up-regulation of pH in kelp forests, with potential benefits for calcifiers, and propose that this mechanism may increase with the projected expansion of Arctic vegetation in response to warming and loss of sea ice.

A Genetically Encoded Ratiometric pH Probe: Wavelength Regulation-Inspired Design of pH Indicators.

PubMed

Berbasova, Tetyana; Tahmasebi Nick, Setare; Nosrati, Meisam; Nossoni, Zahra; Santos, Elizabeth M; Vasileiou, Chrysoula; Geiger, James H; Borhan, Babak

2018-04-12

Mutants of human cellular retinol-binding protein II (hCRBPII) were engineered to bind a julolidine retinal analogue for the purpose of developing a ratiometric pH sensor. The design relied on the electrostatic influence of a titratable amino acid side chain, which affects the absorption and, thus, the emission of the protein/fluorophore complex. The ratio of emissions obtained at two excitation wavelengths that correspond to the absorption of the two forms of the protein/fluorophore complex, leads to a concentration-independent measure of pH. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid functional screening of Streptomyces coelicolor regulators by use of a pH indicator and application to the MarR-like regulator AbsC.

PubMed

Yang, Yung-Hun; Song, Eunjung; Lee, Bo-Rahm; Kim, Eun-jung; Park, Sung-Hee; Kim, Yun-Gon; Lee, Chang-Soo; Kim, Byung-Gee

2010-06-01

To elucidate the function of an unknown regulator in Streptomyces, differences in phenotype and antibiotic production between a deletion mutant and a wild-type strain (WT) were compared. These differences are easily hidden by complex media. To determine the specific nutrient conditions that reveal such differences, we used a multiwell method containing different nutrients along with bromothymol blue. We found several nutrients that provide key information on characterization conditions. By comparing the growth of wild-type and mutant strains on screened nutrients, we were able to measure growth, organic acid production, and antibiotic production for the elucidation of regulator function. As a result of this method, a member of the MarR-like regulator family, SCO5405 (AbsC), was newly characterized to control pyruvate dehydrogenase in Streptomyces coelicolor. Deletion of SCO5405 increased the pH of the culture broth due to decreased production of organic acids such as pyruvate and alpha-ketoglutarate and increased extracellular actinorhodin (ACT) production in minimal medium containing glucose and alanine (MMGA). This method could therefore be a high-throughput method for the characterization of unknown regulators.

A fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic.

PubMed

Chen, Kuncai; He, Rong; Luo, Xiaoyan; Qin, Pengzhe; Tan, Lei; Tang, Youwen; Yang, Zhicong

2017-08-15

This paper demonstrates a new strategy for developing a fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic. The technique provides amino modified Mn-doped ZnS QDs as fluorescent supports, 4-vinylphenylbronic acid as a covalent monomer, N-isopropyl acrylamide as a thermo-responsive monomer in combination with acrylamide as a non-covalent monomer, and glycosyl moiety of a glycopeptide antibiotic as a template to produce fluorescent molecularly imprinted polymer (FMIP) in aqueous solution. The FMIP can alter its functional moieties and structure with pH and temperature stimulation. This allows recognition of target molecules through control of pH and temperature. The fluorescence intensity of the FMIP was enhanced gradually as the concentration of telavancin increased, and showed selective recognition toward the target glycopeptide antibiotic preferentially among other antibiotics. Using the FMIP as a sensing material, good linear correlations were obtained over the concentration range of 3.0-300.0μg/L and with a low limit of detection of 1.0μg/L. The analysis results of telavancin in real samples were consistent with that obtained by liquid chromatography tandem mass spectrometry. Copyright © 2017 Elsevier B.V. All rights reserved.

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. Copyright 2001 Academic Press.

Roles of interstitial fluid pH in diabetes mellitus: Glycolysis and mitochondrial function

PubMed Central

Marunaka, Yoshinori

2015-01-01

The pH of body fluids is one the most important key factors regulating various cell function such as enzyme activity and protein-protein interaction via modification of its binding affinity. Therefore, to keep cell function normal, the pH of body fluids is maintained constant by various systems. Insulin resistance is one of the most important, serious factors making the body condition worse in diabetes mellitus. I have recently found that the pH of body (interstitial) fluids is lower in diabetes mellitus than that in non-diabetic control, and that the lowered pH is one of the causes producing insulin resistance. In this review article, I introduce importance of body (interstitial) fluid pH in regulation of body function, evidence on abnormal regulation of body fluid pH in diabetes mellitus, and relationship between the body fluid pH and insulin resistance. Further, this review proposes perspective therapies on the basis of regulation of body fluid pH including propolis (honeybee product) diet. PMID:25685283

Microbial Diversity in the Midguts of Field and Lab-Reared Populations of the European Corn Borer Ostrinia nubilalis

PubMed Central

Belda, Eugeni; Pedrola, Laia; Peretó, Juli; Martínez-Blanch, Juan F.; Montagud, Arnau; Navarro, Emilio; Urchueguía, Javier; Ramón, Daniel; Moya, Andrés; Porcar, Manuel

2011-01-01

Background Insects are associated with microorganisms that contribute to the digestion and processing of nutrients. The European Corn Borer (ECB) is a moth present world-wide, causing severe economical damage as a pest on corn and other crops. In the present work, we give a detailed view of the complexity of the microorganisms forming the ECB midgut microbiota with the objective of comparing the biodiversity of the midgut-associated microbiota and explore their potential as a source of genes and enzymes with biotechnological applications. Methodological/Principal Findings A high-throughput sequencing approach has been used to identify bacterial species, genes and metabolic pathways, particularly those involved in plant-matter degradation, in two different ECB populations (field-collected vs. lab-reared population with artificial diet). Analysis of the resulting sequences revealed the massive presence of Staphylococcus warneri and Weissella paramesenteroides in the lab-reared sample. This enabled us to reconstruct both genomes almost completely. Despite the apparently low diversity, 208 different genera were detected in the sample, although most of them at very low frequency. By contrast, the natural population exhibited an even higher taxonomic diversity along with a wider array of cellulolytic enzyme families. However, in spite of the differences in relative abundance of major taxonomic groups, not only did both metagenomes share a similar functional profile but also a similar distribution of non-redundant genes in different functional categories. Conclusions/Significance Our results reveal a highly diverse pool of bacterial species in both O. nubilalis populations, with major differences: The lab-reared sample is rich in gram-positive species (two of which have almost fully sequenced genomes) while the field sample harbors mainly gram-negative species and has a larger set of cellulolytic enzymes. We have found a clear relationship between the diet and the midgut

Axillary pH and influence of deodorants.

PubMed

Stenzaly-Achtert, S.; Schölermann, A.; Schreiber, J.; Diec, K. H.; Rippke, F.; Bielfeldt, S.

2000-05-01

BACKGROUND/AIMS: In moist intertriginous regions, such as the armpit, the pH value is physiologically higher than in other skin regions. The regulation of the axillary pH-value was examined in an open study with 48 subjects in three groups with n=16 each. METHODS: In the first 10 days (run-in) the subjects received a standard treatment in the axilla with shaving, cleansing and application of a pH-neutral deodorant. This was followed by a 5 day treatment period with the three test products (pH5 Eucerin(R) Deodorant Roll-on, Deodorant Balsam Spray, Deodorant Cream). The study was concluded by a wash-out period with procedures identical to the run-in phase. The pH was measured with a calibrated pH-meter. RESULTS: A significant pH reduction was shown during the treatment period when compared to the run-in phase. The Deodorant Roll-on induced a reduction of the mean pH values from 6.1 to 5.3, the Deodorant Balsam Spray from 6.5 to 5.7 and the Deodorant Cream from 6.2 to 5.3. During the wash-out period all pH values returned to baseline. CONCLUSION: All of the deodorants tested demonstrated a significant reduction in axillary pH. There is evidence that a high skin pH promotes the growth of several microorganisms that produce malodor. Therefore, the regulation of pH may contribute to the deodorant efficacy of the test products.

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

USDA-ARS?s Scientific Manuscript database

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...

Tissue-specific Proteogenomic Analysis of Plutella xylostella Larval Midgut Using a Multialgorithm Pipeline.

PubMed

Zhu, Xun; Xie, Shangbo; Armengaud, Jean; Xie, Wen; Guo, Zhaojiang; Kang, Shi; Wu, Qingjun; Wang, Shaoli; Xia, Jixing; He, Rongjun; Zhang, Youjun

2016-06-01

The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept that has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multialgorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a data set comprising 2694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. To get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a nonmodel animal. As the first tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest. © 2016 by The American Society for Biochemistry and

Tissue-specific Proteogenomic Analysis of Plutella xylostella Larval Midgut Using a Multialgorithm Pipeline*

PubMed Central

Zhu, Xun; Xie, Shangbo; Armengaud, Jean; Xie, Wen; Guo, Zhaojiang; Kang, Shi; Wu, Qingjun; Wang, Shaoli; Xia, Jixing; He, Rongjun; Zhang, Youjun

2016-01-01

The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept that has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multialgorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a data set comprising 2694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. To get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a nonmodel animal. As the first tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest. PMID:26902207

Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia.

PubMed

Hulikova, Alzbeta; Harris, Adrian L; Vaughan-Jones, Richard D; Swietach, Pawel

2013-04-01

Acid-extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid-extrusion remains unclear. We studied pH-regulation under normoxia and hypoxia in eight cancer cell-lines (HCT116, RT112, MDA-MB-468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH-sensitive fluorophore, cSNARF-1. Hypoxia responses were triggered by pre-incubation in low O(2) or with the 2-oxoglutarate-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport-substrate removal, acid-extrusion flux was dissected into components due to Na(+)/H(+) exchange (NHE) and Na(+)-dependent HCO(3)(-) transport. In half of the cell-lines (HCT116, RT112, MDA-MB-468, MCF10A), acid-extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA-MB-468), NHE-flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4-h hypoxic incubation reduced NHE-flux reversibly with a time-constant of 1-2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48-h hypoxia, inhibition of NHE-flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid-extrusion by Na(+)-dependent HCO(3)(-) transport was hypoxia-insensitive and comparable in all cell lines. This constitutive and stable element of pH-regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell-specific differences in their dynamic response to larger acid loads. Copyright © 2012 Wiley Periodicals, Inc.

Syntheses, structures and properties of four Cd(II) coordination polymers induced by the pH regulator

NASA Astrophysics Data System (ADS)

Xu, Yun; Ding, Fang; Liu, Dong; Yang, Pei-Pei; Zhu, Li-Li

2018-03-01

Four new coordination polymers [Cd2(CHDC)2(APYZ)(H2O)2](H2O) (1), [Cd(HCHDC)2(APYZ) (H2O)] (2), [Cd2(CHDC)2(PYZ)(H2O)2](H2O) (3), and [Cd(HCHDC)2(PYZ)(H2O)] (4) (H2CHDC = 1,4-cyclohexanedicarboxylic acid, APYZ = 2-aminopyrazine, PYZ = pyrazine) have been synthesized under the hydrothermal conditions by changing the pH regulator and the N-containing ligands. The pH regulator impacted on the degree of deprotonation of the 1,4-cyclohexanedicarboxylic acid ligand and resulted in the formation of the two pairs of different networks. Polymers 1 and 3 crystallize in monoclinic, space group P21/c, exhibit two dimensional 63 net, which further formed three-dimensional supramolecular structure by the Csbnd H⋯O hydrogen bond interactions. While polymers 2 and 4 possess one dimensional chain structures and further link into two dimensional layered supramolecular structures by intermolecular hydrogen bonding interactions. From all three conformers of H2CHDC, e,a-cis is consistently present in the Cd coordination polymers. Furthermore, photoluminescence properties of four polymers are also investigated, the luminescent intensity of polymer 1 (or 2) with amino group in pyrazine is dramatically stronger than that of the similar structure of polymer 3 (or 4) without amino group in pyrazine, the results shown that the presence of the amino group from 2-aminopyrazine play a key role in increasing the luminescence properties.

A Non-Invasive Deep Tissue PH Monitor.

DTIC Science & Technology

1995-08-11

disturbances in acid-base regulation may have serious effects on metabolic activity, circulation, and the central nervous system. Currently, acid-base...to tissue ischemia than is arterial pH. Consequently, a non-invasive deep tissue pH monitor has enormous value as a mechanism for rapid and effective ...achieved, and improve our understanding of what physical effects are important to successful non-invasive deep tissue pH monitoring. This last statement

Experimental Evolution of Escherichia coli K-12 at High pH and RpoS Induction.

PubMed

Hamdallah, Issam; Torok, Nadia; Bischof, Katarina M; Majdalani, Nadim; Chadalavada, Sriya; Mdluli, Nonto; Creamer, Kaitlin E; Clark, Michelle; Holdener, Chase; Basting, Preston J; Gottesman, Susan; Slonczewski, Joan L

2018-05-25

Experimental evolution of Escherichia coli K-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS1 knockout of phoB (positive regulator of the phosphate regulon). A phoB :: kanR knockout increased growth at high pH. phoB mutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations in pcnB (poly(A) polymerase) also increased growth at high pH. Three out of four populations showed deletions of torI, an inhibitor of TorR, which activates expression of torCAD (trimethylamine N-oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma RpoS, either in the coding gene or in regulators of RpoS expression. RpoS is required for survival in extreme base. In our microplate assay, rpoS deletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadapters that block degradation (IraM, IraD, and IraP). Other genes with mutations after high pH evolution encode regulators such as yobG ( mgrB ) (PhoPQ regulator), rpoN (nitrogen starvation sigma), malI , and purR ; as well as envelope proteins ompT and yahO Overall, E. coli evolution at high pH selects for mutations in key transcriptional regulators, including phoB and the stationary-phase sigma RpoS. IMPORTANCE Escherichia coli in its native habitat encounters high pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms. Copyright © 2018 American Society for Microbiology.

Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes

PubMed Central

Han, Yeon Soo; Thompson, Joanne; Kafatos, Fotis C.; Barillas-Mury, Carolina

2000-01-01

We present a detailed analysis of the interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei ookinetes during invasion of the mosquito by the parasite. In this mosquito, P.berghei ookinetes invade polarized columnar epithelial cells with microvilli, which do not express high levels of vesicular ATPase. The invaded cells are damaged, protrude towards the midgut lumen and suffer other characteristic changes, including induction of nitric oxide synthase (NOS) expression, a substantial loss of microvilli and genomic DNA fragmentation. Our results indicate that the parasite inflicts extensive damage leading to subsequent death of the invaded cell. Ookinetes were found to be remarkably plastic, to secrete a subtilisin-like serine protease and the GPI-anchored surface protein Pbs21 into the cytoplasm of invaded cells, and to be capable of extensive lateral movement between cells. The epithelial damage inflicted is repaired efficiently by an actin purse-string-mediated restitution mechanism, which allows the epithelium to ‘bud off’ the damaged cells without losing its integrity. A new model, the time bomb theory of ookinete invasion, is proposed and its implications are discussed. PMID:11080150

Effects of soil pH and aluminum on plant respiration

Treesearch

Rakesh Minocha; Subhash C. Minocha

2005-01-01

Interactions among external (soil) pH, cellular pH, and their effects on respiratory metabolism are complex. While the effects of changes in the apoplastic pH on the cytosolic pH are not clearly understood, pH directly affects enzymatic reactions in the cell, and pH-regulated ion uptake has profound indirect effects on cellular respiratory metabolism. A major...

[The diagnostic performance of color Doppler ultrasonography for newborn four cases of midgut volvulus accompanied by intestinal malrotation].

PubMed

Ikeshima, Yukari; Hisano, Katsuya; Morisawa, Takeshi; Inoue, Kozue; Shimamoto, Masahiro; Koujitani, Toshiaki; Yonetani, Masahiko; Yasufuku, Masao

2014-03-01

Midgut volvulus accompanied by intestinal malrotation is classified as a surgical emergency disease of the newborn, which emerges with the bilious vomiting or melena. This report presents four patients of this disease in our hospital, evaluated by color Doppler ultrasonography before surgical operation. All four patients were presented by bilious vomiting at the onset. By color Doppler ultrasonography method, the whirlpool sign which is the view of intestine and superior mesenteric vein rotated around with the axis of superior mesenteric artery, were shown in all cases. This whirlpool sign led to the diagnosis of midgut volvulus accompanied by intestinal malrotation. Intestinal contrast imaging was tested in three patients for the purpose of confirming the diagnosis. Repair of the volvulus and a ladd operation was performed in all four patients, without the excision of intestine because of no intestinal ischemic change. The clinical courses of four cases were good, and all patients were discharged within 17 days. Early diagnosis and timely surgical operation are essential for decreasing the possibility of occurring intestinal ischemic changes and improving clinical outcome after surgical operation. We propose that color Doppler ultrasonography is the powerful tool for the diagnosis of this disease, especially for the newborn, for whom the available diagnostic tests are limited.

Exploring the Midgut Transcriptome and Brush Border Membrane Vesicle Proteome of the Rice Stem Borer, Chilo suppressalis (Walker)

PubMed Central

Peng, Chuanhua; Wang, Xiaoping; Li, Fei; Lin, Yongjun

2012-01-01

The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV). Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac. PMID:22666467

Continuous pH monitoring in a perfused bioreactor system using an optical pH sensor

NASA Technical Reports Server (NTRS)

Jeevarajan, Antony S.; Vani, Sundeep; Taylor, Thomas D.; Anderson, Melody M.

2002-01-01

Monitoring and regulating the pH of the solution in a bioprocess is one of the key steps in the success of bioreactor operation. An in-line optical pH sensor, based on the optical absorption properties of phenol red present in the medium, was developed and tested in this work for use in NASA space bioreactors based on a rotating wall-perfused vessel system supporting a baby hamster kidney (BHK-21) cell culture. The sensor was tested over three 30-day and one 124-day cell runs. The pH sensor initially was calibrated and then used during the entire cell culture interval. The pH reported by the sensor was compared to that measured by a fiber optically coupled Shimadzu spectrophotometer and a blood gas analyzer. The maximum standard error of prediction for all the four cell runs for development pH sensor against BGA was +/-0.06 pH unit and for the fiber optically coupled Shimadzu spectrophotometer against the blood gas analyzer was +/-0.05 pH unit. The pH sensor system performed well without need of recalibration for 124 days. Copyright 2002 Wiley Periodicals, Inc.

Putting the pH into phosphatidic acid signaling

PubMed Central

2011-01-01

The lipid phosphatidic acid (PA) has important roles in cell signaling and metabolic regulation in all organisms. New evidence indicates that PA also has an unprecedented role as a pH biosensor, coupling changes in pH to intracellular signaling pathways. pH sensing is a property of the phosphomonoester headgroup of PA. A number of other potent signaling lipids also contain headgroups with phosphomonoesters, implying that pH sensing by lipids may be widespread in biology. PMID:22136116

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

The PH domain of phosphoinositide-dependent kinase-1 exhibits a novel, phospho-regulated monomer-dimer equilibrium with important implications for kinase domain activation: single-molecule and ensemble studies.

PubMed

Ziemba, Brian P; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J

2013-07-16

Phosphoinositide-dependent kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology, this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric states of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. This study investigates the binding of purified wild-type (WT) and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single-molecule and ensemble measurements. Single-molecule analysis of the brightness of the fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single-molecule analysis of two-dimensional (2D) diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate as a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little penetration of the protein into the bilayer as observed for other PH domains. The 2D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that allows

Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection.

PubMed

Liu, Yan-Xia; Li, Fen-Xiang; Liu, Zhuan-Zhuan; Jia, Zhi-Rong; Zhou, Yan-He; Zhang, Hao; Yan, Hui; Zhou, Xian-Qiang; Chen, Xiao-Guang

2016-06-01

Mosquito microRNAs (miRNAs) are involved in host-virus interaction, and have been reported to be altered by dengue virus (DENV) infection in Aedes albopictus (Diptera: Culicidae). However, little is known about the molecular mechanisms of Aedes albopictus midgut-the first organ to interact with DENV-involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA (mRNA) expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut. © 2016 Institute of Zoology, Chinese Academy of Sciences.

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

PubMed Central

Pinelli, Laurent; Nissant, Antoine; Edwards, Aurélie; Paulais, Marc

2016-01-01

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 Ca2+ 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 Ca2+ 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

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. © 2016 Pinelli et al.

Analysis of Homologs of Cry-toxin Receptor-Related Proteins in the Midgut of a Non-Bt Target, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

PubMed Central

Shao, Ensi; Lin, Li; Liu, Sijun; Zhang, Jiao; Chen, Xuelin; Sha, Li; Huang, Zhipeng; Huang, Biwang; Guan, Xiong

2018-01-01

Abstract The brown planthopper (BPH) Nilaparvata lugens is one of the most destructive insect pests in the rice fields of Asia. Like other hemipteran insects, BPH is not susceptible to Cry toxins of Bacillus thuringiensis (Bt) or transgenic rice carrying Bt cry genes. Lack of Cry receptors in the midgut is one of the main reasons that BPH is not susceptible to the Cry toxins. The main Cry-binding proteins (CBPs) of the susceptible insects are cadherin, aminopeptidase N (APN), and alkaline phosphatase (ALP). In this study, we analyzed and validated de novo assembled transcripts from transcriptome sequencing data of BPH to identify and characterize homologs of cadherin, APN, and ALP. We then compared the cadherin-, APN-, and ALP-like proteins of BPH to previously reported CBPs to identify their homologs in BPH. The sequence analysis revealed that at least one cadherin, one APN, and two ALPs of BPH contained homologous functional domains identified from the Cry-binding cadherin, APN, and ALP, respectively. Quantitative real-time polymerase chain reaction used to verify the expression level of each putative Cry receptor homolog in the BPH midgut indicated that the CBPs homologous APN and ALP were expressed at high or medium-high levels while the cadherin was expressed at a low level. These results suggest that homologs of CBPs exist in the midgut of BPH. However, differences in key motifs of CBPs, which are functional in interacting with Cry toxins, may be responsible for insusceptibility of BPH to Cry toxins. PMID:29415259

Does transgenic Cry1Ac + CpTI cotton pollen affect hypopharyngeal gland development and midgut proteolytic enzyme activity in the honey bee Apis mellifera L. (Hymenoptera, Apidae)?

PubMed

Han, Peng; Niu, Chang-Ying; Biondi, Antonio; Desneux, Nicolas

2012-11-01

The transgenic Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) cotton cultivar CCRI41 is increasingly used in China and potential side effects on the honey bee Apis mellifera L. have been documented recently. Two studies have assessed potential lethal and sublethal effects in young bees fed with CCRI41 cotton pollen but no effect was observed on learning capacities, although lower feeding activity in exposed honey bees was noted (antifeedant effect). The present study aimed at providing further insights into potential side effects of CCRI41 cotton on honey bees. Emerging honey bees were exposed to different pollen diets using no-choice feeding protocols (chronic exposure) in controlled laboratory conditions and we aimed at documenting potential mechanisms underneath the CCRI41 antifeedant effect previously reported. Activity of midgut proteolytic enzyme of young adult honey bees fed on CCRI41 cotton pollen were not significantly affected, i.e. previously observed antifeedant effect was not linked to disturbed activity of the proteolytic enzymes in bees' midgut. Hypopharyngeal gland development was assessed by quantifying total extractable proteins from the glands. Results suggested that CCRI41 cotton pollen carries no risk to hypopharyngeal gland development of young adult honey bees. In the two bioassays, honey bees exposed to 1 % soybean trypsin inhibitor were used as positive controls for both midgut proteolytic enzymes and hypopharyngeal gland proteins quantification, and bees exposed to 48 ppb (part per billion) (i.e. 48 ng g(-1)) imidacloprid were used as controls for exposure to a sublethal concentration of toxic product. The results show that the previously reported antifeedant effect of CCRI41 cotton pollen on honey bees is not linked to effects on their midgut proteolytic enzymes or on the development of their hypopharyngeal glands. The results of the study are discussed in the framework of risk assessment of transgenic crops on honey bees.

Molecular characterization of sodium/proton exchanger 3 (NHE3) from the yellow fever vector, Aedes aegypti.

PubMed

Pullikuth, Ashok K; Aimanova, Karlygash; Kang'ethe, Wanyoike; Sanders, Heather R; Gill, Sarjeet S

2006-09-01

Transport across insect epithelia is thought to depend on the activity of a vacuolar-type proton ATPase (V-ATPase) that energizes ion transport through a secondary proton/cation exchanger. Although several of the subunits of the V-ATPase have been cloned, the molecular identity of the exchanger has not been elucidated. Here, we present the identification of sodium/proton exchanger isoform 3 (NHE3) from yellow fever mosquito, Aedes aegypti (AeNHE3). AeNHE3 localizes to the basal plasma membrane of Malpighian tubule, midgut and the ion-transporting sector of gastric caeca. Midgut expression of NHE3 shows a different pattern of enrichment between larval and adult stages, implicating it in the maintenance of regional pH in the midgut during the life cycle. In all tissues examined, NHE3 predominantly localizes to the basal membrane. In addition the limited expression in intracellular vesicles in the median Malpighian tubules may reflect a potential functional versatility of NHE3 in a tissue-specific manner. The localization of V-ATPase and NHE3, and exclusion of Na+/K+-ATPase from the distal ion-transporting sector of caeca, indicate that the role of NHE3 in ion and pH regulation is intricately associated with functions of V-ATPase. The AeNHE3 complements yeast mutants deficient in yeast NHEs, NHA1 and NHX1. To further examine the functional property of AeNHE3, we expressed it in NHE-deficient fibroblast cells. AeNHE3 expressing cells were capable of recovering intracellular pH following an acid load. The recovery was independent of the large cytoplasmic region of AeNHE3, implying this domain to be dispensable for NHE3 ion transport function. 22Na+ uptake studies indicated that AeNHE3 is relatively insensitive to amiloride and EIPA and is capable of Na+ transport in the absence of the cytoplasmic tail. Thus, the core domain containing the transmembrane regions of NHE3 is sufficient for pH recovery and ion transport. The present data facilitate refinement of the

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.

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

Effects of neem oil (Azadirachta indica A. Juss) on midgut cells of predatory larvae Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae).

PubMed

Scudeler, Elton Luiz; dos Santos, Daniela Carvalho

2013-01-01

The effects of ingested neem oil, a botanical insecticide obtained from the seeds of the neem tree, Azadirachta indica, on the midgut cells of predatory larvae Ceraeochrysa claveri were analyzed. C. claveri were fed on eggs of Diatraea saccharalis treated with neem oil at a concentration of 0.5%, 1% and 2% during throughout the larval period. Light and electron microscopy showed severe damages in columnar cells, which had many cytoplasmic protrusions, clustering and ruptured of the microvilli, swollen cells, ruptured cells, dilatation and vesiculation of rough endoplasmic reticulum, development of smooth endoplasmic reticulum, enlargement of extracellular spaces of the basal labyrinth, intercellular spaces and necrosis. The indirect ingestion of neem oil with prey can result in severe alterations showing direct cytotoxic effects of neem oil on midgut cells of C. claveri larvae. Therefore, the safety of neem oil to non-target species as larvae of C. claveri was refuted, thus the notion that plants derived are safer to non-target species must be questioned in future ecotoxicological studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Comparative analysis of Bacillus thuringiensis toxin binding to gypsy moth, browntail moth, and douglas-fir tussock moth midgut tissue sections using fluorescence microscopy

Treesearch

Algimantas P. Valaitis; John D. Podgwaite

2011-01-01

Many strains of Bacillus thuringiensis (Bt) produce insecticidal proteins, also referred to as Cry toxins, in crystal inclusions during sporulation. When ingested by insects, the Cry toxins bind to receptors on the brush border midgut epithelial cells and create pores in the epithelial gut membranes resulting in the death of...

Contribution of midgut bacteria to blood digestion and egg production in aedes aegypti (diptera: culicidae) (L.)

PubMed Central

2011-01-01

Background The insect gut harbors a variety of microorganisms that probably exceed the number of cells in insects themselves. These microorganisms can live and multiply in the insect, contributing to digestion, nutrition, and development of their host. Recent studies have shown that midgut bacteria appear to strengthen the mosquito's immune system and indirectly enhance protection from invading pathogens. Nevertheless, the physiological significance of these bacteria for mosquitoes has not been established to date. In this study, oral administration of antibiotics was employed in order to examine the contribution of gut bacteria to blood digestion and fecundity in Aedes aegypti. Results The antibiotics carbenicillin, tetracycline, spectinomycin, gentamycin and kanamycin, were individually offered to female mosquitoes. Treatment of female mosquitoes with antibiotics affected the lysis of red blood cells (RBCs), retarded the digestion of blood proteins and reduced egg production. In addition, antibiotics did not affect the survival of mosquitoes. Mosquito fertility was restored in the second gonotrophic cycle after suspension of the antibiotic treatment, showing that the negative effects of antibiotics in blood digestion and egg production in the first gonotrophic cycle were reversible. Conclusions The reduction of bacteria affected RBC lysis, subsequently retarded protein digestion, deprived mosquito from essential nutrients and, finally, oocyte maturation was affected, resulting in the production of fewer viable eggs. These results indicate that Ae. aegypti and its midgut bacteria work in synergism to digest a blood meal. Our findings open new possibilities to investigate Ae. aegypti-associated bacteria as targets for mosquito control strategies. PMID:21672186

Full-thickness small intestine necrosis with midgut volvulus, distributed in a patchy fashion, is reversible with moderate blood flow: resumption of normal function to non-viable intestine.

PubMed

Amano, Hizuru; Uchida, Hiroo; Kawashima, Hiroshi; Tanaka, Yujiro; Kishimoto, Hiroshi

2014-08-01

Midgut volvulus is a highly life-threatening condition that carries a high risk of short gut syndrome. We report a case of catastrophic neonatal midgut volvulus in which second-look laparotomy revealed apparently non-viable remnant small intestine but with a moderate blood supply. Full-thickness small intestine necrosis was distributed in a patchy fashion, with non-viable and necrotic areas distributed so widely that no portion of the intestine could be resected. A section of full-thickness necrotic intestine preserved at surgery was able to regenerate, and normal function was restored over a period of 1 month. This case indicated that intestinal resumption may be dependent on blood flow. Even when intestinal viability is questionable, preservation enables the chance of regeneration if moderate blood flow is present.

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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

pH regulation of the kinetic stability of the lipase from Thermomyces lanuginosus.

PubMed

Wang, H; Andersen, K K; Sehgal, P; Hagedorn, J; Westh, P; Borch, K; Otzen, D E

2013-01-08

Thermomyces lanuginosus lipase (TlL) is a kinetically stable protein, resistant toward both denaturation and refolding in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multimillimolar surfactant concentrations but fails to refold from the acid urea-denatured state at submillimolar concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4-6; in this pH range, SDS binds as micellelike clusters. Although hysteresis can be eliminated by reducing disulfide bonds, destabilizing the native state, and lowering the unfolding activation barrier, SDS sensitivity is not directly linked to intrinsic kinetic stability [its resistance to the general chemical denaturant guanidinium chloride (GdmCl)], because TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from approximately -12 to approximately -5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS resistance, because this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.

Evaluation of the protective efficacy of Ornithodoros moubata midgut membrane antigens selected using omics and in silico prediction algorithms.

PubMed

Obolo-Mvoulouga, Prosper; Oleaga, Ana; Manzano-Román, Raúl; Pérez-Sánchez, Ricardo

2018-04-30

The African argasid tick Ornithodoros moubata transmits two important pathogens, the African swine fever virus and the spirochete Borrelia duttoni, the cause of human relapsing fever. To date, only conventional control measures such as widespread application of acaricides, strict control measures, and animal movement restrictions have been implemented to confine these diseases. Vaccines against tick infestations have the potential to be among the most efficacious interventions for the management of these diseases. Plasma membrane-associated proteins upregulated in tick midgut cells in response to blood feeding and digestion are thought to play vital functions in tick physiology and in the transmission of tick-borne pathogens. In addition, their antigenic extracellular regions are easily accessible to antibodies synthesised by immunised hosts, which makes them interesting targets for tick vaccine design. The mialomes (midgut transcriptomes and proteomes) of unfed O. moubata females and of engorged females at 48 h post-feeding have recently been obtained, providing a wealth of predicted midgut protein sequences. In the current study, these mialomes were screened using in silico tools to select predicted antigenic transmembrane proteins that were upregulated after feeding (516 proteins). The functionally annotatable proteins from this list (396 proteins) were then manually inspected following additional criteria in order to select a finite and easy-manageable number of candidate antigens for tick vaccine design. The extracellular antigenic regions of five of these candidates were obtained either as truncated recombinant proteins or as KLH-conjugated synthetic peptides, formulated in Freund's adjuvant, and individually administered to rabbits to assess their immunogenicity and protective potential against infestations by O. moubata and the Iberian species Ornithodoros erraticus. All candidates were highly immunogenic, but provided low protection against the O

Hindgut Innate Immunity and Regulation of Fecal Microbiota through Melanization in Insects*

PubMed Central

Shao, Qimiao; Yang, Bing; Xu, Qiuyun; Li, Xuquan; Lu, Zhiqiang; Wang, Chengshu; Huang, Yongping; Söderhäll, Kenneth; Ling, Erjun

2012-01-01

Many insects eat the green leaves of plants but excrete black feces in an as yet unknown mechanism. Insects cannot avoid ingesting pathogens with food that will be specifically detected by the midgut immune system. However, just as in mammals, many pathogens can still escape the insect midgut immune system and arrive in the hindgut, where they are excreted out with the feces. Here we show that the melanization of hindgut content induced by prophenoloxidase, a key enzyme that induces the production of melanin around invaders and at wound sites, is the last line of immune defense to clear bacteria before feces excretion. We used the silkworm Bombyx mori as a model and found that prophenoloxidase produced by hindgut cells is secreted into the hindgut contents. Several experiments were done to clearly demonstrate that the blackening of the insect feces was due to activated phenoloxidase, which served to regulate the number of bacteria in the hindgut. Our analysis of the silkworm hindgut prophenoloxidase discloses the natural secret of why the phytophagous insect feces is black and provides insight into hindgut innate immunity, which is still rather unclear in mammals. PMID:22375003

Carbohydrases in the digestive system of the spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae).

PubMed

Ghamari, Mahboob; Hosseininaveh, Vahid; Darvishzadeh, Ali; Chougule, Nanasaheb P

2014-04-01

The spined soldier bug, Podisus maculiventris, is a generalist predator of insects and has been used in biological control. However, information on the digestion of food in this insect is lacking. Therefore, we have studied the digestive system in P. maculiventris, and further characterized carbohydrases in the digestive tract. The midgut of all developmental stages was composed of anterior, median, and posterior regions. The volumes of the anterior midgut decreased and the median midgut increased in older instars and adults, suggesting a more important role of the median midgut in food digestion. However, carbohydrase activities were predominant in the anterior midgut. In comparing the specific activity of carbohydrases, α-amylase activity was more in the salivary glands (with two distinct activity bands in zymograms), and glucosidase and galactosidase activities were more in the midgut. Salivary α-amylases were detected in the prey hemolymph, demonstrating the role of these enzymes in extra-oral digestion. However, the catalytic efficiency of midgut α-amylase activity was approximately twofold more than that of the salivary gland enzymes, and was more efficient in digesting soluble starch than glycogen. Midgut α-amylases were developmentally regulated, as one isoform was found in first instar compared to three isoforms in fifth instar nymphs. Starvation significantly affected carbohydrase activities in the midgut, and acarbose inhibited α-amylases from both the salivary glands and midgut in vitro and in vivo. The structural diversity and developmental regulation of carbohydrases in the digestive system of P. maculiventris demonstrate the importance of these enzymes in extra-oral and intra-tract digestion, and may explain the capability of the hemipteran to utilize diverse food sources. © 2014 Wiley Periodicals, Inc.

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. © 2012 The Authors Journal compilation © 2012 FEBS.

Proton mediated control of biochemical reactions with bioelectronic pH modulation

NASA Astrophysics Data System (ADS)

Deng, Yingxin; Miyake, Takeo; Keene, Scott; Josberger, Erik E.; Rolandi, Marco

2016-04-01

In Nature, protons (H+) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H+ channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H+ currents and H+ concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H+ between PdHx contacts and solution. The present transducer records bistable pH modulation from an “enzymatic flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. The transducer also controls bioluminescence from firefly luciferase by affecting solution pH.

Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells.

PubMed

Brown, David A; Melvin, James E; Yule, David I

2003-11-01

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.

The chemistry, physiology and pathology of pH in cancer.

PubMed

Swietach, Pawel; Vaughan-Jones, Richard D; Harris, Adrian L; Hulikova, Alzbeta

2014-03-19

Cell survival is conditional on the maintenance of a favourable acid-base balance (pH). Owing to intensive respiratory CO2 and lactic acid production, cancer cells are exposed continuously to large acid-base fluxes, which would disturb pH if uncorrected. The large cellular reservoir of H(+)-binding sites can buffer pH changes but, on its own, is inadequate to regulate intracellular pH. To stabilize intracellular pH at a favourable level, cells control trans-membrane traffic of H(+)-ions (or their chemical equivalents, e.g. ) using specialized transporter proteins sensitive to pH. In poorly perfused tumours, additional diffusion-reaction mechanisms, involving carbonic anhydrase (CA) enzymes, fine-tune control extracellular pH. The ability of H(+)-ions to change the ionization state of proteins underlies the exquisite pH sensitivity of cellular behaviour, including key processes in cancer formation and metastasis (proliferation, cell cycle, transformation, migration). Elevated metabolism, weakened cell-to-capillary diffusive coupling, and adaptations involving H(+)/H(+)-equivalent transporters and extracellular-facing CAs give cancer cells the means to manipulate micro-environmental acidity, a cancer hallmark. Through genetic instability, the cellular apparatus for regulating and sensing pH is able to adapt to extracellular acidity, driving disease progression. The therapeutic potential of disturbing this sequence by targeting H(+)/H(+)-equivalent transporters, buffering or CAs is being investigated, using monoclonal antibodies and small-molecule inhibitors.

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

Strain-specific variation in a soilborne phytopathogenic fungus for the expression of genes involved in pH signal transduction pathway, pathogenesis and saprophytic survival in response to environmental pH changes.

PubMed

Daval, Stéphanie; Lebreton, Lionel; Gracianne, Cécile; Guillerm-Erckelboudt, Anne-Yvonne; Boutin, Morgane; Marchi, Muriel; Gazengel, Kévin; Sarniguet, Alain

2013-12-01

The soilborne fungus Gaeumannomyces graminis var. tritici (Ggt) causes take-all, a wheat root disease. In an original strain-specific way, a previous study indicates that inside the Ggt species, some strains grow preferentially at acidic pH and other strains at neutral/alkaline pH. The most important mechanism for a fungal response to the environmental pH is the Pal pathway which integrates the products of the six pal genes and the transcription factor PacC. To evaluate whether the Ggt strain-specific growth in function of the ambient pH is mediated via the Pal pathway, a transcriptional study of the genes encoding this pathway was carried out. This study provided the first evidence that the pH signalling pathway similar to those described in other fungi operated in Ggt. The pacC gene was induced at neutral pH whatever the strain. In an original way, the expression of Ggt genes coding for the different Pal proteins depended on the strain and on the ambient pH. In the strain growing better at acidic pH, few pal genes were pH-regulated, and some were overexpressed at neutral pH when regulated. In the strain growing better at neutral pH, underexpression of most of the pal genes at neutral pH occurred. The strains displayed higher gene expression in the ambient pH that unfavoured their growth as if it was a compensation system. All pH taken together, a globally weaker Pal transcript level occurred in the strains that were less sensitive to acidic pH, and on the contrary, the strain growing better on neutral pH showed higher Pal mRNA levels. The expression of genes involved in pathogenesis and saprophytic growth was also regulated by the ambient pH and the strain: each gene displayed a specific pH-regulation that was similar between strains. But all pH taken together, the global transcript levels of four out of six genes were higher in the strain growing better on neutral pH. Altogether, for the first time, the results show that inside a species, conditions affecting

Histopathological effects of cypermethrin and Bacillus thuringiensis var. israelensis on midgut of Chironomus calligraphus larvae (Diptera: Chironomidae).

PubMed

Lavarías, Sabrina; Arrighetti, Florencia; Siri, Augusto

2017-06-01

Pesticides are extensively used for the control of agricultural pests and disease vectors, but they also affect non-target organisms. Cypermethrin (CYP) is a synthetic pyrethroid used worldwide. Otherwise, bioinsecticides like Bacillus thuringiensis var. israelensis (Bti) have received great attention as an environmentally benign and desirable alternative. In order to evaluate the toxicity of those pesticides, Chironomus calligraphus was selected due to its high sensitivity to some toxicants. Third and fourth instars larvae were exposed to serial dilutions of CYP and Bti to determine LC 50 values. In order to evaluate the potentially histopathological alterations as biomarkers, after 96-h of exposure, live larvae were fixed for histological analysis of the mid region of digestive tract. The 96-h LC 50 values were 0.52 and 1.506μg/L for CYP and Bti, respectively. Midgut histological structure of the control group showed a single layer of cubical cells with microvilli in their apical surface and a big central nucleus. The midgut epithelium of larvae exposed to a low concentration of CYP (0.037μg/L) showed secretion activity and vacuolization while at high concentration (0.3μg/L) cells showed a greater disorganization and a more developed fat body. On the other hand, Bti caused progressive histological damage in this tissue. Chironomus calligraphus is sensitive to Bti and CYP toxicity like other Chironomus species. The histopathological alterations could be a valuable tool to assess toxicity mechanism of different pesticides. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteomics analysis of Fusarium proliferatum under various initial pH during fumonisin production.

PubMed

Li, Taotao; Gong, Liang; Wang, Yong; Chen, Feng; Gupta, Vijai Kumar; Jian, Qijie; Duan, Xuewu; Jiang, Yueming

2017-07-05

Fusarium proliferatum as a fungal pathogen can produce fumonisin which causes a great threat to animal and human health. Proteomic approach was a useful tool for investigation into mycotoxin biosynthesis in fungal pathogens. In this study, we analyzed the fumonisin content and mycelium proteins of Fusarium proliferatum cultivated under the initial pH5 and 10. Fumonisin production after 10days was significantly induced in culture condition at pH10 than pH5. Ninety nine significantly differently accumulated protein spots under the two pH conditions were detected using two dimensional polyacrylamide gel electrophoresis and 89 of these proteins were successfully identified by MALDI-TOF/TOF and LC-ESI-MS/MS analysis. Among these 89 proteins, 45 were up-regulated at pH10 while 44 were up-accumulated at pH5. At pH10, these proteins were found to involve in the modification of fumonisin backbone including up-regulated polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase, which might contribute to the induction of fumonisin production. At pH5, these up-regulated proteins such as l-amino-acid oxidase, isocitrate dehydrogenase and citrate lyase might inhibit the condensation of fumonisin backbone, resulting in reduced production of fumonisins. These results may help us to understand the molecular mechanism of the fumonisin synthesis in F. proliferatum. To extend our understanding of the mechanism of the fumonisin biosynthesis of F. proliferatum, we reported the fumonisin production in relation to the differential proteins of F. proliferatum mycelium under two pH culture conditions. Among these 89 identified spots, 45 were up-accumulated at pH10 while 44 were up-accumulated at pH5. Our results revealed that increased fumonisin production at pH10 might be related to the induction of fumonisin biosynthesis caused by up-regulation of polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase. Meanwhile, the

Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages

PubMed Central

Canton, Johnathan; Khezri, Rojyar; Glogauer, Michael; Grinstein, Sergio

2014-01-01

Macrophages respond to changes in environmental stimuli by assuming distinct functional phenotypes, a phenomenon referred to as macrophage polarization. We generated classically (M1) and alternatively (M2) polarized macrophages—two extremes of the polarization spectrum—to compare the properties of their phagosomes. Specifically, we analyzed the regulation of the luminal pH after particle engulfment. The phagosomes of M1 macrophages had a similar buffering power and proton (equivalent) leakage permeability but significantly reduced proton-pumping activity compared with M2 phagosomes. As a result, only the latter underwent a rapid and profound acidification. By contrast, M1 phagosomes displayed alkaline pH oscillations, which were caused by proton consumption upon dismutation of superoxide, followed by activation of a voltage- and Zn2+-sensitive permeation pathway, likely HV1 channels. The paucity of V-ATPases in M1 phagosomes was associated with, and likely caused by, delayed fusion with late endosomes and lysosomes. The delayed kinetics of maturation was, in turn, promoted by the failure of M1 phagosomes to acidify. Thus, in M1 cells, elimination of pathogens through deployment of the microbicidal NADPH oxidase is given priority at the expense of delayed acidification. By contrast, M2 phagosomes proceed to acidify immediately in order to clear apoptotic bodies rapidly and effectively. PMID:25165138

Ileocolic intussusception mimicking the imaging appearance of midgut volvulus as a result of extrinsic duodenal obstruction.

PubMed

Gasparini, Flavia F; Navarro, Oscar M; Dasgupta, Roshni; Gerstle, J Ted; Thorner, Paul S; Manson, David E

2005-12-01

Duodenal obstruction caused by ileocolic intussusception in the absence of intestinal malrotation is extremely rare. We present and discuss the imaging findings in an infant with an intussusception secondary to a duplication cyst in whom sonography also showed inversion of the orientation of the mesenteric vessels and a distended stomach. A contrast medium study revealed a proximal duodenal obstruction with a beak appearance suggestive of midgut volvulus. At surgery, an ileocolic intussusception causing duodenal obstruction without concomitant malrotation or volvulus was found. The combination of duodenal obstruction and abnormal relationship of the mesenteric vessels as a result of ileocolic intussusception has not previously been reported in the literature.

pH measurement and a rational and practical pH control strategy for high throughput cell culture system.

PubMed

Zhou, Haiying; Purdie, Jennifer; Wang, Tongtong; Ouyang, Anli

2010-01-01

The number of therapeutic proteins produced by cell culture in the pharmaceutical industry continues to increase. During the early stages of manufacturing process development, hundreds of clones and various cell culture conditions are evaluated to develop a robust process to identify and select cell lines with high productivity. It is highly desirable to establish a high throughput system to accelerate process development and reduce cost. Multiwell plates and shake flasks are widely used in the industry as the scale down model for large-scale bioreactors. However, one of the limitations of these two systems is the inability to measure and control pH in a high throughput manner. As pH is an important process parameter for cell culture, this could limit the applications of these scale down model vessels. An economical, rapid, and robust pH measurement method was developed at Eli Lilly and Company by employing SNARF-4F 5-(-and 6)-carboxylic acid. The method demonstrated the ability to measure the pH values of cell culture samples in a high throughput manner. Based upon the chemical equilibrium of CO(2), HCO(3)(-), and the buffer system, i.e., HEPES, we established a mathematical model to regulate pH in multiwell plates and shake flasks. The model calculates the required %CO(2) from the incubator and the amount of sodium bicarbonate to be added to adjust pH to a preset value. The model was validated by experimental data, and pH was accurately regulated by this method. The feasibility of studying the pH effect on cell culture in 96-well plates and shake flasks was also demonstrated in this study. This work shed light on mini-bioreactor scale down model construction and paved the way for cell culture process development to improve productivity or product quality using high throughput systems. Copyright 2009 American Institute of Chemical Engineers

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.

Ca2+-associated triphasic pH changes in mitochondria during brown adipocyte activation.

PubMed

Hou, Yanyan; Kitaguchi, Tetsuya; Kriszt, Rókus; Tseng, Yu-Hua; Raghunath, Michael; Suzuki, Madoka

2017-08-01

Brown adipocytes (BAs) are endowed with a high metabolic capacity for energy expenditure due to their high mitochondria content. While mitochondrial pH is dynamically regulated in response to stimulation and, in return, affects various metabolic processes, how mitochondrial pH is regulated during adrenergic stimulation-induced thermogenesis is unknown. We aimed to reveal the spatial and temporal dynamics of mitochondrial pH in stimulated BAs and the mechanisms behind the dynamic pH changes. A mitochondrial targeted pH-sensitive protein, mito-pHluorin, was constructed and transfected to BAs. Transfected BAs were stimulated by an adrenergic agonist, isoproterenol. The pH changes in mitochondria were characterized by dual-color imaging with indicators that monitor mitochondrial membrane potential and heat production. The mechanisms of pH changes were studied by examining the involvement of electron transport chain (ETC) activity and Ca 2+ profiles in mitochondria and the intracellular Ca 2+ store, the endoplasmic reticulum (ER). A triphasic mitochondrial pH change in BAs upon adrenergic stimulation was revealed. In comparison to a thermosensitive dye, we reveal that phases 1 and 2 of the pH increase precede thermogenesis, while phase 3, characterized by a pH decrease, occurs during thermogenesis. The mechanism of pH increase is partially related to ETC. In addition, the pH increase occurs concurrently with an increase in mitochondrial Ca 2+ . This Ca 2+ increase is contributed to by an influx from the ER, and it is further involved in mitochondrial pH regulation. We demonstrate that an increase in mitochondrial pH is implicated as an early event in adrenergically stimulated BAs. We further suggest that this pH increase may play a role in the potentiation of thermogenesis.

Pleiotropic function of DLX3 in amelogenesis: from regulating pH and keratin expression to controlling enamel rod decussation.

PubMed

Duverger, Olivier; Morasso, Maria I

2018-12-01

DLX3 is essential for tooth enamel development and is so far the only transcription factor known to be mutated in a syndromic form of amelogenesis imperfecta. Through conditional deletion of Dlx3 in the dental epithelium in mouse, we have previously established the involvement of DLX3 in enamel pH regulation, as well as in controlling the expression of sets of keratins that contribute to enamel rod sheath formation. Here, we show that the decussation pattern of enamel rods was lost in conditional knockout animals, suggesting that DLX3 controls the coordinated migration of ameloblasts during enamel secretion. We further demonstrate that DLX3 regulates the expression of some components of myosin II complexes potentially involved in driving the movement of ameloblasts that leads to enamel rod decussation.

Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis

PubMed Central

2009-01-01

Background The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis. Results Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 ± 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions. Conclusions Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense. PMID:20025733

Improving pH sensitivity by field-induced charge regulation in flexible biopolymer electrolyte gated oxide transistors

NASA Astrophysics Data System (ADS)

Liu, Ning; Gan, Lu; Liu, Yu; Gui, Weijun; Li, Wei; Zhang, Xiaohang

2017-10-01

Electrical manipulation of charged ions in electrolyte-gated transistors is crucial for enhancing the electric-double-layer (EDL) gating effect, thereby improving their sensing abilities. Here, indium-zinc-oxide (IZO) based thin-film-transistors (TFTs) are fabricated on flexible plastic substrate. Acid doped chitosan-based biopolymer electrolyte is used as the gate dielectric, exhibiting an extremely high EDL capacitance. By regulating the dynamic EDL charging process with special gate potential profiles, the EDL gating effect of the chitosan-gated TFT is enhanced, and then resulting in higher pH sensitivities. An extremely high sensitivity of ∼57.8 mV/pH close to Nernst limit is achieved when the gate bias of the TFT sensor sweeps at a rate of 10 mV/s. Additionally, an enhanced sensitivity of 2630% in terms of current variation with pH range from 11 to 3 is realized when the device is operated in the ion depletion mode with a negative gate bias of -0.7 V. Robust ionic modulation is demonstrated in such chitosan-gated sensors. Efficiently driving the charged ions in the chitosan-gated IZO-TFT provides a new route for ultrasensitive, low voltage, and low-cost biochemical sensing technologies.

The chemistry, physiology and pathology of pH in cancer

PubMed Central

Swietach, Pawel; Vaughan-Jones, Richard D.; Harris, Adrian L.; Hulikova, Alzbeta

2014-01-01

Cell survival is conditional on the maintenance of a favourable acid–base balance (pH). Owing to intensive respiratory CO2 and lactic acid production, cancer cells are exposed continuously to large acid–base fluxes, which would disturb pH if uncorrected. The large cellular reservoir of H+-binding sites can buffer pH changes but, on its own, is inadequate to regulate intracellular pH. To stabilize intracellular pH at a favourable level, cells control trans-membrane traffic of H+-ions (or their chemical equivalents, e.g. ) using specialized transporter proteins sensitive to pH. In poorly perfused tumours, additional diffusion-reaction mechanisms, involving carbonic anhydrase (CA) enzymes, fine-tune control extracellular pH. The ability of H+-ions to change the ionization state of proteins underlies the exquisite pH sensitivity of cellular behaviour, including key processes in cancer formation and metastasis (proliferation, cell cycle, transformation, migration). Elevated metabolism, weakened cell-to-capillary diffusive coupling, and adaptations involving H+/H+-equivalent transporters and extracellular-facing CAs give cancer cells the means to manipulate micro-environmental acidity, a cancer hallmark. Through genetic instability, the cellular apparatus for regulating and sensing pH is able to adapt to extracellular acidity, driving disease progression. The therapeutic potential of disturbing this sequence by targeting H+/H+-equivalent transporters, buffering or CAs is being investigated, using monoclonal antibodies and small-molecule inhibitors. PMID:24493747

Different host complement systems and their interactions with saliva from Lutzomyia longipalpis (Diptera, Psychodidae) and Leishmania infantum promastigotes.

PubMed

Mendes-Sousa, Antonio Ferreira; Nascimento, Alexandre Alves Sousa; Queiroz, Daniel Costa; Vale, Vladimir Fazito; Fujiwara, Ricardo Toshio; Araújo, Ricardo Nascimento; Pereira, Marcos Horácio; Gontijo, Nelder Figueiredo

2013-01-01

Lutzomyia longipalpis is the vector of Leishmania infantum in the New World, and its saliva inhibits classical and alternative human complement system pathways. This inhibition is important in protecting the insect´s midgut from damage by the complement. L. longipalpis is a promiscuous blood feeder and must be protected against its host's complement. The objective of this study was to investigate the action of salivary complement inhibitors on the sera of different host species, such as dogs, guinea pigs, rats and chickens, at a pH of 7.4 (normal blood pH) and 8.15 (the midgut pH immediately after a blood meal). We also investigated the role of the chicken complement system in Leishmania clearance in the presence and absence of vector saliva. The saliva was capable of inhibiting classical pathways in dogs, guinea pigs and rats at both pHs. The alternative pathway was not inhibited except in dogs at a pH of 8.15. The chicken classical pathway was inhibited only by high concentrations of saliva and it was better inhibited by the midgut contents of sand flies. Neither the saliva nor the midgut contents had any effect on the avian alternative pathway. Fowl sera killed L. infantum promastigotes, even at a low concentration (2%), and the addition of L. longipalpis saliva did not protect the parasites. The high body temperature of chickens (40°C) had no effect on Leishmania viability during our assays. Salivary inhibitors act in a species-specific manner. It is important to determine their effects in the natural hosts of Leishmania infantum because they act on canid and rodent complements but not on chickens (which do not harbour the parasite). Moreover, we concluded that the avian complement system is the probable mechanism through which chickens eliminate Leishmania and that their high body temperature does not influence this parasite.

Regulating the local pH level of titanium via Mg-Fe layered double hydroxides films for enhanced osteogenesis.

PubMed

Li, Qianwen; Wang, Donghui; Qiu, Jiajun; Peng, Feng; Liu, Xuanyong

2018-05-01

Hard tissue implant materials which can cause a suitable alkaline microenvironment are thought to be beneficial for stimulating osteoblast differentiation while suppressing osteoclast generation. To make the local pH around the interface between materials and cells controllable, we prepared a series of Mg-Fe layered double hydroxide (LDH) films on acid-etched pure titanium surfaces via hydrothermal treatment. By adjusting the Mg/Fe proportion ratio, the interlayer spacing of Mg-Fe LDHs was regulated, making their OH- exchange abilities adjustable, and this ultimately resulted in a microenvironment with a controllable pH value. In vitro experiments demonstrated that the Mg-Fe LDH film-modified titanium surface possessed good biocompatibility and osteogenic activity, especially the Mg-Fe LDH film with Mg/Fe proportion ratio of 4, which could form a suitable alkaline microenvironment for the growth and osteogenetic differentiation of stem cells. These results demonstrate the potential application of the prepared Mg-Fe LDH films in enhancing the osteogenesis of implant materials while providing a new way into the design of controllable alkaline environment.

Separate Gating Mechanisms Mediate the Regulation of K2P Potassium Channel TASK-2 by Intra- and Extracellular pH*

PubMed Central

Niemeyer, María Isabel; Cid, L. Pablo; Peña-Münzenmayer, Gaspar; Sepúlveda, Francisco V.

2010-01-01

TASK-2 (KCNK5 or K2P5.1) is a background K+ channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pHo) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pHi) is independent from that under the command of pHo. It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pHo had normal pHi-dependent gating. Increasing extracellular K+ concentration acid shifts pHo activity curve of TASK-2 yet did not affect pHi gating of TASK-2. pHo modulation of TASK-2 is voltage-dependent, whereas pHi gating was not altered by membrane potential. These results suggest that pHo, which controls a selectivity filter external gate, and pHi act at different gating processes to open and close TASK-2 channels. We speculate that pHi regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys245) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pHi. We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pHi-dependence curve of TASK-2 as expected from its lower pKa. We conclude that intracellular pH, together with pHo, is a critical determinant of TASK-2 activity and therefore of its physiological function. PMID:20351106

Molecular aspects of bacterial pH sensing and homeostasis

PubMed Central

Krulwich, Terry A.; Sachs, George; Padan, Etana

2011-01-01

Diverse mechanisms for pH-sensing and cytoplasmic pH homeostasis enable most bacteria to tolerate or grow at external pH values that are outside the cytoplasmic pH range they must maintain for growth. The most extreme cases are exemplified by the extremophiles that inhabit environments whose pH is below 3 or above 11. Here we describe how recent insights into the structure and function of key molecules and their regulators reveal novel strategies of bacterial pH-homeostasis. These insights may help us better target certain pathogens and better harness the capacities of environmental bacteria. PMID:21464825

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

PubMed

Chotiwan, Nunya; Andre, Barbara G; Sanchez-Vargas, Irma; Islam, M Nurul; Grabowski, Jeffrey M; Hopf-Jannasch, Amber; Gough, Erik; Nakayasu, Ernesto; Blair, Carol D; Belisle, John T; Hill, Catherine A; Kuhn, Richard J; Perera, Rushika

2018-02-01

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted

Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

PubMed

Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

2016-09-01

The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. Copyright © 2016 Elsevier B.V. All

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes

PubMed Central

Chotiwan, Nunya; Andre, Barbara G.; Sanchez-Vargas, Irma; Islam, M. Nurul; Grabowski, Jeffrey M.; Hopf-Jannasch, Amber; Gough, Erik; Nakayasu, Ernesto; Blair, Carol D.; Hill, Catherine A.; Kuhn, Richard J.

2018-01-01

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted

NBCe1 (SLC4A4) a potential pH Regulator in Enamel Organ Cells during Enamel Development in the Mouse

PubMed Central

Jalali, R; Guo, J; Zandieh-Doulabi, B; Bervoets, TJM; Paine, ML; Boron, W; Parker, M; Bijvelds, MJC; Medina, JF; DenBesten, PK; Bronckers, ALJJ

2016-01-01

During 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 co-transporting HCO3− with Na+. Mutation in SLC4A4 (coding for the Na+ bicarbonate co-transporter NBCe1) induces developmental defects in human and murine enamel. We hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in mouse wild-type dental epithelium and examined the effect of 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 recognize isoforms A/B/D/E and isoform C was seen in ameloblasts in secretory stage, no or very low staining in early maturation-stage but moderately to high staining in late maturation-stage. The papillary layer showed the opposite pattern and immunostained prominently at early maturation-stage but gradually showed less staining at mid- and late maturation-stage. In NBCe1−/− mice ameloblasts were disorganized, the enamel thin and severely hypomineralized. Enamel organs of CFTR−/− and AE2a,b−/− mice (believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Our data show that expression of NBCe1 in ameloblast and papillary layer cell depends on developmental stage and possibly responds to pH changes. PMID:25012520

Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

PubMed Central

Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

2010-01-01

BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

Regulation of gene expression in roots of the pH-sensitive Vaccinium corymbosum and the pH-tolerant Vaccinium arboreum in response to near neutral pH stress using RNA-Seq.

PubMed

Payá-Milans, Miriam; Nunez, Gerardo H; Olmstead, James W; Rinehart, Timothy A; Staton, Margaret

2017-08-07

Blueberries are one of the few horticultural crops adapted to grow in acidic soils. Neutral to basic soil pH is detrimental to all commonly cultivated blueberry species, including Vaccinium corymbosum (VC). In contrast, the wild species V. arboreum (VA) is able to tolerate a wider range of soil pH. To assess the molecular mechanisms involved in near neutral pH stress response, plants from pH-sensitive VC (tetraploid) and pH-tolerant VA (diploid) were grown at near neutral pH 6.5 and at the preferred pH of 4.5. Transcriptome sequencing of root RNA was performed for 4 biological replications per species x pH level interaction, for a total of 16 samples. Reads were mapped to the reference genome from diploid V. corymbosum, transforming ~55% of the reads to gene counts. A quasi-likelihood F test identified differential expression due to pH stress in 337 and 4867 genes in VA and VC, respectively. Both species shared regulation of genes involved in nutrient homeostasis and cell wall metabolism. VA and VC exhibited differential regulation of signaling pathways related to abiotic/biotic stress, cellulose and lignin biosynthesis, and nutrient uptake. The specific responses in VA likely facilitate tolerance to higher soil pH. In contrast, response in VC, despite affecting a greater number of genes, is not effective overcoming the stress induced by pH. Further inspection of those genes with differential expression that are specific in VA may provide insight on the mechanisms towards tolerance.

A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

PubMed Central

Chang, Xiaoxiao; Donnelly, Linda; Sun, Daoyang; Rao, Jingping; Reid, Michael S.; Jiang, Cai-Zhong

2014-01-01

Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO), and ABA (NCED) biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA) and abiotic stresses (dehydration, NaCl and cold). Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence. PMID:24551088

A Petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence.

PubMed

Chang, Xiaoxiao; Donnelly, Linda; Sun, Daoyang; Rao, Jingping; Reid, Michael S; Jiang, Cai-Zhong

2014-01-01

Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO), and ABA (NCED) biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA) and abiotic stresses (dehydration, NaCl and cold). Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence.

Intracellular pH in sperm physiology.

PubMed

Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

2014-08-01

Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. Copyright © 2014 Elsevier Inc. All rights reserved.

Physiological protective action of dissolved organic carbon on ion regulation and nitrogenous waste excretion of zebrafish (Danio rerio) exposed to low pH in ion-poor water.

PubMed

Duarte, Rafael M; Wood, Chris M; Val, Adalberto L; Smith, D Scott

2018-06-11

Dissolved organic carbon (DOC) represents a heterogeneous group of naturally-occurring molecules in aquatic environments, and recent studies have evidenced that optically dark DOCs can exert some positive effects on ionoregulatory homeostasis of aquatic organisms in acidic waters. We investigated the effects of Luther Marsh DOC, a dark allochthonous DOC, on ion regulation and N-waste excretion of zebrafish acutely exposed to either neutral or low pH in ion-poor water. In the first experiment, simultaneous exposure to pH 4.0 and DOC greatly attenuated the stimulation of Na + diffusive losses (J out Na ), and prevented the blockade of Na + uptake (J in Na ) seen in zebrafish exposed to pH 4.0 alone, resulting in much smaller disturbances in Na + net losses (J net Na ). DOC also attenuated the stimulation of net Cl - losses (J net Cl ) and ammonia excretion (J net Amm ) during acidic challenge. In the second experiment, zebrafish acclimated to DOC displayed similar regulation of J in Na and J out Na , and, therefore, reduced J net Na at pH 4.0, effects which persisted even when DOC was no longer present. Protective effects of prior acclimation to DOC on J net Cl and J net Amm at pH 4.0 also occurred, but were less marked than those on Na + balance. Urea fluxes were unaffected by the experimental treatments. Overall, these effects were clearly beneficial to the ionoregulatory homeostasis of zebrafish at low pH, and were quite similar to those seen in a recent parallel study using darker DOC from the upper Rio Negro. This suggests that dark allochthonous DOCs share some chemical properties that render fish tolerant to ionoregulatory disturbances during acidic challenge.

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prashek, Jennifer; Bouyain, Samuel; Fu, Mingui

De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this studymore » we show that isolated PH and START domains interact with each other. The crystal structure of a PH–START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH–START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine–rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.« less

A digestive prolyl carboxypeptidase in Tenebrio molitor larvae.

PubMed

Goptar, Irina A; Shagin, Dmitry A; Shagina, Irina A; Mudrik, Elena S; Smirnova, Yulia A; Zhuzhikov, Dmitry P; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

2013-06-01

Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP. Published by Elsevier Ltd.

Meet EPA Scientist Dermont Bouchard, Ph.D.

EPA Pesticide Factsheets

EPA Scientist Dermont Bouchard, Ph.D., is working to better understand how tiny nanomaterials might be released into the environment. His research helps regulators and other decision-makers lower risks and better protect human health and the environment

Antennally mediated negative feedback regulation of pheromone production in the pine engraver beetle, Ips pini

NASA Astrophysics Data System (ADS)

Ginzel, Matthew D.; Bearfield, Jeremy C.; Keeling, Christopher I.; McCormack, Colin C.; Blomquist, Gary J.; Tittiger, Claus

2007-01-01

Bark beetles use monoterpenoid aggregation pheromones to coordinate host colonization and mating. These chemical signals are produced de novo in midgut cells via the mevalonate pathway, and pheromone production may be regulated by a negative feedback system mediated through the antennae. In this study, we explored the effect of antennectomy on pheromone production and transcript levels of key mevalonate pathway genes in juvenile hormone III-treated male pine engraver beetles, Ips pini (Say). Antennectomized males produced significantly greater amounts of pheromone than podectomized males and those with intact antennae. Likewise, mRNA levels of three mevalonate pathway genes important in pheromone biosynthesis were measured by quantitative real-time PCR and found to be induced to a greater extent with antennectomy, suggesting a transcriptional regulation of pheromone production.

The Pochonia chlamydosporia serine protease gene vcp1 is subject to regulation by carbon, nitrogen and pH: implications for nematode biocontrol.

PubMed

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.

Regulation of cellular pH: From molecules to membranes

NASA Astrophysics Data System (ADS)

Grabe, Michael David

The vacuolar H+-ATPase (V-ATPase) is a universal class of proton pumps responsible for creating and maintaining acidic milieus in both intracellular and extracellular spaces. In the first chapter, I develop a mechanochemical model of this enzyme based upon the counter-rotation of adjacent subunits. The mathematical approach details a general integrated method for describing the mechanical and chemical reactions that occur in motor systems. A novel escapement is proposed for how the protons cross the protein-bilayer interface, and it is shown how this movement couples to ATP hydrolysis. This model reproduces a variety of experimental data while providing a framework for understanding the function of the enzyme's subunits. Specifically, it explains how ATP hydrolysis can uncouple from proton movement, which has important consequences for cellular energetics and pH regulation. Until now only an equilibrium theory of organelle acidification has been proposed; however, recent experiments show that large proton leaks prevent many cellular compartments from reaching thermodynamic equilibrium. The characterization of the V-ATPase is used in the second chapter in order to develop a unified model of organelle acidification based on the interplay of ion pumps and channels and the physical characteristics of the organelle. This model successfully describes the time dependent acidification of many different organelle systems. It accurately predicts both the electrical and concentration dependent terms of the chemical potential. In conjunction with fluorescence experiments, I determined the first measurements of the proton permeability of organelles along the secretory pathway. These measurements allowed me to make the first estimates of the number of V-ATPases in each compartment by analyzing the resting pH's of the respective organelles. I found a decrease in permeability from the endoplasmic reticulum (ER) (51 x 10-4 cm/s) to the Golgi (21 x 10-4 cm/s) to the mature secretory

PhMYB4 fine-tunes the floral volatile signature of Petunia x hybrida through PhC4H.

PubMed

Colquhoun, Thomas A; Kim, Joo Young; Wedde, Ashlyn E; Levin, Laura A; Schmitt, Kyle C; Schuurink, Robert C; Clark, David G

2011-01-01

In Petunia × hybrida cv 'Mitchell Diploid' (MD), floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis is controlled spatially, developmentally, and daily at molecular, metabolic, and biochemical levels. Multiple genes have been shown to encode proteins that either directly catalyse a biochemical reaction yielding FVBP compounds or are involved in metabolite flux prior to the formation of FVBP compounds. It was hypothesized that multiple transcription factors are involved in the precise regulation of all necessary genes, resulting in the specific volatile signature of MD flowers. After acquiring all available petunia transcript sequences with homology to Arabidopsis thaliana R2R3-MYB transcription factors, PhMYB4 (named for its close identity to AtMYB4) was identified, cloned, and characterized. PhMYB4 transcripts accumulate to relatively high levels in floral tissues at anthesis and throughout open flower stages, which coincides with the spatial and developmental distribution of FVBP production and emission. Upon RNAi suppression of PhMYB4 (ir-PhMYB4) both petunia cinnamate-4-hydroxylase (PhC4H1 and PhC4H2) gene transcript levels were significantly increased. In addition, ir-PhMYB4 plants emit higher levels of FVBP compounds derived from p-coumaric acid (isoeugenol and eugenol) compared with MD. Together, these results indicate that PhMYB4 functions in the repression of C4H transcription, indirectly controlling the balance of FVBP production in petunia floral tissue (i.e. fine-tunes).

In Vivo and In Vitro Binding of Vip3Aa to Spodoptera frugiperda Midgut and Characterization of Binding Sites by 125I Radiolabeling

PubMed Central

Chakroun, Maissa

2014-01-01

Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the binding protocol for the 125I-Vip3Aa to S. frugiperda brush border membrane vesicles (BBMV) allowed the determination of binding parameters of Vip3A proteins for the first time. Heterologous competition using Vip3Ad, Vip3Ae, and Vip3Af as competitor proteins showed that they share the same binding site with Vip3Aa. In contrast, when using Cry1Ab and Cry1Ac as competitors, no competitive binding was observed, which makes them appropriate candidates to be used in combination with Vip3A proteins in transgenic crops. PMID:25002420

Measurement of the Extracellular pH of Adherently Growing Mammalian Cells with High Spatial Resolution Using a Voltammetric pH Microsensor.

PubMed

Munteanu, Raluca-Elena; Stǎnicǎ, Luciana; Gheorghiu, Mihaela; Gáspár, Szilveszter

2018-05-15

There are only a few tools suitable for measuring the extracellular pH of adherently growing mammalian cells with high spatial resolution, and none of them is widely used in laboratories around the world. Cell biologists very often limit themselves to measuring the intracellular pH with commercially available fluorescent probes. Therefore, we built a voltammetric pH microsensor and investigated its suitability for monitoring the extracellular pH of adherently growing mammalian cells. The voltammetric pH microsensor consisted of a 37 μm diameter carbon fiber microelectrode modified with reduced graphene oxide and syringaldazine. While graphene oxide was used to increase the electrochemically active surface area of our sensor, syringaldazine facilitated pH sensing through its pH-dependent electrochemical oxidation and reduction. The good sensitivity (60 ± 2.5 mV/pH unit), reproducibility (coefficient of variation ≤3% for the same pH measured with 5 different microsensors), and stability (pH drift around 0.05 units in 3 h) of the built voltammetric pH sensors were successfully used to investigate the acidification of the extracellular space of both cancer cells and normal cells. The results indicate that the developed pH microsensor and the perfected experimental protocol based on scanning electrochemical microscopy can reveal details of the pH regulation of cells not attainable with pH sensors lacking spatial resolution or which cannot be reproducibly positioned in the extracellular space.

Ligand Accessibility and Bioactivity of a Hormone-Dendrimer Conjugate Depend on pH and pH History

PubMed Central

Kim, Sung Hoon; Madak-Erdogan, Zeynep; Bae, Sung Chul; Carlson, Kathryn E.; Mayne, Christopher G.; Granick, Steve; Katzenellenbogen, Benita S.; Katzenellenbogen, John A.

2016-01-01

Estrogen conjugates with a polyamidoamine (PAMAM) dendrimer have shown remarkably selective regulation of the non-genomic actions of estrogens in target cells. In response to pH changes, however, these estrogen-dendrimer conjugates (EDCs) display a major morphological transition that alters the accessibility of the estrogen ligands that compromises the bioactivity of the EDC. A sharp break in dynamic behavior near pH 7 occurs for three different ligands on the surface of a PAMAM-G6 dendrimer: a fluorophore (tetramethylrhodamine, TMR) and two estrogens (17α-ethynylestradiol and diphenolic acid). Collisional quenching and time-resolved fluorescence anisotropy experiments with TMR-PAMAM reveal high ligand shielding above pH 7 and low shielding below pH 7. Furthermore, when pH was cycled from 8.5 (conditions of ligand-PAMAM conjugation) to 4.5 (e.g., endosome/lysosome) and through 6.5 (e.g., hypoxic environment) back to pH 8.5, the 17α-ethynylestradiol and diphenolic acid PAMAM conjugates experience a dramatic, irreversible loss in cell stimulatory activity; dynamic NMR studies indicate that the hormonal ligands had become occluded within the more hydrophobic core of the PAMAM dendrimer. Thus, the active state of these estrogen-dendrimer conjugates appears to be metastable. This pH-dependent irreversible masking of activity is of considerable relevance to the design of drug conjugates with amine-bearing PAMAM dendrimers. PMID:26186415

Acidic Extracellular pH Promotes Activation of Integrin αvβ3

PubMed Central

Paradise, Ranjani K.; Lauffenburger, Douglas A.; Van Vliet, Krystyn J.

2011-01-01

Acidic extracellular pH is characteristic of the cell microenvironment in several important physiological and pathological contexts. Although it is well established that acidic extracellular pH can have profound effects on processes such as cell adhesion and migration, the underlying molecular mechanisms are largely unknown. Integrin receptors physically connect cells to the extracellular matrix, and are thus likely to modulate cell responses to extracellular conditions. Here, we examine the role of acidic extracellular pH in regulating activation of integrin αvβ3. Through computational molecular dynamics simulations, we find that acidic extracellular pH promotes opening of the αvβ3 headpiece, indicating that acidic pH can thereby facilitate integrin activation. This prediction is consistent with our flow cytometry and atomic force microscope-mediated force spectroscopy assays of integrin αvβ3 on live cells, which both demonstrate that acidic pH promotes activation at the intact cell surface. Finally, quantification of cell morphology and migration measurements shows that acidic extracellular pH affects cell behavior in a manner that is consistent with increased integrin activation. Taken together, these computational and experimental results suggest a new and complementary mechanism of integrin activation regulation, with associated implications for cell adhesion and migration in regions of altered pH that are relevant to wound healing and cancer. PMID:21283814

Atrial natriuretic peptide: a magic bullet for cancer therapy targeting Wnt signaling and cellular pH regulators.

PubMed

Serafino, A; Pierimarchi, P

2014-01-01

Atrial natriuretic peptide (ANP) is a cardiac hormone playing a crucial role in cardiovascular homeostasis mainly through blood volume and pressure regulation. In the last years, the new property ascribed to ANP of inhibiting tumor growth both in vitro and in vivo has made this peptide an attractive candidate for anticancer therapy. The molecular mechanism underlying the anti-proliferative effect of ANP has been mainly related to its interaction with the specific receptors NPRs, through which this natriuretic hormone inhibits some metabolic targets critical for cancer development, including the Ras-MEK1⁄2-ERK1⁄2 kinase cascade, functioning as a multikinase inhibitor. In this review we summarize the current knowledge on this topic, focusing on our recent data demonstrating that the antitumor activity of this natriuretic hormone is also mediated by a concomitant effect on the Wnt/β-catenin pathway and on the pH regulation ability of cancer cells, through a Frizzled-related mechanism. This peculiarity of simultaneously targeting two processes crucial for neoplastic transformation and solid tumor survival reinforces the utility of ANP for the development of both preventive and therapeutic strategies.

Inositol Pentakisphosphate Isomers Bind PH Domains with Varying Specificity and Inhibit Phosphoinositide Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Jackson; S Al-Saigh; C Schultz

2011-12-31

PH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. Myo-Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similaritymore » of inositol phosphates we were interested in examining the specificity of PH domains towards the family of myo-inositol pentakisphosphate isomers. In work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different myo-inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does. These results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting myo-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathway.« less

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.

Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium

PubMed Central

Walker, Nancy M.; Liu, Jinghua; Stein, Sydney R.; Stefanski, Casey D.; Strubberg, Ashlee M.

2015-01-01

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl− and HCO3− efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3−)-loading proteins and upregulation of the basolateral membrane HCO3−-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl−/HCO3− exchange with maximized gradients, it also had increased intracellular Cl− concentration relative to wild-type. Pharmacological reduction of intracellular Cl− concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl− and HCO3− efflux, which impairs pHi regulation by Ae2. Retention of Cl− and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. PMID:26542396

pH regulation in barnacle muscle fibers: dependence on extracellular sodium and bicarbonate.

PubMed

Boron, W F; McCormick, W C; Roos, A

1981-01-01

Intracellular pH (pHi) regulation was studied in barnacle muscle fibers with pH-sensitive microelectrodes. The cells were acid loaded, and the subsequent recovery of pHi was monitored. The rate of recovery was reduced by one-third when external Na+ ([Na+]o) was replaced by Li+, but recovery was completely abolished when Na+ was replaced by choline or N-methyl-D-glucamine. In other experiments, varying amounts of Na+ were replaced by choline, and the acid extrusion rate, derived from the recovery rate of pHi, was calculated at a single value of pHi, 6.80. The dependence of the acid extrusion rate on [Na+]o could be described by Michaelis-Menten kinetics; at pHo (extracellular) = 8.0 and [HCO3-]o (extracellular) = 10 mM, the apparent Km and Vmax were 59 mM and 1.3 mmol x l(-1) x min-1. When [HCO3-]o was reduced to 2.5 mM at the same pHo, Km did not change significantly, but Vmax was substantially reduced. On the other hand, when pHo was reduced to 7.4 at constant [HCO3-]o, Vmax changed only slightly, but Km increased substantially. In similar experiments, we examined the dependence of the acid extrusion rate on [HCO3-]o. At pHo = 8.0 and [Na+]o = 440 mM, the apparent Km and Vmax were 4.1 mM and 2.1 mmol x 1-1 x min-1. When pHo was reduced to 7.4, Vmax was not altered, but Km substantially increased. The kinetic data are discussed in terms of the role of pHo, [Na+]o, and [HCO3-]o in the pHi-regulating system.

Acidic pH sensing in the bacterial cytoplasm is required for Salmonella virulence.

PubMed

Choi, Jeongjoon; Groisman, Eduardo A

2016-09-01

pH regulates gene expression, biochemical activities and cellular behaviors. A mildly acidic pH activates the master virulence regulatory system PhoP/PhoQ in the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The sensor PhoQ harbors an extracytoplasmic domain implicated in signal sensing, and a cytoplasmic domain controlling activation of the regulator PhoP. We now report that, surprisingly, a decrease in Salmonella's own cytoplasmic pH induces transcription of PhoP-activated genes even when the extracytoplasmic pH remains neutral. Amino acid substitutions in PhoQ's cytoplasmic domain hindered activation by acidic pH and attenuated virulence in mice, but did not abolish activation by low Mg(2+) or the antimicrobial peptide C18G. Conversely, removal of PhoQ's extracytoplasmic domains prevented the response to the latter PhoQ-activating signals but not to acidic pH. PhoP-dependent genes were minimally induced by acidic pH in the non-pathogenic species Salmonella bongori but were activated by low Mg(2+) and C18G as in pathogenic S. enterica. Our findings indicate that the sensor PhoQ enables S. enterica to respond to both host- and bacterial-derived signals that alter its cytoplasmic pH. © 2016 John Wiley & Sons Ltd.

17-4 PH and 15-5 PH

NASA Technical Reports Server (NTRS)

Johnson, Howard T.

1995-01-01

17-4 PH and 15-5 PH are extremely useful and versatile precipitation-hardening stainless steels. Armco 17-4 PH is well suited for the magnetic particle inspection requirements of Aerospace Material Specification. Armco 15-5 PH and 17-4 PH are produced in billet, plate, bar, and wire. Also, 15-5 PH is able to meet the stringent mechanical properties required in the aerospace and nuclear industries. Both products are easy to heat treat and machine, making them very useful in many applications.

Fluorescent probes and nanoparticles for intracellular sensing of pH values

NASA Astrophysics Data System (ADS)

Shi, Wen; Li, Xiaohua; Ma, Huimin

2014-12-01

Intracellular pH regulates a number of cell metabolism processes and its sensing is thus of great importance for cell studies. Among various methods, fluorescent probes have been widely used for sensing intracellular pH values because of their high sensitivity and spatiotemporal resolution capability. In this article, the development of fluorescent probes with good practicability in sensing intracellular pH values and pH variation during 2009 - 2014 is reviewed. These fluorescence probes are divided into two kinds: small molecules and nanoparticles. Photophysical properties, advantages/disadvantages and applications of the two kinds of probes are discussed in detail.

Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

PubMed Central

Zha, Wenjun; Peng, Xinxin; Chen, Rongzhi; Du, Bo; Zhu, Lili; He, Guangcun

2011-01-01

Background RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. Methodology/Principal Findings The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. Conclusions Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants. PMID:21655219

Variant vicilins from a resistant Vigna unguiculata lineage (IT81D-1053) accumulate inside Callosobruchus maculatus larval midgut epithelium.

PubMed

Oliveira, Gabriel B; Kunz, Daniele; Peres, Tanara V; Leal, Rodrigo B; Uchôa, Adriana F; Samuels, Richard I; Macedo, Maria Lígia R; Carlini, Célia R; Ribeiro, Alberto F; Grangeiro, Thalles B; Terra, Walter R; Xavier-Filho, José; Silva, Carlos P

2014-02-01

It has been demonstrated that variant vicilins are the main resistance factor of cowpea seeds (Vigna unguiculata) against attack by the cowpea beetle Callosobruchus maculatus. There is evidence that the toxic properties of these storage proteins may be related to their interaction with glycoproteins and other microvillar membrane constituents along the digestive tract of the larvae. New findings have shown that following interaction with the microvilli, the vicilins are absorbed across the intestinal epithelium and thus reach the internal environment of the larvae. In the present paper we studied the insecticidal activity of the variant vicilins purified from a resistant cowpea variety (IT81D-1053). Bioassays showed that the seeds of this genotype affected larval growth, causing developmental retardation and 100% mortality. By feeding C. maculatus larvae on susceptible and IT81D-1053 derived vicilins (FITC labelled or unlabelled), followed by fluorescence and immunogold cytolocalization, we were able to demonstrate that both susceptible and variant forms are internalized in the midgut cells and migrate inside vesicular structures from the apex to the basal portion of the enterocytes. However, when larvae were fed with the labelled vicilins for 24h and then returned to a control diet, the concentration of the variant form remained relatively high, suggesting that variant vicilins are not removed from the cells at the same rate as the non-variant vicilins. We suggest that the toxic effects of variant vicilins on midgut cells involve the binding of these proteins to the cell surface followed by internalization and interference with the normal physiology of the enterocytes, thereby affecting larval development in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

DNA damage in haemocytes and midgut gland cells of Steatoda grossa (Theridiidae) spiders exposed to food contaminated with cadmium.

PubMed

Stalmach, Monika; Wilczek, Grażyna; Wilczek, Piotr; Skowronek, Magdalena; Mędrzak, Monika

2015-03-01

The aim of this study was to assess the genotoxic effects of Cd on haemocytes and midgut gland cells of web-building spiders, Steatoda grossa (Theridiidae), exposed to the metal under laboratory conditions. Analyzes were conducted on adult females and males, fed for four weeks with cadmium-contaminated Drosophila hydei flies, grown on a medium suplemented with 0.25 mM CdCl2. The comet assay, providing a quantitative measure of DNA strand breaks, was used to evaluate the DNA damage caused by the metal. Cadmium content was measured in whole spider bodies by the AAS method. Metal body burden was significantly lower in females (0.25 µgg(-1) dry weight) than in males (3.03 µgg(-1) dry weight), suggesting that females may have more effective mechanisms controlling the uptake of metal, via the digestive tract, or its elimination from the body. Irrespectively of sex, spiders fed prey contaminated with cadmium showed significantly higher values of comet parameters: tail DNA (TDNA), tail length (TL) and olive tail moment (OTM), in comparison with the control. In midgut gland cells, the level of DNA damage was higher for males than females, while in haemocytes the genotoxic effect of cadmium was greater in females. The obtained results indicate that in spiders cadmium displays strong genotoxic effects and may cause DNA damage even at low concentrations, however the severity of damage seems to be sex- and internal organ-dependent. The comet assay can be considered a sensitive tool for measuring the deleterious effect of cadmium on DNA integrity in spiders. Copyright © 2014 Elsevier Inc. All rights reserved.

Conserved mechanisms of tumorigenesis in the Drosophila adult midgut.

PubMed

Martorell, Òscar; Merlos-Suárez, Anna; Campbell, Kyra; Barriga, Francisco M; Christov, Christo P; Miguel-Aliaga, Irene; Batlle, Eduard; Casanova, Jordi; Casali, Andreu

2014-01-01

Whereas the series of genetic events leading to colorectal cancer (CRC) have been well established, the precise functions that these alterations play in tumor progression and how they disrupt intestinal homeostasis remain poorly characterized. Activation of the Wnt/Wg signaling pathway by a mutation in the gene APC is the most common trigger for CRC, inducing benign lesions that progress to carcinomas due to the accumulation of other genetic alterations. Among those, Ras mutations drive tumour progression in CRC, as well as in most epithelial cancers. As mammalian and Drosophila's intestines share many similarities, we decided to explore the alterations induced in the Drosophila midgut by the combined activation of the Wnt signaling pathway with gain of function of Ras signaling in the intestinal stem cells. Here we show that compound Apc-Ras clones, but not clones bearing the individual mutations, expand as aggressive intestinal tumor-like outgrowths. These lesions reproduce many of the human CRC hallmarks such as increased proliferation, blockade of cell differentiation and cell polarity and disrupted organ architecture. This process is followed by expression of tumoral markers present in human lesions. Finally, a metabolic behavioral assay shows that these flies suffer a progressive deterioration in intestinal homeostasis, providing a simple readout that could be used in screens for tumor modifiers or therapeutic compounds. Taken together, our results illustrate the conservation of the mechanisms of CRC tumorigenesis in Drosophila, providing an excellent model system to unravel the events that, upon mutation in Apc and Ras, lead to CRC initiation and progression.

Ligand accessibility and bioactivity of a hormone–dendrimer conjugate depend on pH and pH history

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sung Hoon; Madak-Erdogan, Zeynep; Bae, Sung Chul

Estrogen conjugates with a polyamidoamine (PAMAM) dendrimer have shown remarkably selective regulation of the nongenomic actions of estrogens in target cells in this paper. In response to pH changes, however, these estrogen–dendrimer conjugates (EDCs) display a major morphological transition that alters the accessibility of the estrogen ligands that compromises the bioactivity of the EDC. A sharp break in dynamic behavior near pH 7 occurs for three different ligands on the surface of a PAMAM-G6 dendrimer: a fluorophore (tetramethylrhodamine [TMR]) and two estrogens (17α-ethynylestradiol and diphenolic acid). Collisional quenching and time-resolved fluorescence anisotropy experiments with TMR–PAMAM revealed high ligand shielding abovemore » pH 7 and low shielding below pH 7. Furthermore, when the pH was cycled from 8.5 (conditions of ligand–PAMAM conjugation) to 4.5 (e.g., endosome/lysosome) and through 6.5 (e.g., hypoxic environment) back to pH 8.5, the 17α-ethynylestradiol– and diphenolic acid–PAMAM conjugates experienced a dramatic, irreversible loss in cell stimulatory activity; dynamic NMR studies indicated that the hormonal ligands had become occluded within the more hydrophobic core of the PAMAM dendrimer. Thus, the active state of these estrogen–dendrimer conjugates appears to be metastable. Finally, this pH-dependent irreversible masking of activity is of considerable relevance to the design of drug conjugates with amine-bearing PAMAM dendrimers.« less

Ligand accessibility and bioactivity of a hormone–dendrimer conjugate depend on pH and pH history

DOE PAGES

Kim, Sung Hoon; Madak-Erdogan, Zeynep; Bae, Sung Chul; ...

2015-07-17

Estrogen conjugates with a polyamidoamine (PAMAM) dendrimer have shown remarkably selective regulation of the nongenomic actions of estrogens in target cells in this paper. In response to pH changes, however, these estrogen–dendrimer conjugates (EDCs) display a major morphological transition that alters the accessibility of the estrogen ligands that compromises the bioactivity of the EDC. A sharp break in dynamic behavior near pH 7 occurs for three different ligands on the surface of a PAMAM-G6 dendrimer: a fluorophore (tetramethylrhodamine [TMR]) and two estrogens (17α-ethynylestradiol and diphenolic acid). Collisional quenching and time-resolved fluorescence anisotropy experiments with TMR–PAMAM revealed high ligand shielding abovemore » pH 7 and low shielding below pH 7. Furthermore, when the pH was cycled from 8.5 (conditions of ligand–PAMAM conjugation) to 4.5 (e.g., endosome/lysosome) and through 6.5 (e.g., hypoxic environment) back to pH 8.5, the 17α-ethynylestradiol– and diphenolic acid–PAMAM conjugates experienced a dramatic, irreversible loss in cell stimulatory activity; dynamic NMR studies indicated that the hormonal ligands had become occluded within the more hydrophobic core of the PAMAM dendrimer. Thus, the active state of these estrogen–dendrimer conjugates appears to be metastable. Finally, this pH-dependent irreversible masking of activity is of considerable relevance to the design of drug conjugates with amine-bearing PAMAM dendrimers.« less

D120 and K152 within the PH Domain of T Cell Adapter SKAP55 Regulate Plasma Membrane Targeting of SKAP55 and LFA-1 Affinity Modulation in Human T Lymphocytes

PubMed Central

Witte, Amelie; Meineke, Bernhard; Sticht, Jana; Philipsen, Lars; Kuropka, Benno; Müller, Andreas J.; Freund, Christian

2017-01-01

ABSTRACT The β2-integrin lymphocyte function-associated antigen 1 (LFA-1) is needed for the T cell receptor (TCR)-induced activation of LFA-1 to promote T cell adhesion and interaction with antigen-presenting cells (APCs). LFA-1-mediated cell-cell interactions are critical for proper T cell differentiation and proliferation. The Src kinase-associated phosphoprotein of 55 kDa (SKAP55) is a key regulator of TCR-mediated LFA-1 signaling (inside-out/outside-in signaling). To gain an understanding of how SKAP55 controls TCR-mediated LFA-1 activation, we assessed the functional role of its pleckstrin homology (PH) domain. We identified two critical amino acid residues within the PH domain of SKAP55, aspartic acid 120 (D120) and lysine 152 (K152). D120 facilitates the retention of SKAP55 in the cytoplasm of nonstimulated T cells, while K152 promotes SKAP55 membrane recruitment via actin binding upon TCR triggering. Importantly, the K152-dependent interaction of the PH domain with actin promotes the binding of talin to LFA-1, thus facilitating LFA-1 activation. These data suggest that K152 and D120 within the PH domain of SKAP55 regulate plasma membrane targeting and TCR-mediated activation of LFA-1. PMID:28052935

Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.

PubMed

Pastor-Soler, Nuria; Beaulieu, Valerie; Litvin, Tatiana N; Da Silva, Nicolas; Chen, Yanqiu; Brown, Dennis; Buck, Jochen; Levin, Lonny R; Breton, Sylvie

2003-12-05

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.

Proton mediated control of biochemical reactions with bioelectronic pH modulation

DOE PAGES

Deng, Yingxin; Miyake, Takeo; Keene, Scott; ...

2016-04-07

In Nature, protons (H +) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H + channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H + currents and H + concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H + between PdH x contacts and solution. The present transducer records bistable pH modulation from an “enzymaticmore » flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. Furthermore, the transducer also controls bioluminescence from firefly luciferase by affecting solution pH.« less

Proton mediated control of biochemical reactions with bioelectronic pH modulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Yingxin; Miyake, Takeo; Keene, Scott

In Nature, protons (H +) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H + channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H + currents and H + concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H + between PdH x contacts and solution. The present transducer records bistable pH modulation from an “enzymaticmore » flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. Furthermore, the transducer also controls bioluminescence from firefly luciferase by affecting solution pH.« less

Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase.

PubMed

Schewe, Bettina; Schmälzlin, Elmar; Walz, Bernd

2008-03-01

Blowfly salivary gland cells have a vacuolar-type H(+)-ATPase (V-ATPase) in their apical membrane that energizes secretion of a KCl-rich saliva upon stimulation with serotonin (5-hydroxytryptamine, 5-HT). We have used BCECF to study microfluometrically whether V-ATPase and carbonic anhydrase (CA) are involved in intracellular pH (pH(i)) regulation, and we have localized CA activity by histochemistry. We show: (1) mean pH(i) in salivary gland cells is 7.5+/-0.3 pH units (N=96), higher than that expected from passive H(+) distribution; (2) low 5-HT concentrations (0.3-3 nmol l(-1)) induce a dose-dependent acidification of up to 0.2 pH units, with 5-HT concentrations >10 nmol l(-1), causing monophasic or multiphasic pH changes; (3) the acidifying effect of 5-HT is mimicked by bath application of cAMP, forskolin or IBMX; (4) salivary gland cells exhibit CA activity; (5) CA inhibition with acetazolamide and V-ATPase inhibition with concanamycin A lead to a slow acidification of steady-state pH(i); (6) 5-HT stimuli in the presence of acetazolamide induce an alkalinization that can be decreased by simultaneous application of the V-ATPase inhibitor concanamycin A; (7) concanamycin A removes alkali-going components from multiphasic 5-HT-induced pH changes; (8) NHE activity and a Cl(-)-dependent process are involved in generating 5-HT-induced pH changes; (9) the salivary glands probably contain a Na(+)-driven amino acid transporter. We conclude that V-ATPase and CA contribute to steady-state pH(i) regulation and 5-HT-induced outward H(+) pumping does not cause an alkalinization of pH(i) because of cytosolic H(+) accumulation attributable to stimulated cellular respiration and AE activity, masking the alkalizing effect of V-ATPase-mediated acid extrusion.

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

PubMed Central

Collier, Daniel M.; Peterson, Zerubbabel J.; Blokhin, Ilya O.; Benson, Christopher J.; Snyder, Peter M.

2012-01-01

A growing body of evidence suggests that the extracellular domain of the epithelial Na+ channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na+ self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γE455 is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with βE446A generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na+ self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH. PMID:23060445

In Memoriam: Amar J.S. Klar, Ph.D. | Center for Cancer Research

Cancer.gov

In Memoriam: Amar J.S. Klar, Ph.D. The Center for Cancer Research mourns the recent death of colleague and friend Amar J.S. Klar, Ph.D.  Dr. Klar was a much-liked and respected member of the NCI community as part of the Gene Regulation and Chromosome Biology Laboratory since 1988.

PhOBF1, a petunia ocs element binding factor, plays an important role in antiviral RNA silencing.

PubMed

Sun, Daoyang; Li, Shaohua; Niu, Lixin; Reid, Michael S; Zhang, Yanlong; Jiang, Cai-Zhong

2017-02-01

Virus-induced gene silencing (VIGS) is a common reverse genetics strategy for characterizing the function of genes in plants. The detailed mechanism governing RNA silencing efficiency triggered by viruses is largely unclear. Here, we reveal that a petunia (Petunia hybrida) ocs element binding factor, PhOBF1, one of the basic leucine zipper (bZIP) transcription factors, was up-regulated by Tobacco rattle virus (TRV) infection. Simultaneous silencing of PhOBF1 and a reporter gene, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV-based VIGS led to a failure of the development of leaf photobleaching or the white-corollas phenotype. PhOBF1 silencing caused down-regulation of RNA silencing-related genes, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs). After inoculation with the TRV-PhPDS, PhOBF1-RNAi lines exhibited a substantially impaired PDS silencing efficiency, whereas overexpression of PhOBF1 resulted in a recovery of the silencing phenotype (photobleaching) in systemic leaves. A compromised resistance to TRV and Tobacco mosaic virus was found in PhOBF1-RNAi lines, while PhOBF1-overexpressing lines displayed an enhanced resistance to their infections. Compared with wild-type plants, PhOBF1-silenced plants accumulated lower levels of free salicylic acid (SA), salicylic acid glucoside, and phenylalanine, contrarily to higher levels of those in plants overexpressing PhOBF1. Furthermore, transcripts of a number of genes associated with the shikimate and phenylpropanoid pathways were decreased or increased in PhOBF1-RNAi or PhOBF1-overexpressing lines, respectively. Taken together, the data suggest that PhOBF1 regulates TRV-induced RNA silencing efficiency through modulation of RDRs, DCLs, and AGOs mediated by the SA biosynthesis pathway. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Water balance creates a threshold in soil pH at the global scale.

PubMed

Slessarev, E W; Lin, Y; Bingham, N L; Johnson, J E; Dai, Y; Schimel, J P; Chadwick, O A

2016-11-21

Soil pH regulates the capacity of soils to store and supply nutrients, and thus contributes substantially to controlling productivity in terrestrial ecosystems. However, soil pH is not an independent regulator of soil fertility-rather, it is ultimately controlled by environmental forcing. In particular, small changes in water balance cause a steep transition from alkaline to acid soils across natural climate gradients. Although the processes governing this threshold in soil pH are well understood, the threshold has not been quantified at the global scale, where the influence of climate may be confounded by the effects of topography and mineralogy. Here we evaluate the global relationship between water balance and soil pH by extracting a spatially random sample (n = 20,000) from an extensive compilation of 60,291 soil pH measurements. We show that there is an abrupt transition from alkaline to acid soil pH that occurs at the point where mean annual precipitation begins to exceed mean annual potential evapotranspiration. We evaluate deviations from this global pattern, showing that they may result from seasonality, climate history, erosion and mineralogy. These results demonstrate that climate creates a nonlinear pattern in soil solution chemistry at the global scale; they also reveal conditions under which soils maintain pH out of equilibrium with modern climate.

Water balance creates a threshold in soil pH at the global scale

NASA Astrophysics Data System (ADS)

Slessarev, E. W.; Lin, Y.; Bingham, N. L.; Johnson, J. E.; Dai, Y.; Schimel, J. P.; Chadwick, O. A.

2016-12-01

Soil pH regulates the capacity of soils to store and supply nutrients, and thus contributes substantially to controlling productivity in terrestrial ecosystems. However, soil pH is not an independent regulator of soil fertility—rather, it is ultimately controlled by environmental forcing. In particular, small changes in water balance cause a steep transition from alkaline to acid soils across natural climate gradients. Although the processes governing this threshold in soil pH are well understood, the threshold has not been quantified at the global scale, where the influence of climate may be confounded by the effects of topography and mineralogy. Here we evaluate the global relationship between water balance and soil pH by extracting a spatially random sample (n = 20,000) from an extensive compilation of 60,291 soil pH measurements. We show that there is an abrupt transition from alkaline to acid soil pH that occurs at the point where mean annual precipitation begins to exceed mean annual potential evapotranspiration. We evaluate deviations from this global pattern, showing that they may result from seasonality, climate history, erosion and mineralogy. These results demonstrate that climate creates a nonlinear pattern in soil solution chemistry at the global scale; they also reveal conditions under which soils maintain pH out of equilibrium with modern climate.

Near-future pH conditions severely impact calcification, metabolism and the nervous system in the pteropod Heliconoides inflatus.

PubMed

Moya, Aurelie; Howes, Ella L; Lacoue-Labarthe, Thomas; Forêt, Sylvain; Hanna, Bishoy; Medina, Mónica; Munday, Philip L; Ong, Jue-Sheng; Teyssié, Jean-Louis; Torda, Gergely; Watson, Sue-Ann; Miller, David J; Bijma, Jelle; Gattuso, Jean-Pierre

2016-12-01

Shelled pteropods play key roles in the global carbon cycle and food webs of various ecosystems. Their thin external shell is sensitive to small changes in pH, and shell dissolution has already been observed in areas where aragonite saturation state is ~1. A decline in pteropod abundance has the potential to disrupt trophic networks and directly impact commercial fisheries. Therefore, it is crucial to understand how pteropods will be affected by global environmental change, particularly ocean acidification. In this study, physiological and molecular approaches were used to investigate the response of the Mediterranean pteropod, Heliconoides inflatus, to pH values projected for 2100 under a moderate emissions trajectory (RCP6.0). Pteropods were subjected to pH T 7.9 for 3 days, and gene expression levels, calcification and respiration rates were measured relative to pH T 8.1 controls. Gross calcification decreased markedly under low pH conditions, while genes potentially involved in calcification were up-regulated, reflecting the inability of pteropods to maintain calcification rates. Gene expression data imply that under low pH conditions, both metabolic processes and protein synthesis may be compromised, while genes involved in acid-base regulation were up-regulated. A large number of genes related to nervous system structure and function were also up-regulated in the low pH treatment, including a GABA A receptor subunit. This observation is particularly interesting because GABA A receptor disturbances, leading to altered behavior, have been documented in several other marine animals after exposure to elevated CO 2 . The up-regulation of many genes involved in nervous system function suggests that exposure to low pH could have major effects on pteropod behavior. This study illustrates the power of combining physiological and molecular approaches. It also reveals the importance of behavioral analyses in studies aimed at understanding the impacts of low pH on

Down-regulation of aminopeptidase N and ABC transporter subfamily G transcripts in Cry1Ab and Cry1Ac resistant Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae)

PubMed Central

Zhang, Tiantao; Coates, Brad S.; Wang, Yueqin; Wang, Yidong; Bai, Shuxiong; Wang, Zhenying; He, Kanglai

2017-01-01

The Asian corn borer (ACB), Ostrinia furnacalis (Lepidoptera: Crambidae), is a highly destructive pest of cultivated maize throughout East Asia. Bacillus thuringiensis (Bt) crystalline protein (Cry) toxins cause mortality by a mechanism involving pore formation or signal transduction following toxin binding to receptors along the midgut lumen of susceptible insects, but this mechanism and mutations therein that lead to resistance are not fully understood. In the current study, quantitative comparisons were made among midgut expressed transcripts from O. furnacalis susceptible (ACB-BtS) and laboratory selected strains resistant to Cry1Ab (ACB-AbR) and Cry1Ac toxins (ACB-AcR) when feeding on non-Bt diet. From a combined de novo transcriptome assembly of 83,370 transcripts, ORFs of ≥ 100 amino acids were predicted and annotated for 28,940 unique isoforms derived from 12,288 transcripts. Transcriptome-wide expression estimated from RNA-seq read depths predicted significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as a putative ATP binding cassette transporter group G (abcg) gene in both ACB-AbR and -AcR (log2[fold-change] ≥ 1.36; P < 0.0001). The transcripts that were most highly differentially regulated in both ACB-AbR and -AcR compared to ACB-BtS (log2[fold-change] ≥ 2.0; P < 0.0001) included up- and down-regulation of serine proteases, storage proteins and cytochrome P450 monooxygenases, as well as up-regulation of genes with predicted transport function. This study predicted the significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as abccg gene in both ACB-AbR and -AcR. These data are important for the understanding of systemic differences between Bt resistant and susceptible genotypes. PMID:28808417

Arrestin-related proteins mediate pH signaling in fungi.

PubMed

Herranz, Silvia; Rodríguez, José M; Bussink, Henk-Jan; Sánchez-Ferrero, Juan C; Arst, Herbert N; Peñalva, Miguel A; Vincent, Olivier

2005-08-23

Metazoan arrestins bind to seven-transmembrane (7TM) receptors to regulate function. Aspergillus nidulans PalF, a protein involved in the fungal ambient pH signaling pathway, contains arrestin N-terminal and C-terminal domains and binds strongly to two different regions within the C-terminal cytoplasmic tail of the 7TM, putative pH sensor PalH. Upon exposure to alkaline ambient pH, PalF is phosphorylated and, like mammalian beta-arrestins, ubiquitinated in a signal-dependent and 7TM protein-dependent manner. Substitution in PalF of a highly conserved arrestin N-terminal domain Ser residue prevents PalF-PalH interaction and pH signaling in vivo. Thus, PalF is the first experimentally documented fungal arrestin-related protein, dispelling the notion that arrestins are restricted to animal proteomes. Epistasis analyses demonstrate that PalF posttranslational modification is partially dependent on the 4TM protein PalI but independent of the remaining pH signal transduction pathway proteins PalA, PalB, and PalC, yielding experimental evidence bearing on the order of participation of the six components of the pH signal transduction pathway. Our data strongly implicate PalH as an ambient pH sensor, possibly with the cooperation of PalI.

Identification of different trypanosome species in the mid-guts of tsetse flies of the Malanga (Kimpese) sleeping sickness focus of the Democratic Republic of Congo.

PubMed

Simo, Gustave; Silatsa, Barberine; Flobert, Njiokou; Lutumba, Pascal; Mansinsa, Philemon; Madinga, Joule; Manzambi, Emile; De Deken, Reginald; Asonganyi, Tazoacha

2012-09-19

The Malanga sleeping sickness focus of the Democratic Republic of Congo has shown an epidemic evolution of disease during the last century. However, following case detection and treatment, the prevalence of the disease decreased considerably. No active survey has been undertaken in this focus for a couple of years. To understand the current epidemiological status of sleeping sickness as well as the animal African trypanosomiasis in the Malanga focus, we undertook the identification of tsetse blood meals as well as different trypanosome species in flies trapped in this focus. Pyramidal traps were use to trap tsetse flies. All flies caught were identified and live flies were dissected and their mid-guts collected. Fly mid-gut was used for the molecular identification of the blood meal source, as well as for the presence of different trypanosome species. About 949 Glossina palpalis palpalis were trapped; 296 (31.2%) of which were dissected, 60 (20.3%) blood meals collected and 57 (19.3%) trypanosome infections identified. The infection rates were 13.4%, 5.1%, 3.5% and 0.4% for Trypanosoma congolense savannah type, Trypanosoma brucei s.l., Trypanosoma congolense forest type and Trypanosoma vivax, respectively. Three mixed infections including Trypanosoma brucei s.l. and Trypanosoma congolense savannah type, and one mixed infection of Trypanosoma vivax and Trypanosoma congolense savannah type were identified. Eleven Trypanosoma brucei gambiense infections were identified; indicating an active circulation of this trypanosome subspecies. Of all the identified blood meals, about 58.3% were identified as being taken on pigs, while 33.3% and 8.3% were from man and other mammals, respectively. The presence of Trypanosoma brucei in tsetse mid-guts associated with human blood meals is indicative of an active transmission of this parasite between tsetse and man. The considerable number of pig blood meals combined with the circulation of Trypanosoma brucei gambiense in this focus

Effects of pH and Carbon Source on Synechococcus PCC 7002 Cultivation: Biomass and Carbohydrate Production with Different Strategies for pH Control.

PubMed

De Farias Silva, Carlos Eduardo; Sforza, Eleonora; Bertucco, Alberto

2017-02-01

Synechococcus PCC 7002 is an interesting species in view of industrial production of carbohydrates. The cultivation performances of this species are strongly affected by the pH of the medium, which also influences the carbohydrate accumulation. In this work, different methods of pH control were analyzed, in order to obtain a higher production of both Synechococcus biomass and carbohydrates. To better understand the influence of pH on growth and carbohydrate productivity, manual and automatic pH regulation in CO 2 and bicarbonate system were applied. The pH value of 8.5 resulted the best to achieve both of these goals. From an industrial point of view, an alternative way to maintain the pH practically constant during the entire period of cultivation is the exploitation of the bicarbonate-CO 2 buffer system, with the double aim to maintain the pH in the viability range and also to provide the amount of carbon required by growth. In this condition, a high concentration of biomass (6 g L -1 ) and carbohydrate content (around 60 %) were obtained, which are promising in view of a potential use for bioethanol production. The chemical equilibrium of C-N-P species was also evaluated by applying the ionic balance equations, and a relation between the sodium bicarbonate added in the medium and the equilibrium value of pH was discussed.

Wnt/β-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse.

PubMed

Engert, Silvia; Burtscher, Ingo; Liao, W Perry; Dulev, Stanimir; Schotta, Gunnar; Lickert, Heiko

2013-08-01

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse β-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of β-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for β-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of β-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/β-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/β-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.

Neem oil (Azadirachta indica A. Juss) affects the ultrastructure of the midgut muscle of Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae).

PubMed

Scudeler, Elton Luiz; Garcia, Ana Silvia Gimenes; Pinheiro, Patricia Fernanda Felipe; Santos, Daniela Carvalho Dos

2017-01-01

Cytomorphological changes, by means of ultrastructural analyses, have been used to determine the effects of the biopesticide neem oil on the muscle fibers of the midgut of the predator Ceraeochrysa claveri. Insects, throughout the larval period, were fed eggs of Diatraea saccharalis treated with neem oil at a concentration of 0.5%, 1% or 2%. In the adult stage, the midgut was collected from female insects at two stages of adulthood (newly emerged and at the start of oviposition) and processed for ultrastructural analyses. In the newly emerged insects obtained from neem oil treatments, muscle fibers showed a reduction of myofilaments as well as swollen mitochondria and an accumulation of membranous structures. Muscular fibers responded to those cellular injuries with the initiation of detoxification mechanisms, in which acid phosphatase activity was observed in large vesicles located at the periphery of the muscle fiber. At the start of oviposition in the neem oil treated insects, muscle fibers exhibited signs of degeneration, containing vacant areas in which contractile myofilaments were reduced or completely absent, and an accumulation of myelin structures, a dilatation of cisternae of sarcoplasmic reticulum, and mitochondrial swelling and cristolysis were observed. Enzymatic activity for acid phosphatase was present in large vesicles, indicating that mechanisms of lytic activity during the cell injury were utilized but insufficient for recovery from all the cellular damage. The results indicate that the visceral muscle layer is also the target of action of neem oil, and the cytotoxic effects observed may compromise the function of that organ. Copyright © 2016 Elsevier GmbH. All rights reserved.

Histopathological Effects of the Yen-Tc Toxin Complex from Yersinia entomophaga MH96 (Enterobacteriaceae) on the Costelytra zealandica (Coleoptera: Scarabaeidae) Larval Midgut

PubMed Central

Hares, Michelle C.; Jones, Sandra A.; Harper, Lincoln A.; Vernon, James R.; Harland, Duane P.; Jackson, Trevor A.; Hurst, Mark R. H.

2012-01-01

Yersinia entomophaga MH96, which was originally isolated from the New Zealand grass grub, Costelytra zealandica, produces an orally active proteinaceous toxin complex (Yen-Tc), and this toxin is responsible for mortality in a range of insect species, mainly within the Coleoptera and Lepidoptera. The genes encoding Yen-Tc are members of the toxin complex (Tc) family, with orthologs identified in several other bacterial species. As the mechanism of Yen-Tc activity remains unknown, a histopathological examination of C. zealandica larvae was undertaken in conjunction with cultured cells to identify the effects of Yen-Tc and to distinguish the contributions that its individual subunit components make upon intoxication. A progressive series of events that led to the deterioration of the midgut epithelium was observed. Additionally, experiments using a cell culture assay system were carried out to determine the cellular effects of intoxication on cells after topical application and the transient expression of Yen-Tc and its individual components. While observations were broadly consistent with those previously reported for other Tc family members, some differences were noted. In particular, the distinct stepwise disintegration of the midgut shared features associated with both apoptosis and necrotic programmed cell death pathways. Second, we observed, for the first time, a contribution of toxicity from two chitinases associated with the Yen-Tc complex. Our findings were suggestive of the activities encoded within the subunit components of Yen-Tc targeting different sites along putative programmed cell death pathways. Given the observed broad host range for Yen-Tc, these targeted loci are likely to be widely shared among insects. PMID:22544254

Comparative proteomic analysis reveals the suppressive effects of dietary high glucose on the midgut growth of silkworm.

PubMed

Feng, Fan; Chen, Liang; Lian, Chaoqun; Xia, Hengchuan; Zhou, Yang; Yao, Qin; Chen, Keping

2014-08-28

The silkworm, Bombyx mori, is an important model of lepidoptera insect, and it has been used for several models of human diseases. In human being, long-term high-sugar diet can induce the occurrence of diabetes and other related diseases. Interestingly, our experiments revealed the high glucose diet also has a suppressive effect on the development of silkworms. To investigate the molecular mechanism by which high-glucose diet inhibited the midgut growth in silkworms, we employed comparative proteomic analysis to globally identify proteins differentially expressed in normal and high-glucose diet group silkworms. In all, 28 differently proteins were suppressed and 5 proteins induced in high-glucose diet group. Gene ontology analysis showed that most of these differently proteins are mainly involved in metabolic process, catalytic and cellular process. A development related protein, imaginal disk growth factor (IDGF), was further confirmed by western blot exclusively expressing in the normal diet group silkworms. Taken together, our data suggests that IDGF plays a critical role in impairing the development of silkworms by a high-glucose diet. Glucose has been thought to play essential roles in growth and development of silkworm. In this paper, we certified firstly that high-glucose diet can suppress the growth of silkworm, and comparative proteomic was employed to reveal the inhibition mechanism. Moreover, an important regulation related protein (IDGF) was found to involve in this inhibition process. These results will help us get a deeper understanding of the relationship between diet and healthy. Furthermore, IDGF may be the critical protein for reducing the blood sugar in silkworm, and it may be used for screening human hypoglycemic drug. The work has not been submitted elsewhere for publication, in whole or in part, and all the authors have approved the manuscript. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Active subsite properties, subsite residues and targeting to lysosomes or midgut lumen of cathepsins L from the beetle Tenebrio molitor.

PubMed

Damasceno, Ticiane F; Dias, Renata O; de Oliveira, Juliana R; Salinas, Roberto K; Juliano, Maria A; Ferreira, Clelia; Terra, Walter R

2017-10-01

Cathepsins L are the major digestive peptidases in the beetle Tenebrio molitor. Two digestive cathepsins L (TmCAL2 and TmCAL3) from it had their 3D structures solved. The aim of this paper was to study in details TmCAL3 specificity and properties and relate them to its 3D structure. Recombinant TmCAL3 was assayed with 64 oligopeptides with different amino acid replacements in positions P2, P1, P1' and P2'. Results showed that TmCAL3 S2 specificity differs from the human enzyme and that its specificities also explain why on autoactivation two propeptide residues remain in the enzyme. Data on free energy of binding and of activation showed that S1 and S2' are mainly involved in substrate binding, S1' acts in substrate binding and catalysis, whereas S2 is implied mainly in catalysis. Enzyme subsite residues were identified by docking with the same oligopeptide used for kinetics. The subsite hydrophobicities were calculated from the efficiency of hydrolysis of different amino acid replacements in the peptide and from docking data. The results were closer for S1 and S2' than for S1' and S2, indicating that the residue subsites that were more involved in transition state binding are different from those binding the substrate seen in docking. Besides TmCAL1-3, there are nine other cathepsins L, most of them more expressed at midgut. They are supposed to be directed to lysosomes by a Drosophila-like Lerp receptor and/or motifs in their prodomains. The mannose 6-phosphate lysosomal sorting machinery is absent from T. molitor transcriptome. Cathepsin L direction to midgut contents seems to depend on overexpression. Copyright © 2017 Elsevier Ltd. All rights reserved.

pH Reversible Encapsulation of Oppositely Charged Colloids Mediated by Polyelectrolytes

PubMed Central

2017-01-01

We report the first example of reversible encapsulation of micron-sized particles by oppositely charged submicron smaller colloids. The reversibility of this encapsulation process is regulated by pH-responsive poly(acrylic acid) (PAA) present in solution. The competitive adsorption between the small colloids and the poly(acrylic acid) on the surface of the large colloids plays a key role in the encapsulation behavior of the system. pH offers an experimental knob to tune the electrostatic interactions between the two oppositely charged particle species via regulation of the charge density of the poly(acrylic acid). This results in an increased surface coverage of the large colloids by the smaller colloids when decreasing pH. Furthermore, the poly(acrylic acid) also acts as a steric barrier limiting the strength of the attractive forces between the oppositely charged particle species, thereby enabling detachment of the smaller colloids. Finally, based on the pH tunability of the encapsulation behavior and the ability of the small colloids to detach, reversible encapsulation is achieved by cycling pH in the presence of the PAA polyelectrolytes. The role of polyelectrolytes revealed in this work provides a new and facile strategy to control heteroaggregation behavior between oppositely charged colloids, paving the way to prepare sophisticated hierarchical assemblies. PMID:28419800

Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae)

NASA Astrophysics Data System (ADS)

Keeling, Christopher I.; Blomquist, Gary J.; Tittiger, Claus

In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.

Effect of Induced Oxidative Stress and Herbal Extracts on Acid Phosphatase Activity in Lysosomal and Microsomal Fractions of Midgut Tissue of the Silkworm, Bombyx mori

PubMed Central

Gaikwad, Y. B.; Gaikwad, S. M.; Bhawane, G. P.

2010-01-01

Lysosomal and microsomal acid phosphatase activity was estimated in midgut tissue of silkworm larvae, Bombyx mori L. (Lepidoptera: Bombycidae), after induced oxidative stress by D-galactose. The larvae were simultaneously were treated with ethanolic extracts of Bacopa monniera and Lactuca sativa to study their antioxidant properties. Lipid peroxidation and fluorescence was measured to analyze extent of oxidative stress. The ethanolic extract of Lactuca sativa was found to be more effective in protecting membranes against oxidative stress than Bacopa monniera. PMID:20874583

Studies on Proteinases from Some Blood-Sucking Insects,

DTIC Science & Technology

ovinus, and Pediculus humanus, but not in those of Cimex lectularius or Rhodnius prolixus. The trypsin and chymotrypsin have been partially... Cimex and Rhodnius appear to have a high molecular weight proteinase with optimal activity at pH 5 in their midguts. (Author)

Effect of air breathing on acid-base and ion regulation after exhaustive exercise and during low pH exposure in the bowfin, Amia calva.

PubMed

Gonzalez, R J; Milligan, L; Pagnotta, A; McDonald, D G

2001-01-01

To explore a potential conflict between air breathing and acid-base regulation in the bowfin (Amia calva), we examined how individuals with access to air differed from fish without air access in their response to acidosis. After exhaustive exercise, bowfin with access to air recovered significantly more slowly from the acidosis than fish without air access. While arterial blood pH (pH(a)) of fish without air access recovered to resting levels by 8 h, pH(a) was still significantly depressed in fish having access to air. In addition, Pco(2) was slightly more elevated in fish having air access than those without it. Fish with access to air still had a significant metabolic acid load after 8-h recovery, while those without air access completely cleared the load within 4 h. These results suggest that bowfin with access to air were breathing air and, consequently, were less able to excrete CO(2) and H(+) and experienced a delayed recovery. In contrast, during exposure to low pH, air breathing seemed to have a protective effect on acid-base status in bowfin. During exposure to low pH water, bowfin with access to air developed a much milder acidosis than bowfin without air access. The more severe acidosis in fish without air access was caused by an increased rate of lactic acid production. It appears that enhanced O(2) delivery allowed air-breathing bowfin to avoid acidosis-induced anaerobic metabolism and lactic acid production. In addition, during low pH exposure, plasma Na(+) and Cl(-) concentrations of fish without air access fell slightly more rapidly than those in fish with air access, indicating that the branchial ventilatory changes associated with air breathing limited, to some degree, ion losses associated with low pH exposure.

Surveillance of Nicotine and pH in Cigarette and Cigar Filler

PubMed Central

Lawler, Tameka S.; Stanfill, Stephen B.; deCastro, B. Rey; Lisko, Joseph G.; Duncan, Bryce W.; Richter, Patricia; Watson, Clifford H.

2017-01-01

Objective We examined differences between nicotine concentrations and pH in cigarette and cigar tobacco filler. Methods Nicotine and pH levels for 50 cigarette and 75 cigar brands were measured. Non-mentholated and mentholated cigarette products were included in the analysis along with several cigar types as identified by the manufacturer: large cigars, pipe tobacco cigars, cigarillos, mini cigarillos, and little cigars. Results There were significant differences found between pH and nicotine for cigarette and cigar tobacco products. Mean nicotine concentrations in cigarettes (19.2 mg/g) and large cigars (15.4 mg/g) were higher than the other cigars types, especially the pipe tobacco cigars (8.79 mg/g). The mean pH for cigarettes was pH 5.46. Large cigars had the highest mean pH value (pH 6.10) and pipe tobacco cigars had the lowest (pH 5.05). Conclusions Although cigarettes are the most common combustible tobacco product used worldwide, cigar use remains popular. Our research provides a means to investigate the possibility of distinguishing the 2 tobacco product types and offers information on nicotine and pH across a wide range of cigarette and cigar varieties that may be beneficial to help establish tobacco policies and regulations across product types. PMID:28989950

Histidine residues in the Na+-coupled ascorbic acid transporter-2 (SVCT2) are central regulators of SVCT2 function, modulating pH sensitivity, transporter kinetics, Na+ cooperativity, conformational stability, and subcellular localization.

PubMed

Ormazabal, Valeska; Zuñiga, Felipe A; Escobar, Elizabeth; Aylwin, Carlos; Salas-Burgos, Alexis; Godoy, Alejandro; Reyes, Alejandro M; Vera, Juan Carlos; Rivas, Coralia I

2010-11-19

Na(+)-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His(109), His(203), His(206), His(269), and His(413), are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na(+) cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His(413), localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na(+) and loss of Na(+) cooperativity, which leads to a decreased V(max) without altering the transport K(m); (ii) exofacial histidine residues His(203), His(206), and His(413) may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K(m); and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.

A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system.

PubMed

Raimondo, Joseph V; Joyce, Bradley; Kay, Louise; Schlagheck, Theresa; Newey, Sarah E; Srinivas, Shankar; Akerman, Colin J

2013-01-01

Within the nervous system, intracellular Cl(-) and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission, and network excitability. Cl(-) and pH are often co-regulated, and network activity results in the movement of both Cl(-) and H(+). Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl(-) and pH sensors have been described previously, these either lack ion specificity or are unsuitable for neuronal use. Here we present ClopHensorN-a new genetically-encoded ratiometric Cl(-) and pH sensor that is optimized for the nervous system. We demonstrate the ability of ClopHensorN to dissociate and simultaneously quantify Cl(-) and H(+) concentrations under a variety of conditions. In addition, we establish the sensor's utility by characterizing activity-dependent ion dynamics in hippocampal neurons.

Characterization of a beta-glycosidase highly active on disaccharides and of a beta-galactosidase from Tenebrio molitor midgut lumen.

PubMed

Ferreira, Alexandre H P; Terra, Walter R; Ferreira, Clélia

2003-02-01

The midgut of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae has four beta-glycosidases. The properties of two of these enzymes (betaGly1 and betaGly2) have been described elsewhere. In this paper, the characterization of the other two glycosidases (betaGly3 and betaGly4) is described. BetaGly3 has one active site, hydrolyzes disaccharides, cellodextrins, synthetic substrates and beta-glucosides produced by plants. The enzyme is inhibited by amygdalin, cellotriose, cellotetraose and cellopentaose in high concentrations, probably due to transglycosylation. betaGly3 hydrolyzes beta 1,4-glycosidic linkages with a catalytic rate independent of the substrate polymerization degree (k(int)) of 11.9 s(-1). Its active site is formed by four subsites, where subsites +1 and -1 bind glucose residues with higher affinity than subsite +2. The main role of betaGly3 seems to be disaccharide hydrolysis. BetaGly4 is a beta-galactosidase, since it has highest activity against beta-galactosides. It can also hydrolyze fucosides, but not glucosides, and has Triton X-100 as a non-essential activator (K(a)=15 microM, pH 4.5). betaGly4 has two active sites that can hydrolyze p-nitrophenyl beta-galactoside (NPbetaGal). The one hydrolyzing NPbetaGal with more efficiency is also active against methylumbellipheryl beta-D-galactoside and lactose. The other active site hydrolyzes NPbetaFucoside and binds NPbetaGal weakly. BetaGly4 hydrolyzes hydrophobic substrates with high catalytical efficiency and is able to bind octyl-beta-thiogalactoside in its active site with high affinity. The betaGly4 physiological role is supposed to be the hydrolysis of galactolipids that are found in membranes from vegetal tissues. As the enzyme has a hydrophobic site where Triton X-100 can bind, it might be activated by membrane lipids, thus becoming fully active only at the surface of cell membranes.

The Production Rate and Employment of Ph.D. Astronomers

NASA Astrophysics Data System (ADS)

Metcalfe, Travis S.

2008-02-01

In an effort to encourage self-regulation of the astronomy job market, I examine the supply of, and demand for, astronomers over time. On the supply side, I document the production rate of Ph.D. astronomers from 1970 to 2006 using the UMI Dissertation Abstracts database, along with data from other independent sources. I compare the long-term trends in Ph.D. production with federal astronomy research funding over the same time period, and I demonstrate that additional funding is correlated with higher subsequent Ph.D. production. On the demand side, I monitor the changing patterns of employment using statistics about the number and types of jobs advertised in the AAS Job Register from 1984 to 2006. Finally, I assess the sustainability of the job market by normalizing this demand by the annual Ph.D. production. The most recent data suggest that there are now annual advertisements for about one postdoctoral job, half a faculty job, and half a research/support position for every new domestic Ph.D. recipient in astronomy and astrophysics. The average new astronomer might expect to hold up to 3 jobs before finding a steady position.

Treatment of Salmonella enterica serovar Enteritidis with a sublethal concentration of trisodium phosphate or alkaline pH induces thermotolerance.

PubMed

Sampathkumar, Balamurugan; Khachatourians, George G; Korber, Darren R

2004-08-01

The responses of Salmonella enterica serovar Enteritidis to a sublethal dose of trisodium phosphate (TSP) and its equivalent alkaline pH made with NaOH were examined. Pretreatment of S. enterica serovar Enteritidis cells with 1.5% TSP or pH 10.0 solutions resulted in a significant increase in thermotolerance, resistance to 2.5% TSP, resistance to high pH, and sensitivity to acid and H(2)O(2). Protein inhibition studies with chloramphenicol revealed that thermotolerance, unlike resistance to high pH, was dependent on de novo protein synthesis. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of total cellular proteins from untreated control cells resolved as many as 232 proteins, of which 22 and 15% were absent in TSP- or alkaline pH-pretreated cells, respectively. More than 50% of the proteins that were either up- or down-regulated by TSP pretreatment were also up- or down-regulated by alkaline pH pretreatment. Sodium dodecyl sulfate-PAGE analysis of detergent-insoluble outer membrane proteins revealed the up-regulation of at least four proteins. Mass spectrometric analysis showed the up-regulated proteins to include those involved in the transport of small hydrophilic molecules across the cytoplasmic membrane and those that act as chaperones and aid in the export of newly synthesized proteins by keeping them in open conformation. Other up-regulated proteins included common housekeeping proteins like those involved in amino acid biosynthesis, nucleotide metabolism, and aminoacyl-tRNA biosynthesis. In addition to the differential expression of proteins following TSP or alkaline pH treatment, changes in membrane fatty acid composition were also observed. Alkaline pH- or TSP-pretreated cells showed a higher saturated and cyclic to unsaturated fatty acid ratio than did the untreated control cells. These results suggest that the cytoplasmic membrane could play a significant role in the induction of thermotolerance and resistance to other stresses following TSP

The Metalloprotease of Listeria monocytogenes Is Regulated by pH▿

PubMed Central

Forster, Brian M.; Bitar, Alan Pavinski; Slepkov, Emily R.; Kota, Karthik J.; Sondermann, Holger; Marquis, Hélène

2011-01-01

Listeria monocytogenes is an intracytosolic bacterial pathogen. Among the factors contributing to escape from vacuoles are a phosphatidylcholine phospholipase C (PC-PLC) and a metalloprotease (Mpl). Both enzymes are translocated across the bacterial membrane as inactive proproteins, whose propeptides serve in part to maintain them in association with the bacterium. We have shown that PC-PLC maturation is regulated by Mpl and pH and that Mpl maturation occurs by autocatalysis. In this study, we tested the hypothesis that Mpl activity is pH regulated. To synchronize the effect of pH on bacteria, the cytosolic pH of infected cells was manipulated immediately after radiolabeling de novo-synthesized bacterial proteins. Immunoprecipitation of secreted Mpl from host cell lysates revealed the presence of the propeptide and catalytic domain in samples treated at pH 6.5 but not at pH 7.3. The zymogen was present in small amounts under all conditions. Since proteases often remain associated with their respective propeptide following autocatalysis, we aimed at determining whether pH regulates autocatalysis or secretion of the processed enzyme. For this purpose, we used an Mpl construct that contains a Flag tag at the N terminus of its catalytic domain and antibodies that can distinguish N-terminal and non-N-terminal Flag. By fluorescence microscopy, we observed the Mpl zymogen associated with the bacterium at physiological pH but not following acidification. Mature Mpl was not detected in association with the bacterium at either pH. Using purified proteins, we determined that processing of the PC-PLC propeptide by mature Mpl is also pH sensitive. These results indicate that pH regulates the activity of Mpl on itself and on PC-PLC. PMID:21803995

Neuroserpin Differentiates Between Forms of Tissue Type Plasminogen Activator via pH Dependent Deacylation

PubMed Central

Carlson, Karen-Sue B.; Nguyen, Lan; Schwartz, Kat; Lawrence, Daniel A.; Schwartz, Bradford S.

2016-01-01

Tissue-type plasminogen activator (t-PA), initially characterized for its critical role in fibrinolysis, also has key functions in both physiologic and pathologic processes in the CNS. Neuroserpin (NSP) is a t-PA specific serine protease inhibitor (serpin) found almost exclusively in the CNS that regulates t-PA’s proteolytic activity and protects against t-PA mediated seizure propagation and blood–brain barrier disruption. This report demonstrates that NSP inhibition of t-PA varies profoundly as a function of pH within the biologically relevant pH range for the CNS, and reflects the stability, rather than the formation of NSP: t-PA acyl-enzyme complexes. Moreover, NSP differentiates between the zymogen-like single chain form (single chain t-PA, sct-PA) and the mature protease form (two chain t-PA, tct-PA) of t-PA, demonstrating different pH profiles for protease inhibition, different pH ranges over which catalytic deacylation occurs, and different pH dependent profiles of deacylation rates for each form of t-PA. NSP’s pH dependent inhibition of t-PA is not accounted for by differential acylation, and is specific for the NSP-t-PA serpin-protease pair. These results demonstrate a novel mechanism for the differential regulation of the two forms of t-PA in the CNS, and suggest a potential specific regulatory role for CNS pH in controlling t-PA proteolytic activity. PMID:27378851

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

PubMed Central

Crankshaw, D L; Hetnarski, K; Wilkinson, C F

1979-01-01

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes. Images Fig. 3. PMID:117798

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

PubMed

Crankshaw, D L; Hetnarski, K; Wilkinson, C F

1979-09-01

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

Histone Core Phosphorylation Regulates DNA Accessibility*

PubMed Central

Brehove, Matthew; Wang, Tao; North, Justin; Luo, Yi; Dreher, Sarah J.; Shimko, John C.; Ottesen, Jennifer J.; Luger, Karolin; Poirier, Michael G.

2015-01-01

Nucleosome unwrapping dynamics provide transient access to the complexes involved in DNA transcription, repair, and replication, whereas regulation of nucleosome unwrapping modulates occupancy of these complexes. Histone H3 is phosphorylated at tyrosine 41 (H3Y41ph) and threonine 45 (H3T45ph). H3Y41ph is implicated in regulating transcription, whereas H3T45ph is involved in DNA replication and apoptosis. These modifications are located in the DNA-histone interface near where the DNA exits the nucleosome, and are thus poised to disrupt DNA-histone interactions. However, the impact of histone phosphorylation on nucleosome unwrapping and accessibility is unknown. We find that the phosphorylation mimics H3Y41E and H3T45E, and the chemically correct modification, H3Y41ph, significantly increase nucleosome unwrapping. This enhances DNA accessibility to protein binding by 3-fold. H3K56 acetylation (H3K56ac) is also located in the same DNA-histone interface and increases DNA unwrapping. H3K56ac is implicated in transcription regulation, suggesting that H3Y41ph and H3K56ac could function together. We find that the combination of H3Y41ph with H3K56ac increases DNA accessibility by over an order of magnitude. These results suggest that phosphorylation within the nucleosome DNA entry-exit region increases access to DNA binding complexes and that the combination of phosphorylation with acetylation has the potential to significantly influence DNA accessibility to transcription regulatory complexes. PMID:26175159

Dipeptidyl peptidase 4 - An important digestive peptidase in Tenebrio molitor larvae.

PubMed

Tereshchenkova, Valeriia F; Goptar, Irina A; Kulemzina, Irina A; Zhuzhikov, Dmitry P; Serebryakova, Marina V; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

2016-09-01

Dipeptidyl peptidase 4 (DPP 4) is a proline specific serine peptidase that plays an important role in different regulatory processes in mammals. In this report, we isolated and characterized a unique secreted digestive DPP 4 from the anterior midgut of a stored product pest, Tenebrio molitor larvae (TmDPP 4), with a biological function different than that of the well-studied mammalian DPP 4. The sequence of the purified enzyme was confirmed by mass-spectrometry, and was identical to the translated RNA sequence found in a gut EST database. The purified peptidase was characterized according to its localization in the midgut, and substrate specificity and inhibitor sensitivity were compared with those of human recombinant DPP 4 (rhDPP 4). The T. molitor enzyme was localized mainly in the anterior midgut of the larvae, and 81% of the activity was found in the fraction of soluble gut contents, while human DPP 4 is a membrane enzyme. TmDPP 4 was stable in the pH range 5.0-9.0, with an optimum activity at pH 7.9, similar to human DPP 4. Only specific inhibitors of serine peptidases, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suppressed TmDPP 4 activity, and the specific dipeptidyl peptidase inhibitor vildagliptin was most potent. The highest rate of TmDPP 4 hydrolysis was found for the synthetic substrate Arg-Pro-pNA, while Ala-Pro-pNA was a better substrate for rhDPP 4. Related to its function in the insect midgut, TmDPP 4 efficiently hydrolyzed the wheat storage proteins gliadins, which are major dietary proteins of T. molitor. Published by Elsevier Ltd.

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

PubMed Central

Gjetting, Kisten Sisse Krag; Ytting, Cecilie Karkov; Schulz, Alexander; Fuglsang, Anja Thoe

2012-01-01

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots. PMID:22407646

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor.

PubMed

Gjetting, Kisten Sisse Krag; Ytting, Cecilie Karkov; Schulz, Alexander; Fuglsang, Anja Thoe

2012-05-01

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.