Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

PubMed

Guo, Jinya; Miao, Yansong; Cai, Yi

2017-01-01

Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

Progressive quality control of secretory proteins in the early secretory compartment by ERp44

PubMed Central

Sannino, Sara; Anelli, Tiziana; Cortini, Margherita; Masui, Shoji; Degano, Massimo; Fagioli, Claudio; Inaba, Kenji; Sitia, Roberto

2014-01-01

ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval via interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here we show that also conserved histidines in the C-terminal tail regulate ERp44 in vivo. Mutants lacking these histidines are hyperactive in retaining substrates. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon over-expression of different partners. The ensuing gradients may help optimising folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes. PMID:25097228

Ancestry and evolution of a secretory pathway serpin

PubMed Central

2008-01-01

Background The serpin (serine protease inhibitor) superfamily constitutes a class of functionally highly diverse proteins usually encompassing several dozens of paralogs in mammals. Though phylogenetic classification of vertebrate serpins into six groups based on gene organisation is well established, the evolutionary roots beyond the fish/tetrapod split are unresolved. The aim of this study was to elucidate the phylogenetic relationships of serpins involved in surveying the secretory pathway routes against uncontrolled proteolytic activity. Results Here, rare genomic characters are used to show that orthologs of neuroserpin, a prominent representative of vertebrate group 3 serpin genes, exist in early diverging deuterostomes and probably also in cnidarians, indicating that the origin of a mammalian serpin can be traced back far in the history of eumetazoans. A C-terminal address code assigning association with secretory pathway organelles is present in all neuroserpin orthologs, suggesting that supervision of cellular export/import routes by antiproteolytic serpins is an ancient trait, though subtle functional and compartmental specialisations have developed during their evolution. The results also suggest that massive changes in the exon-intron organisation of serpin genes have occurred along the lineage leading to vertebrate neuroserpin, in contrast with the immediately adjacent PDCD10 gene that is linked to its neighbour at least since divergence of echinoderms. The intron distribution pattern of closely adjacent and co-regulated genes thus may experience quite different fates during evolution of metazoans. Conclusion This study demonstrates that the analysis of microsynteny and other rare characters can provide insight into the intricate family history of metazoan serpins. Serpins with the capacity to defend the main cellular export/import routes against uncontrolled endogenous and/or foreign proteolytic activity represent an ancient trait in eukaryotes that

Ancestry and evolution of a secretory pathway serpin.

PubMed

Kumar, Abhishek; Ragg, Hermann

2008-09-15

The serpin (serine protease inhibitor) superfamily constitutes a class of functionally highly diverse proteins usually encompassing several dozens of paralogs in mammals. Though phylogenetic classification of vertebrate serpins into six groups based on gene organisation is well established, the evolutionary roots beyond the fish/tetrapod split are unresolved. The aim of this study was to elucidate the phylogenetic relationships of serpins involved in surveying the secretory pathway routes against uncontrolled proteolytic activity. Here, rare genomic characters are used to show that orthologs of neuroserpin, a prominent representative of vertebrate group 3 serpin genes, exist in early diverging deuterostomes and probably also in cnidarians, indicating that the origin of a mammalian serpin can be traced back far in the history of eumetazoans. A C-terminal address code assigning association with secretory pathway organelles is present in all neuroserpin orthologs, suggesting that supervision of cellular export/import routes by antiproteolytic serpins is an ancient trait, though subtle functional and compartmental specialisations have developed during their evolution. The results also suggest that massive changes in the exon-intron organisation of serpin genes have occurred along the lineage leading to vertebrate neuroserpin, in contrast with the immediately adjacent PDCD10 gene that is linked to its neighbour at least since divergence of echinoderms. The intron distribution pattern of closely adjacent and co-regulated genes thus may experience quite different fates during evolution of metazoans. This study demonstrates that the analysis of microsynteny and other rare characters can provide insight into the intricate family history of metazoan serpins. Serpins with the capacity to defend the main cellular export/import routes against uncontrolled endogenous and/or foreign proteolytic activity represent an ancient trait in eukaryotes that has been maintained

Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

PubMed Central

Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

2012-01-01

The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

PubMed Central

Courel, Maïté; Vasquez, Michael S.; Hook, Vivian Y.; Mahata, Sushil K.; Taupenot, Laurent

2008-01-01

Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative α-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane. PMID:18299326

Progressive quality control of secretory proteins in the early secretory compartment by ERp44.

PubMed

Sannino, Sara; Anelli, Tiziana; Cortini, Margherita; Masui, Shoji; Degano, Massimo; Fagioli, Claudio; Inaba, Kenji; Sitia, Roberto

2014-10-01

ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes. © 2014. Published by The Company of Biologists Ltd.

Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

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

Faddy, Helen M.; Smart, Chanel E.; Xu, Ren

2008-04-09

The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold duringmore » lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.« less

An evolving paradigm for the secretory pathway?

PubMed Central

Lippincott-Schwartz, Jennifer

2011-01-01

The paradigm that the secretory pathway consists of a stable endoplasmic reticulum and Golgi apparatus, using discrete transport vesicles to exchange their contents, gained important support from groundbreaking biochemical and genetic studies during the 1980s. However, the subsequent development of new imaging technologies with green fluorescent protein introduced data on dynamic processes not fully accounted for by the paradigm. As a result, we may be seeing an example of how a paradigm is evolving to account for the results of new technologies and their new ways of describing cellular processes. PMID:22039065

The secretory pathway at 50: a golden anniversary for some momentous grains of silver.

PubMed

Matlin, Karl S; Caplan, Michael J

2017-01-15

The secretory pathway along which newly synthesized secretory and membrane proteins traffic through the cell was revealed in two articles published 50 years ago. This discovery was the culmination of decades of effort to unite the power of biochemical and morphological methodologies in order to elucidate the dynamic nature of the cell's biosynthetic machinery. The secretory pathway remains a central paradigm of modern cell biology. Its elucidation 50 years ago inspired tremendous multidisciplinary and on-going efforts to understand the machinery that makes it run, the adaptations that permit it to serve the needs of specialized cell types, and the pathological consequences that arise when it is perturbed. © 2017 Matlin and Caplan. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The plant secretory pathway seen through the lens of the cell wall.

PubMed

van de Meene, A M L; Doblin, M S; Bacic, Antony

2017-01-01

Secretion in plant cells is often studied by looking at well-characterised, evolutionarily conserved membrane proteins associated with particular endomembrane compartments. Studies using live cell microscopy and fluorescent proteins have illuminated the highly dynamic nature of trafficking, and electron microscopy studies have resolved the ultrastructure of many compartments. Biochemical and molecular analyses have further informed about the function of particular proteins and endomembrane compartments. In plants, there are over 40 cell types, each with highly specialised functions, and hence potential variations in cell biological processes and cell wall structure. As the primary function of secretion in plant cells is for the biosynthesis of cell wall polysaccharides and apoplastic transport complexes, it follows that utilising our knowledge of cell wall glycosyltransferases (GTs) and their polysaccharide products will inform us about secretion. Indeed, this knowledge has led to novel insights into the secretory pathway, including previously unseen post-TGN secretory compartments. Conversely, our knowledge of trafficking routes of secretion will inform us about polarised and localised deposition of cell walls and their constituent polysaccharides/glycoproteins. In this review, we look at what is known about cell wall biosynthesis and the secretory pathway and how the different approaches can be used in a complementary manner to study secretion and provide novel insights into these processes.

Identification of a novel trafficking pathway exporting a replication protein, Orc2 to nucleus via classical secretory pathway in Plasmodium falciparum.

PubMed

Sharma, Rahul; Sharma, Bhumika; Gupta, Ashish; Dhar, Suman Kumar

2018-05-01

Malaria parasites use an extensive secretory pathway to traffic a number of proteins within itself and beyond. In higher eukaryotes, Endoplasmic Reticulum (ER) membrane bound transcription factors such as SREBP are reported to get processed en route and migrate to nucleus under the influence of specific cues. However, a protein constitutively trafficked to the nucleus via classical secretory pathway has not been reported. Herein, we report the presence of a novel trafficking pathway in an apicomplexan, Plasmodium falciparum where a homologue of an Origin Recognition Complex 2 (Orc2) goes to the nucleus following its association with the ER. Our work highlights the unconventional role of ER in protein trafficking and reports for the first time an ORC homologue getting trafficked through such a pathway to the nucleus where it may be involved in DNA replication and other ancillary functions. Such trafficking pathways may have a profound impact on the cell biology of a malaria parasite and have significant implications in strategizing new antimalarials. Copyright © 2018 Elsevier B.V. All rights reserved.

Golgi-to-plastid trafficking of proteins through secretory pathway: Insights into vesicle-mediated import toward the plastids.

PubMed

Baslam, Marouane; Oikawa, Kazusato; Kitajima-Koga, Aya; Kaneko, Kentaro; Mitsui, Toshiaki

2016-09-01

The diversity of protein targeting pathways to plastids and their regulation in response to developmental and metabolic status is a key issue in the regulation of cellular function in plants. The general import pathways that target proteins into and across the plastid envelope with changes in gene expression are critical for plant development by regulating the response to physiological and metabolic changes within the cell. Glycoprotein targeting to complex plastids involves routing through the secretory pathway, among others. However, the mechanisms of trafficking via this system remain poorly understood. The present article discusses our results in site-specific N-glycosylation of nucleotide pyrophosphatase/phosphodiesterases (NPPs) glycoproteins and highlights protein delivery in Golgi/plastid pathway via the secretory pathway. Furthermore, we outline the hypotheses that explain the mechanism for importing vesicles trafficking with nucleus-encoded proteins into plastids.

A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway

PubMed Central

Westerbeck, Jason W.

2015-01-01

ABSTRACT Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system which require residues in the HD. Here, we use the known structural data from SARS-CoV E to infer the residues important for ion channel activity and the oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells and the residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. IMPORTANCE CoVs are important human pathogens with significant zoonotic potential, as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including the use of attenuated viruses that lack the CoV E protein or express E-protein mutants. However, no approved vaccines or antiviral therapeutics exist. We previously reported that the

Secretory pathway Ca2+ -ATPases promote in vitro microcalcifications in breast cancer cells.

PubMed

Dang, Donna; Prasad, Hari; Rao, Rajini

2017-11-01

Calcification of the breast is often an outward manifestation of underlying molecular changes that drive carcinogenesis. Up to 50% of all non-palpable breast tumors and 90% of ductal carcinoma in situ present with radiographically dense mineralization in mammographic scans. However, surprisingly little is known about the molecular pathways that lead to microcalcifications in the breast. Here, we report on a rapid and quantitative in vitro assay to monitor microcalcifications in breast cancer cell lines, including MCF7, MDA-MB-231, and Hs578T. We show that the Secretory Pathway Ca 2+ -ATPases SPCA1 and SPCA2 are strongly induced under osteogenic conditions that elicit microcalcifications. SPCA gene expression is significantly elevated in breast cancer subtypes that are associated with microcalcifications. Ectopic expression of SPCA genes drives microcalcifications and is dependent on pumping activity. Conversely, knockdown of SPCA expression significantly attenuates formation of microcalcifications. We propose that high levels of SPCA pumps may initiate mineralization in the secretory pathway by elevating luminal Ca 2+ . Our new findings offer mechanistic insight and functional implications on a widely observed, yet poorly understood radiographic signature of breast cancer. © 2017 Wiley Periodicals, Inc.

Analysis of Protein Localization and Secretory Pathway Function Using the Yeast "Saccharomyces Cerevisiae"

ERIC Educational Resources Information Center

Vallen, Elizabeth

2002-01-01

The isolation and characterization of mutants has been crucial in understanding a number of processes in the field of cell biology. In this exercise, students examine the effects of mutations in the secretory pathway on protein localization. Yeast strains deficient for synthesis of histidinol dehydrogenase are transformed with a plasmid encoding a…

Calcium-containing phosphopeptides pave the secretory pathway for efficient protein traffic and secretion in fungi.

PubMed

Martín, Juan F

2014-09-10

Casein phosphopeptides (CPPs) containing chelated calcium drastically increase the secretion of extracellular homologous and heterologous proteins in filamentous fungi. Casein phosphopeptides released by digestion of alpha - and beta-casein are rich in phosphoserine residues (SerP). They stimulate enzyme secretion in the gastrointestinal tract and enhance the immune response in mammals, and are used as food supplements. It is well known that casein phosphopeptides transport Ca2+ across the membranes and play an important role in Ca2+ homeostasis in the cells. Addition of CPPs drastically increases the production of heterologous proteins in Aspergillus as host for industrial enzyme production. Recent proteomics studies showed that CPPs alter drastically the vesicle-mediated secretory pathway in filamentous fungi, apparently because they change the calcium concentration in organelles that act as calcium reservoirs. In the organelles calcium homeostasis a major role is played by the pmr1 gene, that encodes a Ca2+/Mn2+ transport ATPase, localized in the Golgi complex; this transporter controls the balance between intra-Golgi and cytoplasmic Ca2+ concentrations. A Golgi-located casein kinase (CkiA) governs the ER to Golgi directionality of the movement of secretory proteins by interacting with the COPII coat of secretory vesicles when they reach the Golgi. Mutants defective in the casein-2 kinase CkiA show abnormal targeting of some secretory proteins, including cytoplasmic membrane amino acid transporters that in ckiA mutants are miss-targeted to vacuolar membranes. Interestingly, addition of CPPs increases a glyceraldehyde-3-phpshate dehydrogenase protein that is known to associate with microtubules and act as a vesicle/membrane fusogenic agent. In summary, CPPs alter the protein secretory pathway in fungi adapting it to a deregulated protein traffic through the organelles and vesicles what results in a drastic increase in secretion of heterologous and also of

Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT20 cells.

PubMed

Tooze, J; Tooze, S A; Fuller, S D

1987-09-01

Murine hepatitis virus (strain A59), (MHV-A59) is a coronavirus that buds into pre-Golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. The fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. MHV-A59 also infects, albeit inefficiently, AtT20 cells, murine pituitary tumor cells with a regulated as well as a constitutive exocytic pathway. Here we examine AtT20 cells at early times after the infection, when the Golgi apparatus retains its morphological and biochemical integrity. We observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same Golgi stacks and accumulate in the trans-Golgi network. Their pathways diverge at this site, the condensed secretory proteins including the ACTH going to the secretory granules and the coronavirus to post-Golgi transport vesicles devoid of ACTH. On very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-Golgi vesicles. We conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein ACTH, diverge at the exit from the trans-Golgi network.

Copper transport into the secretory pathway is regulated by oxygen in macrophages

PubMed Central

White, Carine; Kambe, Taiho; Fulcher, Yan G.; Sachdev, Sherri W.; Bush, Ashley I.; Fritsche, Kevin; Lee, Jaekwon; Quinn, Thomas P.; Petris, Michael J.

2009-01-01

Summary Copper is an essential nutrient for a variety of biochemical processes; however, the redox properties of copper also make it potentially toxic in the free form. Consequently, the uptake and intracellular distribution of this metal is strictly regulated. This raises the issue of whether specific pathophysiological conditions can promote adaptive changes in intracellular copper distribution. In this study, we demonstrate that oxygen limitation promotes a series of striking alterations in copper homeostasis in RAW264.7 macrophage cells. Hypoxia was found to stimulate copper uptake and to increase the expression of the copper importer, CTR1. This resulted in increased copper delivery to the ATP7A copper transporter and copper-dependent trafficking of ATP7A to cytoplasmic vesicles. Significantly, the ATP7A protein was required to deliver copper into the secretory pathway to ceruloplasmin, a secreted copperdependent enzyme, the expression and activity of which were stimulated by hypoxia. However, the activities of the alternative targets of intracellular copper delivery, superoxide dismutase and cytochrome c oxidase, were markedly reduced in response to hypoxia. Collectively, these findings demonstrate that copper delivery into the biosynthetic secretory pathway is regulated by oxygen availability in macrophages by a selective increase in copper transport involving ATP7A. PMID:19351718

Characterization of new mutants in the early part of the yeast secretory pathway isolated by a (/sup 3/H)mannose suicide selection

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

Newman, A.P.; Ferro-Novick, S.

We have adapted a (/sup 3/H)mannose suicide selection to identify mutations in additional genes which function in the early part of the yeast secretory pathway. Thus far this protocol has led to the identification of two new genes which are implicated in this process, as well as additional alleles of previously identified genes. The new mutants, bet1 and bet2, are temperature sensitive for growth and protein transport. Thin section analysis has revealed the accumulation of a network of endoplasmic reticulum (ER) at the restrictive temperature (37/sup 0/C). Precursors of exported proteins that accumulate in the cell at 37/sup 0/C aremore » terminally core glycosylated. These observations suggest that the transport of precursors is blocked subsequent to translocation into the ER but before entry into the Golgi apparatus. The bet1 and bet2 mutants define two new complementation groups which have the same properties as previously identified ER-accumulating mutants. This and previous findings suggest that protein exit from the ER and entry into the Golgi apparatus is a complex process requiring at least 11 genes.« less

A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

PubMed

Hood-Degrenier, Jennifer K

2008-01-01

The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

The cargo receptor p24A facilitates calcium sensing receptor maturation and stabilization in the early secretory pathway

PubMed Central

Stepanchick, Ann; Breitwieser, Gerda E.

2010-01-01

The calcium sensing receptor (CaSR) is a Family 3/C G protein-coupled receptor with slow and partial targeting to the plasma membrane in both native and heterologous cells. We identified cargo receptor family member p24A in yeast two-hybrid screens with the CaSR carboxyl terminus. Interactions were confirmed by immunoprecipitation of either p24A or CaSR in transiently transfected HEK293 cells. Only the immaturely glycosylated form of CaSR interacts with p24A. Dissociation likely occurs in the endoplasmic reticulum Golgi intermediate compartment (ERGIC) or cis-Golgi, since only the uncleaved form of a CaSR mutant sensitive to the trans-Golgi enzyme furin was coimmunoprecipitated with p24A. p24A and p24A(ΔGOLD) significantly increased total and plasma membrane CaSR protein but p24A(FF/AA) did not. The CaSR carboxyl terminus distal to T868 is required for differential sensitivity to p24A and its mutants. Interaction with p24A therefore increases CaSR stability in the ER and enhances plasma membrane targeting. Neither wt Sar1p or the T39N mutant increased CaSR maturation or abundance while the H79G mutant increased abundance but prevented maturation of CaSR. These results suggest that p24A is the limiting factor in CaSR trafficking in the early secretory pathway, and that cycling between the ER and ERGIC protects CaSR from degradation. PMID:20361938

The Ca2+/H+ antiporter TMEM165 expression, localization in the developing, lactating and involuting mammary gland parallels the secretory pathway Ca2+ATPase (SPCA1)

USDA-ARS?s Scientific Manuscript database

Plasma membrane Ca2+-ATPase 2 (PMCA2) knockout mice showed that ~ 60 % of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca2+-ATPase’s 1 and/or 2 (SP...

Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway

PubMed Central

Hatori, Yuta; Yan, Ye; Schmidt, Katharina; Furukawa, Eri; Hasan, Nesrin M.; Yang, Nan; Liu, Chin-Nung; Sockanathan, Shanthini; Lutsenko, Svetlana

2016-01-01

Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transition from a broad range of redox states to a uniformly reducing cytosol facilitates reduction of the copper chaperone Atox1, liberating its metal-binding site. Concomitantly, expression of Atox1 and its partner, a copper transporter ATP7A, is upregulated. These events produce a higher flux of copper through the secretory pathway that balances copper in the cytosol and increases supply of the cofactor to copper-dependent enzymes, expression of which is elevated in differentiated neurons. Direct link between glutathione oxidation and copper compartmentalization allows for rapid metabolic adjustments essential for normal neuronal function. PMID:26879543

Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway.

PubMed

Hatori, Yuta; Yan, Ye; Schmidt, Katharina; Furukawa, Eri; Hasan, Nesrin M; Yang, Nan; Liu, Chin-Nung; Sockanathan, Shanthini; Lutsenko, Svetlana

2016-02-16

Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transition from a broad range of redox states to a uniformly reducing cytosol facilitates reduction of the copper chaperone Atox1, liberating its metal-binding site. Concomitantly, expression of Atox1 and its partner, a copper transporter ATP7A, is upregulated. These events produce a higher flux of copper through the secretory pathway that balances copper in the cytosol and increases supply of the cofactor to copper-dependent enzymes, expression of which is elevated in differentiated neurons. Direct link between glutathione oxidation and copper compartmentalization allows for rapid metabolic adjustments essential for normal neuronal function.

The golgin GMAP-210 is required for efficient membrane trafficking in the early secretory pathway

PubMed Central

Roboti, Peristera; Sato, Keisuke; Lowe, Martin

2015-01-01

Golgins are coiled-coil proteins that participate in membrane-tethering events at the Golgi complex. Golgin-mediated tethering is thought to be important for vesicular trafficking and Golgi organization. However, the degree to which individual golgins contribute to these processes is poorly defined, and it has been proposed that golgins act in a largely redundant manner. Previous studies on the golgin GMAP-210 (also known as TRIP11), which is mutated in the rare skeletal disorder achondrogenesis type 1A, have yielded conflicting results regarding its involvement in trafficking. Here, we re-investigated the trafficking role of GMAP-210, and found that it is indeed required for efficient trafficking in the secretory pathway. GMAP-210 acts at both the endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) and Golgi complex during anterograde trafficking, and is also required for retrograde trafficking to the ER. Using co-depletion experiments, we also found that GMAP-210 acts in a partially redundant manner with the golgin GM130 to ensure efficient anterograde cargo delivery to the cis-Golgi. In summary, our results indicate a role for GMAP-210 in several trafficking steps at the ER–Golgi interface, some of which are partially redundant with another golgin, namely GM130 (also known as GOLGA2). PMID:25717001

The complete general secretory pathway in gram-negative bacteria.

PubMed Central

Pugsley, A P

1993-01-01

The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins. PMID:8096622

Sorting of the neuroendocrine secretory protein Secretogranin II into the regulated secretory pathway: role of N- and C-terminal alpha-helical domains.

PubMed

Courel, Maïté; Vasquez, Michael S; Hook, Vivian Y; Mahata, Sushil K; Taupenot, Laurent

2008-04-25

Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative alpha-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane.

Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition

PubMed Central

Stoffel, Wilhelm; Hammels, Ina; Jenke, Bitta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Schauss, Astrid; Etich, Julia; Heilig, Juliane; Zaucke, Frank

2016-01-01

Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus–pituitary–somatotropic hypoplasia. The unbiased smpd3−/− mouse mutant and derived smpd3−/− primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3−/− mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition. PMID:27882938

Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition.

PubMed

Stoffel, Wilhelm; Hammels, Ina; Jenke, Bitta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Schauss, Astrid; Etich, Julia; Heilig, Juliane; Zaucke, Frank

2016-11-24

Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus-pituitary-somatotropic hypoplasia. The unbiased smpd3-/- mouse mutant and derived smpd3-/- primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3-/- mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition.

Quality control in the secretory assembly line.

PubMed Central

Helenius, A

2001-01-01

As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway. PMID:11260794

A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

ERIC Educational Resources Information Center

Hood-DeGrenier, Jennifer K.

2008-01-01

The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

The regulated secretory pathway and human disease: insights from gene variants and single nucleotide polymorphisms.

PubMed

Lin, Wei-Jye; Salton, Stephen R

2013-01-01

The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired.

CREB3L1-mediated functional and structural adaptation of the secretory pathway in hormone-stimulated thyroid cells.

PubMed

García, Iris A; Torres Demichelis, Vanina; Viale, Diego L; Di Giusto, Pablo; Ezhova, Yulia; Polishchuk, Roman S; Sampieri, Luciana; Martinez, Hernán; Sztul, Elizabeth; Alvarez, Cecilia

2017-12-15

Many secretory cells increase the synthesis and secretion of cargo proteins in response to specific stimuli. How cells couple increased cargo load with a coordinate rise in secretory capacity to ensure efficient transport is not well understood. We used thyroid cells stimulated with thyrotropin (TSH) to demonstrate a coordinate increase in the production of thyroid-specific cargo proteins and ER-Golgi transport factors, and a parallel expansion of the Golgi complex. TSH also increased expression of the CREB3L1 transcription factor, which alone caused amplified transport factor levels and Golgi enlargement. Furthermore, CREB3L1 potentiated the TSH-induced increase in Golgi volume. A dominant-negative CREB3L1 construct hampered the ability of TSH to induce Golgi expansion, implying that this transcription factor contributes to Golgi expansion. Our findings support a model in which CREB3L1 acts as a downstream effector of TSH to regulate the expression of cargo proteins, and simultaneously increases the synthesis of transport factors and the expansion of the Golgi to synchronize the rise in cargo load with the amplified capacity of the secretory pathway. © 2017. Published by The Company of Biologists Ltd.

A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum

PubMed Central

1987-01-01

We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation. PMID:3305520

The Regulated Secretory Pathway and Human Disease: Insights from Gene Variants and Single Nucleotide Polymorphisms

PubMed Central

Lin, Wei-Jye; Salton, Stephen R.

2013-01-01

The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired. PMID:23964269

Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

NASA Astrophysics Data System (ADS)

Rietdorf, Jens; Stephens, David J.; Squire, Anthony; Simpson, Jeremy; Shima, David T.; Paccaud, Jean-Pierre; Bastiaens, Philippe I.; Pepperkok, Rainer

2000-04-01

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

PubMed Central

Bowen, Aaron B; Bourke, Ashley M; Hiester, Brian G; Hanus, Cyril

2017-01-01

Neurons face the challenge of regulating the abundance, distribution and repertoire of integral membrane proteins within their immense, architecturally complex dendritic arbors. While the endoplasmic reticulum (ER) supports dendritic translation, most dendrites lack the Golgi apparatus (GA), an essential organelle for conventional secretory trafficking. Thus, whether secretory cargo is locally trafficked in dendrites through a non-canonical pathway remains a fundamental question. Here we define the dendritic trafficking itinerary for key synaptic molecules in rat cortical neurons. Following ER exit, the AMPA-type glutamate receptor GluA1 and neuroligin 1 undergo spatially restricted entry into the dendritic secretory pathway and accumulate in recycling endosomes (REs) located in dendrites and spines before reaching the plasma membrane. Surprisingly, GluA1 surface delivery occurred even when GA function was disrupted. Thus, in addition to their canonical role in protein recycling, REs also mediate forward secretory trafficking in neuronal dendrites and spines through a specialized GA-independent trafficking network. PMID:28875935

Relationships of the early insulin secretory response and oral disposition index with gastric emptying in subjects with normal glucose tolerance.

PubMed

Marathe, Chinmay S; Rayner, Christopher K; Lange, Kylie; Bound, Michelle; Wishart, Judith; Jones, Karen L; Kahn, Steven E; Horowitz, Michael

2017-02-01

The oral disposition index, the product of the early insulin secretory response during an oral glucose tolerance test and insulin sensitivity, is used widely for both the prediction of, and evaluation of the response to interventions, in type 2 diabetes. Gastric emptying, which determines small intestinal exposure of nutrients, modulates postprandial glycemia. The aim of this study was to determine whether the insulin secretory response and the disposition index (DI) related to gastric emptying in subjects with normal glucose tolerance. Thirty-nine subjects consumed a 350 mL drink containing 75 g glucose labeled with 99m Tc-sulfur colloid. Gastric emptying (by scintigraphy), blood glucose (G) and plasma insulin (I) were measured between t  = 0-120 min. The rate of gastric emptying was derived from the time taken for 50% emptying ( T 50 ) and expressed as kcal/min. The early insulin secretory response was estimated by the ratio of the change in insulin (∆I 0-30 ) to that of glucose at 30 min (∆G 0-30 ) represented as ∆I 0-30 /∆G 0-30 Insulin sensitivity was estimated as 1/fasting insulin and the DI was then calculated as ∆I 0-30 /∆G 0-30  × 1/fasting insulin. There was a direct relationship between ∆G 0-30 and gastric emptying ( r  = 0.47, P  = 0.003). While there was no association of either ∆I 0-30 ( r  = -0.16, P  = 0.34) or fasting insulin ( r  = 0.21, P  = 0.20), there were inverse relationships between the early insulin secretory response ( r  = -0.45, P  = 0.004) and the DI ( r  = -0.33, P  = 0.041), with gastric emptying. We conclude that gastric emptying is associated with both insulin secretion and the disposition index in subjects with normal glucose tolerance, such that when gastric emptying is relatively more rapid, both the early insulin secretory response and the disposition index are less. These findings should be interpreted as "hypothesis generating" and provide the rationale for longitudinal studies to

Pro-hormone Secretogranin II Regulates Dense Core Secretory Granule Biogenesis in Catecholaminergic Cells*

PubMed Central

Courel, Maïté; Soler-Jover, Alex; Rodriguez-Flores, Juan L.; Mahata, Sushil K.; Elias, Salah; Montero-Hadjadje, Maïté; Anouar, Youssef; Giuly, Richard J.; O'Connor, Daniel T.; Taupenot, Laurent

2010-01-01

Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H+-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network. PMID:20061385

Pro-hormone secretogranin II regulates dense core secretory granule biogenesis in catecholaminergic cells.

PubMed

Courel, Maïté; Soler-Jover, Alex; Rodriguez-Flores, Juan L; Mahata, Sushil K; Elias, Salah; Montero-Hadjadje, Maïté; Anouar, Youssef; Giuly, Richard J; O'Connor, Daniel T; Taupenot, Laurent

2010-03-26

Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H(+)-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network.

The evolution of plant secretory structures and emergence of terpenoid chemical diversity.

PubMed

Lange, Bernd Markus

2015-01-01

Secretory structures in terrestrial plants appear to have first emerged as intracellular oil bodies in liverworts. In vascular plants, internal secretory structures, such as resin ducts and laticifers, are usually found in conjunction with vascular bundles, whereas subepidermal secretory cavities and epidermal glandular trichomes generally have more complex tissue distribution patterns. The primary function of plant secretory structures is related to defense responses, both constitutive and induced, against herbivores and pathogens. The ability to sequester secondary (or specialized) metabolites and defense proteins in secretory structures was a critical adaptation that shaped plant-herbivore and plant-pathogen interactions. Although this review places particular emphasis on describing the evolution of pathways leading to terpenoids, it also assesses the emergence of other metabolite classes to outline the metabolic capabilities of different plant lineages.

Regulated and constitutive protein targeting can be distinguished by secretory polarity in thyroid epithelial cells

PubMed Central

1991-01-01

We have studied concurrent apical/basolateral and regulated/constitutive secretory targeting in filter-grown thyroid epithelial monolayers in vitro, by following the exocytotic routes of two newly synthesized endogenous secretory proteins, thyroglobulin (Tg) and p500. Tg is a regulated secretory protein as indicated by its acute secretory response to secretagogues. Without stimulation, pulse-labeled Tg exhibits primarily two kinetically distinct routes: less than or equal to 80% is released in an apical secretory phase which is largely complete by 6-10 h, with most of the remaining Tg retained in intracellular storage from which delayed apical discharge is seen. The rapid export observed for most Tg is unlikely to be because of default secretion, since its apical polarity is preserved even during the period (less than or equal to 10 h) when p500 is released basolaterally by a constitutive pathway unresponsive to secretagogues. p500 also exhibits a second, kinetically distinct secretory route: at chase times greater than 10 h, a residual fraction (less than or equal to 8%) of p500 is secreted with an apical preponderance similar to that of Tg. It appears that this fraction of p500 has failed to be excluded from the regulated pathway, which has a predetermined apical polarity. From these data we hypothesize that a targeting hierarchy may exist in thyroid epithelial cells such that initial sorting to the regulated pathway may be a way of insuring apical surface delivery from one of two possible exocytotic routes originating in the immature storage compartment. PMID:1991788

Unconventional secretion of FABP4 by endosomes and secretory lysosomes.

PubMed

Villeneuve, Julien; Bassaganyas, Laia; Lepreux, Sebastien; Chiritoiu, Marioara; Costet, Pierre; Ripoche, Jean; Malhotra, Vivek; Schekman, Randy

2018-02-05

An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum-Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion. © 2018 Villeneuve et al.

A senescence secretory switch mediated by PI3K/AKT/mTOR activation controls chemoprotective endothelial secretory responses

PubMed Central

Bent, Eric H.; Gilbert, Luke A.; Hemann, Michael T.

2016-01-01

Cancer therapy targets malignant cells that are surrounded by a diverse complement of nonmalignant stromal cells. Therapy-induced damage of normal cells can alter the tumor microenvironment, causing cellular senescence and activating cancer-promoting inflammation. However, how these damage responses are regulated (both induced and resolved) to preserve tissue homeostasis and prevent chronic inflammation is poorly understood. Here, we detail an acute chemotherapy-induced secretory response that is self-limiting in vitro and in vivo despite the induction of cellular senescence. We used tissue-specific knockout mice to demonstrate that endothelial production of the proinflammatory cytokine IL-6 promotes chemoresistance and show that the chemotherapeutic doxorubicin induces acute IL-6 release through reactive oxygen species-mediated p38 activation in vitro. Doxorubicin causes endothelial senescence but, surprisingly, without a typical senescence secretory response. We found that endothelial cells repress senescence-associated inflammation through the down-regulation of PI3K/AKT/mTOR signaling and that reactivation of this pathway restores senescence-associated inflammation. Thus, we describe a mechanism by which damage-associated paracrine secretory responses are restrained to preserve tissue homeostasis and prevent chronic inflammation. PMID:27566778

Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

PubMed Central

Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

2013-01-01

The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

[Expansion of secretory cells in the fallopian tubal epithelium in the early stages of the pathogenesis of ovarian serous carcinomas].

PubMed

Asaturova, A V; Ezhova, L S; Faizullina, N M; Adamyan, L V; Khabas, G N; Sannikova, M V

to investigate the frequency of the types of fallopian tubal secretory cell expansion (SCE) in diseases of the reproductive organs and to determine the immunophenotype and biological role of the cells in the early stages of the pathogenesis of high-grade ovarian serous carcinomas (HGOSC). The investigation enrolled 287 patients with extraovarian diseases and ovarian serous tumors varying in grade, whose fallopian tubes were morphologically and immunohistochemically examined using p53, Ki-67, PAX2, Bcl-2, beta-catenin, and ALDH1 markers. The material was statistically processed applying the Mann-Whitney test and χ2 test. The rate of secretory cell proliferation (SCP) (more than 10 consecutive secretory cells) and that of secretory cell overgrowth (SCO) (more than 30 consecutive secretory cells) increase with age in all investigated reproductive system diseases. The rate of SCP in the corpus fimbriatum of the patients with HGOSC was 5.9 times higher than that in those with extraovarian disease (p<0.01); when comparing the same patient groups, that of SCO was 3.4 times higher (p<0.05). The immunohistochemical characteristics of the investigated lesions (in scores) were as follows: PAX2 was expressed in the intact epithelium (2.8), in SCP (1.3), in SCO (1.2), in serous tubal intraepithelial carcinoma (STIC) (1.0), and in HGOSC (0.9); Bcl-2 was in the intact epithelium (2.2), in SCP (2.1), STIC (0.9), and in HGOSC (0.6), β-catenin was in the intact epithelium (0.5), in SCP (2.85), in SCO (2.95), in STIC (0.6), and in HGOSC (0.5); ALDH1 was in the intact epithelium (0.5), in SCP (2.91), in SCO (2.92), in STIC (1.2), and in HGOSC (0.6). There were statistically significant differences with a 95% confidence interval (p<0.05) for: 1) PAX2 between the intact epithelium and pathology (fallopian tube lesions and HGOSC); 2) Bcl-2 between the intact epithelium and SCE (SCP and SCO) and between SCE and HGOSC; 3) beta-catenin between the intact epithelium and SCE (SCP and SCO

The Exosome Secretory Pathway Transports Amyloid Precursor Protein Carboxyl-terminal Fragments from the Cell into the Brain Extracellular Space*

PubMed Central

Perez-Gonzalez, Rocio; Gauthier, Sebastien A.; Kumar, Asok; Levy, Efrat

2012-01-01

In vitro studies have shown that neuronal cell cultures secrete exosomes containing amyloid-β precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-β (Aβ). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphology were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were observed in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the number of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs associated with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and Aβ, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted Aβ, is harmful to the brain. PMID:23129776

[Semiquantitative measurement of progesterone receptors in luteal-phase-defect endometrial cells during secretory phase].

PubMed

Ma, Q; Han, Z; Huang, W

1998-03-01

To investigate the changes of endometrial progesterone receptor (PR) of luteal-phase-defect (LPD) patients during the secretory phase, thirteen patients with complaints of infertility or habitual abortion were studied. During the early-mid secretory phase, endometrial tissue was obtained by dilatation and curettage (D & C) for histological and receptor study: meanwhile serum E2, P, FSH, LH and PRL were measured. Based on histologic diagnosis, the patients were divided into two groups: the LPD group (n = 7) and the normal control group(n = 6). PR content was determined by immunohisto-chemical (IHC) assay. The results showed that during the early-mid luteal phase a significantly low PR content on endometrial glandular nucleus was observed in LPD group, compared with normal control(6.75 +/- 2.57 vs 9.50 +/- 1.64 P < 0.05), but no difference in serum progesterone was noted between the two groups. These findings suggest that during early-mid secretory phase, PR content on endometrial glandular nucleus decreases in LPD cases, which results in deficient response of endometrium to proper stimulus of progesterone. This change may cause endometrial secretory deficiency and blockade of embreyo implantation. That is why infertility or habitual abortion happened.

The progamic phase of an early-divergent angiosperm, Annona cherimola (Annonaceae)

PubMed Central

Lora, J.; Hormaza, J. I.; Herrero, M.

2010-01-01

Background and Aims Recent studies of reproductive biology in ancient angiosperm lineages are beginning to shed light on the early evolution of flowering plants, but comparative studies are restricted by fragmented and meagre species representation in these angiosperm clades. In the present study, the progamic phase, from pollination to fertilization, is characterized in Annona cherimola, which is a member of the Annonaceae, the largest extant family among early-divergent angiosperms. Beside interest due to its phylogenetic position, this species is also an ancient crop with a clear niche for expansion in subtropical climates. Methods The kinetics of the reproductive process was established following controlled pollinations and sequential fixation. Gynoecium anatomy, pollen tube pathway, embryo sac and early post-fertilization events were characterized histochemically. Key Results A plesiomorphic gynoecium with a semi-open carpel shows a continuous secretory papillar surface along the carpel margins, which run from the stigma down to the obturator in the ovary. The pollen grains germinate in the stigma and compete in the stigma-style interface to reach the narrow secretory area that lines the margins of the semi-open stylar canal and is able to host just one to three pollen tubes. The embryo sac has eight nuclei and is well provisioned with large starch grains that are used during early cellular endosperm development. Conclusions A plesiomorphic simple gynoecium hosts a simple pollen–pistil interaction, based on a support–control system of pollen tube growth. Support is provided through basipetal secretory activity in the cells that line the pollen tube pathway. Spatial constraints, favouring pollen tube competition, are mediated by a dramatic reduction in the secretory surface available for pollen tube growth at the stigma–style interface. This extramural pollen tube competition contrasts with the intrastylar competition predominant in more recently derived

Dynamic Regulation of Ero1α and Peroxiredoxin 4 Localization in the Secretory Pathway*

PubMed Central

Kakihana, Taichi; Araki, Kazutaka; Vavassori, Stefano; Iemura, Shun-ichiro; Cortini, Margherita; Fagioli, Claudio; Natsume, Tohru; Sitia, Roberto; Nagata, Kazuhiro

2013-01-01

In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis. PMID:23979138

ER stress and Parkinson's disease: Pathological inputs that converge into the secretory pathway.

PubMed

Mercado, Gabriela; Castillo, Valentina; Soto, Paulina; Sidhu, Anita

2016-10-01

The major clinical feature of Parkinson's disease (PD) is impairment in motor control as a result of extensive dopaminergic neuron loss in the substantia nigra pars compacta. The central pathological hallmark of PD is the formation of neuronal cytoplasmic inclusions of insoluble proteins called Lewy bodies, of which fibrillar aggregates of misfolded αSynuclein are the major components. Despite intense research on the pathogenic mechanism that trigger neuronal loss and disease progression, the neurogenesis of PD remains unknown. However, studies on genetics of PD have identified specific genes and proteins linked to this disease. Genetic mutations linked with different forms of familial PD have unveiled a closer relationship between pathology and impairments at different points in the secretory pathway. Accumulation of misfolded/unfolded proteins in the endoplasmic reticulum and disruptions in protein clearance mechanisms result in activation of an adaptive stress pathway known as the unfolded protein response (UPR). UPR signaling is mediated by three stress sensors that induce independent and convergent signaling branches that help to maintain homeostasis, or eventually trigger cell death under chronic stress conditions. Signs of ER stress are observed in post-mortem tissue from sporadic human PD cases and in most animal models of the disease, implicating all three branches of this cellular response. However, the exact contribution of the UPR in the progression of PD or in dopaminergic neuron survival is not yet well understood. A large number of studies reveal a clear activation of the UPR in toxicological models resembling sporadic PD, where ATF6, XBP1 and CHOP have a functional role in controlling dopaminergic neuron survival in neurotoxin-based models of PD in vivo. Also pharmacological and gene therapy approaches aimed to target different points of this pathway have revealed an important functional role in PD pathogenesis. This article is part of a Special

A Dynamic Study of Protein Secretion and Aggregation in the Secretory Pathway

PubMed Central

Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; Anelli, Tiziana

2014-01-01

Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC. PMID:25279560

A dynamic study of protein secretion and aggregation in the secretory pathway.

PubMed

Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; Sitia, Roberto; Anelli, Tiziana

2014-01-01

Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

Evidence for possible non-canonical pathway(s) driven early-onset colorectal cancer in India

PubMed Central

Raman, Ratheesh; Kotapalli, Viswakalyan; Adduri, Raju; Gowrishankar, Swarnalata; Bashyam, Leena; Chaudhary, Ajay; Vamsy, Mohana; Patnaik, Sujith; Srinivasulu, Mukta; Sastry, Regulagadda; Rao, Subramanyeshwar; Vasala, Anjayneyulu; Kalidindi, NarasimhaRaju; Pollack, Jonathan; Murthy, Sudha; Bashyam, Murali

2012-01-01

Two genetic instability pathways viz. chromosomal instability, driven primarily by APC mutation induced deregulated Wnt signaling, and microsatellite instability (MSI) caused by mismatch repair (MMR) inactivation, together account for greater than 90% of late-onset colorectal cancer. Our understanding of early-onset sporadic CRC is however comparatively limited. In addition, most seminal studies have been performed in the western population and analyses of tumorigenesis pathway(s) causing CRC in developing nations have been rare. We performed a comparative analysis of early and late-onset CRC from India with respect to common genetic aberrations including Wnt, KRAS and p53 (constituting the classical CRC progression sequence) in addition to MSI. Our results revealed the absence of Wnt and MSI in a significant proportion of early-onset as against late-onset CRC in India. In addition, KRAS mutation frequency was significantly lower in early-onset CRC indicating that a significant proportion of CRC in India may follow tumorigenesis pathways distinct from the classical CRC progression sequence. Our study has therefore revealed the possible existence of non-canonical tumorigenesis pathways in early-onset CRC in India. PMID:23168910

Implementing Guided Pathways: Early Insights from the AACC Pathways Colleges. Executive Summary

ERIC Educational Resources Information Center

Jenkins, Davis; Lahr, Hana; Fink, John

2017-01-01

This summary is a digest of key findings from "Implementing Guided Pathways: Early Insights From the AACC Pathways Colleges," a report based on Community College Research Center's (CCRC's) research on the 30 colleges involved in the American Association of Community Colleges' (AACC) Pathways Project. The research presented in this report…

COPI selectively drives maturation of the early Golgi

PubMed Central

Papanikou, Effrosyni; Day, Kasey J; Austin, Jotham; Glick, Benjamin S

2015-01-01

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins. DOI: http://dx.doi.org/10.7554/eLife.13232.001 PMID:26709839

COPI selectively drives maturation of the early Golgi

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

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

COPI selectively drives maturation of the early Golgi

DOE PAGES

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham; ...

2015-12-28

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

Uncoupling of Secretion From Growth in Some Hormone Secretory Tissues

PubMed Central

2014-01-01

Context: Most syndromes with benign primary excess of a hormone show positive coupling of hormone secretion to size or proliferation in the affected hormone secretory tissue. Syndromes that lack this coupling seem rare and have not been examined for unifying features among each other. Evidence Acquisition: Selected clinical and basic features were analyzed from original reports and reviews. We examined indices of excess secretion of a hormone and indices of size of secretory tissue within the following three syndromes, each suggestive of uncoupling between these two indices: familial hypocalciuric hypercalcemia, congenital diazoxide-resistant hyperinsulinism, and congenital primary hyperaldosteronism type III (with G151E mutation of the KCNJ5 gene). Evidence Synthesis: Some unifying features among the three syndromes were different from features present among common tumors secreting the same hormone. The unifying and distinguishing features included: 1) expression of hormone excess as early as the first days of life; 2) normal size of tissue that oversecretes a hormone; 3) diffuse histologic expression in the hormonal tissue; 4) resistance to treatment by subtotal ablation of the hormone-secreting tissue; 5) causation by a germline mutation; 6) low potential of the same mutation to cause a tumor by somatic mutation; and 7) expression of the mutated molecule in a pathway between sensing of a serum metabolite and secretion of hormone regulating that metabolite. Conclusion: Some shared clinical and basic features of uncoupling of secretion from size in a hormonal tissue characterize three uncommon states of hormone excess. These features differ importantly from features of common hormonal neoplasm of that tissue. PMID:25004249

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

PubMed Central

de Wilde, Adriaan H.; Wannee, Kazimier F.; Scholte, Florine E. M.; Goeman, Jelle J.; ten Dijke, Peter; Snijder, Eric J.

2015-01-01

ABSTRACT To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although

Ovulation in Drosophila is controlled by secretory cells of the female reproductive tract

PubMed Central

Sun, Jianjun; Spradling, Allan C

2013-01-01

How oocytes are transferred into an oviduct with a receptive environment remains poorly known. We found that glands of the Drosophila female reproductive tract, spermathecae and/or parovaria, are required for ovulation and to promote sperm storage. Reducing total secretory cell number by interferring with Notch signaling during development blocked ovulation. Knocking down expression after adult eclosion of the nuclear hormone receptor Hr39, a master regulator of gland development, slowed ovulation and blocked sperm storage. However, ovulation (but not sperm storage) continued when only canonical protein secretion was compromised in adult glands. Our results imply that proteins secreted during adulthood by the canonical secretory pathway from female reproductive glands are needed to store sperm, while a non-canonical glandular secretion stimulates ovulation. Our results suggest that the reproductive tract signals to the ovary using glandular secretions, and that this pathway has been conserved during evolution. DOI: http://dx.doi.org/10.7554/eLife.00415.001 PMID:23599892

Reduced β-Cell Secretory Capacity in Pancreatic-Insufficient, but Not Pancreatic-Sufficient, Cystic Fibrosis Despite Normal Glucose Tolerance.

PubMed

Sheikh, Saba; Gudipaty, Lalitha; De Leon, Diva D; Hadjiliadis, Denis; Kubrak, Christina; Rosenfeld, Nora K; Nyirjesy, Sarah C; Peleckis, Amy J; Malik, Saloni; Stefanovski, Darko; Cuchel, Marina; Rubenstein, Ronald C; Kelly, Andrea; Rickels, Michael R

2017-01-01

Patients with pancreatic-insufficient cystic fibrosis (PI-CF) are at increased risk for developing diabetes. We determined β-cell secretory capacity and insulin secretory rates from glucose-potentiated arginine and mixed-meal tolerance tests (MMTTs), respectively, in pancreatic-sufficient cystic fibrosis (PS-CF), PI-CF, and normal control subjects, all with normal glucose tolerance, in order to identify early pathophysiologic defects. Acute islet cell secretory responses were determined under fasting, 230 mg/dL, and 340 mg/dL hyperglycemia clamp conditions. PI-CF subjects had lower acute insulin, C-peptide, and glucagon responses compared with PS-CF and normal control subjects, indicating reduced β-cell secretory capacity and α-cell function. Fasting proinsulin-to-C-peptide and proinsulin secretory ratios during glucose potentiation were higher in PI-CF, suggesting impaired proinsulin processing. In the first 30 min of the MMTT, insulin secretion was lower in PI-CF compared with PS-CF and normal control subjects, and glucagon-like peptide 1 and gastric inhibitory polypeptide were lower compared with PS-CF, and after 180 min, glucose was higher in PI-CF compared with normal control subjects. These findings indicate that despite "normal" glucose tolerance, adolescents and adults with PI-CF have impairments in functional islet mass and associated early-phase insulin secretion, which with decreased incretin responses likely leads to the early development of postprandial hyperglycemia in CF. © 2017 by the American Diabetes Association.

Microbiota promote secretory cell determination in the intestinal epithelium by modulating host Notch signaling.

PubMed

Troll, Joshua V; Hamilton, M Kristina; Abel, Melissa L; Ganz, Julia; Bates, Jennifer M; Stephens, W Zac; Melancon, Ellie; van der Vaart, Michiel; Meijer, Annemarie H; Distel, Martin; Eisen, Judith S; Guillemin, Karen

2018-02-23

Resident microbes promote many aspects of host development, although the mechanisms by which microbiota influence host tissues remain unclear. We showed previously that the microbiota is required for allocation of appropriate numbers of secretory cells in the zebrafish intestinal epithelium. Because Notch signaling is crucial for secretory fate determination, we conducted epistasis experiments to establish whether the microbiota modulates host Notch signaling. We also investigated whether innate immune signaling transduces microbiota cues via the Myd88 adaptor protein. We provide the first evidence that microbiota-induced, Myd88-dependent signaling inhibits host Notch signaling in the intestinal epithelium, thereby promoting secretory cell fate determination. These results connect microbiota activity via innate immune signaling to the Notch pathway, which also plays crucial roles in intestinal homeostasis throughout life and when impaired can result in chronic inflammation and cancer. © 2018. Published by The Company of Biologists Ltd.

Secretory products of helminth parasites as immunomodulators.

PubMed

Harnett, William

2014-07-01

Parasitic helminths release molecules into their environment, which are generally referred to as excretory-secretory products or ES. ES derived from a wide range of nematodes, trematodes and cestodes have been studied during the past 30-40 years, their characterization evolving from simple biochemical procedures such as SDS-PAGE in the early days to sophisticated proteomics in the 21st century. Study has incorporated investigation of ES structure, potential as vaccines, immunodiagnostic utility, functional activities and immunomodulatory properties. Immunomodulation by ES is increasingly the area of most intensive research with a number of defined helminth products extensively analyzed with respect to the nature of their selective effects on cells of the immune system as well as the molecular mechanisms, which underlie these immunomodulatory effects. As a consequence, we are now beginning to learn the identities of the receptors that ES employ and are increasingly acquiring detailed knowledge of the signalling pathways that they interact with and subvert. Such information is contributing to the growing idea that the anti-inflammatory properties of a number of ES products makes them suitable starting points for the development of novel drugs for treating human inflammatory disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae.

PubMed

Yano, Akira; Kikuchi, Sayaka; Nakagawa, Yuko; Sakamoto, Yuichi; Sato, Toshitsugu

2009-01-01

The shiitake mushroom, Lentinula edodes, has an extracelluar secretory-type laccase, Lcc1, and a fruiting-body-accumulation-type laccase, Lcc4. We previously reported the production of Lcc1 by plant cells, but had difficulty producing Lcc4. Here, we report the production of Lcc1 and Lcc4 by Aspergillus oryzae and the extracellular secretory production of Lcc4 using a modified secretion signal peptide (SP) from Lcc1. Sp-Lcc4 produced by A. oryzae had biochemical activities similar to Lcc4 produced by L. edodes. Lcc1 did not react with beta-(3,4-dihydroxyphenol) alanine (DOPA), but Lcc4 from L. edodes and A. oryzae could oxidize DOPA. K(M) values for the substrates 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate), 2,6-dimethoxyphenol, guaiacol, pyrogallol, and catechol were similar for Lcc4 and Sp-Lcc4. In conclusion, a non-secretory-type fungal laccase is secreted into the culture media with its original enzymatic properties by exploiting modified secretory signal peptide. 2008 Elsevier GmbH.

Unremitting Cell Proliferation in the Secretory Phase of Eutopic Endometriosis

PubMed Central

Franco-Murillo, Yanira; Miranda-Rodríguez, José Antonio; Rendón-Huerta, Erika; Montaño, Luis F.; Cornejo, Gerardo Velázquez; Gómez, Lucila Poblano; Valdez-Morales, Francisco Javier; Gonzalez-Sanchez, Ignacio

2014-01-01

Objective: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. Design: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3β), and phosphorylated glycogen synthase kinase 3 beta (pGSK3β), throughout the menstrual cycle. Results: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3β was identical in all samples and cycle phases, whereas pAkt and pGSK3β, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. Conclusion: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways. PMID:25194152

Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway.

PubMed

Wilcox, C A; Fuller, R S

1991-10-01

The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.

Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway

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

Xiang, Yong; Karaveg, Khanita; Moremen, Kelley W.

2016-11-17

Asn-linked glycosylation of newly synthesized polypeptides occurs in the endoplasmic reticulum of eukaryotic cells. Glycan structures are trimmed and remodeled as they transit the secretory pathway, and processing intermediates play various roles as ligands for folding chaperones and signals for quality control and intracellular transport. Key steps for the generation of these trimmed intermediates are catalyzed by glycoside hydrolase family 47 (GH47) α-mannosidases that selectively cleave α1,2-linked mannose residues. Despite the sequence and structural similarities among the GH47 enzymes, the molecular basis for residue-specific cleavage remains obscure. The present studies reveal enzyme–substrate complex structures for two related GH47 α-mannosidases andmore » provide insights into how these enzymes recognize the same substrates differently and catalyze the complementary glycan trimming reactions necessary for glycan maturation.« less

The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

PubMed Central

Stalder, Danièle; Novick, Peter J.

2016-01-01

Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling. PMID:26700316

The Secretory System of Arabidopsis

PubMed Central

Bassham, Diane C.; Brandizzi, Federica; Otegui, Marisa S.; Sanderfoot, Anton A.

2008-01-01

Over the past few years, a vast amount of research has illuminated the workings of the secretory system of eukaryotic cells. The bulk of this work has been focused on the yeast Saccharomyces cerevisiae, or on mammalian cells. At a superficial level, plants are typical eukaryotes with respect to the operation of the secretory system; however, important differences emerge in the function and appearance of endomembrane organelles. In particular, the plant secretory system has specialized in several ways to support the synthesis of many components of the complex cell wall, and specialized kinds of vacuole have taken on a protein storage role—a role that is intended to support the growing seedling, but has been co-opted to support human life in the seeds of many crop plants. In the past, most research on the plant secretory system has been guided by results in mammalian or fungal systems but recently plants have begun to stand on their own as models for understanding complex trafficking events within the eukaryotic endomembrane system. PMID:22303241

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway.

PubMed

Garcia, Angelo L; Han, Shan-Kuo; Janssen, William G; Khaing, Zin Z; Ito, Timothy; Glucksman, Marc J; Benson, Deanna L; Salton, Stephen R J

2005-12-16

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.

Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

PubMed Central

Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

2014-01-01

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-μ chains in the early secretory pathway.

PubMed

Giannone, Chiara; Fagioli, Claudio; Valetti, Caterina; Sitia, Roberto; Anelli, Tiziana

2017-02-03

The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-μ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant μ chains lacking the Cμ1 domain (μ∆). Our data reveal that μ∆ lacking 563 glycans (μ∆5) form larger intracellular aggregates than μ∆ and are not secreted. Like μ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, μ∆ lacking 402 glycans (μ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (μ∆4-5). These results suggest that the two C-terminal Ig-μ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers.

Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-μ chains in the early secretory pathway

PubMed Central

Giannone, Chiara; Fagioli, Claudio; Valetti, Caterina; Sitia, Roberto; Anelli, Tiziana

2017-01-01

The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-μ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant μ chains lacking the Cμ1 domain (μ∆). Our data reveal that μ∆ lacking 563 glycans (μ∆5) form larger intracellular aggregates than μ∆ and are not secreted. Like μ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, μ∆ lacking 402 glycans (μ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (μ∆4–5). These results suggest that the two C-terminal Ig-μ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers. PMID:28157181

Heritable temperament pathways to early callous-unemotional behaviour.

PubMed

Waller, Rebecca; Trentacosta, Christopher J; Shaw, Daniel S; Neiderhiser, Jenae M; Ganiban, Jody M; Reiss, David; Leve, Leslie D; Hyde, Luke W

2016-12-01

Early callous-unemotional behaviours identify children at risk for antisocial behaviour. Recent work suggests that the high heritability of callous-unemotional behaviours is qualified by interactions with positive parenting. To examine whether heritable temperament dimensions of fearlessness and low affiliative behaviour are associated with early callous-unemotional behaviours and whether parenting moderates these associations. Using an adoption sample (n = 561), we examined pathways from biological mother self-reported fearlessness and affiliative behaviour to child callous-unemotional behaviours via observed child fearlessness and affiliative behaviour, and whether adoptive parent observed positive parenting moderated pathways. Biological mother fearlessness predicted child callous-unemotional behaviours via earlier child fearlessness. Biological mother low affiliative behaviour predicted child callous-unemotional behaviours, although not via child affiliative behaviours. Adoptive mother positive parenting moderated the fearlessness to callous-unemotional behaviour pathway. Heritable fearlessness and low interpersonal affiliation traits contribute to the development of callous-unemotional behaviours. Positive parenting can buffer these risky pathways. © The Royal College of Psychiatrists 2016.

Heritable temperament pathways to early callous–unemotional behaviour

PubMed Central

Waller, Rebecca; Trentacosta, Christopher J.; Shaw, Daniel S.; Neiderhiser, Jenae M.; Ganiban, Jody M.; Reiss, David; Leve, Leslie D.; Hyde, Luke W.

2016-01-01

Background Early callous–unemotional behaviours identify children at risk for antisocial behaviour. Recent work suggests that the high heritability of callous–unemotional behaviours is qualified by interactions with positive parenting. Aims To examine whether heritable temperament dimensions of fearlessness and low affiliative behaviour are associated with early callous–unemotional behaviours and whether parenting moderates these associations. Method Using an adoption sample (n = 561), we examined pathways from biological mother self-reported fearlessness and affiliative behaviour to child callous–unemotional behaviours via observed child fearlessness and affiliative behaviour, and whether adoptive parent observed positive parenting moderated pathways. Results Biological mother fearlessness predicted child callous–unemotional behaviours via earlier child fearlessness. Biological mother low affiliative behaviour predicted child callous–unemotional behaviours, although not via child affiliative behaviours. Adoptive mother positive parenting moderated the fearlessness to callous–unemotional behaviour pathway. Conclusions Heritable fearlessness and low interpersonal affiliation traits contribute to the development of callous–unemotional behaviours. Positive parenting can buffer these risky pathways. PMID:27765772

Sorafenib Impedes Rift Valley Fever Virus Egress by Inhibiting Valosin-Containing Protein Function in the Cellular Secretory Pathway.

PubMed

Brahms, Ashwini; Mudhasani, Rajini; Pinkham, Chelsea; Kota, Krishna; Nasar, Farooq; Zamani, Rouzbeh; Bavari, Sina; Kehn-Hall, Kylene

2017-11-01

There is an urgent need for therapeutic development to combat infections caused by Rift Valley fever virus (RVFV), which causes devastating disease in both humans and animals. In an effort to repurpose drugs for RVFV treatment, our previous studies screened a library of FDA-approved drugs. The most promising candidate identified was the hepatocellular and renal cell carcinoma drug sorafenib. Mechanism-of-action studies indicated that sorafenib targeted a late stage in virus infection and caused a buildup of virions within cells. In addition, small interfering RNA (siRNA) knockdown studies suggested that nonclassical targets of sorafenib are important for the propagation of RVFV. Here we extend our previous findings to identify the mechanism by which sorafenib inhibits the release of RVFV virions from the cell. Confocal microscopy imaging revealed that glycoprotein Gn colocalizes and accumulates within the endoplasmic reticulum (ER) and the transport of Gn from the Golgi complex to the host cell membrane is reduced. Transmission electron microscopy demonstrated that sorafenib caused virions to be present inside large vacuoles inside the cells. p97/valosin-containing protein (VCP), which is involved in membrane remodeling in the secretory pathway and a known target of sorafenib, was found to be important for RVFV egress. Knockdown of VCP resulted in decreased RVFV replication, reduced Gn Golgi complex localization, and increased Gn ER accumulation. The intracellular accumulation of RVFV virions was also observed in cells transfected with siRNA targeting VCP. Collectively, these data indicate that sorafenib causes a disruption in viral egress by targeting VCP and the secretory pathway, resulting in a buildup of virions within dilated ER vesicles. IMPORTANCE In humans, symptoms of RVFV infection mainly include a self-limiting febrile illness. However, in some cases, infected individuals can also experience hemorrhagic fever, neurological disorders, liver failure, and

Subcellular location of secretory proteins retained in the liver during the ethanol-induced inhibition of hepatic protein secretion in the rat

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

Volentine, G.D.; Tuma, D.J.; Sorrell, M.F.

1986-01-01

Ethanol administration inhibits the secretion of proteins by the liver, resulting in their hepatocellular retention. Experiments were designed in this study to determine the subcellular location of the retained secretory proteins. Ethanol was administered acutely to nonfasted rats by gastric intubation, whereas control animals received an isocaloric dose of glucose. Two hours after intubation, when maximum blood ethanol levels (45 mM) were observed, (/sup 3/H)leucine and (/sup 14/C)fucose were injected simultaneously into the dorsal vein of the penis. The labelling of secretory proteins was determined in the liver and plasma at various time periods after label injection. Ethanol treatment decreasedmore » the secretion of both leucine- and fucose-labeled proteins into the plasma. This inhibition of secretion was accompanied by a corresponding increase in the hepatic retention of both leucine- and fucose-labeled immunoprecipitable secretory proteins. At the time of maximum inhibition of secretion, leucine labeled secretory proteins located in the Golgi apparatus represented about 50% of the accumulated secretory proteins in the livers of the ethanol-treated rats, whereas the remainder was essentially equally divided among the rough and smooth endoplasmic reticulum and cytosol. Because fucose is incorporated into secretory proteins almost exclusively in the Golgi complex, fucose-labeled proteins accumulated in the livers of the ethanol-treated rats mainly in the Golgi apparatus, with the remainder located in the cytosol. These results show that ethanol administration causes an impaired movement of secretory proteins along the secretory pathway, and that secretory proteins accumulate mainly, but not exclusively, in the Golgi apparatus.« less

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells.

PubMed

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells

PubMed Central

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to

The secretory pathway: exploring yeast diversity.

PubMed

Delic, Marizela; Valli, Minoska; Graf, Alexandra B; Pfeffer, Martin; Mattanovich, Diethard; Gasser, Brigitte

2013-11-01

Protein secretion is an essential process for living organisms. In eukaryotes, this encompasses numerous steps mediated by several hundred cellular proteins. The core functions of translocation through the endoplasmic reticulum membrane, primary glycosylation, folding and quality control, and vesicle-mediated secretion are similar from yeasts to higher eukaryotes. However, recent research has revealed significant functional differences between yeasts and mammalian cells, and even among diverse yeast species. This review provides a current overview of the canonical protein secretion pathway in the model yeast Saccharomyces cerevisiae, highlighting differences to mammalian cells as well as currently unresolved questions, and provides a genomic comparison of the S. cerevisiae pathway to seven other yeast species where secretion has been investigated due to their attraction as protein production platforms, or for their relevance as pathogens. The analysis of Candida albicans, Candida glabrata, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Schizosaccharomyces pombe reveals that many - but not all - secretion steps are more redundant in S. cerevisiae due to duplicated genes, while some processes are even absent in this model yeast. Recent research obviates that even where homologous genes are present, small differences in protein sequence and/or differences in the regulation of gene expression may lead to quite different protein secretion phenotypes. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism

USGS Publications Warehouse

Ramirez-Gomez, F.; Greene, W.; Rego, K.; Hansen, J.D.; Costa, G.; Kataria, P.; Bromage, E.S.

2012-01-01

The gene encoding IgH δ has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated δ-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory δ transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory δ transcript resulted in two δ-H chains, which incorporated Cμ1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory δ mRNA and IgD+ plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.

Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding.

PubMed

de Castro, Minique Hilda; de Klerk, Daniel; Pienaar, Ronel; Rees, D Jasper G; Mans, Ben J

2017-08-10

Ticks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding. The sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open

Porosome: The Universal Secretory Portal in Cells

NASA Astrophysics Data System (ADS)

Jena, Bhanu

2012-10-01

In the past 50 years it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the ``all or none'' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. Moreover, it makes no sense for mammalian cells to possess such `all or none' mechanism of cell secretion, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuoles in paramecium, or the various types of secretory structures in bacteria. Therefore, in 1993 in a News and Views article in Nature, E. Neher wrote ``It seems terribly wasteful that, during the release of hormones and neurotransmitters from a cell, the membrane of a vesicle should merge with the plasma membrane to be retrieved for recycling only seconds or minutes later.'' This conundrum in the molecular mechanism of cell secretion was finally resolved in 1997 following discovery of the ``Porosome,'' the universal secretory machinery in cells. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release inravesicular contents to the outside during cell secretion. In the past decade, the composition of the porosome, its structure and dynamics at nm resolution and in real time, and its functional reconstitution into artificial lipid membrane, have all been elucidated. Since porosomes in exocrine and neuroendocrine cells measure 100-180 nm

Cellular and molecular mechanism for secretory autophagy.

PubMed

Kimura, Tomonori; Jia, Jingyue; Claude-Taupin, Aurore; Kumar, Suresh; Choi, Seong Won; Gu, Yuexi; Mudd, Michal; Dupont, Nicolas; Jiang, Shanya; Peters, Ryan; Farzam, Farzin; Jain, Ashish; Lidke, Keith A; Adams, Christopher M; Johansen, Terje; Deretic, Vojo

2017-06-03

Macroautophagy/autophagy plays a role in unconventional secretion of leaderless cytosolic proteins. Whether and how secretory autophagy diverges from conventional degradative autophagy is unclear. We have shown that the prototypical secretory autophagy cargo IL1B/IL-1β (interleukin 1 β) is recognized by TRIM16, and that this first to be identified secretory autophagy receptor interacts with the R-SNARE SEC22B to jointly deliver cargo to the MAP1LC3B-II-positive sequestration membranes. Cargo secretion is unaffected by knockdowns of STX17, a SNARE catalyzing autophagosome-lysosome fusion as a prelude to cargo degradation. Instead, SEC22B in combination with plasma membrane syntaxins completes cargo secretion. Thus, secretory autophagy diverges from degradative autophagy by using specialized receptors and a dedicated SNARE machinery to bypass fusion with lysosomes.

Heritable and Nonheritable Pathways to Early Callous-Unemotional Behaviors.

PubMed

Hyde, Luke W; Waller, Rebecca; Trentacosta, Christopher J; Shaw, Daniel S; Neiderhiser, Jenae M; Ganiban, Jody M; Reiss, David; Leve, Leslie D

2016-09-01

Callous-unemotional behaviors in early childhood signal higher risk for trajectories of antisocial behavior and callous-unemotional traits that culminate in later diagnoses of conduct disorder, antisocial personality disorder, and psychopathy. Studies demonstrate high heritability of callous-unemotional traits, but little research has examined specific heritable pathways to early callous-unemotional behaviors. Studies also indicate that positive parenting protects against the development of callous-unemotional traits, but genetically informed designs have not been used to confirm that these relationships are not the product of gene-environment correlations. In a sample of adopted children and their biological and adoptive mothers, the authors tested novel heritable and nonheritable pathways to preschool callous-unemotional behaviors. In an adoption cohort of 561 families, history of severe antisocial behavior assessed in biological mothers and observations of adoptive mother positive reinforcement at 18 months were examined as predictors of callous-unemotional behaviors at 27 months. Despite limited or no contact with offspring, biological mother antisocial behavior predicted early callous-unemotional behaviors. Adoptive mother positive reinforcement protected against early callous-unemotional behaviors. High levels of adoptive mother positive reinforcement buffered the effects of heritable risk for callous-unemotional behaviors posed by biological mother antisocial behavior. The findings elucidate heritable and nonheritable pathways to early callous-unemotional behaviors. The results provide a specific heritable pathway to callous-unemotional behaviors and compelling evidence that parenting is an important nonheritable factor in the development of callous-unemotional behaviors. The finding that positive reinforcement buffered heritable risk for callous-unemotional behaviors has important translational implications for the prevention of trajectories to serious

Secretory Structure, Histochemistry and Phytochemistry Analyses of Stimulant Plant

NASA Astrophysics Data System (ADS)

Umah, C.; Dorly; Sulistyaningsih, Y. C.

2017-03-01

Plants that are used as stimulant supposed to contains various metabolit compounds that are produced or secreted by secretory structures. This study aimed to identify the secretory structure of plant used as stimulant and chemical compounds accumulated in it. The secretory structure and its histochemistry were observed on plant material that are used as herbal ingredient. Phytochemical content was analyzed by using a qualitative test. The result showed that the idioblast cells and secretory cavities were found in the leaves of Decaspermum fruticosum, and Polyalthia rumphii. Most idioblast cells contained lipophilic substances and terpenoids or alkaloids, while secretory cavity contained alkaloid. Phytochemical analysis for D. fruticosum, and P. rumphii contain terpenoids, phenols, steroids, and flavonoids

Influence of experimental hypokinesia on gastric secretory function

NASA Technical Reports Server (NTRS)

Markova, O. O.; Vavryshchuk, V. I.; Rozvodovskyy, V. I.; Proshcheruk, V. A.

1980-01-01

The gastric secretory function of rats was studied in 4, 8, 16 and 30 day hypokinesia. Inhibition of both the gastric juice secretory and acid producing functions was found. The greatest inhibition was observed on day 8 of limited mobility. By days 16 and 30 of the experiment, a tendency of the gastric secretory activity to return to normal was observed, although it remained reduced.

A Role for Serglycin Proteoglycan in Mast Cell Apoptosis Induced by a Secretory Granule-mediated Pathway*

PubMed Central

Melo, Fabio Rabelo; Waern, Ida; Rönnberg, Elin; Åbrink, Magnus; Lee, David M.; Schlenner, Susan M.; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Turk, Boris; Wernersson, Sara; Pejler, Gunnar

2011-01-01

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin−/− cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis. PMID:21123167

Cells in 3D-reconstitutued eccrine sweat gland cell spheroids differentiate into gross cystic disease fluid protein 15-expressing dark secretory cells and carbonic anhydrase II-expressing clear secretory cells.

PubMed

Li, Haihong; Chen, Liyun; Zhang, Mingjun; Zhang, Bingna

2017-07-01

Secretory coils of eccrine sweat glands are composed of myoepithelial cells, dark secretory cells and clear secretory cells. The two types of cells play important roles in sweat secretion. In our previous study, we demonstrated that the 3D-reconstituted eccrine sweat gland cell spheroids differentiate into secretory coil-like structures. However, whether the secretory coil-like structures further differentiate into dark secretory cells and clear secretory cells were is still unknown. In this study, we detected the differentiation of clear and dark secretory cells in the 3D-reconstituted eccrine sweat gland cell spheroids using the dark secretory cell-specific marker, GCDFP-15, and clear secretory cell-specific marker, CAII by immunofluorescence staining. Results showed that there were both GCDFP-15- and CAII-expressing cells in 12-week-old 3D spheroids, similar to native eccrine sweat glands, indicating that the spheroids possess a cellular structure capable of sweat secretion. We conclude that the 12-week 3D spheroids may have secretory capability. Copyright © 2017 Elsevier GmbH. All rights reserved.

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

PubMed

Jutras, Philippe V; D'Aoust, Marc-André; Couture, Manon M-J; Vézina, Louis-Philippe; Goulet, Marie-Claire; Michaud, Dominique; Sainsbury, Frank

2015-09-01

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Parsing the Effects Violence Exposure in Early Childhood: Modeling Developmental Pathways

PubMed Central

Carter, Alice S.; Ford, Julian D.

2012-01-01

Objective To prospectively examine pathways from early childhood violence exposure and trauma-related symptoms to school-age emotional health. Methods A longitudinal, birth cohort (N = 437) was assessed with parent reports of lifetime violence exposure and trauma-related symptoms at 3 years of age and later, internalizing and externalizing symptoms, and social competence at school age. Results Early family and neighborhood violence correlated significantly with early trauma-related symptoms and also significantly predicted school-age internalizing and externalizing symptoms and poorer competence, independent of sociodemographic risk and past-year violence exposure. Longitudinal pathways were significantly mediated by arousal and avoidance symptoms at 3 years of age, which increased risk for clinically significant emotional problems and lower competence at school age (adjusted odds ratios = 3.1–6.1, p < 0.01). Conclusions Trauma-related symptoms may mediate developmental pathways from early violence exposure to later emotional health. Interventions that prevent or reduce early trauma-related symptoms may ameliorate the long-term deleterious impact of violence exposure. PMID:21903730

Secretory IgA: Designed for Anti-Microbial Defense

PubMed Central

Brandtzaeg, Per

2013-01-01

Prevention of infections by vaccination remains a compelling goal to improve public health. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion – a term coined for non-inflammatory antibody shielding of internal body surfaces, mediated principally by secretory immunoglobulin A (SIgA). The exported antibodies are polymeric, mainly IgA dimers (pIgA), produced by local plasma cells (PCs) stimulated by antigens that target the mucose. SIgA was early shown to be complexed with an epithelial glycoprotein – the secretory component (SC). A common SC-dependent transport mechanism for pIgA and pentameric IgM was then proposed, implying that membrane SC acts as a receptor, now usually called the polymeric Ig receptor (pIgR). From the basolateral surface, pIg-pIgR complexes are taken up by endocytosis and then extruded into the lumen after apical cleavage of the receptor – bound SC having stabilizing and innate functions in the secretory antibodies. Mice deficient for pIgR show that this is the only receptor responsible for epithelial export of IgA and IgM. These knockout mice show a variety of defects in their mucosal defense and changes in their intestinal microbiota. In the gut, induction of B-cells occurs in gut-associated lymphoid tissue, particularly the Peyer’s patches and isolated lymphoid follicles, but also in mesenteric lymph nodes. PC differentiation is accomplished in the lamina propria to which the activated memory/effector B-cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue but by different homing receptors. This compartmentalization is a challenge for mucosal vaccination, as are the mechanisms used by the mucosal immune system to discriminate between commensal symbionts (mutualism), pathobionts, and overt pathogens (elimination). PMID:23964273

New features of triacylglycerol biosynthetic pathways of peanut seeds in early developmental stages.

PubMed

Yu, Mingli; Liu, Fengzhen; Zhu, Weiwei; Sun, Meihong; Liu, Jiang; Li, Xinzheng

2015-11-01

The peanut (Arachis hypogaea L.) is one of the three most important oil crops in the world due to its high average oil content (50 %). To reveal the biosynthetic pathways of seed oil in the early developmental stages of peanut pods with the goal of improving the oil quality, we presented a method combining deep sequencing analysis of the peanut pod transcriptome and quantitative real-time PCR (RT-PCR) verification of seed oil-related genes. From the sequencing data, approximately 1500 lipid metabolism-associated Unigenes were identified. The RT-PCR results quantified the different expression patterns of these triacylglycerol (TAG) synthesis-related genes in the early developmental stages of peanut pods. Based on these results and analysis, we proposed a novel construct of the metabolic pathways involved in the biosynthesis of TAG, including the Kennedy pathway, acyl-CoA-independent pathway and proposed monoacylglycerol pathway. It showed that the biosynthetic pathways of TAG in the early developmental stages of peanut pods were much more complicated than a simple, unidirectional, linear pathway.

A Novel Apoptosis Pathway that is Defective in Early Breast Cancer

DTIC Science & Technology

2005-04-01

AD Award Number: DAMD17-02-1-0612 TITLE: A Novel Apoptosis Pathway that is Defective in Early Breast Cancer PRINCIPAL INVESTIGATOR: Scott Cramer...Defective in Early DAMD17-02-1-0612 Breast Cancer 6. AUTHOR(S) Scott Cramer, Ph.D. 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING...These experiments were intended to determine why breast cancer cells are resistant to this apoptosis pathway BODY. As outlined in the previous

Isolated Rat Hepatocyte Couplets: A Primary Secretory Unit for Electrophysiologic Studies of Bile Secretory Function

NASA Astrophysics Data System (ADS)

Graf, J.; Gautam, A.; Boyer, J. L.

1984-10-01

Hepatocyte couplets were isolated by collagenase perfusion from rat liver. Between adjacent cells, the bile canaliculus forma a closed space into which secretion occurs. As in intact liver, Mg2+-ATPase is localized at the canalicular lumen, the organic anion fluorescein is excreted, and secretion is modified by osmotic gradients. By passing a microelectrode through one cell into the canalicular vacuole, a transepithelial potential profile was obtained. In 27 cell couplets the steady-state intracellular (-26.3 ± 5.3 mV) and intracanalicular (-5.9 ± 3.3 mV) potentials were recorded at 37 degrees C with reference to the external medium. Input resistances were determined within the cell (86 ± 23 MΩ ) and in the bile canalicular lumen (32 ± 17 MΩ ) by passing current pulses through the microelectrode. These data define electrical driving forces for ion transport across the sinusoidal, canalicular, and paracellular barriers and indicate ion permeation across a leaky paracellular junctional pathway. These findings indicate that the isolated hepatocyte couplet is an effective model for electrophysiologic studies of bile secretory function.

β-Cell secretory defects are present in pancreatic insufficient cystic fibrosis with 1-hour oral glucose tolerance test glucose ≥155 mg/dL.

PubMed

Nyirjesy, Sarah C; Sheikh, Saba; Hadjiliadis, Denis; De Leon, Diva D; Peleckis, Amy J; Eiel, Jack N; Kubrak, Christina; Stefanovski, Darko; Rubenstein, Ronald C; Rickels, Michael R; Kelly, Andrea

2018-06-08

Patients with pancreatic insufficient cystic fibrosis (PI-CF) meeting standard criteria for normal glucose tolerance display impaired β-cell secretory capacity and early-phase insulin secretion defects. We sought evidence of impaired β-cell secretory capacity, a measure of functional β-cell mass, among those with early glucose intolerance (EGI), defined as 1-hour oral glucose tolerance test (OGTT) glucose ≥155 mg/dL (8.6 mmol/L). A cross-sectional study was conducted in the Penn and CHOP Clinical & Translational Research Centers. PI-CF categorized by OGTT as normal (PI-NGT: 1-hour glucose <155 mg/dL and 2-hour <140 mg/dL [7.8 mmol/L]; n = 13), PI-EGI (1-hour ≥155 mg/dL and 2-hour <140 mg/dL; n = 13), impaired (PI-IGT: 2-hour ≥140 and <200 mg/dL [11.1 mmol/L]; n = 8), and diabetic (cystic fibrosis-related diabetes, CFRD: 2-hour ≥200 mg/dL; n = 8) participated. Post-prandial glucose tolerance and insulin secretion, and β-cell secretory capacity and demand were derived from mixed-meal tolerance tests (MMTTs), and glucose-potentiated arginine (GPA) tests, respectively. PI-EGI had elevated post-prandial glucose with reduced early-phase insulin secretion during MMTT compared to PI-NGT (P < .05). PI-EGI also exhibited impaired acute insulin and C-peptide responses to GPA (P < .01 vs PI-NGT), measures of β-cell secretory capacity. Proinsulin secretory ratios were higher under hyperglycemic clamp conditions in PI-IGT and CFRD (P < .05 vs PI-NGT), and correlated with 1-hour glucose in PI-CF (P < .01). PI-CF patients with 1-hour OGTT glucose ≥155 mg/dL already manifest impaired β-cell secretory capacity with associated early-phase insulin secretion defects. Avoiding hyperglycemia in patients with EGI may be important for preventing excessive insulin demand indicated by disproportionately increased proinsulin secretion. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Secretory structure and histochemistry test of some Zingiberaceae plants

NASA Astrophysics Data System (ADS)

Indriyani, Serafinah

2017-11-01

A secretory structure is a structure that produces a plant's metabolite substances. Secretory structures are grouped into an internal and external. Zingiberaceae plants are known as traditional medicine plants and as spice plants due to secretory structures in their tissues. The objective of the research were to describe the secretory structure of Zingiberaceae plants and to discover the qualitatively primary metabolite substances in plant's tissues via histochemistry test. The research was conducted by observation descriptive design, quantitative data including the density of secretory cells per mm². The quantitative data were analyzed by ANOVA and continued by Duncan at α = 5 %. The results showed that the secretory structures in leaves, rhizome, and the root of 14 species of Zingiberaceae plants are found in the mesophyll of leaves and cortex, and also pith in rhizome and roots. The type of secretory structure is internal. Within the root of Zingiber cassumunar Roxb.(bengle), Curcuma domestica Val. (kunyit), Curcuma zedoaria (Berg.) Roscoe (kunyit putih), Zingiber zerumbet (L.) J.E. Smith (lempuyang), Alpiniapurpurata K. Schum (lengkuas merah), and Curcuma aeruginosa Val. (temu ireng) were found amylum grains, while in Kaemferia galanga L. (kencur), Boesen bergiapandurata L. (temu kunci), and Curcuma xanthorrhiza Roxb. (temulawak) there were no amylum grains in the root as well as in the leaves. The roots of bengle had the greatest density of amylum grain, it had 248.1 ± 9.8 secretory cells of amylum grains per mm². Lipids (oil droplets) were found in the root of bengle, Zingiber officinale Roxb. Var. emprit (jahe emprit), Zingiber officinale Roxb. Var. Gajah (jahe gajah), Zingiber officinale Roxb. Var. Rubrum (jahe merah), Keampferia angustifolia L. (kunci pepet), kunyit, kunyit putih, lempuyang, lengkua smerah, Curcuma aeruginosa Val. (temu ireng), and Curcuma mangga Val. and van Zijp (temu mangga); the root of lempuyang had the greatest density of oil

Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics.

PubMed

Wang, Li; Cui, Jing; Hu, Dan Dan; Liu, Ruo Dan; Wang, Zhong Quan

2014-01-22

The excretory-secretory (ES) proteins of Trichinella spiralis muscle larvae (ML) come mainly from the excretory granules of the stichosome and the cuticles (membrane proteins), are directly exposed to the host's immune system, and are the main target antigens, which induce the immune responses. Although the ES proteins are the most commonly used diagnostic antigens for trichinellosis, their main disadvantage are the false negative results during the early stage of infection. The aim of this study was to identify early specific diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. Two-dimensional electrophoresis (2-DE) combined with Western blot were used to screen the early diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. The protein spots recognized by the sera from BALB/c mice infected with T. spiralis at 18 days post-infection (dpi) were identified by MALDI-TOF/TOF-MS and putatively annotated using GO terms obtained from the InterPro databases. The ES proteins were analyzed by 2-DE, and more than 33 protein spots were detected with molecular weight varying from 40 to 60 kDa and isoelectric point (pI) from 4 to 7. When probed with the sera from infected mice at 18 dpi, 21 protein spots were recognized and then identified, and they were characterized to correlate with five different proteins of T. spiralis, including two serine proteases, one deoxyribonuclease (DNase) II, and two kinds of trypsin. The five proteins were functionally categorized into molecular function and biological process according to GO hierarchy. 2-DE and Western blot combined with MALDI-TOF/TOF-MS were used to screen the diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. The five proteins of T. spiralis identified (two serine proteases, DNase II and two kinds of trypsin) might be the early specific diagnostic antigens of trichinellosis.

Ethnic variations in pathways into early intervention services for psychosis.

PubMed

Ghali, Sharif; Fisher, Helen L; Joyce, John; Major, Barnaby; Hobbs, Lorna; Soni, Sujata; Chisholm, Brock; Rahaman, Nikola; Papada, Peggy; Lawrence, Jo; Bloy, Sally; Marlowe, Karl; Aitchison, Katherine J; Power, Paddy; Johnson, Sonia

2013-04-01

Ethnic variations have previously been identified in the duration of untreated psychosis (DUP) and pathways into psychiatric services. These have not been examined in the context of early intervention services, which may alter these trajectories. To explore ethnic differences in the nature and duration of pathways into early intervention services. In a naturalistic cohort study, data were collected for 1024 individuals with psychotic disorders accepted for case management by eight London early intervention services. Duration of untreated psychosis was prolonged in the White British group compared with most other ethnic groups. White British individuals were more likely to make contact with their general practitioner and less likely to be seen within emergency medical services. All Black patient groups were more likely than their White British counterparts to experience involvement of criminal justice agencies. Variations continue to exist in how and when individuals from different ethnic groups access early intervention services. These may account for disparities in DUP.

Inhibition of the adrenomedullin/nitric oxide signaling pathway in early diabetic retinopathy.

PubMed

Blom, Jan J; Giove, Thomas J; Favazza, Tara L; Akula, James D; Eldred, William D

2011-06-01

The nitric oxide (NO) signaling pathway is integrally involved in visual processing and changes in the NO pathway are measurable in eyes of diabetic patients. The small peptide adrenomedullin (ADM) can activate a signaling pathway to increase the enzyme activity of neuronal nitric oxide synthase (nNOS). ADM levels are elevated in eyes of diabetic patients and therefore, ADM may play a role in the pathology of diabetic retinopathy. The goal of this research was to test the effects of inhibiting the ADM/NO signaling pathway in early diabetic retinopathy. Inhibition of this pathway decreased NO production in high-glucose retinal cultures. Treating diabetic mice with the PKC β inhibitor ruboxistaurin for 5 weeks lowered ADM mRNA levels and ADM-like immunoreactivity and preserved retinal function as assessed by electroretinography. The results of this study indicate that inhibiting the ADM/NO signaling pathway prevents neuronal pathology and functional losses in early diabetic retinopathy.

Ursodeoxycholic acid attenuates colonic epithelial secretory function

PubMed Central

Kelly, Orlaith B; Mroz, Magdalena S; Ward, Joseph B J; Colliva, Carolina; Scharl, Michael; Pellicciari, Roberto; Gilmer, John F; Fallon, Padraic G; Hofmann, Alan F; Roda, Aldo; Murray, Frank E; Keely, Stephen J

2013-01-01

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl− secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl− secretory responses to the Ca2+ and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n= 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na+/K+-ATPase activity and basolateral K+ channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg−1) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50–200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea. PMID:23507881

Cellular responses to the expression of unstable secretory proteins in the filamentous fungus Aspergillus oryzae.

PubMed

Yokota, Jun-Ichi; Shiro, Daisuke; Tanaka, Mizuki; Onozaki, Yasumichi; Mizutani, Osamu; Kakizono, Dararat; Ichinose, Sakurako; Shintani, Tomoko; Gomi, Katsuya; Shintani, Takahiro

2017-03-01

Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels in Aspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for the hrdA gene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein in A. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.

Oxytocin Pathways in the Intergenerational Transmission of Maternal Early Life Stress

PubMed Central

Toepfer, Philipp; Heim, Christine; Entringer, Sonja; Binder, Elisabeth; Wadhwa, Pathik; Buss, Claudia

2017-01-01

Severe stress in early life, such as childhood abuse and neglect, constitutes a major risk factor in the etiology of psychiatric disorders and somatic diseases. Importantly, these long-term effects may impact the next generation. The intergenerational transmission of maternal early life stress (ELS) may occur via pre-and postnatal pathways, such as alterations in maternal-fetal-placental stress physiology, maternal depression during pregnancy and postpartum, as well as impaired mother-offspring interactions. The neuropeptide oxytocin (OT) has gained considerable attention for its role in modulating all of these assumed transmission pathways. Moreover, central and peripheral OT signaling pathways are highly sensitive to environmental exposures and may be compromised by ELS with implications for these putative transmission mechanisms. Together, these data suggest that OT pathways play an important role in the intergenerational transmission of maternal ELS in humans. By integrating recent studies on gene-environment interactions and epigenetic modifications in OT pathway genes, the present review aims to develop a conceptual framework of intergenerational transmission of maternal ELS that emphasizes the role of OT. PMID:28027955

The effect of hepatocyte growth factor on secretory functions in human eosinophils.

PubMed

Yamauchi, Yumiko; Ueki, Shigeharu; Konno, Yasunori; Ito, Wataru; Takeda, Masahide; Nakamura, Yuka; Nishikawa, Junko; Moritoki, Yuki; Omokawa, Ayumi; Saga, Tomoo; Hirokawa, Makoto

2016-12-01

Hepatocyte growth factor (HGF), originally identified as a potent mitogen for mature hepatocytes, is now recognized as a humoral mediator in inflammatory and immune responses. Previous studies indicated that HGF negatively regulated allergic airway inflammation. In view of eosinophils playing a role in the pathogenesis of asthma, especially in airway remodeling as a rich source of pro-fibrogenic mediators, the effects of HGF on the different types of eosinophil secretory functions were examined in this study. We found that HGF significantly inhibited IL-5-induced secretion of TGF-β and VEGF from human eosinophils. The inhibitory effect is not associated with TGF-β transcription; rather, it is associated with ultrastructural granule emptying and loss of intracellular TGF-β contents, indicating HGF inhibits the process of piecemeal degranulation. The effect of HGF on extracellular trap cell death (ETosis) that mediates cytolytic degranulation was also investigated; however, immobilized IgG- or phorbol myristate acetate-induced ETosis was only minimally attenuated by HGF. These results reveal the effect of HGF on the distinct pathways of eosinophil secretory functions and also provide novel insights into the role of HGF in the pathogenesis of allergic inflammation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Acquisition of Lubrol insolubility, a common step for growth hormone and prolactin in the secretory pathway of neuroendocrine cells.

PubMed

Lee, M S; Zhu, Y L; Chang, J E; Dannies, P S

2001-01-05

Rat prolactin in the dense cores of secretory granules of the pituitary gland is a Lubrol-insoluble aggregate. In GH(4)C(1) cells, newly synthesized rat prolactin and growth hormone were soluble, but after 30 min about 40% converted to a Lubrol-insoluble form. Transport from the endoplasmic reticulum is necessary for conversion to Lubrol insolubility, since incubating cells with brefeldin A or at 15 degrees C reduced formation of insoluble rat (35)S-prolactin. Formation of Lubrol-insoluble aggregates has protein and cell specificity; newly synthesized human growth hormone expressed in AtT20 cells underwent a 40% conversion to Lubrol insolubility with time, but albumin did not, and human growth hormone expressed in COS cells underwent less than 10% conversion to Lubrol insolubility. del32-46 growth hormone, a naturally occurring form of growth hormone, and P89L growth hormone underwent conversion, although they were secreted more slowly, indicating that there is some tolerance in structural requirements for aggregation. An intracellular compartment with an acidic pH is not necessary for conversion to Lubrol insolubility, because incubation with chloroquine or bafilomycin slowed, but did not prevent, the conversion. GH(4)C(1) cells treated with estradiol, insulin, and epidermal growth factor accumulate more secretory granules and store more prolactin, but not more growth hormone, than untreated cells; Lubrol-insoluble aggregates of prolactin and growth hormone formed to the same extent in hormone-treated or untreated GH(4)C(1) cells, but prolactin was retained longer in hormone-treated cells. These findings indicate that aggregation alone is not sufficient to cause retention of secretory granule proteins, and there is an additional selective process.

The use of lectins as markers for differentiated secretory cells in planarians.

PubMed

Zayas, Ricardo M; Cebrià, Francesc; Guo, Tingxia; Feng, Junjie; Newmark, Phillip A

2010-11-01

Freshwater planarians have reemerged as excellent models to investigate mechanisms underlying regeneration. The introduction of molecular tools has facilitated the study of planarians, but cell- and tissue-specific markers are still needed to examine differentiation of most cell types. Here we report the utility of fluorescent lectin-conjugates to label tissues in the planarian Schmidtea mediterranea. We show that 16 lectin-conjugates stain planarian cells or tissues; 13 primarily label the secretory cells, their cytoplasmic projections, and terminal pores. Thus, we examined regeneration of the secretory system using lectin markers and functionally characterized two genes expressed in the secretory cells: marginal adhesive gland-1 (mag-1) and Smed-reticulocalbin1 (Smed-rcn1). RNAi knockdown of these genes caused a dramatic reduction of secretory cell lectin staining, suggesting a role for mag-1 and Smed-rcn1 in secretory cell differentiation. Our results provide new insights into planarian secretory system regeneration and add new markers for labeling several planarian tissues. © 2010 Wiley-Liss, Inc.

A New Approach of Short Wave Protection against Middle Cerebral Artery Occlusion/Reperfusion Injury via Attenuation of Golgi Apparatus Stress by Inhibition of Downregulation of Secretory Pathway Ca(2+)-ATPase Isoform 1 in Rats.

PubMed

Fan, Yongmei; Zhang, Changjie; Li, Ting; Peng, Wenna; Yin, Jing; Li, Xiaofao; Kong, Ying; Lan, Chunna; Wang, Rumi; Hu, Zhiping

2016-07-01

Short wave (SW), a pattern of electromagnetic therapy, achieves an oscillating electromagnetic field. It has been reported that it may have a potential effect on cerebral injury. The present study was designed to investigate the potential role and possible mechanism of SW in focal cerebral ischemia/reperfusion (I/R) injury in rats. Secretory pathway Ca(2+)/Mn(2+) ATPase isoform 1 is a major component of Golgi apparatus stress. It has been reported as representative of Golgi apparatus stress. Up to 120 minutes of middle cerebral artery occlusion (MCAO) and reperfusion injury was induced in male Sprague-Dawley rats. Different sessions of SW daily were administered over head after reperfusion from day 1 to day 7. Functional recovery scores, survival rates, infarct volume analysis, electron microscope test, and western blotting studies were used to analyze the therapy. SW protected against neuronal death and apoptosis in cornu ammon 1 region of hippocampus by reducing neuronal deficit, infarct volume, and ultrastructure. SW partly inhibited upregulation of caspase3. In addition, the expression of secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) was upregulated by SW. Our data indicate that SW can be protected against focal cerebral I/R injury, and the influence on Golgi apparatus stress might provide us a new perspective in further study. To the authors' knowledge, this is the first report using SW to increase expression of SPCA1 indicating modulate Golgi apparatus stress in MCAO and reperfusion model. Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product.

PubMed

Thuwajit, Chanitra; Thuwajit, Peti; Uchida, Kazuhiko; Daorueang, Daoyot; Kaewkes, Sasithorn; Wongkham, Sopit; Miwa, Masanao

2006-06-14

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product. NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR. Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05). O. viverrini ES product stimulates the significant changes of gene expression in several functional categories

A tissue engineered human endometrial stroma that responds to cues for secretory differentiation, decidualization and menstruation

PubMed Central

Schutte, Stacey C.; Taylor, Robert N.

2012-01-01

Objective To show the responsiveness of a tissue engineered human endometrial stroma to combinations of hormones mimicking the secretory and menstrual phases of the cycle. Design In vitro experimental study Setting University uterine biology research laboratory Cells Telomerase immortalized human endometrial stromal cells Interventions The stromal cells were cultured in monolayers (2D) or encapsulated in a collagen I hydrogel (3D) to create a simplified tissue engineered stroma. The cells and tissues were exposed to hormone treatments mimicking early and late secretory phases, decidualization and steroid withdrawal conditions to recapitulate menstruation. Main Outcome Measure(s) Morphological and biochemical markers of decidualization and collagenase activity Result(s) The 3D tissue is capable of manifesting changes in morphology and biochemical markers of decidualization similar to 2D culture and characteristic of endometrial stroma in vivo. Unlike 2D culture, the 3D tissue responded to steroid withdrawal by increased collagenase activity and tissue breakdown. Conclusion(s) 3D tissue engineered endometrial stroma can mimic secretory and menstrual phases of the cycle and may be useful for studying uterine receptivity and menstruation in a physiological endocrine environment. PMID:22306710

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules.

PubMed

Kasai, Kazuo; Fujita, Takuji; Gomi, Hiroshi; Izumi, Tetsuro

2008-07-01

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic beta cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.

Heritable and non-heritable pathways to early callous-unemotional behaviors

PubMed Central

Hyde, Luke W.; Waller, Rebecca; Trentacosta, Christopher J.; Shaw, Daniel S.; Neiderhiser, Jenae M.; Ganiban, Jody M.; Reiss, David; Leve, Leslie D.

2016-01-01

Objective Callous-unemotional behaviors in early childhood identify children at high risk for severe trajectories of antisocial behavior and callous-unemotional traits that culminate in later diagnoses of conduct disorder, antisocial personality disorder, and psychopathy. Studies have demonstrated high heritability of callous-unemotional traits, but little research has examined specific heritable pathways to earlier callous-unemotional behaviors. Additionally, studies indicate that positive parenting protects against the development of callous-unemotional traits, but genetically informed designs have not been used to confirm that these relationships are not the product of gene-environment correlations. Method Using an adoption cohort of 561 families, biological mothers reported their history of severe antisocial behavior. Observations of adoptive mother positive reinforcement at 18 months were examined as predictors of callous-unemotional behaviors when children were 27 months old. Results Biological mother antisocial behavior predicted early callous-unemotional behaviors despite having no or limited contact with offspring. Adoptive mother positive reinforcement protected against early callous-unemotional behaviors in children not genetically related to the parent. High levels of adoptive mother positive reinforcement buffered the effects of heritable risk for callous-unemotional behaviors posed by biological mother antisocial behavior. Conclusions The findings elucidate heritable and non-heritable pathways to early callous-unemotional behaviors. The results provide a specific heritable pathway to callous-unemotional behaviors and compelling evidence that parenting is an important non-heritable factor in the development of callous-unemotional behaviors. As positive reinforcement buffered heritable risk for callous-unemotional behaviors, these findings have important translational implications for the prevention of trajectories to serious antisocial behavior. PMID

Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

PubMed Central

Balic, Anamaria; Mina, Mina

2011-01-01

Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

PubMed Central

Garg, Abhishek D; Krysko, Dmitri V; Verfaillie, Tom; Kaczmarek, Agnieszka; Ferreira, Gabriela B; Marysael, Thierry; Rubio, Noemi; Firczuk, Malgorzata; Mathieu, Chantal; Roebroek, Anton J M; Annaert, Wim; Golab, Jakub; de Witte, Peter; Vandenabeele, Peter; Agostinis, Patrizia

2012-01-01

Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC-IIhigh) and functional stimulation (NOhigh, IL-10absent, IL-1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress. PMID:22252128

Pathways to Early Violent Death: The Voices of Serious Violent Youth Offenders

PubMed Central

Richardson, Joseph B.; Brown, Jerry; Van Brakle, Michelle

2013-01-01

Quantitative studies have uncovered factors associated with early violent death among youth offenders detained in the juvenile justice system, but little is known about the contextual factors associated with pathways to early violent death among youths detained in adult jails. We interviewed young Black male serious violent youth offenders detained in an adult jail to understand their experience of violence. Their narratives reveal how the code of the street, informal rules that govern interpersonal violence among poor inner-city Black male youths, increases the likelihood of violent victimization. Youth offenders detained in adult jails have the lowest rate of service provision among all jail populations. We have addressed how services for youth offenders can be improved to reduce the pathways to early violent death. PMID:23678923

Pathways to early violent death: the voices of serious violent youth offenders.

PubMed

Richardson, Joseph B; Brown, Jerry; Van Brakle, Michelle

2013-07-01

Quantitative studies have uncovered factors associated with early violent death among youth offenders detained in the juvenile justice system, but little is known about the contextual factors associated with pathways to early violent death among youths detained in adult jails. We interviewed young Black male serious violent youth offenders detained in an adult jail to understand their experience of violence. Their narratives reveal how the code of the street, informal rules that govern interpersonal violence among poor inner-city Black male youths, increases the likelihood of violent victimization. Youth offenders detained in adult jails have the lowest rate of service provision among all jail populations. We have addressed how services for youth offenders can be improved to reduce the pathways to early violent death.

Association of a GPI-anchored protein with detergent-resistant membranes facilitates its trafficking through the early secretory pathway.

PubMed

Hein, Zeynep; Hooper, Nigel M; Naim, Hassan Y

2009-01-15

Membrane microdomains are implicated in the trafficking and sorting of several membrane proteins. In particular GPI-anchored proteins cluster into Triton X-100 resistant, cholesterol- and sphingolipid-rich membrane microdomains and are sorted to the apical membrane. A growing body of evidence has pointed to the existence of other types of microdomains that are insoluble in detergents, such as Lubrol WX and Tween-20. Here, we report on the role of detergent-resistant membranes formed at early stages in the biosynthesis of membrane dipeptidase (MDP), a GPI-anchored protein, on its trafficking and sorting. Pulse-chase experiments revealed a retarded maturation rate of the GPI-anchor deficient mutant (MDPDeltaGPI) as compared to the wild type protein (wtMDP). However, Golgi to cell surface delivery rate did not show a significant difference between the two variants. On the other hand, early biosynthetic forms of wtMDP were partially insoluble in Tween-20, while MDPDeltaGPI was completely soluble. The lack of association of MDPDeltaGPI with detergent-resistant membranes prior to maturation in the Golgi and the reduction in its trafficking rate strongly suggest the existence of an early trafficking control mechanisms for membrane proteins operating at a level between the endoplasmic reticulum and the cis-Golgi.

Pathways through which health influences early retirement: a qualitative study.

PubMed

de Wind, Astrid; Geuskens, Goedele A; Reeuwijk, Kerstin G; Westerman, Marjan J; Ybema, Jan Fekke; Burdorf, Alex; Bongers, Paulien M; van der Beek, Allard J

2013-04-03

Due to the aging of the population, there is a societal need for workers to prolong their working lives. In the Netherlands, many employees still leave the workforce before the official retirement age of 65. Previous quantitative research showed that poor self-perceived health is a risk factor of (non-disability) early retirement. However, little is known on how poor health may lead to early retirement, and why poor health leads to early retirement in some employees, but not in others. Therefore, the present qualitative study aims to identify in which ways health influences early retirement. Face-to-face semi-structured interviews were conducted with 30 employees (60-64 years) who retired before the official retirement age of 65. Participants were selected from the Study on Transitions in Employment, Ability and Motivation. The interviews were transcribed verbatim, a summary was made including a timeline, and the interviews were open coded. In 15 of the 30 persons, health played a role in early retirement. Both poor and good health influenced early retirement. For poor health, four pathways were identified. First, employees felt unable to work at all due to health problems. Second, health problems resulted in a self-perceived (future) decline in the ability to work, and employees chose to retire early. Third, employees with health problems were afraid of a further decline in health, and chose to retire early. Fourth, employees with poor health retired early because they felt pushed out by their employer, although they themselves did not experience a reduced work ability. A good health influenced early retirement, since persons wanted to enjoy life while their health still allowed to do so. The financial opportunity to retire sometimes triggered the influence of poor health on early retirement, and often triggered the influence of good health. Employees and employers barely discussed opportunities to prolong working life. Poor and good health influence early

Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic

PubMed Central

Hsu, Ann; Siegler, Karen E.

2017-01-01

ABSTRACT Amino acid sequence differences in the variable region of immunoglobulin (Ig) cause wide variations in secretion outputs. To address how a primary sequence difference comes to modulate Ig secretion, we investigated the biosynthetic process of 2 human IgG2κ monoclonal antibodies (mAbs) that differ only by one amino acid in the light chain complementarity-determining region 1 while showing ∼20-fold variance in secretion titer. Although poorly secreted, the lower-secreting mAb of the 2 was by no means defective in terms of its folding stability, antigen binding, and in vitro biologic activity. However, upon overexpression in HEK293 cells, the low-secreting mAb revealed a high propensity to aggregate into enlarged globular structures called Russell bodies (RBs) in the endoplasmic reticulum. While Golgi morphology was affected by the formation of RBs, secretory pathway membrane traffic remained operational in those cells. Importantly, cellular protein synthesis was severely suppressed in RB-positive cells through the phosphorylation of eIF2α. PERK-dependent signaling was implicated in this event, given the upregulation and nuclear accumulation of downstream effectors such as ATF4 and CHOP. These findings illustrated that the underlining process of poor Ig secretion in RB-positive cells was due to downregulation of Ig synthesis instead of a disruption or blockade of secretory pathway trafficking. Therefore, RB formation signifies an end of active Ig production at the protein translation level. Consequently, depending on how soon and how severely an antibody-expressing cell develops the RB phenotype, the productive window of Ig secretion can vary widely among the cells expressing different mAbs. PMID:28379093

Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

PubMed

Ito, Sayaka; Kobayashi, Yoshihiko; Yamamoto, Yuki; Kimura, Koji; Okuda, Kiyoshi

2016-11-01

Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

Localization of DNA and RNA in eosinophil secretory granules.

PubMed

Behzad, Ali R; Walker, David C; Abraham, Thomas; McDonough, John; Mahmudi-Azer, Salahadin; Chu, Fanny; Shaheen, Furquan; Hogg, James C; Paré, Peter D

2010-01-01

Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent ultrastructural evidence suggests that protein synthesis may also take place within eosinophilic granules. We used 2 different methods to detect the presence of DNA and RNA in eosinophil secretory granules. Using bromodeoxyuridine, a thymidine analogue, and bromouridine, a uracil analogue, we labeled the DNA and RNA in eosinophils in vivo in rabbits. Immunoelectron microscopy to localize these molecules was performed on ultrathin sections of blood and bone marrow eosinophils using monoclonal anti-bromodeoxyuridine antibody with IgG as a control. The immunogold grain density was measured in each subcellular compartment within the eosinophils and analyzed using image analysis software. A combination of DNA/CD63 immunofluorescence staining and a fluorescently labeled molecular probe that stains RNA was used to examine the presence of DNA and RNA in the secretory granules of human blood eosinophils. The mean density of bromodeoxyuridine-labeled DNA and bromouridine-labeled RNA immunogold grains in the secretory granules of blood and bone marrow eosinophils were significantly higher (p < 0.0005) than cytoplasmic or background staining. We also demonstrated the existence of DNA and RNA in the CD63-positive secretory granules of human peripheral blood eosinophils by means of immunofluorescent staining and a fluorescently labeled molecular probe. These results provide evidence that eosinophil granules are the site of DNA and RNA synthesis and suggest the potential for a new role(s) for eosinophil-secretory granules. Copyright 2009 S. Karger AG, Basel.

Imaging Polarized Secretory Traffic at the Immune Synapse in Living T Lymphocytes.

PubMed

Calvo, Víctor; Izquierdo, Manuel

2018-01-01

Immune synapse (IS) formation by T lymphocytes constitutes a crucial event involved in antigen-specific, cellular and humoral immune responses. After IS formation by T lymphocytes and antigen-presenting cells, the convergence of secretory vesicles toward the microtubule-organizing center (MTOC) and MTOC polarization to the IS are involved in polarized secretion at the synaptic cleft. This specialized mechanism appears to specifically provide the immune system with a fine strategy to increase the efficiency of crucial secretory effector functions of T lymphocytes, while minimizing non-specific, cytokine-mediated stimulation of bystander cells, target cell killing and activation-induced cell death. The molecular bases involved in the polarized secretory traffic toward the IS in T lymphocytes have been the focus of interest, thus different models and several imaging strategies have been developed to gain insights into the mechanisms governing directional secretory traffic. In this review, we deal with the most widely used, state-of-the-art approaches to address the molecular mechanisms underlying this crucial, immune secretory response.

Altered white matter in early visual pathways of humans with amblyopia.

PubMed

Allen, Brian; Spiegel, Daniel P; Thompson, Benjamin; Pestilli, Franco; Rokers, Bas

2015-09-01

Amblyopia is a visual disorder caused by poorly coordinated binocular input during development. Little is known about the impact of amblyopia on the white matter within the visual system. We studied the properties of six major visual white-matter pathways in a group of adults with amblyopia (n=10) and matched controls (n=10) using diffusion weighted imaging (DWI) and fiber tractography. While we did not find significant differences in diffusion properties in cortico-cortical pathways, patients with amblyopia exhibited increased mean diffusivity in thalamo-cortical visual pathways. These findings suggest that amblyopia may systematically alter the white matter properties of early visual pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

JAG1-Mediated Notch Signaling Regulates Secretory Cell Differentiation of the Human Airway Epithelium.

PubMed

Gomi, Kazunori; Staudt, Michelle R; Salit, Jacqueline; Kaner, Robert J; Heldrich, Jonna; Rogalski, Allison M; Arbelaez, Vanessa; Crystal, Ronald G; Walters, Matthew S

2016-08-01

Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.

The HEART Pathway randomized trial: identifying emergency department patients with acute chest pain for early discharge.

PubMed

Mahler, Simon A; Riley, Robert F; Hiestand, Brian C; Russell, Gregory B; Hoekstra, James W; Lefebvre, Cedric W; Nicks, Bret A; Cline, David M; Askew, Kim L; Elliott, Stephanie B; Herrington, David M; Burke, Gregory L; Miller, Chadwick D

2015-03-01

The HEART Pathway is a decision aid designed to identify emergency department patients with acute chest pain for early discharge. No randomized trials have compared the HEART Pathway with usual care. Adult emergency department patients with symptoms related to acute coronary syndrome without ST-elevation on ECG (n=282) were randomized to the HEART Pathway or usual care. In the HEART Pathway arm, emergency department providers used the HEART score, a validated decision aid, and troponin measures at 0 and 3 hours to identify patients for early discharge. Usual care was based on American College of Cardiology/American Heart Association guidelines. The primary outcome, objective cardiac testing (stress testing or angiography), and secondary outcomes, index length of stay, early discharge, and major adverse cardiac events (death, myocardial infarction, or coronary revascularization), were assessed at 30 days by phone interview and record review. Participants had a mean age of 53 years, 16% had previous myocardial infarction, and 6% (95% confidence interval, 3.6%-9.5%) had major adverse cardiac events within 30 days of randomization. Compared with usual care, use of the HEART Pathway decreased objective cardiac testing at 30 days by 12.1% (68.8% versus 56.7%; P=0.048) and length of stay by 12 hours (9.9 versus 21.9 hours; P=0.013) and increased early discharges by 21.3% (39.7% versus 18.4%; P<0.001). No patients identified for early discharge had major adverse cardiac events within 30 days. The HEART Pathway reduces objective cardiac testing during 30 days, shortens length of stay, and increases early discharges. These important efficiency gains occurred without any patients identified for early discharge suffering MACE at 30 days. URL: http://www.clinicaltrials.gov. Unique Identifier: NCT01665521. © 2015 American Heart Association, Inc.

The non-canonical BMP and Wnt/β-catenin signaling pathways orchestrate early tooth development

PubMed Central

Yuan, Guohua; Yang, Guobin; Zheng, Yuqian; Zhu, Xiaojing; Chen, Zhi; Zhang, Zunyi; Chen, YiPing

2015-01-01

BMP and Wnt signaling pathways play a crucial role in organogenesis, including tooth development. Despite extensive studies, the exact functions, as well as if and how these two pathways act coordinately in regulating early tooth development, remain elusive. In this study, we dissected regulatory functions of BMP and Wnt pathways in early tooth development using a transgenic noggin (Nog) overexpression model (K14Cre;pNog). It exhibits early arrested tooth development, accompanied by reduced cell proliferation and loss of odontogenic fate marker Pitx2 expression in the dental epithelium. We demonstrated that overexpression of Nog disrupted BMP non-canonical activity, which led to a dramatic reduction of cell proliferation rate but did not affect Pitx2 expression. We further identified a novel function of Nog by inhibiting Wnt/β-catenin signaling, causing loss of Pitx2 expression. Co-immunoprecipitation and TOPflash assays revealed direct binding of Nog to Wnts to functionally prevent Wnt/β-catenin signaling. In situ PLA and immunohistochemistry on Nog mutants confirmed in vivo interaction between endogenous Nog and Wnts and modulation of Wnt signaling by Nog in tooth germs. Genetic rescue experiments presented evidence that both BMP and Wnt signaling pathways contribute to cell proliferation regulation in the dental epithelium, with Wnt signaling also controlling the odontogenic fate. Reactivation of both BMP and Wnt signaling pathways, but not of only one of them, rescued tooth developmental defects in K14Cre;pNog mice, in which Wnt signaling can be substituted by transgenic activation of Pitx2. Our results reveal the orchestration of non-canonical BMP and Wnt/β-catenin signaling pathways in the regulation of early tooth development. PMID:25428587

α2-COP is involved in early secretory traffic in Arabidopsis and is required for plant growth

PubMed Central

Gimeno-Ferrer, Fátima; Pastor-Cantizano, Noelia; Bernat-Silvestre, César; Selvi-Martínez, Pilar; Vera-Sirera, Francisco; Gao, Caiji; Perez-Amador, Miguel Angel; Jiang, Liwen; Aniento, Fernando

2017-01-01

Abstract COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (α-, β-, β′-, γ-, δ-, ε- and ζ-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of γ and δ, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of α2-COP, an Arabidopsis α-COP isoform. The α2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24δ5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The α2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the α2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the α2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of α2-COP affects the expression of secretory pathway genes. PMID:28025315

Pathways through which health influences early retirement: a qualitative study

PubMed Central

2013-01-01

Background Due to the aeging of the population, there is a societal need for workers to prolong their working lives. In the Netherlands, many employees still leave the workforce before the official retirement age of 65. Previous quantitative research showed that poor self-perceived health is a risk factor of (non-disability) early retirement. However, little is known on how poor health may lead to early retirement, and why poor health leads to early retirement in some employees, but not in others. Therefore, the present qualitative study aims to identify in which ways health influences early retirement. Methods Face-to-face semi-structured interviews were conducted with 30 employees (60–64 years) who retired before the official retirement age of 65. Participants were selected from the Study on Transitions in Employment, Ability and Motivation. The interviews were transcribed verbatim, a summary was made including a timeline, and the interviews were open coded. Results In 15 of the 30 persons, health played a role in early retirement. Both poor and good health influenced early retirement. For poor health, four pathways were identified. First, employees felt unable to work at all due to health problems. Second, health problems resulted in a self-perceived (future) decline in the ability to work, and employees chose to retire early. Third, employees with health problems were afraid of a further decline in health, and chose to retire early. Fourth, employees with poor health retired early because they felt pushed out by their employer, although they themselves did not experience a reduced work ability. A good health influenced early retirement, since persons wanted to enjoy life while their health still allowed to do so. The financial opportunity to retire sometimes triggered the influence of poor health on early retirement, and often triggered the influence of good health. Employees and employers barely discussed opportunities to prolong working life. Conclusions

21 CFR 866.5380 - Free secretory component immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5380 Free secretory component immuno-logical test system. (a) Identification. A free... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Free secretory component immuno-logical test...

Resveratrol reduces senescence-associated secretory phenotype by SIRT1/NF-κB pathway in gut of the annual fish Nothobranchius guentheri.

PubMed

Liu, Shan; Zheng, Zhaodi; Ji, Shuhua; Liu, Tingting; Hou, Yanhan; Li, Shasha; Li, Guorong

2018-06-13

Senescent cells display a senescence-associated secretory phenotype (SASP), which contributes to aging. Resveratrol, an activator of SIRT1, has anti-aging, anti-inflammatory, anti-oxidant, anti-free radical and other pharmacological effects. The genus of the annual fish Nothobranchius has become an emerging animal model for studying aging. However, the underlying mechanism for resveratrol to delay aging by SASP regulation has not been elucidated in vertebrates. In this study, the annual fish N. guentheri were fed with resveratrol for long-term treatment. The results showed that resveratrol reversed intensive senescence-associated β-galactosidase activity with aging process, down-regulated levels of SASP-associated proinflammatory cytokines IL-8 and TNFα, and up-regulated expression of anti-inflammatory cytokine IL-10 in gut of the fish. Resveratrol increased SIRT1 expression, and inhibited NF-κB by decreasing RelA/p65, Ac-RelA/p65 and p-IκBα levels and by increasing the interaction between SIRT1 and RelA/p65. Moreover, resveratrol reversed the decline of intestinal epithelial cells (IECs) and intestinal stem cells (ISCs) caused by aging in gut of the fish. Together, our results implied that resveratrol inhibited SASP through SIRT1/NF-κB signaling pathway and delayed aging of the annual fish N. guentheri. Copyright © 2018. Published by Elsevier Ltd.

Secretory expression of a heterologous protein, Aiio-AIO6BS, in Bacillus subtilis via a non-classical secretion pathway.

PubMed

Pan, Xingliang; Yang, Yalin; Liu, Xuewei; Li, Dong; Li, Juan; Guo, Xiaoze; Zhou, Zhigang

2016-09-16

The quenching enzyme AIO6 (AiiO-AIO6) has been reported as a feed additive preparation for application in aquaculture and biological control of pathogenic Aeromonas hydrophila. We developed an economical strategy to express AIO6BS (AiiO-AIO6BS, codon optimized AIO6 in Bacillus subtilis) in Bacillus subtilis for facilitating its widespread application. Promoter p43 without the signal peptide was used for secretory expression of AIO6BS in B. subtilis. Western blotting analysis demonstrated that AIO6BS was successfully expressed and secreted into the cell culture. Expression analysis of AIO6BS in the single or double mutant of the lytC and lytD genes for cell autolysis in B. subtilis 1A751 and cell autolysis-resistant engineered strain LM2531 derived from the wild type 168 indicated that the release of the heterologous protein AIO6BS was not simply mediated by cell lysis. Expression level of AIO6BS did not change in the mutants of B. subtilis that harbored mutations in the secA, tatAC, or ecsA genes compared with that in the parent wild type strain. These results suggested the AIO6BS protein was likely secreted via a non-classical secretion pathway. The expression analysis of the various N- or C-terminal truncated gene products indicated that AIO6BS probably acts as an export signal to direct its self-secretion across the cell membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

PubMed Central

2012-01-01

Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins. PMID:22234238

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway.

PubMed

Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Flórez, Martha G; Jami, Mohammad S; Paniagua, Miguel A; Martín, Juan F

2012-01-10

The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

Feasibility of a Clinical Pathway with Early Oral Intake and Discharge for Laparoscopic Gastrectomy.

PubMed

Nakagawa, M; Tomii, C; Inokuchi, M; Otsuki, S; Kojima, K

2017-12-01

Although some studies have reported the safety of early oral intake after gastrectomy, it still remains controversial. This study focused on the feasibility of a clinical pathway with early oral intake and discharge setting for exclusively laparoscopic distal gastrectomy. A clinical pathway was applied to 403 patients until December 2014. In the protocol, patients are allowed to take a sip of water and a soft diet on the first and second days after the operation, respectively, and the discharge day is set as the fifth to seventh day after the operation. Clinicopathological variables were prospectively collected, and risk factors for discharge variances were analyzed. The completion rate of the clinical pathway was 76.9%. There were five re-admissions (1.2%). The overall morbidity rate was 18% ( n = 72), and major complications (Clavien-Dindo IIIa or greater) occurred in 13 patients (3%). Complications were the causes for discharge variances in 68 cases (73%), while the attending surgeons' judgment was the cause in 25 cases (27%). On multivariate analysis, age (odds ratio = 2.23, 95% confidence interval = 1.38-3.60, p = 0.001) and operative time (odds ratio = 2.38, 95% confidence interval = 1.45-3.98, p = 0.001) were independent risk factors for discharge variances. A high completion rate of a clinical pathway with early oral intake and discharge setting for laparoscopic distal gastrectomy was achievable with an acceptably low re-admission rate. Laparoscopic distal gastrectomy is recommended as a first step for a clinical pathway with an early oral intake and discharge protocol.

Glycosaminoglycan synthesis by adult rat submandibular salivary-gland secretory units.

PubMed

Cutler, L S; Christian, C P; Rendell, J K

1987-01-01

The synthesis of glycosaminoglycans (GAG) by a preparation of purified, functional submandibular-gland secretory units (acini and intercalated ducts) was examined. Such units were isolated from Sprague-Dawley rats by digestion of minced gland with hyaluronidase and collagenase followed by gentle sieving of the digest through a graded series of Teflon screens. They incorporated amino acids into exocrine proteins which could be released by stimulation with isoproterenol as in vivo, indicating their functional integrity. Secretory units, incubated for 2 h in medium containing [35S]-sodium sulphate alone or in combination with [3H]-glucosamine, were then washed, homogenized and digested in pronase. The resulting material was then sequentially digested by specific enzymic and chemical procedures and analysed by chromatography on Sephadex G-50 columns to identify the various GAG synthesized. Secretory units synthesized a GAG mixture which was 20-25 per cent hyaluronic acid, 70-75 per cent heparan sulphate, and only 3-5 per cent chondroitin or dermatan sulphates, similar to that synthesized in vivo. No GAG was present in the secretory material, suggesting that all the GAG synthesized was destined for the basement membrane or cell surface.

Early Childhood Predictors of Low-Income Boys’ Pathways to Antisocial Behavior in Childhood, Adolescence, and Early Adulthood

PubMed Central

Shaw, Daniel S.; Gilliam, Mary

2016-01-01

Guided by a bridging model of pathways leading to low-income boys’ early-starting and persistent trajectories of antisocial behavior, the current paper reviews evidence supporting the model from early childhood through early adulthood. Using primarily a cohort of 310 low-income boys of families recruited from WIC centers in a large metropolitan area followed from infancy to early adulthood, and smaller cohorts of boys and girls followed through early childhood, we provide evidence supporting the critical role of parenting, maternal depression, and other proximal family risk factors in early childhood that are prospectively linked to trajectories of parent-reported conduct problems in early and middle childhood, youth-reported antisocial behavior during adolescence and early adulthood, as well as court-reported violent offending in adolescence. The findings are discussed in terms of the need to identify at-risk boys in early childhood and methods and platforms for engaging families in health care settings not previously used to implement preventive mental health services. PMID:28026042

Early Diagnosis of Clear Cell Kidney Cancer via VHL/HIF Pathway Regulated-Circulating microRNA

DTIC Science & Technology

2016-05-01

Award Number: W81XWH-11-1-0715 TITLE: Early Diagnosis of Clear Cell Kidney Cancer via VHL/HIF Pathway-Regulated Circulating microRNA PRINCIPAL...TITLE AND SUBTITLE Sa. CONTRACT NUMBER Early Diagnosis of Clear Cell Kidney Cancer via VHL/HIF Pathway- Regulated Circulating microRNA Sb. GRANT NUMBER...panel of diagnostic miRNAs that are measurable in serum and will be able to identify kidney cancer in its earliest stages. We hypothesized that serum

Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase

PubMed Central

Ace, Christopher I; Okulicz, William C

2004-01-01

Background In the endometrium the steroid hormone progesterone (P), acting through its nuclear receptors, regulates the expression of specific target genes and gene networks required for endometrial maturation. Proper endometrial maturation is considered a requirement for embryo implantation. Endometrial receptivity is a complex process that is spatially and temporally restricted and the identity of genes that regulate receptivity has been pursued by a number of investigators. Methods In this study we have used high density oligonucleotide microarrays to screen for changes in mRNA transcript levels between normal proliferative and adequate secretory phases in Rhesus monkey artificial menstrual cycles. Biotinylated cRNA was prepared from day 13 and days 21–23 of the reproductive cycle and transcript levels were compared by hybridization to Affymetrix HG-U95A arrays. Results Of ~12,000 genes profiled, we identified 108 genes that were significantly regulated during the shift from a proliferative to an adequate secretory endometrium. Of these genes, 39 were up-regulated at days 21–23 versus day 13, and 69 were down-regulated. Genes up-regulated in P-dominant tissue included: secretoglobin (uteroglobin), histone 2A, polo-like kinase (PLK), spermidine/spermine acetyltransferase 2 (SAT2), secretory leukocyte protease inhibitor (SLPI) and metallothionein 1G (MT1G), all of which have been previously documented as elevated in the Rhesus monkey or human endometrium during the secretory phase. Genes down-regulated included: transforming growth factor beta-induced (TGFBI or BIGH3), matrix metalloproteinase 11 (stromelysin 3), proenkephalin (PENK), cysteine/glycine-rich protein 2 (CSRP2), collagen type VII alpha 1 (COL7A1), secreted frizzled-related protein 4 (SFRP4), progesterone receptor membrane component 1 (PGRMC1), chemokine (C-X-C) ligand 12 (CXCL12) and biglycan (BGN). In addition, many novel/unknown genes were also identified. Validation of array data was performed

Nordihydroguaiaretic acid blocks protein transport in the secretory pathway causing redistribution of Golgi proteins into the endoplasmic reticulum.

PubMed

Fujiwara, T; Takami, N; Misumi, Y; Ikehara, Y

1998-01-30

We have investigated the effect of nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, on the intracellular protein transport and the structure of the Golgi complex. Pulse-chase experiments and immunoelectron microscopy showed that NDGA strongly inhibits the transport of newly synthesized secretory proteins to the Golgi complex resulting in their accumulation in the endoplasmic reticulum (ER). Despite their retention in the ER, oligosaccharides of secretory and ER-resident proteins were processed to endoglycosidase H-resistant forms, raising the possibility that oligosaccharide-processing enzymes are redistributed from the Golgi to the ER. Morphological observations further revealed that alpha-mannosidase II (a cis/medial-Golgi marker), but not TGN38 (a trans-Golgi network marker), rapidly redistributes to the ER in the presence of NDGA, resulting in the disappearance of the characteristic Golgi structure. Upon removal of the drug, the Golgi complex was reassembled into the normal structure as judged by perinuclear staining of alpha-mannosidase II and by restoration of the secretion. These effects of NDGA are quite similar to those of brefeldin A. However, unlike brefeldin A, NDGA did not cause a dissociation of beta-coatomer protein, a subunit of coatomer, from the Golgi membrane. On the contrary, NDGA exerted the stabilizing effect on beta-coatomer protein/membrane interaction against the dissociation caused by brefeldin A and ATP depletion. Taken together, these results indicate that NDGA is a potent agent disrupting the structure and function of the Golgi complex with a mechanism different from those known for other drugs reported so far.

Early risk factors and developmental pathways to chronic high inhibition and social anxiety disorder in adolescence.

PubMed

Essex, Marilyn J; Klein, Marjorie H; Slattery, Marcia J; Goldsmith, H Hill; Kalin, Ned H

2010-01-01

Evidence suggests that chronic high levels of behavioral inhibition are a precursor of social anxiety disorder. The authors sought to identify early risk factors for, and developmental pathways to, chronic high inhibition among school-age children and the association of chronic high inhibition with social anxiety disorder by adolescence. A community sample of 238 children was followed from birth to grade 9. Mothers, teachers, and children reported on the children's behavioral inhibition from grades 1 to 9. Lifetime history of psychiatric disorders was available for the subset of 60 (25%) children who participated in an intensive laboratory assessment at grade 9. Four early risk factors were assessed: female gender; exposure to maternal stress during infancy and the preschool period; and at age 4.5 years, early manifestation of behavioral inhibition and elevated afternoon salivary cortisol levels. All four risk factors predicted greater and more chronic inhibition from grades 1 to 9, and together they defined two developmental pathways. The first pathway, in girls, was partially mediated by early evidence of behavioral inhibition and elevated cortisol levels at age 4.5 years. The second pathway began with exposure to early maternal stress and was also partially mediated by childhood cortisol levels. By grade 9, chronic high inhibition was associated with a lifetime history of social anxiety disorder. Chronic high levels of behavioral inhibition are associated with social anxiety disorder by adolescence. The identification of two developmental pathways suggests the potential importance of considering both sets of risk factors in developing preventive interventions for social anxiety disorder.

Antigenic analyses of tissues and excretory and secretory products from Strongylus vulgaris.

PubMed

Wynne, E; Slocombe, J O; Wilkie, B N

1981-07-01

Rabbit antisera were prepared against veronal buffered saline extracts of L4 and L5 Strongylus vulgaris, adult S. vulgaris and adult Strongylus equinus retrieved from naturally infected horses. In agar gel diffusion with these antisera, adult S vulgaris and S. equinus each appeared to have at least one unique antigen; larval S. vulgaris appeared to have two species-specific and two stage-specific antigens. There were several common antigens. Excretory and secretory products were collected also from L4 and L5 an maintained over several days in tissue culture fluid. In agar gel diffusion against the above rabbit antisera, a stage-specific antigen was found also in excretory and secretory products. In addition, excretory and secretory products had three antigens in common with adult and larval S. vulgaris, but only one of these was common to adult S. equinus. The excretory and secretory products appear, therefore, to have two species-specific and one stage-specific antigens.

Conserved Genetic Pathways Controlling the Development of the Diffuse Endocrine System in Vertebrates and Drosophila

PubMed Central

Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina

2014-01-01

The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. PMID:20005229

The knottin-like Blufensin family regulates genes involved in nuclear import and the secretory pathway in barley-powdery mildew interactions

PubMed Central

Xu, Weihui; Meng, Yan; Surana, Priyanka; Fuerst, Greg; Nettleton, Dan; Wise, Roger P.

2015-01-01

Plants have evolved complex regulatory mechanisms to control a multi-layered defense response to microbial attack. Both temporal and spatial gene expression are tightly regulated in response to pathogen ingress, modulating both positive and negative control of defense. BLUFENSINs, small knottin-like peptides in barley, wheat, and rice, are highly induced by attack from fungal pathogens, in particular, the obligate biotrophic fungus, Blumeria graminis f. sp. hordei (Bgh), causal agent of barley powdery mildew. Previous research indicated that Blufensin1 (Bln1) functions as a negative regulator of basal defense mechanisms. In the current report, we show that BLN1 and BLN2 can both be secreted to the apoplast and Barley stripe mosaic virus (BSMV)-mediated overexpression of Bln2 increases susceptibility of barley to Bgh. Bimolecular fluorescence complementation (BiFC) assays signify that BLN1 and BLN2 can interact with each other, and with calmodulin. We then used BSMV-induced gene silencing to knock down Bln1, followed by Barley1 GeneChip transcriptome analysis, to identify additional host genes influenced by Bln1. Analysis of differential expression revealed a gene set enriched for those encoding proteins annotated to nuclear import and the secretory pathway, particularly Importin α1-b and Sec61 γ subunits. Further functional analysis of these two affected genes showed that when silenced, they also reduced susceptibility to Bgh. Taken together, we postulate that Bln1 is co-opted by Bgh to facilitate transport of disease-related host proteins or effectors, influencing the establishment of Bgh compatibility on its barley host. PMID:26089830

EARLY CHILDHOOD PREDICTORS OF LOW-INCOME BOYS' PATHWAYS TO ANTISOCIAL BEHAVIOR IN CHILDHOOD, ADOLESCENCE, AND EARLY ADULTHOOD.

PubMed

Shaw, Daniel S; Gilliam, Mary

2017-01-01

Guided by a bridging model of pathways leading to low-income boys' early starting and persistent trajectories of antisocial behavior, the current article reviews evidence supporting the model from early childhood through early adulthood. Using primarily a cohort of 310 low-income boys of families recruited from Women, Infants, and Children Nutrition Supplement centers in a large metropolitan area followed from infancy to early adulthood and a smaller cohort of boys and girls followed through early childhood, we provide evidence supporting the critical role of parenting, maternal depression, and other proximal family risk factors in early childhood that are prospectively linked to trajectories of parent-reported conduct problems in early and middle childhood, youth-reported antisocial behavior during adolescence and early adulthood, and court-reported violent offending in adolescence. The findings are discussed in terms of the need to identify at-risk boys in early childhood and methods and platforms for engaging families in healthcare settings not previously used to implement preventive mental health services. © 2016 Michigan Association for Infant Mental Health.

Early development of the circumferential axonal pathway in mouse and chick spinal cord.

PubMed

Holley, J A

1982-03-10

The early development of the circumferential axonal pathway in the brachial and lumbar spinal cord of mouse and chick embryos was studied by scanning and transmission electron microscopy. The cellular processes which comprise this pathway grow in the transverse plane and along the lateral margin of the marginal zone (i.e., circumferentially oriented), as typified by the early embryonic commissural axons. The first formative event observed was in the ventrolateral margin of the primitive spinal cord ventricular zone. Cellular processes were found near the external limiting membrane that appeared to grow a variable distance either dorsally or ventrally. Later in development, presumptive motor column neurons migrated into the ventrolateral region, distal to these early circumferentially oriented processes. Concurrently, other circumferentially oriented perikarya and processes appeared along the dorsolateral margin. Due to their aligned sites of origin and parallel growth, the circumferential processes formed a more or less continuous line or pathway, which in about 10% of the scanned specimens could be followed along the entire lateral margin of the embryonic spinal cord. Several specimens later in development had two sets of aligned circumferential processes in the ventral region. Large numbers of circumferential axons were then found to follow the preformed pathway by fasciculation, after the primitive motor column had become established. Since the earliest circumferential processes appeared to differentiate into axons and were found nearly 24 hours prior to growth of most circumferential axons, their role in guidance as pioneering axons was suggested.

Involvement of Dynein and Spectrin with Early Melanosome Transport and Melanosomal Protein Trafficking

PubMed Central

Watabe, Hidenori; Valencia, Julio C.; Le Pape, Elodie; Yamaguchi, Yuji; Nakamura, Masayuki; Rouzaud, François; Hoashi, Toshihiko; Kawa, Yoko; Mizoguchi, Masako; Hearing, Vincent J.

2007-01-01

Melanosomes are unique membrane-bound organelles specialized for the synthesis and distribution of melanin. Mechanisms involved in the trafficking of proteins to melanosomes and in the transport of mature pigmented melanosomes to the dendrites of melanocytic cells are being characterized but details about those processes during early stages of melanosome maturation are not well understood. Early melanosomes must remain in the perinuclear area until critical components are assembled. In this study, we characterized the processing of two distinct melanosomal proteins, TYR and Pmel17, to elucidate protein processing in early or late steps of the secretory pathway, respectively, and to determine mechanisms underlying the subcellular localization and transport of early melanosomes. We used immunological, biochemical and molecular approaches to demonstrate that the movement of early melanosomes in the perinuclear area depends primarily on microtubules but not on actin filaments. In contrast, the trafficking of TYR and Pmel17 depends on cytoplasmic dynein and its interaction with the spectrin/ankyrin system which is involved with the sorting of cargo from the plasma membrane. These results provide important clues towards understanding the processes involved with early events in melanosome formation and transport. PMID:17687388

Genetic moderation of multiple pathways linking early cumulative socioeconomic adversity and young adults' cardiometabolic disease risk.

PubMed

Wickrama, Kandauda A S; Lee, Tae Kyoung; O'Neal, Catherine Walker

2018-02-01

Recent research suggests that psychosocial resources and life stressors are mediating pathways explaining socioeconomic variation in young adults' health risks. However, less research has examined both these pathways simultaneously and their genetic moderation. A nationally representative sample of 11,030 respondents with prospective data collected over 13 years from the National Study of Adolescent to Adult Health was examined. First, the association between early cumulative socioeconomic adversity and young adults' (ages 25-34) cardiometabolic disease risk, as measured by 10 biomarkers, through psychosocial resources (educational attainment) and life stressors (accelerated transition to adulthood) was examined. Second, moderation of these pathways by the serotonin transporter linked polymorphic region gene (5-HTTLPR) was examined. There was evidence for the association between early socioeconomic adversity and young adults' cardiometabolic disease risk directly and indirectly through educational attainment and accelerated transitions. These direct and mediating pathways were amplified by the 5-HTTLPR polymorphism. These findings elucidate how early adversity can have an enduring influence on young adults' cardiometabolic disease risk directly and indirectly through psychosocial resources and life stressors and their genetic moderation. This information suggests that effective intervention and prevention programs should focus on early adversity, youth educational attainment, and their transition to young adulthood.

Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

PubMed Central

González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

2013-01-01

Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

Distribution Profile of Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channels in α and β Cells of Pancreas: Dominant Localization in Secretory Granules and Common Error in Identification of Secretory Granule Membranes.

PubMed

Hur, Yong Suk; Yoo, Seung Hyun

2015-01-01

The α and β cells of pancreatic islet release important hormones in response to intracellular Ca increases that result from Ca releases through the inositol 1,4,5-trisphoshate receptor (IP3R)/Ca channels. Yet no systematic studies on distribution of IP3R/Ca channels have been done, prompting us to investigate the distribution of all 3 IP3R isoforms. Immunogold electron microscopy was performed to determine the presence and the relative concentrations of all 3 IP3R isoforms in 2 major organelles secretory granules (SGs) and the endoplasmic reticulum of α and β cells of rat pancreas. All 3 IP3R isoforms were present in SG membranes of both cells, and the IP3R concentrations in SGs were ∼2-fold higher than those in the endoplasmic reticulum. Moreover, large halos shown in the electron microscope images of insulin-containing SGs of β cells were gap spaces that resulted from separation of granule membranes from the surrounding cytoplasm. These results strongly suggest the important roles of SGs in IP3-induced, Ca-dependent regulatory secretory pathway in pancreas. Moreover, the accurate location of SG membranes of β cells was further confirmed by the location of another integral membrane protein synaptotagmin V and of membrane phospholipid PI(4,5)P2.

Excretory-secretory product of third-stage Gnathostoma spinigerum larvae induces apoptosis in human peripheral blood mononuclear cells.

PubMed

Viseshakul, Nareerat; Dechkhajorn, Wilanee; Benjathummarak, Surachet; Nuamtanong, Supaporn; Maneerat, Yaowapa

2017-10-01

Human gnathostomiasis caused by third-stage Gnathostoma spinigerum larvae (G. spinigerum L3) is an important zoonotic disease in tropical areas of the world. The excretory-secretory products (ES) that are excreted by infective larva play a significant role in host immune evasion and tissue destruction. To investigate the poorly understood mechanisms of G. spinigerum L3 pathogenesis, we focused on the potential effect of ES on inducing apoptosis in human immune cells by using human peripheral blood mononuclear cells (PBMCs) as a model. Early and late apoptosis of PBMCs were assessed following the exposure of these cells to G. spinigerum L3 ES (0.1, 0.5, and 1.0 μg/ml) for 6-48 h. The apoptotic cells were identified by flow cytometric staining of PBMC with FITC-annexin V and propidium iodide. The expression of regulatory genes related to apoptosis mechanisms in ES-treated PBMCs was investigated using a Human Apoptosis RT 2 Profiler™ PCR Array. The results showed significant levels of early phase apoptosis at 18 h and of late phase apoptosis at 24 h. We speculate that this apoptosis in PBMCs occurs via the extrinsic pathway. Apoptosis in the ES-induced PBMCs was observed as quickly as 90 min after exposure, and the highest effect was observed at 18-24 h. Furthermore, ES can trigger apoptosis lasting for 48 h. Our findings expand the understanding of one of the mechanisms involved, immune-evasive strategy mechanism used by G. spinigerum larvae during human gnathostomiasis.

HAI-2 stabilizes, inhibits and regulates SEA-cleavage-dependent secretory transport of matriptase.

PubMed

Nonboe, Annika W; Krigslund, Oliver; Soendergaard, Christoffer; Skovbjerg, Signe; Friis, Stine; Andersen, Martin N; Ellis, Vincent; Kawaguchi, Makiko; Kataoka, Hiroaki; Bugge, Thomas H; Vogel, Lotte K

2017-06-01

It has recently been shown that hepatocyte growth factor activator inhibitor-2 (HAI-2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI-2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI-2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non-SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto-cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI-2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI-2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI-2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Developmental pathways from childhood conduct problems to early adult depression: findings from the ALSPAC cohort.

PubMed

Stringaris, Argyris; Lewis, Glyn; Maughan, Barbara

2014-07-01

Pathways from early-life conduct problems to young adult depression remain poorly understood. To test developmental pathways from early-life conduct problems to depression at age 18. Data (n = 3542) came from the Avon Longitudinal Study of Parents and Children (ALSPAC). Previously derived conduct problem trajectories (ages 4-13 years) were used to examine associations with depression from ages 10 to 18 years, and the role of early childhood factors as potential confounders. Over 43% of young adults with depression in the ALSPAC cohort had a history of child or adolescent conduct problems, yielding a population attributable fraction of 0.15 (95% CI 0.08-0.22). The association between conduct problems and depression at age 18 was considerable even after adjusting for prior depression (odds ratio 1.55, 95% CI 1.24-1.94). Early-onset persistent conduct problems carried the highest risk for later depression. Irritability characterised depression for those with a history of conduct problems. Early-life conduct problems are robustly associated with later depressive disorder and may be useful targets for early intervention. Royal College of Psychiatrists.

Educational Pathways and Change in Crime Between Adolescence and Early Adulthood

PubMed Central

Swisher, Raymond R.; Dennison, Christopher R.

2016-01-01

Objectives This article examines the relationship between intergenerational educational pathways and change in crime. Moreover, it examines the potential mediating roles of family and employment transitions, economic stressors, and social psychological factors. Method Data from the National Longitudinal Study of Adolescent to Adult Health (N = 14,742) and negative binomial models are used to assess associations between educational pathways (i.e., upward, downward, and stable) and change in crime between adolescence and early adulthood. Selection effects are assessed with lagged dependent variables and controls for self-control, grades, and the Add Health Picture Vocabulary Test. Results Intergenerational educational pathways are significantly associated with changes in crime. Downward educational pathways were predictive of increases in crime, whereas upward pathways were associated with decreases in crime. These associations were partly mediated by family transitions, and more strongly by economic stressors. These results were robust to controls for selection related variables. Conclusions This study is among the first to examine the relationship between intergenerational educational pathways and crime in the United States. Both upward and downward changes in educational attainments were found to be significant for crime. These findings are notable given the continuing expansion of higher education as well as concerns regarding increasing stratification and downward mobility in the United States. PMID:28348441

Spatial Relationships between Markers for Secretory and Endosomal Machinery in Human Cytomegalovirus-Infected Cells versus Those in Uninfected Cells▿†

PubMed Central

Das, Subhendu; Pellett, Philip E.

2011-01-01

Human cytomegalovirus (HCMV) induces extensive remodeling of the secretory apparatus to form the cytoplasmic virion assembly compartment (cVAC), where virion tegumentation and envelopment take place. We studied the structure of the cVAC by confocal microscopy to assess the three-dimensional distribution of proteins specifically associated with individual secretory organelles. In infected cells, early endosome antigen 1 (EEA1)-positive vesicles are concentrated at the center of the cVAC and, as previously seen, are distinct from structures visualized by markers for the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network (TGN). EEA1-positive vesicles can be strongly associated with markers for recycling endosomes, to a lesser extent with markers associated with components of the endosomal sorting complex required for transport III (ESCRT III) machinery, and then with markers of late endosomes. In comparisons of uninfected and infected cells, we found significant changes in the structural associations and colocalization of organelle markers, as well as in net organelle volumes. These results provide new evidence that the HCMV-induced remodeling of the membrane transport apparatus involves much more than simple relocation and expansion of preexisting structures and are consistent with the hypothesis that the shift in identity of secretory organelles in HCMV-infected cells results in new functional profiles. PMID:21471245

Isolation of intact sub-dermal secretory cavities from Eucalyptus

PubMed Central

2010-01-01

Background The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils. Results Leaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h), with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories. Conclusions The protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain. PMID:20807444

Aldosterone stimulates vacuolar H+-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway

PubMed Central

Winter, Christian; Kampik, Nicole B.; Vedovelli, Luca; Rothenberger, Florina; Păunescu, Teodor G.; Stehberger, Paul A.; Brown, Dennis; John, Hubert

2011-01-01

Urinary acidification in the collecting duct is mediated by the activity of H+-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H+-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H+-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific Gαq inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca2+ with BAPTA, and blockade of protein kinase C prevented the stimulation of H+-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H+-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H+-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H+-ATPase activity. Thus, the nongenomic modulation of H+-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a Gαq protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H+-ATPase activity and contribute to final urinary acidification. PMID:21832245

Conserved genetic pathways controlling the development of the diffuse endocrine system in vertebrates and Drosophila.

PubMed

Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina L

2010-05-01

The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. Copyright 2009. Published by Elsevier Inc.

Membrane Tension Inhibits Rapid and Slow Endocytosis in Secretory Cells.

PubMed

Wu, Xin-Sheng; Elias, Sharon; Liu, Huisheng; Heureaux, Johanna; Wen, Peter J; Liu, Allen P; Kozlov, Michael M; Wu, Ling-Gang

2017-12-05

Endocytosis generates spherical or ellipsoid-like vesicles from the plasma membrane, which recycles vesicles that fuse with the plasma member during exocytosis in neurons and endocrine secretory cells. Although tension in the plasma membrane is generally considered to be an important factor in regulating endocytosis, whether membrane tension inhibits or facilitates endocytosis remains debated in the endocytosis field, and has been rarely studied for vesicular endocytosis in secretory cells. Here we report that increasing membrane tension by adjusting osmolarity inhibited both the rapid (a few seconds) and slow (tens of seconds) endocytosis in calyx-type nerve terminals containing conventional active zones and in neuroendocrine chromaffin cells. We address the mechanism of this phenomenon by computational modeling of the energy barrier that the system must overcome at the stage of membrane budding by an assembling protein coat. We show that this barrier grows with increasing tension, which may slow down or prevent membrane budding. These results suggest that in live secretory cells, membrane tension exerts inhibitory action on endocytosis. Published by Elsevier Inc.

Developmental pathways from childhood conduct problems to early adult depression: findings from the ALSPAC cohort

PubMed Central

Stringaris, Argyris; Lewis, Glyn; Maughan, Barbara

2014-01-01

Background Pathways from early-life conduct problems to young adult depression remain poorly understood. Aims To test developmental pathways from early-life conduct problems to depression at age 18. Method Data (n = 3542) came from the Avon Longitudinal Study of Parents and Children (ALSPAC). Previously derived conduct problem trajectories (ages 4-13 years) were used to examine associations with depression from ages 10 to 18 years, and the role of early childhood factors as potential confounders. Results Over 43% of young adults with depression in the ALSPAC cohort had a history of child or adolescent conduct problems, yielding a population attributable fraction of 0.15 (95% CI 0.08-0.22). The association between conduct problems and depression at age 18 was considerable even after adjusting for prior depression (odds ratio 1.55, 95% CI 1.24-1.94). Early-onset persistent conduct problems carried the highest risk for later depression. Irritability characterised depression for those with a history of conduct problems. Conclusions Early-life conduct problems are robustly associated with later depressive disorder and may be useful targets for early intervention. PMID:24764545

Stress and resource pathways connecting early socioeconomic adversity to young adults' physical health risk.

PubMed

Wickrama, Kandauda K A S; Lee, Tae Kyoung; O'Neal, Catherine Walker; Kwon, Josephine A

2015-05-01

Although research has established the impact of early stress, including stressful life contexts, and early resources, such as educational attainment, on various adolescent health outcomes, previous research has not adequately investigated "integrative models" incorporating both stress and resource mediational pathways to explain how early socioeconomic adversity impacts physical health outcomes, particularly in early life stages. Data on early childhood/adolescent stress and socioeconomic resources as well as biomarkers indicating physical health status in young adulthood were collected from 11,798 respondents (54 % female) over a 13-year period from youth participating in the National Study of Adolescent Health (Add Health). Physical health risk in young adulthood was measured using a composite index of nine regulatory biomarkers of cardiovascular and metabolic systems. Heterogeneity in stress and socioeconomic resource pathways was assessed using latent class analysis to identify clusters, or classes, of stress and socioeconomic resource trajectories. The influence of early socioeconomic adversity on young adults' physical health risk, as measured by biomarkers, was estimated, and the role of stress and socioeconomic resource trajectory classes as linking mechanisms was assessed. There was evidence for the influence of early socioeconomic adversity on young adults' physical health risk directly and indirectly through stress and socioeconomic resource trajectory classes over the early life course. These findings suggest that health models should be broadened to incorporate both stress and resource experiences simultaneously. Furthermore, these findings have prevention and intervention implications, including the importance of early socioeconomic adversity and key intervention points for "turning" the trajectories of at-risk youth.

Anomaly Detection in Host Signaling Pathways for the Early Prognosis of Acute Infection.

PubMed

Wang, Kun; Langevin, Stanley; O'Hern, Corey S; Shattuck, Mark D; Ogle, Serenity; Forero, Adriana; Morrison, Juliet; Slayden, Richard; Katze, Michael G; Kirby, Michael

2016-01-01

Clinical diagnosis of acute infectious diseases during the early stages of infection is critical to administering the appropriate treatment to improve the disease outcome. We present a data driven analysis of the human cellular response to respiratory viruses including influenza, respiratory syncytia virus, and human rhinovirus, and compared this with the response to the bacterial endotoxin, Lipopolysaccharides (LPS). Using an anomaly detection framework we identified pathways that clearly distinguish between asymptomatic and symptomatic patients infected with the four different respiratory viruses and that accurately diagnosed patients exposed to a bacterial infection. Connectivity pathway analysis comparing the viral and bacterial diagnostic signatures identified host cellular pathways that were unique to patients exposed to LPS endotoxin indicating this type of analysis could be used to identify host biomarkers that can differentiate clinical etiologies of acute infection. We applied the Multivariate State Estimation Technique (MSET) on two human influenza (H1N1 and H3N2) gene expression data sets to define host networks perturbed in the asymptomatic phase of infection. Our analysis identified pathways in the respiratory virus diagnostic signature as prognostic biomarkers that triggered prior to clinical presentation of acute symptoms. These early warning pathways correctly predicted that almost half of the subjects would become symptomatic in less than forty hours post-infection and that three of the 18 subjects would become symptomatic after only 8 hours. These results provide a proof-of-concept for utility of anomaly detection algorithms to classify host pathway signatures that can identify presymptomatic signatures of acute diseases and differentiate between etiologies of infection. On a global scale, acute respiratory infections cause a significant proportion of human co-morbidities and account for 4.25 million deaths annually. The development of clinical

Anomaly Detection in Host Signaling Pathways for the Early Prognosis of Acute Infection

PubMed Central

O’Hern, Corey S.; Shattuck, Mark D.; Ogle, Serenity; Forero, Adriana; Morrison, Juliet; Slayden, Richard; Katze, Michael G.

2016-01-01

Clinical diagnosis of acute infectious diseases during the early stages of infection is critical to administering the appropriate treatment to improve the disease outcome. We present a data driven analysis of the human cellular response to respiratory viruses including influenza, respiratory syncytia virus, and human rhinovirus, and compared this with the response to the bacterial endotoxin, Lipopolysaccharides (LPS). Using an anomaly detection framework we identified pathways that clearly distinguish between asymptomatic and symptomatic patients infected with the four different respiratory viruses and that accurately diagnosed patients exposed to a bacterial infection. Connectivity pathway analysis comparing the viral and bacterial diagnostic signatures identified host cellular pathways that were unique to patients exposed to LPS endotoxin indicating this type of analysis could be used to identify host biomarkers that can differentiate clinical etiologies of acute infection. We applied the Multivariate State Estimation Technique (MSET) on two human influenza (H1N1 and H3N2) gene expression data sets to define host networks perturbed in the asymptomatic phase of infection. Our analysis identified pathways in the respiratory virus diagnostic signature as prognostic biomarkers that triggered prior to clinical presentation of acute symptoms. These early warning pathways correctly predicted that almost half of the subjects would become symptomatic in less than forty hours post-infection and that three of the 18 subjects would become symptomatic after only 8 hours. These results provide a proof-of-concept for utility of anomaly detection algorithms to classify host pathway signatures that can identify presymptomatic signatures of acute diseases and differentiate between etiologies of infection. On a global scale, acute respiratory infections cause a significant proportion of human co-morbidities and account for 4.25 million deaths annually. The development of clinical

Effects of ambroxol on the autophagy-lysosome pathway and mitochondria in primary cortical neurons.

PubMed

Magalhaes, J; Gegg, M E; Migdalska-Richards, A; Schapira, A H

2018-01-23

Glucocerebrosidase (GBA1) mutations are the major genetic risk factor for Parkinson's Disease (PD). The pathogenic mechanism is still unclear, but alterations in lysosomal-autophagy processes are implicated due to reduction of mutated glucocerebrosidase (GCase) in lysosomes. Wild-type GCase activity is also decreased in sporadic PD brains. Small molecule chaperones that increase lysosomal GCase activity have potential to be disease-modifying therapies for GBA1-associated and sporadic PD. Therefore we have used mouse cortical neurons to explore the effects of the chaperone ambroxol. This chaperone increased wild-type GCase mRNA, protein levels and activity, as well as increasing other lysosomal enzymes and LIMP2, the GCase transporter. Transcription factor EB (TFEB), the master regulator of the CLEAR pathway involved in lysosomal biogenesis was also increased upon ambroxol treatment. Moreover, we found macroautophagy flux blocked and exocytosis increased in neurons treated with ambroxol. We suggest that ambroxol is blocking autophagy and driving cargo towards the secretory pathway. Mitochondria content was also found to be increased by ambroxol via peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α). Our data suggest that ambroxol, besides being a GCase chaperone, also acts on other pathways, such as mitochondria, lysosomal biogenesis, and the secretory pathway.

Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

PubMed

Yarimizu, Tohru; Nakamura, Mikiko; Hoshida, Hisashi; Akada, Rinji

2015-02-14

Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences. Mutational analysis of the GLuc signal sequence revealed that the GLuc hydrophobic peptide length was lower limit for effective secretion and that the N-terminal basic residue was indispensable. Deletion of the 16th Glu caused enhanced levels of secreted protein, suggesting that this hydrophilic residue defined the boundary of a hydrophobic peptide stretch. Consequently, we redesigned this domain as a repeat of a single hydrophobic amino acid between the N-terminal Lys and C-terminal Glu. Stretches consisting of Phe, Leu, Ile, or Met were effective for secretion but the number of residues affected secretory activity. A stretch containing sixteen consecutive methionine residues (M16) showed the highest activity; the M16 sequence was therefore utilized for the secretory production of human leukemia inhibitory factor protein in yeast, resulting in enhanced secreted protein yield. We present a new concept for the provision of secretory signal sequence ability in the yeast K. marxianus, determined by the number of residues of a single hydrophobic residue located between N-terminal basic and C-terminal acidic amino acid boundaries.

Autoantibodies to alfa-fodrin in patients with Hashimoto thyroiditis and Sjögren's syndrome: possible markers for a common secretory disorder.

PubMed

Szanto, Antonia; Csipo, Istvan; Horvath, Ildiko; Biro, Edit; Szodoray, Peter; Zeher, Margit

2008-09-01

Presence of autoantibodies to alfa-fodrin was investigated in patients with Sjögren's syndrome (n = 61), Hashimoto thyroiditis (n = 27), Sjögren's syndrome associated with Hashimoto thyroiditis (n = 31) and in healthy persons (n = 77). In each group, level of alfa-fodrin antibodies was higher than in the controls. There was no significant difference in their presence either between patients with Hashimoto thyroiditis with or without Sjögren's syndrome, or-in IgA isotype-between Sjögren's and Hashimoto thyroiditis patients. Correlation was found between the level of IgG alfa-fodrin and anti-thyroglobulin antibodies. Based on these findings, fodrin can be associated with both endocrine and exocrine glandular secretion. Antibodies to alfa-fodrin might have a role in the pathogenesis of Hashimoto thyroiditis concerning the "final common effectory pathway", secretion. Alfa-fodrin antibodies can be good markers of secretory disorders. Assessment of these autoantibodies might help the diagnosis and follow-up of patients with impaired secretory capability of not only autoimmune origin.

Antigenic analyses of tissues and excretory and secretory products from Strongylus vulgaris.

PubMed Central

Wynne, E; Slocombe, J O; Wilkie, B N

1981-01-01

Rabbit antisera were prepared against veronal buffered saline extracts of L4 and L5 Strongylus vulgaris, adult S. vulgaris and adult Strongylus equinus retrieved from naturally infected horses. In agar gel diffusion with these antisera, adult S vulgaris and S. equinus each appeared to have at least one unique antigen; larval S. vulgaris appeared to have two species-specific and two stage-specific antigens. There were several common antigens. Excretory and secretory products were collected also from L4 and L5 an maintained over several days in tissue culture fluid. In agar gel diffusion against the above rabbit antisera, a stage-specific antigen was found also in excretory and secretory products. In addition, excretory and secretory products had three antigens in common with adult and larval S. vulgaris, but only one of these was common to adult S. equinus. The excretory and secretory products appear, therefore, to have two species-specific and one stage-specific antigens. Images Fig. 1 a and b. Fig. 2 a and b. Fig. 3 a and b. Fig. 4 a and b. Fig. 5 a and b. Fig. 6 a and b. Fig. 7 a and b. Fig. 8 a and b. PMID:6804070

Genome Analysis Reveals Interplay between 5′UTR Introns and Nuclear mRNA Export for Secretory and Mitochondrial Genes

PubMed Central

Cenik, Can; Chua, Hon Nian; Zhang, Hui; Tarnawsky, Stefan P.; Akef, Abdalla; Derti, Adnan; Tasan, Murat; Moore, Melissa J.; Palazzo, Alexander F.; Roth, Frederick P.

2011-01-01

In higher eukaryotes, messenger RNAs (mRNAs) are exported from the nucleus to the cytoplasm via factors deposited near the 5′ end of the transcript during splicing. The signal sequence coding region (SSCR) can support an alternative mRNA export (ALREX) pathway that does not require splicing. However, most SSCR–containing genes also have introns, so the interplay between these export mechanisms remains unclear. Here we support a model in which the furthest upstream element in a given transcript, be it an intron or an ALREX–promoting SSCR, dictates the mRNA export pathway used. We also experimentally demonstrate that nuclear-encoded mitochondrial genes can use the ALREX pathway. Thus, ALREX can also be supported by nucleotide signals within mitochondrial-targeting sequence coding regions (MSCRs). Finally, we identified and experimentally verified novel motifs associated with the ALREX pathway that are shared by both SSCRs and MSCRs. Our results show strong correlation between 5′ untranslated region (5′UTR) intron presence/absence and sequence features at the beginning of the coding region. They also suggest that genes encoding secretory and mitochondrial proteins share a common regulatory mechanism at the level of mRNA export. PMID:21533221

Signaling Pathway in Early Brain Injury after Subarachnoid Hemorrhage: News Update.

PubMed

Ji, Chengyuan; Chen, Gang

2016-01-01

The annual incidence of subarachnoid hemorrhage (SAH) caused by intracranial aneurysm rupture is approximately 10.5/10 million people in China, making SAH the third most frequently occurring hemorrhage of the intracranial type after cerebral embolism and hypertensive intracerebral hemorrhage. SAH caused by ruptured aneurysm leads to a mortality rate as high as 67 %, and, because of the sudden onset of this disease, approximately 12-15 % of patients die before they can receive effective treatment. Early brain injury (EBI) is the brain damage occurring within the first 72 h after SAH. Two-thirds of mortality caused by SAH occurs within 48 h, mainly as a result of EBI. With the development of molecular biology and medicine microscopy techniques, various signaling pathways involved in EBI after SAH have been revealed. Understanding these signaling pathways may help clinicians treat EBI after SAH and improve long-term prognosis of SAH patients. This chapter summarizes several important signaling pathways implicated in EBI caused by SAH.

Imaging Ca2+-triggered exocytosis of single secretory granules on plasma membrane lawns from neuroendocrine cells.

PubMed

Lang, Thorsten

2008-01-01

This cell-free assay for exocytosis is particularly useful when spatial information about exocytotic sites and biochemical access to the plasma membrane within less than a minute is required. It is based on the study of plasma membrane lawns from secretory cells exhibiting secretory granules filled with neuropeptide Y-green fluorescent protein (NPY-GFP). The sample is prepared by subjecting NPY-GFP-expressing cells to a brief ultrasound pulse, leaving behind a basal, flat plasma membrane with fluorescent attached secretory organelles. These sheets can then be incubated in defined solutions with the benefit that complete solution changes can be achieved in less than 1 min. Individual secretory granules are monitored in the docked state and during exocytosis by video microscopy.

Proteomics of Dense Core Secretory Vesicles Reveal Distinct Protein Categories for Secretion of Neuroeffectors for Cell-Cell Communication

PubMed Central

Wegrzyn, Jill L.; Bark, Steven J.; Funkelstein, Lydiane; Mosier, Charles; Yap, Angel; Kazemi-Esfarjani, Parasa; La Spada, Albert; Sigurdson, Christina; O’Connor, Daniel T.; Hook, Vivian

2010-01-01

Regulated secretion of neurotransmitters and neurohumoural factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoural factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 distinct proteins in the soluble fraction and 384 distinct membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease. PMID:20695487

JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age

PubMed Central

Xu, Ming; Tchkonia, Tamara; Ding, Husheng; Ogrodnik, Mikolaj; Lubbers, Ellen R.; Pirtskhalava, Tamar; White, Thomas A.; Johnson, Kurt O.; Stout, Michael B.; Mezera, Vojtech; Giorgadze, Nino; Jensen, Michael D.; LeBrasseur, Nathan K.; Kirkland, James L.

2015-01-01

Chronic, low grade, sterile inflammation frequently accompanies aging and age-related diseases. Cellular senescence is associated with the production of proinflammatory chemokines, cytokines, and extracellular matrix (ECM) remodeling proteases, which comprise the senescence-associated secretory phenotype (SASP). We found a higher burden of senescent cells in adipose tissue with aging. Senescent human primary preadipocytes as well as human umbilical vein endothelial cells (HUVECs) developed a SASP that could be suppressed by targeting the JAK pathway using RNAi or JAK inhibitors. Conditioned medium (CM) from senescent human preadipocytes induced macrophage migration in vitro and inflammation in healthy adipose tissue and preadipocytes. When the senescent cells from which CM was derived had been treated with JAK inhibitors, the resulting CM was much less proinflammatory. The administration of JAK inhibitor to aged mice for 10 wk alleviated both adipose tissue and systemic inflammation and enhanced physical function. Our findings are consistent with a possible contribution of senescent cells and the SASP to age-related inflammation and frailty. We speculate that SASP inhibition by JAK inhibitors may contribute to alleviating frailty. Targeting the JAK pathway holds promise for treating age-related dysfunction. PMID:26578790

Amelogenins as Potential Buffers during Secretory-stage Amelogenesis

PubMed Central

Guo, J.; Lyaruu, D.M.; Takano, Y.; Gibson, C.W.; DenBesten, P.K.

2015-01-01

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX-/-) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX-/- mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX-/- mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX-/- mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX-/- mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation. PMID:25535204

Amelogenins as potential buffers during secretory-stage amelogenesis.

PubMed

Guo, J; Lyaruu, D M; Takano, Y; Gibson, C W; DenBesten, P K; Bronckers, A L J J

2015-03-01

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX(-/-)) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX(-/-) mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX(-/-) mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX(-/-) mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX(-/-) mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation. © International & American Associations for Dental Research 2014.

Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins to secretory pathway of Aspergillus oryzae.

PubMed

Hama, Shinji; Tamalampudi, Sriappareddy; Shindo, Naoki; Numata, Takao; Yamaji, Hideki; Fukuda, Hideki; Kondo, Akihiko

2008-07-01

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL-green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL-GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.

Assembly, organization, and function of the COPII coat

PubMed Central

Hughes, Helen

2007-01-01

A full mechanistic understanding of how secretory cargo proteins are exported from the endoplasmic reticulum for passage through the early secretory pathway is essential for us to comprehend how cells are organized, maintain compartment identity, as well as how they selectively secrete proteins and other macromolecules to the extracellular space. This process depends on the function of a multi-subunit complex, the COPII coat. Here we describe progress towards a full mechanistic understanding of COPII coat function, including the latest findings in this area. Much of our understanding of how COPII functions and is regulated comes from studies of yeast genetics, biochemical reconstitution and single cell microscopy. New developments arising from clinical cases and model organism biology and genetics enable us to gain far greater insight in to the role of membrane traffic in the context of a whole organism as well as during embryogenesis and development. A significant outcome of such a full understanding is to reveal how the machinery and processes of membrane trafficking through the early secretory pathway fail in disease states. PMID:18060556

Rac1 Regulates Endometrial Secretory Function to Control Placental Development.

PubMed

Davila, Juanmahel; Laws, Mary J; Kannan, Athilakshmi; Li, Quanxi; Taylor, Robert N; Bagchi, Milan K; Bagchi, Indrani C

2015-08-01

During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal

Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles

NASA Astrophysics Data System (ADS)

Lietz, Christopher B.; Toneff, Thomas; Mosier, Charles; Podvin, Sonia; O'Donoghue, Anthony J.; Hook, Vivian

2018-05-01

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. [Figure not available: see fulltext.

Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles

NASA Astrophysics Data System (ADS)

Lietz, Christopher B.; Toneff, Thomas; Mosier, Charles; Podvin, Sonia; O'Donoghue, Anthony J.; Hook, Vivian

2018-03-01

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. [Figure not available: see fulltext.

The endoplasmic reticulum is a hub to sort proteins toward unconventional traffic pathways and endosymbiotic organelles.

PubMed

Bellucci, Michele; De Marchis, Francesca; Pompa, Andrea

2017-12-18

The discovery that much of the extracellular proteome in eukaryotic cells consists of proteins lacking a signal peptide, which cannot therefore enter the secretory pathway, has led to the identification of alternative protein secretion routes bypassing the Golgi apparatus. However, proteins harboring a signal peptide for translocation into the endoplasmic reticulum can also be transported along these alternative routes, which are still far from being well elucidated in terms of the molecular machineries and subcellular/intermediate compartments involved. In this review, we first try to provide a definition of all the unconventional protein secretion pathways in eukaryotic cells, as those pathways followed by proteins directed to an 'external space' bypassing the Golgi, where 'external space' refers to the extracellular space plus the lumen of the secretory route compartments and the inner space of mitochondria and plastids. Then, we discuss the role of the endoplasmic reticulum in sorting proteins toward unconventional traffic pathways in plants. In this regard, various unconventional pathways exporting proteins from the endoplasmic reticulum to the vacuole, plasma membrane, apoplast, mitochondria, and plastids are described, including the short routes followed by the proteins resident in the endoplasmic reticulum. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A nonchromatographic process for purification of secretory immunoglobulins from caprine whey.

PubMed

Matlschweiger, Alexander; Himmler, Gottfried; Linhart, Clemens; Harasek, Michael; Hahn, Rainer

2017-05-01

Secretory immunoglobulins are an important antibody class being primarily responsible for immunoprotection of mucosal surfaces. A simple, non-chromatographic purification process for secretory immunoglobulins from caprine whey was developed. In the first process step whey was concentrated 30-40-fold on a 500 kDa membrane, thereby increasing the purity from 3% to 15%. The second step consisted of a fractionated PEG precipitation, in which high molecular weight impurities were removed first and in the second stage the secretory immunoglobulins were precipitated, leaving a majority of the low molecular weight proteins in solution. The re-dissolved secretory immunoglobulin fraction had a purity of 43% which could then be increased to 72% by diafiltration at a volume exchange factor of 10. Further increase of purity was only possible at the expense of very high buffer consumption. If diafiltration was performed directly after ultrafiltration, followed by precipitation, the yield was higher but purity was only 54%. Overall, filtration performance was characterized by high concentration polarization, therefore process conditions were set to low trans-membrane pressure and moderate protein concentration. As such purity and to a lesser extent throughput were the major objectives rather than yield, since whey, as a by-product of the dairy industry, is a cheap raw material of almost unlimited supply. Ultra-/diafiltration performance was described well by correlations using dimensionless numbers. Compared with a theoretical model (Graetz/Leveque solution) the flux was slightly overestimated. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:642-653, 2017. © 2017 American Institute of Chemical Engineers.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a)

PubMed Central

Chen, Jialin; De Raeymaecker, Joren; Hovgaard, Jannik Brøndsted; Smaardijk, Susanne; Vandecaetsbeek, Ilse; Wuytack, Frank; Møller, Jesper Vuust; Eggermont, Jan; De Maeyer, Marc; Christensen, Søren Brøgger; Vangheluwe, Peter

2017-01-01

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed. PMID:28264934

Depletion of HPV16 early genes induces autophagy and senescence in a cervical carcinogenesis model, regardless of viral physical state.

PubMed

Hanning, Jennifer E; Saini, Harpreet K; Murray, Matthew J; Caffarel, Maria M; van Dongen, Stijn; Ward, Dawn; Barker, Emily M; Scarpini, Cinzia G; Groves, Ian J; Stanley, Margaret A; Enright, Anton J; Pett, Mark R; Coleman, Nicholas

2013-11-01

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral

Biogenesis of the Secretory Granule: Chromogranin a Coiled-Coil Structure Results in Unusual Physical Properties And Suggests a Mechanism for Granule Core Condensation

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

Mosley, C.A.; Taupenot, L.; Biswas, N.

2009-06-03

binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disrupted regulated secretory protein traffic by approximately 65%, while targeted ablation of the CHGA gene in the mouse reduced chromaffin granule cotransmitter concentrations by approximately 40-80%. These results suggest new roles for secretory protein tertiary structure in hormone and transmitter storage, with implications for secretory cargo condensation (or dense core 'packing' structure) within the regulated pathway.« less

Fine-tuning of the flavonoid and monolignol pathways during apple early fruit development.

PubMed

Baldi, Paolo; Moser, Mirko; Brilli, Matteo; Vrhovsek, Urska; Pindo, Massimo; Si-Ammour, Azeddine

2017-05-01

A coordinated regulation of different branches of the flavonoid pathway was highlighted that may contribute to elucidate the role of this important class of compounds during the early stages of apple fruit development. Apple (Malus × domestica Borkh.) is an economically important fruit appreciated for its organoleptic characteristics and its benefits for human health. The first stages after fruit set represent a very important and still poorly characterized developmental process. To enable the profiling of genes involved in apple early fruit development, we combined the suppression subtractive hybridization (SSH) protocol to next-generation sequencing. We identified and characterized genes induced and repressed during fruit development in the apple cultivar 'Golden Delicious'. Our results showed an opposite regulation of genes coding for enzymes belonging to flavonoid and monolignol pathways, with a strong induction of the former and a simultaneous repression of the latter. Two isoforms of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, key enzymes located at the branching point between flavonoid and monolignol pathways, showed opposite expression patterns during the period in analysis, suggesting a possible regulation mechanism. A targeted metabolomic analysis supported the SSH results and revealed an accumulation of the monomers catechin and epicatechin as well as several forms of procyanidin oligomers in apple fruitlets starting early after anthesis, together with a decreased production of other classes of flavonoids such as some flavonols and the dihydrochalcone phlorizin. Moreover, gene expression and metabolites accumulation of 'Golden Delicious' were compared to a wild apple genotype of Manchurian crabapple (Malus mandshurica (Maxim.) Kom.). Significant differences in both gene expression and metabolites accumulation were found between the two genotypes.

The long-term effects of maternal depression: early childhood physical health as a pathway to offspring depression.

PubMed

Raposa, Elizabeth; Hammen, Constance; Brennan, Patricia; Najman, Jake

2014-01-01

Cross-sectional and retrospective studies have highlighted the long-term negative effects of maternal depression on offspring physical, social, and emotional development, but longitudinal research is needed to clarify the pathways by which maternal depression during pregnancy and early childhood affects offspring outcomes. The current study tested one developmental pathway by which maternal depression during pregnancy might negatively impact offspring mental health in young adulthood, via poor physical health in early childhood. The sample consisted of 815 Australian youth and their mothers who were followed for 20 years. Mothers reported on their own depressive symptoms during pregnancy and offspring early childhood. Youth completed interviews about health-related stress and social functioning at age 20 years, and completed a questionnaire about their own depressive symptoms 2 to 5 years later. Path analysis indicated that prenatal maternal depressive symptoms predicted worse physical health during early childhood for offspring, and this effect was partially explained by ongoing maternal depression in early childhood. Offspring poor physical health during childhood predicted increased health-related stress and poor social functioning at age 20. Finally, increased health-related stress and poor social functioning predicted increased levels of depressive symptoms later in young adulthood. Maternal depression had a significant total indirect effect on youth depression via early childhood health and its psychosocial consequences. Poor physical health in early childhood and its effects on young adults' social functioning and levels of health related stress is one important pathway by which maternal depression has long-term consequences for offspring mental health. Copyright © 2014 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.

Style morphology and pollen tube pathway.

PubMed

Gotelli, M M; Lattar, E C; Zini, L M; Galati, B G

2017-12-01

The style morphology and anatomy vary among different species. Three basic types are: open, closed, and semi-closed. Cells involved in the pollen tube pathway in the different types of styles present abundant endoplasmic reticulum, dictyosomes, mitochondria, and ribosomes. These secretory characteristics are related to the secretion where pollen tube grows. This secretion can be represented by the substances either in the canal or in the intercellular matrix or in the cell wall. Most studies suggest that pollen tubes only grow through the secretion of the canal in open styles. However, some species present pollen tubes that penetrate the epithelial cells of the canal, or grow through the middle lamella between these cells and subepithelial cells. In species with a closed style, a pathway is provided by the presence of an extracellular matrix, or by the thickened cell walls of the stylar transmitting tissue. There are reports in some species where pollen tubes can also penetrate the transmitting tissue cells and continue their growth through the cell lumen. In this review, we define subtypes of styles according to the path of the pollen tube. Style types were mapped on an angiosperm phylogenetic tree following the maximum parsimony principle. In line with this, it could be hypothesized that: the open style appeared in the early divergent angiosperms; the closed type of style originated in Asparagales, Poales, and Eudicots; and the semi-closed style appeared in Rosids, Ericales, and Gentianales. The open style seems to have been lost in core Eudicots, with reversions in some Rosids and Asterids.

The long reach of early adversity: Parenting, stress, and neural pathways to antisocial behavior in adulthood.

PubMed

Gard, Arianna M; Waller, Rebecca; Shaw, Daniel S; Forbes, Erika E; Hariri, Ahmad R; Hyde, Luke W

2017-10-01

Early life adversities including harsh parenting, maternal depression, neighborhood deprivation, and low family economic resources are more prevalent in low-income urban environments and are potent predictors of psychopathology, including, for boys, antisocial behavior (AB). However, little research has examined how these stressful experiences alter later neural function. Moreover, identifying genetic markers of greater susceptibility to adversity is critical to understanding biopsychosocial pathways from early adversity to later psychopathology. Within a sample of 310 low-income boys followed from age 1.5 to 20, multimethod assessments of adversities were examined at age 2 and age 12. At age 20, amygdala reactivity to emotional facial expressions was assessed using fMRI, and symptoms of Antisocial Personality Disorder were assessed via structured clinical interview. Genetic variability in cortisol signaling ( CRHR1 ) was examined as a moderator of pathways to amygdala reactivity. Observed parenting and neighborhood deprivation at age 2 each uniquely predicted amygdala reactivity to emotional faces at age 20 over and above other adversities measured at multiple developmental periods. Harsher parenting and greater neighborhood deprivation in toddlerhood predicted clinically-significant symptoms of AB via less amygdala reactivity to fearful facial expressions and this pathway was moderated by genetic variation in CRHR1 . These results elucidate a pathway linking early adversity to less amygdala reactivity to social signals of interpersonal distress 18 years later, which in turn increased risk for serious AB. Moreover, these findings suggest a genetic marker of youth more susceptible to adversity.

Primary cutaneous secretory carcinoma: A previously overlooked low-grade sweat gland carcinoma.

PubMed

Llamas-Velasco, Mar; Mentzel, Thomas; Rütten, Arno

2018-03-01

Twelve cases of primary cutaneous secretory carcinoma (PCSC) have been published, 9 showing ETV6-NTRK3 translocation, a characteristic finding shared with secretory breast carcinoma and mammary analogue secretory carcinoma. A 34-year-old female presented a solitary nodule on the right groin. Biopsy revealed a secretory carcinoma staining positive with CK7, CAM5.2, mammaglobulin and S100 and negative with GATA3, CK20, podoplanin, calponin and CDX2. ETV6-NTRK3 was demonstrated by Fluorescence in situ hybridization (FISH). PCSC is a rare neoplasm, described in the skin in 2009, that affects more frequently females with a mean age of 42.3 years and it is most commonly located in axilla. Histopathologically, these tumor cells are characterized by bubbly eosinophilic secretions diastase-resistant and bland nuclei and they are arranged in various growth patterns, including microcystic, tubular, solid and papillary. S100, mammoglobin and CK7 are usually positive. We review the main histopathological features to rule out histopathologic mimics such as breast metastasis, salivary tumors, cribriform carcinoma and primary cutaneous adenoid cystic carcinoma. GATA3 negative staining, as in our case, can help to rule out breast metastasis. Moreover, long-term benign follow up (144 months) in this case as well as follow-up data on outcomes from literature review support that PCSC is a low-grade sweat gland carcinoma. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pathogen-induced secretory diarrhea and its prevention.

PubMed

Anand, S; Mandal, S; Patil, P; Tomar, S K

2016-11-01

Secretory diarrhea is a historically known serious health implication around the world which primarily originates through pathogenic microorganisms rather than immunological or genetical disorders. This review highlights infective mechanisms of non-inflammatory secretory diarrhea causing pathogens, known therapeutics and their efficacy against them. These non-inflammatory diarrheal pathogens breach cell barriers, induce inflammation, disrupt fluid secretion across the epithelium by alteration in ion transport by faulting cystic fibrosis transmembrane conductance regulator (CFTR), calcium activated chloride channels and ion exchanger functions. Currently, a variety of prevention strategies have been used to treat these symptoms like use of antibacterial drugs, vaccines, fluid and nutritional therapy, probiotics and prebiotics as adjuncts. In progression of the need for a therapy having quick physiological effects, withdrawing the symptoms with a wide and safe therapeutic index, newer antisecretory agents like potent inhibitors, agonists and herbal remedies are some of the interventions which have come into light through greater understanding of the mechanisms and molecular targets involved in intestinal fluid secretion. Although these therapies have their own pros and cons inside the host, the quest for new antisecretory agents has been a successful elucidation to reduce burden of diarrheal disease.

Early risk pathways to physical versus relational peer aggression: The interplay of externalizing behavior and corporal punishment varies by child sex.

PubMed

Zulauf, Courtney A; Sokolovsky, Alexander W; Grabell, Adam S; Olson, Sheryl L

2018-03-01

Children who aggress against their peers may use physical or relational forms, yet little research has looked at early childhood risk factors and characteristics that uniquely predict high levels of relational versus physical aggression in preadolescence. Accordingly, the main aim of our study was to link early corporal punishment and externalizing behavior to children's physical and relational peer aggression during preadolescence and to examine how these pathways differed by sex. Participants were 193, 3-year-old boys (39%) and girls who were reassessed following the transition to kindergarten (5.5 years) and preadolescence (10.5 years). A series of autoregressive, cross-lagged path analyses were conducted to examine the relationships between child externalizing problems and corporal punishment at ages 3 and 5.5 years, and their association with physical and relational aggression at age 10.5. Multiple group analysis was used to determine whether pathways differed by sex. Three developmental pathways were identified: (i) direct associations between stable childhood externalizing problems and later physical aggression; (ii) a direct pathway from early corporal punishment to preadolescent relational and physical peer aggression; and (iii) an indirect pathway from early corporal punishment to later physical aggression via continuing externalizing problems in middle childhood. Child sex moderated the nature of these pathways, as well as the direction of association between risk and outcome variables. These data advance our understanding of the etiology of distinct forms of peer aggression and highlight the potential for more efficacious prevention and intervention efforts in the early childhood years. © 2018 Wiley Periodicals, Inc.

Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

PubMed

Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

2014-01-01

How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus

PubMed Central

1985-01-01

Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor- mediated endocytosis. MVBs also contained numerous small vesicles, 0.05- 0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins. PMID:3988801

Early Induction of NRF2 Antioxidant Pathway by RHBDF2 Mediates Rapid Cutaneous Wound Healing

PubMed Central

Hosur, Vishnu; Burzenski, Lisa M.; Stearns, Timothy M.; Farley, Michelle L.; Sundberg, John P.; Wiles, Michael V.; Shultz, Leonard D.

2017-01-01

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2cub/cub) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2cub/cub and Rhbdf2+/+ mice at 0h, 15 min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15 min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds. PMID:28268192

Early induction of NRF2 antioxidant pathway by RHBDF2 mediates rapid cutaneous wound healing.

PubMed

Hosur, Vishnu; Burzenski, Lisa M; Stearns, Timothy M; Farley, Michelle L; Sundberg, John P; Wiles, Michael V; Shultz, Leonard D

2017-04-01

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2 cub/cub ) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2 cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2 cub/cub and Rhbdf2 +/+ mice at 0h, 15min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2 cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

Secretory cargo sorting by Ca2+-dependent Cab45 oligomerization at the trans-Golgi network

PubMed Central

Blank, Birgit; Maiser, Andreas; Emin, Derya; Prescher, Jens; Beck, Gisela; Kienzle, Christine; Bartnik, Kira; Habermann, Bianca; Pakdel, Mehrshad; Leonhardt, Heinrich; Lamb, Don C.

2016-01-01

Sorting and export of transmembrane cargoes and lysosomal hydrolases at the trans-Golgi network (TGN) are well understood. However, elucidation of the mechanism by which secretory cargoes are segregated for their release into the extracellular space remains a challenge. We have previously demonstrated that, in a reaction that requires Ca2+, the soluble TGN-resident protein Cab45 is necessary for the sorting of secretory cargoes at the TGN. Here, we report that Cab45 reversibly assembles into oligomers in the presence of Ca2+. These Cab45 oligomers specifically bind secretory proteins, such as COMP and LyzC, in a Ca2+-dependent manner in vitro. In intact cells, mutation of the Ca2+-binding sites in Cab45 impairs oligomerization, as well as COMP and LyzC sorting. Superresolution microscopy revealed that Cab45 colocalizes with secretory proteins and the TGN Ca2+ pump (SPCA1) in specific TGN microdomains. These findings reveal that Ca2+-dependent changes in Cab45 mediate sorting of specific cargo molecules at the TGN. PMID:27138253

Synchronous Parotid (Mammary Analog) Secretory Carcinoma and Acinic Cell Carcinoma: Report of a Case.

PubMed

Mossinelli, C; Pigni, C; Sovardi, F; Occhini, A; Preda, L; Benazzo, M; Morbini, P; Pagella, F

2018-06-06

Mammary analogue secretory carcinoma (MASC) is a recently described low-grade salivary gland malignancy with histologic, immunohistochemical and molecular similarities to secretory carcinoma of the breast, including a specific t(12;15)(p13;q25) resulting in an ETV6-NTRK3 gene fusion. Ultrasound and magnetic resonance imaging frequently document a macrocystic structure. The main differential diagnosis of secretory carcinoma is with low grade acinic cell carcinoma (AciCC). The two can be differentiated with immunohistochemical stains for S100, mammaglobin, carbonic anhydrase VI and DOG-1; the identification of the specific translocation can help to characterize non-typical cases. We report a unique case of synchronous MASC and AciCC presenting in a parotid gland and discuss the implications of the correct identification of the two tumors.

Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin.

PubMed

Lesteberg, Kelsey; Orange, Jordan; Makedonas, George

2017-10-01

Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein, we aimed to determine how new perforin transits to the synapse if not via lytic granules. We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response.

Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin

PubMed Central

Lesteberg, Kelsey E.; Orange, Jordan S.; Makedonas, George

2018-01-01

Background Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein we aimed to determine how new perforin transits to the synapse if not via lytic granules. Results We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. Conclusions The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response. PMID:28822075

Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

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

Somanath, Sangeeta; Partridge, Christopher J.; Marshall, Catriona

Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation.more » These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation. - Highlights: • Snapin mediates granule docking. • Snapin binds SNAP-25. • SNARE complex forms downstream.« less

Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes

PubMed Central

Basso, Manuela; Pozzi, Silvia; Tortarolo, Massimo; Fiordaliso, Fabio; Bisighini, Cinzia; Pasetto, Laura; Spaltro, Gabriella; Lidonnici, Dario; Gensano, Francesco; Battaglia, Elisa; Bendotti, Caterina; Bonetto, Valentina

2013-01-01

Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread. PMID:23592792

Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

PubMed

Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-Ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-Ichiro

2016-10-31

Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

The C2H2 transcription factor regulator of symbiosome differentiation represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula.

PubMed

Sinharoy, Senjuti; Torres-Jerez, Ivone; Bandyopadhyay, Kaustav; Kereszt, Attila; Pislariu, Catalina I; Nakashima, Jin; Benedito, Vagner A; Kondorosi, Eva; Udvardi, Michael K

2013-09-01

Transcription factors (TFs) are thought to regulate many aspects of nodule and symbiosis development in legumes, although few TFs have been characterized functionally. Here, we describe regulator of symbiosome differentiation (RSD) of Medicago truncatula, a member of the Cysteine-2/Histidine-2 (C2H2) family of plant TFs that is required for normal symbiosome differentiation during nodule development. RSD is expressed in a nodule-specific manner, with maximal transcript levels in the bacterial invasion zone. A tobacco (Nicotiana tabacum) retrotransposon (Tnt1) insertion rsd mutant produced nodules that were unable to fix nitrogen and that contained incompletely differentiated symbiosomes and bacteroids. RSD protein was localized to the nucleus, consistent with a role of the protein in transcriptional regulation. RSD acted as a transcriptional repressor in a heterologous yeast assay. Transcriptome analysis of an rsd mutant identified 11 genes as potential targets of RSD repression. RSD interacted physically with the promoter of one of these genes, VAMP721a, which encodes vesicle-associated membrane protein 721a. Thus, RSD may influence symbiosome development in part by repressing transcription of VAMP721a and modifying vesicle trafficking in nodule cells. This establishes RSD as a TF implicated directly in symbiosome and bacteroid differentiation and a transcriptional regulator of secretory pathway genes in plants.

21 CFR 866.5380 - Free secretory component immuno-logical test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... body fluids. Measurement of free secretory component (protein molecules) aids in the diagnosis or... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test...

21 CFR 866.5380 - Free secretory component immuno-logical test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... body fluids. Measurement of free secretory component (protein molecules) aids in the diagnosis or... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test...

21 CFR 866.5380 - Free secretory component immuno-logical test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... body fluids. Measurement of free secretory component (protein molecules) aids in the diagnosis or... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test...

21 CFR 866.5380 - Free secretory component immuno-logical test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... body fluids. Measurement of free secretory component (protein molecules) aids in the diagnosis or... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test...

Contribution of Secretory Antibodies to Intestinal Mucosal Immunity against Helicobacter pylori

PubMed Central

Wijburg, Odilia L. C.; Pedersen, John S.; Walduck, Anna K.; Kwok, Terry; Strugnell, Richard A.; Robins-Browne, Roy M.

2013-01-01

The natural immune response to Helicobacter pylori neither clears infection nor prevents reinfection. However, the ability of secretory antibodies to influence the course of H. pylori infection has not been determined. We compared the natural progression of H. pylori infection in wild-type C57BL/6 mice with that in mice lacking the polymeric immunoglobulin receptor (pIgR) that is essential for the secretion of polymeric antibody across mucosal surfaces. H. pylori SS1-infected wild-type and pIgR knockout (KO) mice were sampled longitudinally for gastrointestinal bacterial load, antibody response, and histological changes. The gastric bacterial loads of wild-type and pIgR KO mice remained constant and comparable at up to 3 months postinfection (mpi) despite SS1-reactive secretory IgA in the intestinal contents of wild-type mice at that time. Conversely, abundant duodenal colonization of pIgR KO animals contrasted with the near-total eradication of H. pylori from the intestine of wild-type animals by 3 mpi. H. pylori was cultured only from the duodenum of those animals in which colonization in the distal gastric antrum was of sufficient density for immunohistological detection. By 6 mpi, the gastric load of H. pylori in wild-type mice was significantly lower than in pIgR KO animals. While there was no corresponding difference between the two mouse strains in gastric pathology results at 6 mpi, reductions in gastric bacterial load correlated with increased gastric inflammation together with an intestinal secretory antibody response in wild-type mice. Together, these results suggest that naturally produced secretory antibodies can modulate the progress of H. pylori infection, particularly in the duodenum. PMID:23918779

Oil Secretory System in Vegetative Organs of Three Arnica Taxa: Essential Oil Synthesis, Distribution and Accumulation.

PubMed

Kromer, Krystyna; Kreitschitz, Agnieszka; Kleinteich, Thomas; Gorb, Stanislav N; Szumny, Antoni

2016-05-01

Arnica, a genus including the medicinal species A. montana, in its Arbo variety, and A. chamissonis, is among the plants richest in essential oils used as pharmaceutical materials. Despite its extensive use, the role of anatomy and histochemistry in the internal secretory system producing the essential oil is poorly understood. Anatomical sections allowed differentiation between two forms of secretory structures which differ according to their distribution in plants. The first axial type is connected to the vascular system of all vegetative organs and forms canals lined with epithelial cells. The second cortical type is represented by elongated intercellular spaces filled with oil formed only between the cortex cells of roots and rhizomes at maturity, with canals lacking an epithelial layer.Only in A. montana rhizomes do secretory structures form huge characteristic reservoirs. Computed tomography illustrates their spatial distribution and fusiform shape. The axial type of root secretory canals is formed at the interface between the endodermis and cortex parenchyma, while, in the stem, they are located in direct contact with veinal parenchyma. The peripheral phloem parenchyma cells are arranged in strands around sieve tube elements which possess a unique ability to accumulate large amounts of oil bodies. The cells of phloem parenchyma give rise to the aforementioned secretory structures while the lipid components (triacylglycerols) stored there support the biosynthesis of essential oils by later becoming a medium in which these oils are dissolved. The results indicate the integrity of axial secretory structures forming a continuous system in vegetative plant organs. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Comparative innate immune interactions of human and bovine secretory IgA with pathogenic and non-pathogenic bacteria.

PubMed

Hodgkinson, Alison J; Cakebread, Julie; Callaghan, Megan; Harris, Paul; Brunt, Rachel; Anderson, Rachel C; Armstrong, Kelly M; Haigh, Brendan

2017-03-01

Secretory IgA (SIgA) from milk contributes to early colonization and maintenance of commensal/symbiotic bacteria in the gut, as well as providing defence against pathogens. SIgA binds bacteria using specific antigenic sites or non-specifically via its glycans attached to α-heavy-chain and secretory component. In our study, we tested the hypothesis that human and bovine SIgA have similar innate-binding activity for bacteria. SIgAs, isolated from human and bovine milk, were incubated with a selection of commensal, pathogenic and probiotic bacteria. Using flow cytometry, we measured numbers of bacteria binding SIgA and their level of SIgA binding. The percentage of bacteria bound by human and bovine SIgA varied from 30 to 90% depending on bacterial species and strains, but was remarkably consistent between human and bovine SIgA. The level of SIgA binding per bacterial cell was lower for those bacteria that had a higher percentage of SIgA-bound bacteria, and higher for those bacteria that had lower percentage of SIgA-bound bacteria. Overall, human and bovine SIgA interacted with bacteria in a comparable way. This contributes to longer term research about the potential benefits of bovine SIgA for human consumers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Qa-SNAREs localized to the trans-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants

PubMed Central

Uemura, Tomohiro; Kim, Hyeran; Saito, Chieko; Ebine, Kazuo; Ueda, Takashi; Schulze-Lefert, Paul; Nakano, Akihiko

2012-01-01

In all eukaryotic cells, a membrane-trafficking system connects the post-Golgi organelles, such as the trans-Golgi network (TGN), endosomes, vacuoles, and the plasma membrane. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. Unlike its roles in animal and yeast cells, the TGN has also been reported to function like early endosomal compartments in plant cells. However, the physiological roles of the TGN functions in plants are not understood. Here, we report a study of the SYP4 group (SYP41, SYP42, and SYP43), which represents the plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) that localizes on the TGN in yeast and animal cells. The SYP4 group regulates the secretory and vacuolar transport pathways in the post-Golgi network and maintains the morphology of the Golgi apparatus and TGN. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance responses to a fungal pathogen. We also reveal a plant cell-specific higher-order role of the SYP4 group in the protection of chloroplasts from salicylic acid-dependent biotic stress. PMID:22307646

Secretory meningioma: clinicopathologic features of eight cases.

PubMed

Nishio, S; Morioka, T; Suzuki, S; Hirano, K; Fukui, M

2001-07-01

The clinical and morphological features of eight patients with meningothelial meningiomas with numerous pseudopsammoma bodies (secretory meningiomas) are presented. The six female and two male patients ranged in age from 43 to 68 years. Tumours were located at the petroclival region in two, the lateral parasellar region in two, the petrous apex in one and the sphenoid ridge in three patients. On magnetic resonance imaging, they were iso or hypointense on T1-weighted images, and hyper or isointense on T 2-weighted images. Peritumoral brain edema was absent in five cases, and was mild to moderate in three cases. Serum carcinoembryonic antigen (CEA) levels were measured preoperatively in three patients, with one having an elevated serum CEA level which re turned to normal following tumour resection. Immunohistochemical analysis on the resected tumour tissues, pseudopsammoma bodies and surrounding tumour cells were shown to be CEA-positive. Ultrastructurally, pseudopsammoma bodies were composed of granular and filamentous materials located predominantly in the intracellular lumina, which were lined by microvilli. While these morphological features of focal epithelial and secretory differentiation of tumour cells call attention to the broad spectrum of differentiation properties of meningiomas, the biological behavior of the eight tumours reported herein corresponded to those of meningiomas in general. Copyright 2001 Harcourt Publishers Ltd.

Novelties in secretory structures and anatomy of Rhynchosia (Fabaceae).

PubMed

De Vargas, Wanderleia; Sartori, Ângela L B; Dias, Edna S

2015-03-01

A comparative anatomical study was carried out on the secretory structures of leaflets from taxa belonging to the genus Rhynchosia - taxa difficult to delimit because of uncertain interspecific relations - in order to evaluate the potential diagnostic value of these anatomical traits for taxonomic assignment. A further objective was to establish consensual denomination for these secretory structures. The new anatomical features found in these taxa were sufficiently consistent to separate the species evaluated. The presence and localization of glandular-punctate structures bulbous-based trichomes, the number of layers in the palisade parenchyma and the arrangement of vascular units distinguish the taxa investigated and these characteristics can be extended to other species of Papilionoideae. The trichomes analyzed were described and classified into five types. Depicted in diagrams, photomicrographs, and by scanning electron microscopy, and listed for the first time at the genus and species levels. The information obtained served to effectively distinguish the taxa investigated among species of Papilonoideae.

Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins

PubMed Central

Calosci, Nicoletta; Chi, Celestine N.; Richter, Barbara; Camilloni, Carlo; Engström, Åke; Eklund, Lars; Travaglini-Allocatelli, Carlo; Gianni, Stefano; Vendruscolo, Michele; Jemth, Per

2008-01-01

The energy landscape theory provides a general framework for describing protein folding reactions. Because a large number of studies, however, have focused on two-state proteins with single well-defined folding pathways and without detectable intermediates, the extent to which free energy landscapes are shaped up by the native topology at the early stages of the folding process has not been fully characterized experimentally. To this end, we have investigated the folding mechanisms of two homologous three-state proteins, PTP-BL PDZ2 and PSD-95 PDZ3, and compared the early and late transition states on their folding pathways. Through a combination of Φ value analysis and molecular dynamics simulations we obtained atomic-level structures of the transition states of these homologous three-state proteins and found that the late transition states are much more structurally similar than the early ones. Our findings thus reveal that, while the native state topology defines essentially in a unique way the late stages of folding, it leaves significant freedom to the early events, a result that reflects the funneling of the free energy landscape toward the native state. PMID:19033470

Implementing Guided Pathways: Early Insights from the AACC Pathways Colleges

ERIC Educational Resources Information Center

Jenkins, Davis; Lahr, Hana; Fink, John

2017-01-01

Across the United States, a growing number of colleges are redesigning their programs and student support services according to the "guided pathways" model. Central to this approach are efforts to clarify pathways to program completion, career advancement, and further education. Equally essential are efforts to help students explore…

Mediated Pathways from Maternal Depression and Early Parenting to Children's Executive Function and Externalizing Behaviour Problems

ERIC Educational Resources Information Center

Baker, Claire; Kuhn, Laura

2018-01-01

Structural equation models were used to examine pathways from maternal depression and early parenting to children's executive function (EF) and externalizing behaviours in the first nationally representative study to obtain direct assessments of children's kindergarten EF skills (i.e., the Early Childhood Longitudinal Study Kindergarten Class of…

Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting.

PubMed

Beuret, Nicole; Hasler, Franziska; Prescianotto-Baschong, Cristina; Birk, Julia; Rutishauser, Jonas; Spiess, Martin

2017-01-26

Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.

The cnidarian nematocyst: a miniature extracellular matrix within a secretory vesicle.

PubMed

Ozbek, Suat

2011-10-01

Nematocysts are the taxon-defining features of all cnidarians including jellyfish, sea anemones, and corals. They are highly sophisticated organelles used for the capture of prey and defense. The nematocyst capsule is produced within a giant post-Golgi vesicle, which is continuously fed by proteins from the secretory pathway. Mature nematocysts consist of a hollow capsule body in which a long tubule is coiled up that, upon discharge, is expelled in a harpoon-like fashion. This is accompanied by the release of a toxin cocktail stored in the capsule matrix. Nematocyst discharge, which is one of the fastest processes in biology, is driven by an extreme osmotic pressure of about 150 bar. The molecular analysis of the nematocyst has from the beginning indicated a collagenous nature of the capsule structure. In particular, a large family of unusual minicollagens has been demonstrated to form the highly resistant scaffold of the capsule. Recent findings on the molecular composition of Hydra nematocysts have confirmed the notion of a specialized extracellular matrix, which is assembled during an intracellular secretion process to form the most complex predatory apparatus at the cellular level.

A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14.

PubMed

Herzig, Yonatan; Sharpe, Hayley J; Elbaz, Yael; Munro, Sean; Schuldiner, Maya

2012-01-01

The endoplasmic reticulum (ER) is the site of synthesis of secreted and membrane proteins. To exit the ER, proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Despite the fundamental role of such cargo receptors in protein traffic, only a few have been identified; their cargo spectrum is unknown and the signals they recognize remain poorly understood. We present here an approach we term "PAIRS" (pairing analysis of cargo receptors), which combines systematic genetic manipulations of yeast with automated microscopy screening, to map the spectrum of cargo for a known receptor or to uncover a novel receptor for a particular cargo. Using PAIRS we followed the fate of ∼150 cargos on the background of mutations in nine putative cargo receptors and identified novel cargo for most of these receptors. Deletion of the Erv14 cargo receptor affected the widest range of cargo. Erv14 substrates have a wide array of functions and structures; however, they are all membrane-spanning proteins of the late secretory pathway or plasma membrane. Proteins residing in these organelles have longer transmembrane domains (TMDs). Detailed examination of one cargo supported the hypothesis that Erv14 dependency reflects the length rather than the sequence of the TMD. The PAIRS approach allowed us to uncover new cargo for known cargo receptors and to obtain an unbiased look at specificity in cargo selection. Obtaining the spectrum of cargo for a cargo receptor allows a novel perspective on its mode of action. The rules that appear to guide Erv14 substrate recognition suggest that sorting of membrane proteins at multiple points in the secretory pathway could depend on the physical properties of TMDs. Such a mechanism would allow diverse proteins to utilize a few receptors without the constraints of evolving location-specific sorting motifs.

Biochemical analysis of secretory proteins synthesized by normal rat pancreas and by pancreatic acinar tumor cells

PubMed Central

1982-01-01

We have examined the secretogogue responsiveness and the pattern of secretory proteins produced by a transplantable rat pancreatic acinar cell tumor. Dispersed tumor cells were found to discharge secretory proteins in vitro when incubated with hormones that act on four different classes of receptors: carbamylcholine, caerulein, secretin- vasoactive intestinal peptide, and bombesin. With all hormones tested, maximal discharge from tumor cells was only about one-half that of control pancreatic lobules, but occurred at the same dose optima except for secretin, whose dose optimum was 10-fold higher. Biochemical analysis of secretory proteins discharged by the tumor cells was carried out by crossed immunoelectrophoresis and by two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis. To establish a baseline for comparison, secretory proteins from normal rat pancreas were identified according to enzymatic activity and correlated with migration position on two-dimensional gels. Our results indicate that a group of basic polypeptides including proelastase, basic trypsinogen, basic chymotrypsinogen, and ribonuclease, two out of three forms of procarboxypeptidase B, and the major lipase species were greatly reduced or absent in tumor cell secretion. In contrast, the amount of acidic chymotrypsinogen was notably increased compared with normal acinar cells. Although the acinar tumor cells are highly differentiated cytologically and express functional receptors for several classes of pancreatic secretagogues, they show quantitative and qualitative differences when compared with normal pancreas with regard to their production of secretory proteins. PMID:6185502

Systemic Lupus Erythematosus and Deficiencies of Early Components of the Complement Classical Pathway

PubMed Central

Macedo, Ana Catarina Lunz; Isaac, Lourdes

2016-01-01

The complement system plays an important role in the innate and acquired immune response against pathogens. It consists of more than 30 proteins found in soluble form or attached to cell membranes. Most complement proteins circulate in inactive forms and can be sequentially activated by the classical, alternative, or lectin pathways. Biological functions, such as opsonization, removal of apoptotic cells, adjuvant function, activation of B lymphocytes, degranulation of mast cells and basophils, and solubilization and clearance of immune complex and cell lysis, are dependent on complement activation. Although the activation of the complement system is important to avoid infections, it also can contribute to the inflammatory response triggered by immune complex deposition in tissues in autoimmune diseases. Paradoxically, the deficiency of early complement proteins from the classical pathway (CP) is strongly associated with development of systemic lupus erythematous (SLE) – mainly C1q deficiency (93%) and C4 deficiency (75%). The aim of this review is to focus on the deficiencies of early components of the CP (C1q, C1r, C1s, C4, and C2) proteins in SLE patients. PMID:26941740

Prenatal alcohol and other early childhood adverse exposures: Direct and indirect pathways to adolescent drinking

PubMed Central

Cornelius, Marie D.; De Genna, Natacha M.; Goldschmidt, Lidush; Larkby, Cynthia; Day, Nancy L.

2016-01-01

We examined direct and indirect pathways between adverse environmental exposures during gestation and childhood and drinking in mid-adolescence. Mothers and their offspring (n = 917 mother/child dyads) were followed prospectively from second trimester to a 16-year follow-up assessment. Interim assessments occurred at delivery, 6, 10, and 14 years. Adverse environmental factors included gestational exposures to alcohol, tobacco, and marijuana, exposures to childhood maltreatment and violence, maternal psychological symptoms, parenting practices, economic and home environments, and demographic characteristics of the mother and child. Indirect effects of early child behavioral characteristics including externalizing, internalizing activity, attention, and impulsivity were also examined. Polytomous logistic regression analyses were used to evaluate direct effects of adverse environmental exposures with level of adolescent drinking. Structural equation modeling (SEM) was applied to simultaneously estimate the relation between early adversity variables, childhood characteristics, and drinking level at age 16 while controlling for significant covariates. Level of drinking among the adolescent offspring was directly predicted by prenatal exposure to alcohol, less parental strictness, and exposures to maltreatment and violence during childhood. Whites and offspring with older mothers were more likely to drink at higher levels. There was a significant indirect effect between childhood exposure to violence and adolescent drinking via childhood externalizing behavior problems. All other hypothesized indirect pathways were not significant. Thus most of the early adversity measures directly predicted adolescent drinking and did not operate via childhood behavioral dysregulation characteristics. These results highlight the importance of adverse environmental exposures on pathways to adolescent drinking. PMID:26994529

Alpha-SNAP functions in insulin exocytosis from mature, but not immature secretory granules in pancreatic beta cells.

PubMed

Nakamichi, Y; Nagamatsu, S

1999-06-24

To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively. There was no difference in percentage labeled-proinsulin release between the control and alpha-SNAP or mutant alpha-SNAP-overexpressed islets. Although percentage labeled-insulin release after a 3-h chase period was significantly increased in alpha-SNAP-overexpressed islets, it was decreased in mutant alpha-SNAP-overexpressed islets. Thus, the results demonstrated that alpha-SNAP overexpression in rat islets primarily increased exocytosis from mature, but not immature insulin secretory granules. On the other hand, in MIN6 cells, alpha-SNAP overexpression scarcely affected glucose-stimulated insulin release; therefore, we examined the effect of mutant alpha-SNAP overexpression as the dominant-negative inhibitor on the newly synthesized proinsulin/insulin release using the same protocol as in the rat islet experiments. alpha-SNAP mutant (1-285) overexpression in MIN6 cells decreased the percentage labeled insulin release from mature secretory granules, but not percentage labeled proinsulin release from immature secretory granules. Thus, our data demonstrate that alpha-SNAP functions mainly in the mature insulin secretory granules in pancreatic beta cells. Copyright 1999 Academic Press.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a).

PubMed

Chen, Jialin; De Raeymaecker, Joren; Hovgaard, Jannik Brøndsted; Smaardijk, Susanne; Vandecaetsbeek, Ilse; Wuytack, Frank; Møller, Jesper Vuust; Eggermont, Jan; De Maeyer, Marc; Christensen, Søren Brøgger; Vangheluwe, Peter

2017-04-28

The Golgi/secretory pathway Ca 2+ /Mn 2+ -transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca 2+ -dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca 2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di- tert -butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Human immunodeficiency virus type 1 envelope proteins traffic toward virion assembly sites via a TBC1D20/Rab1-regulated pathway

PubMed Central

2012-01-01

Background The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here. Findings Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs). Conclusions These findings add TBC1D20 to the network of host factors regulating HIV replication cycle. PMID:22260459

Identify Secretory Protein of Malaria Parasite with Modified Quadratic Discriminant Algorithm and Amino Acid Composition.

PubMed

Feng, Yong-E

2016-06-01

Malaria parasite secretes various proteins in infected red blood cell for its growth and survival. Thus identification of these secretory proteins is important for developing vaccine or drug against malaria. In this study, the modified method of quadratic discriminant analysis is presented for predicting the secretory proteins. Firstly, 20 amino acids are divided into five types according to the physical and chemical characteristics of amino acids. Then, we used five types of amino acids compositions as inputs of the modified quadratic discriminant algorithm. Finally, the best prediction performance is obtained by using 20 amino acid compositions, the sensitivity of 96 %, the specificity of 92 % with 0.88 of Mathew's correlation coefficient in fivefold cross-validation test. The results are also compared with those of existing prediction methods. The compared results shown our method are prominent in the prediction of secretory proteins.

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum

PubMed Central

Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona

2017-01-01

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF4− and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles. PMID:28462015

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum.

PubMed

Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona; Patankar, Swati

2017-01-01

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPx Gl ) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF 4 - and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPx Gl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPx Gl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPx Gl ; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles.

E-cadherin can replace N-cadherin during secretory-stage enamel development.

PubMed

Guan, Xiaomu; Bidlack, Felicitas B; Stokes, Nicole; Bartlett, John D

2014-01-01

N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development. The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. μCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ. The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous

Secretory immunity with special reference to the oral cavity

PubMed Central

Brandtzaeg, Per

2013-01-01

The two principal antibody classes present in saliva are secretory IgA (SIgA) and IgG; the former is produced as dimeric IgA by local plasma cells (PCs) in the stroma of salivary glands and is transported through secretory epithelia by the polymeric Ig receptor (pIgR), also named membrane secretory component (SC). Most IgG in saliva is derived from the blood circulation by passive leakage mainly via gingival crevicular epithelium, although some may be locally produced in the gingiva or salivary glands. Gut-associated lymphoid tissue (GALT) and nasopharynx-associated lymphoid tissue (NALT) do not contribute equally to the pool of memory/effector B cells differentiating to mucosal PCs throughout the body. Thus, enteric immunostimulation may not be the best way to activate the production of salivary IgA antibodies although the level of specific SIgA in saliva may still reflect an intestinal immune response after enteric immunization. It remains unknown whether the IgA response in submandibular/sublingual glands is better related to B-cell induction in GALT than the parotid response. Such disparity is suggested by the levels of IgA in submandibular secretions of AIDS patients, paralleling their highly upregulated intestinal IgA system, while the parotid IgA level is decreased. Parotid SIgA could more consistently be linked to immune induction in palatine tonsils/adenoids (human NALT) and cervical lymph nodes, as supported by the homing molecule profile observed after immune induction at these sites. Several other variables influence the levels of antibodies in salivary secretions. These include difficulties with reproducibility and standardization of immunoassays, the impact of flow rate, acute or chronic stress, protein loss during sample handling, and uncontrolled admixture of serum-derived IgG and monomeric IgA. Despite these problems, saliva is an easily accessible biological fluid with interesting scientific and clinical potentials. PMID:23487566

Identification and staining of distinct populations of secretory organelles in astrocytes.

PubMed

Bezzi, Paola; Volterra, Andrea

2014-05-01

Increasing evidence indicates that astrocytes, the most abundant glial cell type in the brain, respond to an elevation in cytoplasmic calcium concentration ([Ca(2+)]i) by releasing chemical transmitters (also called gliotransmitters) via regulated exocytosis of heterogeneous classes of organelles. By this process, astrocytes exert modulatory influences on neighboring cells and are thought to participate in the control of synaptic circuits and cerebral blood flow. Studying the properties of exocytosis in astrocytes is a challenge, because the cell biological basis of this process is incompletely defined. Astrocytic exocytosis involves multiple populations of secretory vesicles, including synaptic-like microvesicles (SLMVs), dense-core granules (DCGs), and lysosomes. Here we summarize the available information for identifying individual populations of secretory organelles in astrocytes, including DCGs, SLMVs, and lysosomes, and present experimental procedures for specifically staining such populations.

Natural Immunoreactivity of Secretory IgA to Indigenous Strains of Streptococcus mutans From Chinese Spousal Pairs

PubMed Central

Nie, Min; Chen, Dong; Gao, Zhenyan; Wu, Xinyu; Li, Tong

2016-01-01

Background Dental caries is a well-known biofilm-mediated disease initiated by Streptococcus mutans, which should infect and colonize in a milieu perfused with components of the mucosal immune system. Little is known, however, regarding the relationship between the natural secretory IgA activity and S. mutans of a variety of diverse genotypes. Objectives The current study aimed to use spousal pairs to investigate the natural immunoreactivity of salivary secretory IgA to different genotype strains of S. mutans. Patients and Methods Indigenous strains were characterized from nine spousal pairs using polymerase reaction chain (PCR) and arbitrarily primed polymerase chain reaction (AP-PCR) by genotype monitoring. Unstimulated submandibular/sublingual secretions were collected and the concentrations of secretory IgA were determined by the enzyme-linked immunosorbent assay (ELISA). Each saliva sample was examined by Western blot to analyze the immunoreactivity of naturally occurring salivary secretory IgA antibodies for his/her own indigenous strain, spouse’s strain and reference strains including S. mutans GS-5 and Ingbritt (C). Results The results showed that naturally induced salivary IgA antibodies against S. mutans were present in all subjects. Almost all subjects had the similar individual immunoblotting profiles to different genotype strains. Conclusions The current study indicated that the immunoreactivity of secretory IgA might have no direct correlation with the colonization of indigenous flora and rejection of exogenous strains in adults. The relationship of microbes, host and dental caries should be in the light of coevolved microecosystem as a whole, but not caused by one factor alone. PMID:27303613

Secretory proteins in the reproductive tract of the snapping turtle, Chelhydra serpentina.

PubMed

Mahmoud, I Y; Paulson, J R; Dudley, M; Patzlaff, J S; Al-Kindi, A Y A

2004-12-01

SDS-polyacrylamide gel electrophoresis was used to separate the secretory proteins produced by the epithelial and endometrial glands of the uterine tube and uterus in the snapping turtle Chelydra serpentina. The proteins were analyzed throughout the phases of the reproductive cycle from May to August, including preovulatory, ovulatory, postovulatory or luteal, and vitellogenic phases. The pattern of secretory proteins is quite uniform along the length of the uterine tube, and the same is true of the uterus, but the patterns for uterine tube and uterus are clearly different. We identify 13 major proteins in C. serpentina egg albumen. Bands co-migrating with 11 of these are found in the uterine tube, but at most 4 are found in the uterus, suggesting that the majority of the albumen proteins are most likely secreted in the uterine tube, not in the uterus. Although some of the egg albumen proteins are present in the uterine tube only at the time of ovulation, most of the bands corresponding to albumen proteins are present throughout the breeding season even though the snapping turtle is a monoclutch species. These results suggest that the glandular secretory phase in the uterine tube is active and quite homogeneous in function regardless of location or phase of the reproductive cycle.

A Dynamic Analysis of Secretory Granules Containing Proteins Involved In Learning

NASA Astrophysics Data System (ADS)

Prahl, Louis; Simon, Alex; Jacobs, Conor; Fulwiler, Audrey; Hilken, Lindsay; Scalettar, Bethe; Lochner, Janis

2010-10-01

Formation and encoding of long-term memories requires a series of structural changes at synapses, or sites of neuronal communication, in the hippocampus; these changes are mediated by neuromodulatory proteins and serve to strengthen synapses to improve communication. Two prominent neuromodulators, tissue plasminogen activator (tPA) and brain-derived neurotrophic factor (BDNF), are copackaged into secretory granules (SGs) in the body of nerve cells and are transported to distal synapses by motor proteins. At synapses, particularly presynaptic sites, the fate of tPA and BDNF is largely unknown. Motivated by this, and by recent data implicating presynaptic BDNF in early phases of learning, we used fluorescence microscopy to elucidate dynamic properties of presynaptic tPA and BDNF. We find that presynaptic SGs containing tPA and/or BDNF undergo Brownian and anomalous diffusive motion that, in 75% of cases, is so slow that it typically would be classified as immobility. These results suggest that tPA and BDNF are retained at presynaptic sites to facilitate their corelease and role in learning.

Human milk containing specific secretory IgA inhibits binding of Giardia lamblia to nylon and glass surfaces.

PubMed

Samra, H K; Ganguly, N K; Mahajan, R C

1991-06-01

The effects of human milk, containing specific secretory IgA, on the adherence of Giardia lamblia trophozoites in the presence and in the absence of intestinal mucus in vitro were studied. It was found that the trophozoites treated with breast milk, containing specific secretory IgA to G. lamblia, showed a significant decrease (p less than 0.01) in adherence to nylon fibre columns and glass surfaces than did trophozoites treated with milk containing no SIgA antibodies. The adherence to glass surfaces was significantly more (p less than 0.01) in the presence of intestinal mucus than when the mucus was absent. Milk that did not contain specific secretory SIgA to G. lamblia did not decrease the adherence to glass surfaces either in the presence or in the absence of mucus. The fluorescence study revealed the binding of specific secretory IgA on the trophozoite surface. The results suggest that binding of SIgA antibodies in milk to G. lamblia trophozoites inhibits parasite adherence, thus protecting against this infection in breast-fed babies.

The C2H2 Transcription Factor REGULATOR OF SYMBIOSOME DIFFERENTIATION Represses Transcription of the Secretory Pathway Gene VAMP721a and Promotes Symbiosome Development in Medicago truncatula[W][OPEN

PubMed Central

Sinharoy, Senjuti; Torres-Jerez, Ivone; Bandyopadhyay, Kaustav; Kereszt, Attila; Pislariu, Catalina I.; Nakashima, Jin; Benedito, Vagner A.; Kondorosi, Eva; Udvardi, Michael K.

2013-01-01

Transcription factors (TFs) are thought to regulate many aspects of nodule and symbiosis development in legumes, although few TFs have been characterized functionally. Here, we describe REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD) of Medicago truncatula, a member of the Cysteine-2/Histidine-2 (C2H2) family of plant TFs that is required for normal symbiosome differentiation during nodule development. RSD is expressed in a nodule-specific manner, with maximal transcript levels in the bacterial invasion zone. A tobacco (Nicotiana tabacum) retrotransposon (Tnt1) insertion rsd mutant produced nodules that were unable to fix nitrogen and that contained incompletely differentiated symbiosomes and bacteroids. RSD protein was localized to the nucleus, consistent with a role of the protein in transcriptional regulation. RSD acted as a transcriptional repressor in a heterologous yeast assay. Transcriptome analysis of an rsd mutant identified 11 genes as potential targets of RSD repression. RSD interacted physically with the promoter of one of these genes, VAMP721a, which encodes vesicle-associated membrane protein 721a. Thus, RSD may influence symbiosome development in part by repressing transcription of VAMP721a and modifying vesicle trafficking in nodule cells. This establishes RSD as a TF implicated directly in symbiosome and bacteroid differentiation and a transcriptional regulator of secretory pathway genes in plants. PMID:24082011

Keratinocyte secretion of cyclophilin B via the constitutive pathway is regulated through its cyclosporin-binding site.

PubMed

Fearon, Paula; Lonsdale-Eccles, Ann A; Ross, O Kehinde; Todd, Carole; Sinha, Aparna; Allain, Fabrice; Reynolds, Nick J

2011-05-01

Cyclophilin B (CypB) is an endoplasmic reticulum (ER)-resident member of the cyclophilin family of proteins that bind cyclosporin A (CsA). We report that as in other cell types, CypB trafficked from the ER and was secreted by keratinocytes into the media in response to CsA. Concentrations as low as 1 pM of CsA induced secretion of CypB. Using brefeldin A, we showed that CypB is secreted from keratinocytes via the constitutive secretory pathway. We defined that substitution of tryptophan residue 128 in the CsA-binding site of CypB with alanine resulted in dissociation of CypB(W128A)-green fluorescent protein (GFP) from the ER. Photobleaching studies revealed a significant reduction in the diffusible mobility of CypB(W128A)-GFP compared with CypB(WT)-GFP, consistent with redistribution of CypB(W128A)-GFP into secretory vesicles disconnected from the ER/Golgi network. Furthermore, CsA significantly decreased the mobility of CypB(WT)-GFP but not CypB(W128A)-GFP. These studies demonstrate that therapeutically relevant concentrations of CsA regulate secretion of CypB by keratinocytes, and that a key residue within the CsA-binding site of CypB controls retention of CypB within the ER and regulates entry into the secretory pathway. As keratinocytes express CypB receptors (CD147) and CypB exhibits chemotactic properties, these data have implications for the therapeutic effects of CsA in inflammatory skin disease.

Keratinocyte Secretion of Cyclophilin B via the Constitutive Pathway Is Regulated through Its Cyclosporin-Binding Site

PubMed Central

Fearon, Paula; Lonsdale-Eccles, Ann A; Ross, O Kehinde; Todd, Carole; Sinha, Aparna; Allain, Fabrice; Reynolds, Nick J

2011-01-01

Cyclophilin B (CypB) is an endoplasmic reticulum (ER)-resident member of the cyclophilin family of proteins that bind cyclosporin A (CsA). We report that as in other cell types, CypB trafficked from the ER and was secreted by keratinocytes into the media in response to CsA. Concentrations as low as 1 p of CsA induced secretion of CypB. Using brefeldin A, we showed that CypB is secreted from keratinocytes via the constitutive secretory pathway. We defined that substitution of tryptophan residue 128 in the CsA-binding site of CypB with alanine resulted in dissociation of CypBW128A-green fluorescent protein (GFP) from the ER. Photobleaching studies revealed a significant reduction in the diffusible mobility of CypBW128A-GFP compared with CypBWT-GFP, consistent with redistribution of CypBW128A-GFP into secretory vesicles disconnected from the ER/Golgi network. Furthermore, CsA significantly decreased the mobility of CypBWT-GFP but not CypBW128A-GFP. These studies demonstrate that therapeutically relevant concentrations of CsA regulate secretion of CypB by keratinocytes, and that a key residue within the CsA-binding site of CypB controls retention of CypB within the ER and regulates entry into the secretory pathway. As keratinocytes express CypB receptors (CD147) and CypB exhibits chemotactic properties, these data have implications for the therapeutic effects of CsA in inflammatory skin disease. PMID:21270823

Distinct Molecular Events during Secretory Granule Biogenesis Revealed by Sensitivities to Brefeldin A

PubMed Central

Fernandez, Carlos J.; Haugwitz, Michael; Eaton, Benjamin; Moore, Hsiao-Ping H.

1997-01-01

The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse–chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20°C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from ISGs is

Distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin A.

PubMed

Fernandez, C J; Haugwitz, M; Eaton, B; Moore, H P

1997-11-01

The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse-chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20 degrees C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from

A comparison of the binding of secretory component to immunoglobulin A (IgA) in human colostral S-IgA1 and S-IgA2

PubMed Central

Almogren, Adel; Senior, Bernard W; Kerr, Michael A

2007-01-01

A detailed investigation of the binding of secretory component to immunoglobulin A (IgA) in human secretory IgA2 (S-IgA2) was made possible by the development of a new method of purifying S-IgA1, S-IgA2 and free secretory component from human colostrum using thiophilic gel chromatography and chromatography on Jacalin-agarose. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of unreduced pure S-IgA2 revealed that, unlike in S-IgA1, a significant proportion of the secretory component was bound non-covalently in S-IgA2. When S-IgA1 was incubated with a protease purified from Proteus mirabilis the secretory component, but not the α-chain, was cleaved. This is in contrast to serum IgA1, in which the α-chain was cleaved under the same conditions – direct evidence that secretory component does protect the α-chain from proteolytic cleavage in S-IgA. Comparisons between the products of cleavage with P. mirabilis protease of free secretory component and bound secretory component in S-IgA1 and S-IgA2 also indicated that, contrary to the general assumption, the binding of secretory component to IgA is different in S-IgA2 from that in S-IgA1. PMID:17156102

Early adverse experience and substance addiction: dopamine, oxytocin, and glucocorticoid pathways

PubMed Central

Kim, Sohye; Kwok, Stephanie; Mayes, Linda C.; Potenza, Marc N.; Rutherford, Helena J. V.; Strathearn, Lane

2016-01-01

Substance addiction may follow a chronic, relapsing course and critically undermine the physical and psychological well-being of the affected individual and the social units of which the individual is a member. Despite the public health burden associated with substance addiction, treatment options remain suboptimal, with relapses often seen. The present review synthesizes growing insights from animal and human research to shed light upon developmental and neurobiological pathways that may increase susceptibility to addiction. We examine the dopamine system, the oxytocin system, and the glucocorticoid system, as they are particularly relevant to substance addiction. Our aim is to delineate how early adverse experience may induce long-lasting alterations in each of these systems at molecular, neuroendocrine, and behavioral levels and ultimately lead to heightened vulnerability to substance addiction. We further discuss how substance addiction in adulthood may increase the risk of suboptimal caregiving for the next generation, perpetuating the intergenerational cycle of early adverse experiences and addiction. PMID:27508337

The Endoplasmic Reticulum-Associated Degradation Pathways of Budding Yeast

PubMed Central

Thibault, Guillaume; Ng, Davis T.W.

2012-01-01

Protein misfolding is a common cellular event that can produce intrinsically harmful products. To reduce the risk, quality control mechanisms are deployed to detect and eliminate misfolded, aggregated, and unassembled proteins. In the secretory pathway, it is mainly the endoplasmic reticulum-associated degradation (ERAD) pathways that perform this role. Here, specialized factors are organized to monitor and process the folded states of nascent polypeptides. Despite the complex structures, topologies, and posttranslational modifications of client molecules, the ER mechanisms are the best understood among all protein quality-control systems. This is the result of convergent and sometimes serendipitous discoveries by researchers from diverse fields. Although major advances in ER quality control and ERAD came from all model organisms, this review will focus on the discoveries culminating from the simple budding yeast. PMID:23209158

Alterations in molecular pathways in the retina of early experimental glaucoma eyes

PubMed Central

Cao, Li; Wang, Lin; Cull, Grant; Zhou, An

2015-01-01

Glaucoma is a multifactorial, neurodegenerative disease. The molecular mechanisms that underlie the pathophysiological changes in glaucomatous eyes, especially at the early stage of the disease, are poorly understood. Here, we report the findings from a quantitative proteomic analysis of retinas from experimental glaucoma (EG) eyes. An early stage of EG was modeled on unilateral eyes of five nonhuman primates (NHP) by laser treatment-induced elevation of intraocular pressure (IOP). Retinal proteins were extracted from individual EG eyes and their contralateral control eyes of the same animals, respectively, and analyzed by quantitative mass spectrometry (MS). As a result, a total, 475 retinal proteins were confidently identified and quantified. Results of bioinformatic analysis of proteins that showed an increase in the EG eyes suggested changes in apoptosis, DNA damage, immune response, cytoskeleton rearrangement and cell adhesion processes. Interestingly, hemoglobin subunit alpha (HBA) and Ras related C3 botulinum toxin substrate 1 (Rac1) were among the increased proteins. Results of molecular modeling of HBA- and Rac1-associated signaling network implicated the involvement of Mitogen-Activated Protein Kinase (MAPK) pathway in the EG, through which Rac1 may exert a regulatory role on HBA. This is the first observation of this potentially novel signaling network in the NHP retina and in EG. Results of Western blot analyses for Rac1, HBA and a selected MAPK pathway protein indicated synergistic changes in all three proteins in the EG eyes. Further, results of hierarchical cluster analysis of proteomes of control eyes revealed a clear age-proteome relationship, and such relationship appeared disrupted in the EG eyes. In conclusion, our results suggested an increased presence of a potentially novel signaling network at the early stage of glaucoma, and age might be one of the determinant factors in retinal proteomic characteristics under normal conditions. PMID

Pathways from Adolescent Deliberate Self-Poisoning to Early Adult Outcomes: A Six-Year Follow-Up

ERIC Educational Resources Information Center

Aglan, Azza; Kerfoot, Michael; Pickles, Andrew

2008-01-01

Background: Prospective studies show that the adult outcomes of adolescents who deliberately harm themselves are marked by high rates of adversity and psychiatric disorders. The goal of this study was to identify pathways linking childhood risk factors to early adult outcomes of suicidal adolescents. Methods: A clinical sample of 158 adolescents…

Delayed Early Primary Visual Pathway Development in Premature Infants: High Density Electrophysiological Evidence

PubMed Central

Tremblay, Emmanuel; Vannasing, Phetsamone; Roy, Marie-Sylvie; Lefebvre, Francine; Kombate, Damelan; Lassonde, Maryse; Lepore, Franco; McKerral, Michelle; Gallagher, Anne

2014-01-01

In the past decades, multiple studies have been interested in developmental patterns of the visual system in healthy infants. During the first year of life, differential maturational changes have been observed between the Magnocellular (P) and the Parvocellular (P) visual pathways. However, few studies investigated P and M system development in infants born prematurely. The aim of the present study was to characterize P and M system maturational differences between healthy preterm and fullterm infants through a critical period of visual maturation: the first year of life. Using a cross-sectional design, high-density electroencephalogram (EEG) was recorded in 31 healthy preterms and 41 fullterm infants of 3, 6, or 12 months (corrected age for premature babies). Three visual stimulations varying in contrast and spatial frequency were presented to stimulate preferentially the M pathway, the P pathway, or both systems simultaneously during EEG recordings. Results from early visual evoked potentials in response to the stimulation that activates simultaneously both systems revealed longer N1 latencies and smaller P1 amplitudes in preterm infants compared to fullterms. Moreover, preterms showed longer N1 and P1 latencies in response to stimuli assessing the M pathway at 3 months. No differences between preterms and fullterms were found when using the preferential P system stimulation. In order to identify the cerebral generator of each visual response, distributed source analyses were computed in 12-month-old infants using LORETA. Source analysis demonstrated an activation of the parietal dorsal region in fullterm infants, in response to the preferential M pathway, which was not seen in the preterms. Overall, these findings suggest that the Magnocellular pathway development is affected in premature infants. Although our VEP results suggest that premature children overcome, at least partially, the visual developmental delay with time, source analyses reveal abnormal brain

Important first encounter: Service user experience of pathways to care and early detection in first-episode psychosis.

PubMed

Jansen, Jens Einar; Pedersen, Marlene Buch; Hastrup, Lene Halling; Haahr, Ulrik Helt; Simonsen, Erik

2018-04-01

Long duration of untreated psychosis is associated with poor clinical and functional outcomes. However, few systematic attempts have been made to reduce this delay and little is known of service users' experience of early detection efforts. We explored service users' experience of an early detection service and transition to specialized treatment service, including pathway to care, understanding of illness and barriers to adequate assessment and treatment. In-depth interviews were conducted with 10 service users (median age 21, range 18-27, five males and five females) who were diagnosed with a first-episode non-affective psychosis and who were seen by an early detection team (TOP) and currently enrolled in a specialized early intervention service for this disorder (OPUS). Stigma and fear of the 'psychiatric system' were reported as significant barriers to help seeking, while family members were seen as a crucial support. Moreover, the impact of traumatic events on the experience and development of psychosis was highlighted. Finally, participants were relieved by the prospect of receiving help and the early detection team seemed to create a trusting relationship by offering a friendly, 'anti-stigmatized' space, where long-term symptomatology could be disclosed through accurate and validating questioning. Early detection services have two important functions. One is to make accurate assessments and referrals. The other is to instil hope and trust, and to facilitate further treatment by forming an early therapeutic alliance. The findings in this study provide important insights into the way in which early detection efforts and pathways to care are experienced by service users, with direct implications for improving psychiatric services. © 2015 Wiley Publishing Asia Pty Ltd.

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

PubMed Central

Arakel, Eric C.; Richter, Kora P.; Clancy, Anne; Schwappach, Blanche

2016-01-01

Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

Cysteine-Rich Atrial Secretory Protein from the Snail Achatina achatina: Purification and Structural Characterization

PubMed Central

Shabelnikov, Sergey; Kiselev, Artem

2015-01-01

Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported. In this study, we describe an 18.1-kDa, cysteine-rich atrial secretory protein (CRASP) isolated from the terrestrial snail Achatina achatina that has no detectable sequence similarity to any known protein or nucleotide sequence. CRASP is an acidic, 158-residue, N-glycosylated protein composed of eight alpha-helical segments stabilized with five disulphide bonds. A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold. CRASP is most strongly expressed in the atrium in secretory atrial granular cells, and substantial amounts of CRASP are released from the heart upon nerve stimulation. CRASP is detected in the haemolymph of intact animals at nanomolar concentrations. CRASP is the first secretory protein expressed in molluscan atrium to be reported. We propose that CRASP is an example of a taxonomically restricted gene that might be responsible for adaptations specific for terrestrial pulmonates. PMID:26444993

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.

PubMed

Glozman, Rina; Okiyoneda, Tsukasa; Mulvihill, Cory M; Rini, James M; Barriere, Herve; Lukacs, Gergely L

2009-03-23

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

Pathways to Early Coital Debut for Adolescent Girls: A Recursive Partitioning Analysis

PubMed Central

Pearson, Matthew R.; Kholodkov, Tatyana; Henson, James M.; Impett, Emily A.

2011-01-01

The current study examined pathways to early coital debut among early to middle adolescent girls in the United States. In a two-year longitudinal study of 104 adolescent girls, we conducted Recursive Partitioning (RP) analyses to examine the specific factors that were related to engaging in first intercourse by the 10th grade among adolescent girls who had not yet engaged in sexual intercourse by the 8th grade. RP analyses identified subsamples of girls who had low, medium, and high likelihoods of engaging in early coital debut based on six variables (i.e., school aspirations, early physical intimacy experiences, depression, body objectification, body image, and relationship inauthenticity). For example, girls in the lowest likelihood group (3% had engaged in sex by the 10th grade) reported no prior experiences with being touched under their clothes, low body objectification, high aspirations to complete graduate education, and low depressive symptoms; girls in the highest likelihood group (75% had engaged in sex by the 10th grade) also reported no prior experiences with being touched under their clothes but had high levels of body objectification. The implications of these analyses for the development of female adolescent sexuality as well as for advances in quantitative methods are discussed. PMID:21512947

Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome.

PubMed

Yang, Yi; Qin, Meiling; Bao, Puhua; Xu, Wangchao; Xu, Jin

2017-01-01

Autophagy-lysosomal pathway is a cellular protective system to remove aggregated proteins and damaged organelles. Meanwhile, exosome secretion has emerged as a mode to selectively clear the neurotoxic proteins, such as α-synuclein. Mounting evidence suggests that these two cellular processes are coordinated to facilitate the clearance of toxic cellular waste; however the regulators for the transition between these two processes are unclear. Here we show that SCAMP5, a secretory carrier membrane protein significantly induced in the brains of Huntington's disease patients, is quickly and transiently induced by protein stress and autophagic stimulation, and is regulated by the master autophagy transcriptional regulator TFEB. Ironically, SCAMP5 inhibits autophagy flux by blocking the fusion of autophagosomes and lysosomes. Although autophagy is blocked, SCAMP5 does not cause significant protein aggregation in cells. Instead, it promotes the Golgi fragmentation and stimulates the unconventional secretion of the co-localizing α-synuclein via exosome as an exosome component. Therefore, we have identified SCAMP5 as a novel coordinator of autophagy and exosome secretion, which is induced upon protein stress to channel the efficient clearance of toxic proteins via the exosomes rather than autophagy-lysosomal pathway.

The NRF2-KEAP1 Pathway Is an Early Responsive Gene Network in Arsenic Exposed Lymphoblastoid Cells

PubMed Central

Córdova, Emilio J.; Martínez-Hernández, Angélica; Uribe-Figueroa, Laura; Centeno, Federico; Morales-Marín, Mirna; Koneru, Harsha; Coleman, Matthew A.; Orozco, Lorena

2014-01-01

Inorganic arsenic (iAs), a major environmental contaminant, has risen as an important health problem worldwide. More detailed identification of the molecular mechanisms associated with iAs exposure would help to establish better strategies for prevention and treatment. Although chronic iAs exposures have been previously studied there is little to no information regarding the early events of exposure to iAs. To better characterize the early mechanisms of iAs exposure we conducted gene expression studies using sublethal doses of iAs at two different time-points. The major transcripts differentially regulated at 2 hrs of iAs exposure included antioxidants, detoxificants and chaperones. Moreover, after 12 hrs of exposure many of the down-regulated genes were associated with DNA replication and S phase cell cycle progression. Interestingly, the most affected biological pathway by both 2 or 12 hrs of iAs exposure were the Nrf2-Keap1 pathway, represented by the highly up-regulated HMOX1 transcript, which is transcriptionally regulated by the transcription factor Nrf2. Additional Nrf2 targets included SQSTM1 and ABCB6, which were not previously associated with acute iAs exposure. Signalling pathways such as interferon, B cell receptor and AhR route were also responsive to acute iAs exposure. Since HMOX1 expression increased early (20 min) and was responsive to low iAs concentrations (0.1 µM), this gene could be a suitable early biomarker for iAs exposure. In addition, the novel Nrf2 targets SQSTM1 and ABCB6 could play an important and previously unrecognized role in cellular protection against iAs. PMID:24516582

Alpha 1-protease inhibitor moderates human neutrophil elastase-induced emphysema and secretory cell metaplasia in hamsters.

PubMed

Stone, P J; Lucey, E C; Virca, G D; Christensen, T G; Breuer, R; Snider, G L

1990-06-01

A study was undertaken to determine whether emphysema and airway secretory cell metaplasia, induced in hamsters by intratracheal treatment with human neutrophil elastase (HNE), could be moderated by pretreatment with human alpha 1-protease inhibitor (API). API (4.9 mg) was given intratracheally to hamsters 1 h before 0.3 mg HNE. Eight weeks later, lung volumes and pressure-volume relationships were measured in the anaesthetized animals. Mean linear intercepts and secretory cell indices were measured in lung sections. API given 1 h before HNE moderated the development of bronchial secretory cell metaplasia. The severity of emphysema was reduced by 75%. Clearance studies indicated that 80% of the functional activity of instilled API could be lavaged from the lungs after 1 h, indicating a 4 h half-life in the lavageable compartment of the lungs. We calculate that for 50% protection from emphysema the molar ratio of lavageable API to HNE at the time of HNE instillation was 4.8 as compared with 0.78 for 50% inhibition of elastolytic activity in vitro, indicating that API is only 16% as efficient in vivo as compared with its in vitro HNE inhibitory effectiveness. Nevertheless, we conclude that human API given intratracheally is efficacious against HNE-induced emphysema and secretory cell metaplasia.

A Pathway to Enhancing Professionalism: Building a Bridge between TAFE and University Early Childhood Qualifications

ERIC Educational Resources Information Center

Whitington, Victoria; Ebbeck, Marjory; Diamond, Alexandra; Yim, Hoi Yin Bonnie

2009-01-01

It has been argued that a key strategy to improve developmental and educational outcomes for young children is to increase the number of childcare staff with early childhood university degrees (Saracho & Spodek, 2007). In order to upgrade the qualifications of staff, a number of Australian universities provide pathways that enable graduates of…

Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

PubMed

Park, Soonhong; Ahuja, Malini; Kim, Min Seuk; Brailoiu, G Cristina; Jha, Archana; Zeng, Mei; Baydyuk, Maryna; Wu, Ling-Gang; Wassif, Christopher A; Porter, Forbes D; Zerfas, Patricia M; Eckhaus, Michael A; Brailoiu, Eugen; Shin, Dong Min; Muallem, Shmuel

2016-02-01

Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV. © 2015 The Authors.

Functional anatomy reveals secretory activity in papillose anthers of a buzz-pollinated Solanum species (Cyphomandra clade - Solanaceae).

PubMed

Falcão, B F; Stehmann, J R

2018-03-30

Pollination in Solanum (Solanaceae) species is commonly performed by female bees, which vibrate anthers to extract pollen. Another pollen removal type is by male euglossine bees, milking the anthers when searching for floral scents produced by secretory tissues (osmophorous) at the swollen connective of the anthers of species in the Cyphomandra clade. Some species of this clade, however, are buzz-pollinated and present papillate anthers that should also have secretory activity, a hypothesis here tested. The anthers of Solanum luridifuscescens were fixed at different stages of development and analysed under light microscopy, SEM and TEM. Histochemical tests for the detection of starch and lipids were done. Epidermal cells of the abaxial surface of the anthers were visibly papillose, had large nuclei and dense cytoplasm rich in organelles such as mitochondria and plastids, typical features of secretory tissues. In this site, lipid droplets were detected, concomitantly with starch consumption, compatible with the secretory process in osmophores. No exudate or accumulation of substances was seen on the surface; in agreement with a previous pollination study performed in field conditions, where no pollinators were observed collecting floral scents, only pollen. The histochemical and structural analyses have evidenced the lipidic composition of the secretion, strongly pointing to terpenes as the secreted compounds. Ours findings show that papillae of the anthers have secretory activities that produce lipophilic compounds. This does not result in resources for bees, but could be an evolutionary step to the development of more specialised anthers in the Cyphomandra clade. © 2018 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

Morphological and secretory characterization of extrafloral nectaries in plants of coastal Veracruz, Mexico.

PubMed

Díaz-Castelazo, Cecilia; Rico-Gray, Victor; Ortega, Fernando; Angeles, Guillermo

2005-12-01

Morphological descriptions of the extrafloral nectaries (EFNs) of certain plant species are common in the literature, but they rarely relate morphology with histology, gland distribution and secretory attributes. In this study a morphological/secretory characterization of EFNs occurring on several plant species in a tropical coastal community is made and the implications of gland attributes discussed from a functional perspective. The morphology and nectar secretion of the EFNs of 20 plant species are characterized through scanning electron microscopy, histochemical detection of reducing sugars (Fehling's reagent) and nectar volume/concentration estimates. Sixty-five per cent of plant species in coastal communities had EFNs on vegetative structures and 35 % of species had glands on reproductive and vegetative organs. The Fabaceae is the plant family with the most species with EFNs and most diversity of gland morphologies. Four types of vascularized nectaries and four of glandular trichomes are described; sugar-secreting trichomes are characterized using Fehling's technique, and the first descriptions of unicellular and peltate trichomes functioning as EFNs are provided. Glands of ten plant species and six genera are described for the first time. Four plant species possess more than one morphological type of EFN. Eleven species have EFNs in more than one location or organ. More complex glands secrete more nectar, but are functionally homologous to the aggregations of numerous secretory trichomes on specific and valuable plant organs. Important diversity of EFN morphology was foundin the coastal plant community studied. Both vascularized and non-vascularized EFNs are observed in plants and, for the latter, previously non-existent morpho-secretory characterizations are provided with a methodological approach to study them. It is recommended that studies relating EFN attributes (i.e. morphology, distribution) with their differential visitation by insects (i.e. ants

Post-secretory fate of host defence components in mucus.

PubMed

Salathe, Matthias; Forteza, Rosanna; Conner, Gregory E

2002-01-01

Airway mucus is a complex mixture of secretory products that provide a multifaceted defence against infection. Among many antimicrobial substances, mucus contains a peroxidase identical to milk lactoperoxidase (LPO) that is produced by goblet cells and submucosal glands. Airway secretions contain the substrates for LPO, namely thiocyanate and hydrogen peroxide, at concentrations sufficient for production of the biocidal compound hypothiocyanite, a fact confirmed by us in vitro. In vivo, inhibition of airway LPO in sheep significantly inhibits bacterial clearance, suggesting that the LPO system is a major contributor to host defences. Since secretory products including LPO are believed to be steadily removed by mucociliary clearance, their amount and availability on the surface is thought to be controlled solely by secretion. In contrast to this paradigm, new data suggest that LPO and other substances are retained at the ciliary border of the airway epithelium by binding to surface-associated hyaluronan, thereby providing an apical, fully active enzyme pool. Thus, hyaluronan, secreted from submucosal gland cells, plays a previously unrecognized pivotal role in mucosal host defence by retaining LPO and possibly other substances important for first line host defence at the apical surface 'ready for use' and protected from ciliary clearance.

Separation of rat pituitary secretory granules by continuous flow electrophoresis

NASA Technical Reports Server (NTRS)

Hayes, Daniel; Exton, Carrie; Salada, Thomas; Shellenberger, Kathy; Waddle, Jenny; Hymer, W. C.

1990-01-01

The separation of growth hormone-containing cytoplasmic secretory granules from the rat pituitary gland by continuous flow electrophoresis is described. The results are consistent with the hypothesis that granule subpopulations can be separated due to differences in surface charge; these, in turn, may be related to the oligomeric state of the hormone.

Discovering and annotating fish early life-stage (FELS) adverse outcome pathways: Putting the research strategy into practice

EPA Science Inventory

In May 2012, a HESI-sponsored expert workshop yielded a proposed research strategy for systematically discovering, characterizing, and annotating fish early life-stage (FELS) adverse outcome pathways (AOPs) as well as prioritizing AOP development in light of current restrictions ...

An efficient delivery of DAMPs on the cell surface by the unconventional secretion pathway

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

Zhu, Haiyan; Wang, Lan; Ruan, Yuanyuan

2011-01-21

Research highlights: {yields} Hsp60 transported to cell surface through the classical secretory pathway was modified with N-glycosylation. {yields} HSAPB-N18 could efficiently deliver Hsp60 to the cell surface via the unconventional secretory pathway. {yields} Cell surface Hsp60 delivered by HASPB-N18 has a proper conformation. {yields} HASPB-N18 is an efficient delivery signal for other DAMP molecules such as Hsp70 and HMGB1. -- Abstract: Damage-associated molecular patterns (DAMPs) are signals released from dying cells evoking the immune system response in several inflammatory disorders. In normal situations, many of DAMPs are nuclear or cytosolic proteins with defined intracellular function, but they could be foundmore » on the cell surface following tissue injury. The biological function of the translocated DAMPs is still not well known and an efficient delivery of these molecules on the cell surface is required to clarify their biological effects. In this study, we demonstrated that an unclassical secretory signal peptide, N-terminal 18 amino acids of HASPB (HASPB-N18), could efficiently deliver Hsp60, Hsp70, and HMGB1 on the cell surface. Furthermore, the delivery of these molecules on the cell surface by HASPB-N18 is not limited to a special cell line because several cell lines could use this delivery signal to deliver these molecules on the cell surface. Moreover, we demonstrated that Hsp60 on the cell surface delivered by HASPB-N18 could be recognized by a soluble form of LOX-1, which implies that DAMPs on the cell surface delivered by HASPB-N18 have a proper conformation during transport. Therefore, delivery of DAMPs by HASPB-N18 is a reliable model to further understand the biological significance of DAMPs on the cell surface.« less

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism.

PubMed

Wang, Rong; Yu, Zhen; Sunchu, Bharath; Shoaf, James; Dang, Ivana; Zhao, Stephanie; Caples, Kelsey; Bradley, Lynda; Beaver, Laura M; Ho, Emily; Löhr, Christiane V; Perez, Viviana I

2017-06-01

Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-β-galactosidase (β-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β-gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags.

PubMed

Celińska, Ewelina; Borkowska, Monika; Białas, Wojciech; Korpys, Paulina; Nicaud, Jean-Marc

2018-06-01

Upon expression of a given protein in an expression host, its secretion into the culture medium or cell-surface display is frequently advantageous in both research and industrial contexts. Hence, engineering strategies targeting folding, trafficking, and secretion of the proteins gain considerable interest. Yarrowia lipolytica has emerged as an efficient protein expression platform, repeatedly proved to be a competitive secretor of proteins. Although the key role of signal peptides (SPs) in secretory overexpression of proteins and their direct effect on the final protein titers are widely known, the number of reports on manipulation with SPs in Y. lipolytica is rather scattered. In this study, we assessed the potential of ten different SPs for secretion of two heterologous proteins in Y. lipolytica. Genomic and transcriptomic data mining allowed us to select five novel, previously undescribed SPs for recombinant protein secretion in Y. lipolytica. Their secretory potential was assessed in comparison with known, widely exploited SPs. We took advantage of Golden Gate approach, for construction of expression cassettes, and micro-volume enzymatic assays, for functional screening of large libraries of recombinant strains. Based on the adopted strategy, we identified novel secretory tags, characterized their secretory capacity, indicated the most potent SPs, and suggested a consensus sequence of a potentially robust synthetic SP to expand the molecular toolbox for engineering Y. lipolytica.

Evaluation of whole mount in situ hybridization as a tool for pathway-based toxicological research in early-life stage fathead minnows

EPA Science Inventory

Early-life stage fish can be more sensitive to chemical exposure than mature, adult fish. Therefore, defining adverse outcome pathways (AOPs) relevant to early-life stages is critical for linking perturbations of key events during fish development to potential adverse outcomes of...

Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows

EPA Science Inventory

Early-life stage fish can be more sensitive to chemical exposure than adult fish. Therefore, determining possible adverse outcome pathways (AOPs) for early-life stages is crucial. To determine chemical effects and/or mechanisms of action in exposed fish embryos and larvae, whole-...

Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes, and down-regulation of the secretory pathway.

PubMed

Marsolais, Frédéric; Pajak, Agnieszka; Yin, Fuqiang; Taylor, Meghan; Gabriel, Michelle; Merino, Diana M; Ma, Vanessa; Kameka, Alexander; Vijayan, Perumal; Pham, Hai; Huang, Shangzhi; Rivoal, Jean; Bett, Kirstin; Hernández-Sebastià, Cinta; Liu, Qiang; Bertrand, Annick; Chapman, Ralph

2010-06-16

A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

The Role of Secretory Immunoglobulin A in the Natural Sensing of Commensal Bacteria by Mouse Peyer's Patch Dendritic Cells*

PubMed Central

Rol, Nicolas; Favre, Laurent; Benyacoub, Jalil; Corthésy, Blaise

2012-01-01

The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer's patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c+CD11b+CD8− dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria. PMID:23027876

The secretory IgA system of lung secretions in chronic obstructive bronchitis: comparison of sputum with secretions obtained during fibreoptic bronchoscopy.

PubMed Central

Wiggins, J; Hill, S L; Stockley, R A

1984-01-01

The constituents of the secretory immunoglobulin A system (dimeric IgA, total secretory component and free secretory component) were measured in sputum sol phase, tracheal aspirates, and bronchoalveolar lavage fluids from 15 patients undergoing fibreoptic bronchoscopy. All of the proteins showed a progressive decrease in concentration from sputum to the bronchoalveolar lavage fluids (2p less than 0.001). Standardisation of samples by means of protein concentration ratios showed that all secretions were generally similar in respect of their secretory IgA profiles, although major differences remained in some individual patients. The between patient variability of the results was generally reduced by the use of protein concentration ratios, allowing closer comparison between subjects. When the secretion albumin concentration was used as a standard, however, it increased the variability of the sputum sol phase IgA components (2p less than 0.01), whereas it decreased the variability of the IgA components in the bronchoalveolar lavage fluid (2p less than 0.05). The role of albumin as a standard protein for assessing the secretory IgA system in lung secretions remains uncertain. PMID:6463931

Unprecedented multiplicity of Ig transmembrane and secretory mRNA forms in the cartilaginous fish.

PubMed

Rumfelt, Lynn L; Diaz, Marilyn; Lohr, Rebecca L; Mochon, Evonne; Flajnik, Martin F

2004-07-15

In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark, Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth constant (C(H)) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgW(short)) having an unusual Sec tail, which is orthologous to skate IgX(short) cDNA. The IgW and IgW(short) Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and the truncated variants may translate as more stable protein at the cell surface.

AKT delays the early-activated apoptotic pathway in UVB-irradiated keratinocytes via BAD translocation.

PubMed

Claerhout, Sofie; Decraene, David; Van Laethem, An; Van Kelst, Sofie; Agostinis, Patrizia; Garmyn, Marjan

2007-02-01

Upon irradiation with a high dose of UVB, keratinocytes undergo apoptosis as a protective mechanism. In previous work, we demonstrated the existence of an early-activated UVB-induced apoptotic pathway in growth factor-depleted human keratinocytes, which can be substantially delayed by the exclusive supplementation of IGF-1. We now show that in human keratinocytes, IGF-1 inhibits the onset of UVB-triggered apoptosis through a transcriptional independent, AKT-mediated mechanism, involving BAD serine 136 phosphorylation. Our results show that the early UVB-induced apoptosis in growth factor-depleted human keratinocytes is exclusively triggered through the mitochondrial pathway. It is accompanied by BAX translocation, cytochrome c release, and procaspase-9 cleavage, but not by procaspase-8 or BID cleavage. In human keratinocytes, IGF-1 supplementation inhibits these events in a transcription-independent manner. Both IGF-1 supplementation and the transduction of a membrane-targeted form of AKT result in a shift of the BH3-only protein BAD from the mitochondria to the cytoplasm, paralleled by an increase of AKT-specific Ser136 phospho-BAD bound to 14-3-3zeta protein. These data indicate that AKT-induced BAD phosphorylation and its subsequent cytoplasmic sequestration by 14-3-3zeta is a major mechanism responsible for the postponement of UVB-induced apoptosis in human keratinocytes.

Electron microprobe analysis of human labial gland secretory granules in cystic fibrosis

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

Izutsu, K.; Johnson, D.; Schubert, M.

1985-06-01

X-ray microanalysis of freeze-dried labial gland cryosections revealed that Na concentration was doubled and the Ca/S concentration ratio was decreased in secretory granules of labial glands from patients with cystic fibrosis (CF) when compared with glands from normal subjects. Other results suggested that the decrease in the Ca/S concentration ratio resulted from an increase in S concentration. These findings imply that mucous granules in labial saliva showed a CF-related increase in Na and S content, and such changes would be expected to affect the rheology of the mucus after exocytosis. In contrast with a previous study in human parotid glands,more » no evidence was found for CF-related changes in cytoplasmic or nuclear Na, K, and Ca concentrations. Significant elemental differences were found between secretory granules and nuclei and cytoplasm of control cells.« less

Quantitative parameters of seminiferous epithelium in secretory and excretory oligoazoospermia.

PubMed

Francavilla, S; Martini, M; Properzi, G; Cordeschi, G

1990-01-01

Testicular biopsy specimens from infertile men (sperm count, less than 10(6)/ml) were evaluated on 1-micron thick sections, and counts of stem cells and differentiated spermatogonia, primary spermatocytes, early and late spermatids, and Sertoli cells were compared to counts in six fertile men. Biopsy specimens were also compared for the appearance of seminiferous tubule wall, blood vessels, and interstitium. Infertile men were grouped according to the following diagnoses: hypospermatogenesis (n = 5), spermatocyte arrest of spermatogenesis (n = 5), and obstruction of the genital tract (n = 7). A low productivity of spermatogenesis in cases of hypospermatogenesis appeared to be due to an exaggerated degeneration of primary spermatocytes and to a yield of abnormal spermatids. A block of meiosis in spermatocyte arrest was associated with a degeneration of primary spermatocytes and with a reduced number of staminal spermatogonia. Abnormal spermiogenesis was observed in cases of obstruction of the genital tract and was associated with an increase in stem cell spermatogonia. A thickening of seminiferous tubule and blood vessel walls could be responsible for the limited functional capacity of Sertoli cells, causing altered spermiogenesis in cases of excretory azoospermia. A severe primitive failure of Sertoli cells in secretory oligoazoospermia could account for a deranged maturation and degeneration of premeiotic and postmeiotic germ cells.

Pathway to a Phenocopy: Heat Stress Effects in Early Embryogenesis

PubMed Central

Crews, Sarah M.; McCleery, W. Tyler; Hutson, M. Shane

2015-01-01

Background Heat shocks applied at the onset of gastrulation in early Drosophila embryos frequently lead to phenocopies of U-shaped mutants – having characteristic failures in the late morphogenetic processes of germband retraction and dorsal closure. The pathway from non-specific heat stress to phenocopied abnormalities is unknown. Results Drosophila embryos subjected to 30-min, 38-°C heat shocks at gastrulation appear to recover and restart morphogenesis. Post-heat-shock development appears normal, albeit slower, until a large fraction of embryos develop amnioserosa holes (diameters > 100 μm). These holes are positively correlated with terminal U-shaped phenocopies. They initiate between amnioserosa cells and open over tens of minutes by evading normal wound healing responses. They are not caused by tissue-wide increases in mechanical stress or decreases in cell-cell adhesion, but instead appear to initiate from isolated apoptosis of amnioserosa cells. Conclusions The pathway from heat shock to U-shaped phenocopies involves the opening of one or more large holes in the amnioserosa that compromise its structural integrity and lead to failures in morphogenetic processes that rely on amnioserosa-generated tensile forces. The proposed mechanism by which heat shock leads to hole initiation and expansion is heterochonicity – i.e., disruption of morphogenetic coordination between embryonic and extra-embryonic cell types. PMID:26498920

Novel secretory granule morphology in physically fixed pancreatic islets.

PubMed

Dudek, R W; Boyne, A F; Charles, T M

1984-09-01

Protein A-gold immunocytochemistry has been applied to physically fixed beta cells from rat islets of Langerhans. The punctate nature of the gold particles permits improved resolution of the antigenic sites without obscuring the fine ultrastructural preservation obtained by physical fixation. There is a filamentous material within the halo of the secretory granules that is not preserved by aqueous, chemical fixation. When viewed in stereo the filaments appear as an annular cobweb or a series of wheel spokes attached to a centrally located hub (the dense core of the granule). The filaments demonstrate insulin-like immunoreactivity using the protein A-gold technique. The immunoreactivity appears to be restricted to the filaments and the surface of the dense cores. This may be a consequence of the preservation of a solid, insolubilized core state that resists penetration by the antibody and/or the protein A-gold complex. However, the evidence that there is a halo pool of insulin which is separate from the massive core aggregate suggests that i) correspondingly massive exocytotic pits may not be as mandatory for insulin release as has been assumed and ii) the complex kinetics of insulin secretion may be, in part, a reflection of multiple insulin compartments within secretory granules.

Cysteine Cathepsins in the Secretory Vesicle Produce Active Peptides: Cathepsin L Generates Peptide Neurotransmitters and Cathepsin B Produces Beta-Amyloid of Alzheimer’s Disease

PubMed Central

Hook, Vivian; Funkelstein, Lydiane; Wegrzyn, Jill; Bark, Steven; Kindy, Mark; Hook, Gregory

2011-01-01

Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles has been demonstrated as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β-amyloid (Aβ) peptides that accumulate in Alzheimer’s disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrasts with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin function. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. PMID:21925292

WT1 controls antagonistic FGF and BMP-pSMAD pathways in early renal progenitors.

PubMed

Motamedi, Fariba Jian; Badro, Danielle A; Clarkson, Michael; Lecca, M Rita; Bradford, Stephen T; Buske, Fabian A; Saar, Kathrin; Hübner, Norbert; Brändli, André W; Schedl, Andreas

2014-07-17

Kidney organogenesis requires the tight control of proliferation, differentiation and apoptosis of renal progenitor cells. How the balance between these cellular decisions is achieved remains elusive. The Wilms' tumour suppressor Wt1 is required for progenitor survival, but the molecular cause for renal agenesis in mutants is poorly understood. Here we demonstrate that lack of Wt1 abolishes fibroblast growth factor (FGF) and induces BMP/pSMAD signalling within the metanephric mesenchyme. Addition of recombinant FGFs or inhibition of pSMAD signalling rescues progenitor cell apoptosis induced by the loss of Wt1. We further show that recombinant BMP4, but not BMP7, induces an apoptotic response within the early kidney that can be suppressed by simultaneous addition of FGFs. These data reveal a hitherto unknown sensitivity of early renal progenitors to pSMAD signalling, establishes FGF and pSMAD signalling as antagonistic forces in early kidney development and places WT1 as a key regulator of pro-survival FGF signalling pathway genes.

Gene expression profiling reveals different molecular patterns in G-protein coupled receptor signaling pathways between early- and late-onset preeclampsia.

PubMed

Liang, Mengmeng; Niu, Jianmin; Zhang, Liang; Deng, Hua; Ma, Jian; Zhou, Weiping; Duan, Dongmei; Zhou, Yuheng; Xu, Huikun; Chen, Longding

2016-04-01

Early-onset preeclampsia and late-onset preeclampsia have been regarded as two different phenotypes with heterogeneous manifestations; To gain insights into the pathogenesis of the two traits, we analyzed the gene expression profiles in preeclamptic placentas. A whole genome-wide microarray was used to determine the gene expression profiles in placental tissues from patients with early-onset (n = 7; <34 weeks), and late-onset (n = 8; >36 weeks) preeclampsia and their controls who delivered preterm (n = 5; <34 weeks) or at term (n = 5; >36 weeks). Genes were termed differentially expressed if they showed a fold-change ≥ 2 and q-value < 0.05. Quantitative real-time reverse transcriptase PCR was used to verify the results. Western blotting was performed to verify the expressions of secreted genes at the protein level. Six hundred twenty-seven genes were differentially expressed in early-compared with late-onset preeclampsia (177 genes were up-regulated and 450 were down-regulated). Gene ontology analysis identified significant alterations in several biological processes; the top two were immune response and cell surface receptor linked signal transduction. Among the cell surface receptor linked signal transduction-related, differentially expressed genes, those involved in the G-protein coupled receptor protein signaling pathway were significantly enriched. G-protein coupled receptor signaling pathway related genes, such as GPR124 and MRGPRF, were both found to be down-regulated in early-onset preeclampsia. The results were consistent with those of western blotting that the abundance of GPR124 was lower in early-onset compared with late-onset preeclampsia. The different gene expression profiles reflect the different levels of transcription regulation between the two conditions and supported the hypothesis that they are separate disease entities. Moreover, the G-protein coupled receptor signaling pathway related genes may contribute to the mechanism underlying early

PtdIns(4,5)P2 is not required for secretory granule docking.

PubMed

Omar-Hmeadi, Muhmmad; Gandasi, Nikhil R; Barg, Sebastian

2018-06-01

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P 2 ) by recruitment of a 5'-phosphatase strongly inhibited Ca 2+ -dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by α-toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P 2 accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The temporal characteristics of the early and late stages of the L- and M-cone pathways that signal color

PubMed Central

Petrova, Daniela; Henning, G. Bruce; Stockman, Andrew

2013-01-01

Flickering long-wavelength light appears more yellow than steady light of the same average intensity. The hue change is consistent with distortion of the visual signal at some nonlinear site (or sites) that produces temporal components not present in the original stimulus (known as distortion products). We extracted the temporal attenuation characteristics of the early (prenonlinearity) and late (post-nonlinearity) filter stages in the L- and M-cone chromatic pathway by varying the input stimulus to manipulate the distortion products and the measuring of the observers' sensitivity to them. The early, linear, filter stage acts like a band-pass filter peaking at 10–15 Hz with substantial sensitivity losses at both lower and higher frequencies. Its characteristics are consistent with nonlinearity being early in the visual pathway but following surround inhibition. The late stage, in contrast, acts like a low-pass filter with a cutoff frequency around 3 Hz. The response of the early stage speeds up with radiance, but the late stage does not. A plausible site for the nonlinearity, which modelling suggests may be smoothly compressive but with a hard limit at high input levels, is after surround inhibition from the horizontal cells. PMID:23457358

α-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion

PubMed Central

Geng, Xuehui; Lou, Haiyan; Wang, Jian; Li, Lehong; Swanson, Alexandra L.; Sun, Ming; Beers-Stolz, Donna; Watkins, Simon; Perez, Ruth G.

2011-01-01

α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic β-cells, α-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for α-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for α-synuclein localization to insulin granules in β-cells, immunoprecipitation with Western blot analysis for interaction between α-synuclein and KATP channels, and ELISA assays for the effect of altering α-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between α-synuclein knockout and wild-type β-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-α-synuclein antibodies labeled secretory organelles within β-cells. Anti-α-synuclein antibodies colocalized with KATP channel, anti-insulin, and anti-C-peptide antibodies. α-Synuclein coimmunoprecipitated in complexes with KATP channels. Expression of α-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. α-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the β-cell surface membranes observed in these islets. These findings demonstrate that α-synuclein interacts with KATP channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. α-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases. PMID:20858756

Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in Tetrahymena thermophila.

PubMed

Bowman, G R; Turkewitz, A P

2001-12-01

The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation.

Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in Tetrahymena thermophila.

PubMed Central

Bowman, G R; Turkewitz, A P

2001-01-01

The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation. PMID:11779800

Novel Monoclonal Antibodies for Studies of Human and Rhesus Macaque Secretory Component and Human J-Chain

PubMed Central

Zhang, Ruijun; Alam, S. Munir; Yu, Jae-Sung; Scearce, Richard; Lockwood, Bradley; Hwang, Kwan-Ki; Parks, Robert; Permar, Sallie; Brandtzaeg, Per; Haynes, Barton F.

2016-01-01

Immunoglobulin A (IgA) antibodies exist in monomeric, dimeric, and secretory forms. Dimerization of IgA depends on a 15-kD polypeptide termed “joining (J) chain,” which is also part of the binding site for an epithelial glycoprotein called “secretory component (SC),” whether this after apical cleavage on secretory epithelia is ligand bound in secretory IgA (SIgA) or in a free form. Uncleaved membrane SC, also called the “polymeric Ig receptor,” is thus crucial for transcytotic export of SIgA to mucosal surfaces, where it interacts with and modulates commensal bacteria and mediates protective immune responses against exogenous pathogens. To evaluate different forms of IgA, we have produced mouse monoclonal antibodies (MAbs) against human J-chain and free SC. We found that J-chain MAb 9A8 and SC MAb 9H7 identified human dimeric IgA and SIgA in enzyme-linked immunoassay and western blot analysis, as well as functioning in immunohistochemistry to identify cytoplasmic IgA of intestinal lamina propria plasmablasts/plasma cells and crypt epithelium of distal human intestine. Finally, we demonstrated that SC MAb 9H7 cross-reacted with rhesus macaque SIgA. These novel reagents should be of use in the study of the biology of various forms of IgA in humans and SIgA in macaques, as well as in monitoring the production and/or isolation of these forms of IgA. PMID:27386924

Longitudinal Examination of the Bullying-Sexual Violence Pathway across Early to Late Adolescence: Implicating Homophobic Name-Calling.

PubMed

Espelage, Dorothy L; Basile, Kathleen C; Leemis, Ruth W; Hipp, Tracy N; Davis, Jordan P

2018-03-02

The Bully-Sexual Violence Pathway theory has indicated that bullying perpetration predicts sexual violence perpetration among males and females over time in middle school, and that homophobic name-calling perpetration moderates that association among males. In this study, the Bully-Sexual Violence Pathway theory was tested across early to late adolescence. Participants included 3549 students from four Midwestern middle schools and six high schools. Surveys were administered across six time points from Spring 2008 to Spring 2013. At baseline, the sample was 32.2% White, 46.2% African American, 5.4% Hispanic, and 10.2% other. The sample was 50.2% female. The findings reveal that late middle school homophobic name-calling perpetration increased the odds of perpetrating sexual violence in high school among early middle school bullying male and female perpetrators, while homophobic name-calling victimization decreased the odds of high school sexual violence perpetration among females. The prevention of bullying and homophobic name-calling in middle school may prevent later sexual violence perpetration.

Morphological and Secretory Characterization of Extrafloral Nectaries in Plants of Coastal Veracruz, Mexico

PubMed Central

DÍAZ-CASTELAZO, CECILIA; RICO-GRAY, VICTOR; ORTEGA, FERNANDO; ÁNGELES, GUILLERMO

2005-01-01

• Background and Aims Morphological descriptions of the extrafloral nectaries (EFNs) of certain plant species are common in the literature, but they rarely relate morphology with histology, gland distribution and secretory attributes. In this study a morphological/secretory characterization of EFNs occurring on several plant species in a tropical coastal community is made and the implications of gland attributes discussed from a functional perspective. • Methods The morphology and nectar secretion of the EFNs of 20 plant species are characterized through scanning electron microscopy, histochemical detection of reducing sugars (Fehling's reagent) and nectar volume/concentration estimates. • Key Results Sixty-five per cent of plant species in coastal communities had EFNs on vegetative structures and 35 % of species had glands on reproductive and vegetative organs. The Fabaceae is the plant family with the most species with EFNs and most diversity of gland morphologies. Four types of vascularized nectaries and four of glandular trichomes are described; sugar-secreting trichomes are characterized using Fehling's technique, and the first descriptions of unicellular and peltate trichomes functioning as EFNs are provided. Glands of ten plant species and six genera are described for the first time. Four plant species possess more than one morphological type of EFN. Eleven species have EFNs in more than one location or organ. More complex glands secrete more nectar, but are functionally homologous to the aggregations of numerous secretory trichomes on specific and valuable plant organs. • Conclusion Important diversity of EFN morphology was foundin the coastal plant community studied. Both vascularized and non-vascularized EFNs are observed in plants and, for the latter, previously non-existent morpho-secretory characterizations are provided with a methodological approach to study them. It is recommended that studies relating EFN attributes (i.e. morphology

Salivary Secretory Disorders, Inducing Drugs, and Clinical Management

PubMed Central

Miranda-Rius, Jaume; Brunet-Llobet, Lluís; Lahor-Soler, Eduard; Farré, Magí

2015-01-01

Background: Salivary secretory disorders can be the result of a wide range of factors. Their prevalence and negative effects on the patient's quality of life oblige the clinician to confront the issue. Aim: To review the salivary secretory disorders, inducing drugs and their clinical management. Methods: In this article, a literature search of these dysfunctions was conducted with the assistance of a research librarian in the MEDLINE/PubMed Database. Results: Xerostomia, or dry mouth syndrome, can be caused by medication, systemic diseases such as Sjögren's Syndrome, glandular pathologies, and radiotherapy of the head and neck. Treatment of dry mouth is aimed at both minimizing its symptoms and preventing oral complications with the employment of sialogogues and topical acting substances. Sialorrhea and drooling, are mainly due to medication or neurological systemic disease. There are various therapeutic, pharmacologic, and surgical alternatives for its management. The pharmacology of most of the substances employed for the treatment of salivary disorders is well-known. Nevertheless, in some cases a significant improvement in salivary function has not been observed after their administration. Conclusion: At present, there are numerous frequently prescribed drugs whose unwanted effects include some kind of salivary disorder. In addition, the differing pathologic mechanisms, and the great variety of existing treatments hinder the clinical management of these patients. The authors have designed an algorithm to facilitate the decision making process when physicians, oral surgeons, or dentists face these salivary dysfunctions. PMID:26516310

Secretory response induced by essential oils on airway surface fluid: a pharmacological MRI study.

PubMed

Nicolato, Elena; Boschi, Federico; Marzola, Pasquina; Sbarbati, Andrea

2009-07-30

Using pharmacological magnetic resonance imaging, we have performed an in vivo evaluation of the secretory response induced by essential oils in the rat airway. Aim of the work was to establish a computerized method to assess the efficacy of volatile compounds in spatially localized areas without the bias derived by subjective evaluation. Magnetic resonance experiments were carried out using a 4.7 T horizontal magnet. In the trachea, airway surface fluid was easily identified for its high intensity signal. The tracheal glands were also easily visible. The oesophageal lumen was usually collapsed and was identifiable only in the presence of intraluminal liquid. Scotch pine essential oil inhalation significantly increased the surface fluid in the middle portion of the trachea and the increase was visible at both 5 and 10 min. A lesser secretory response was detected after rosemary essential oil inhalation even though the response was significant with respect to the control in particular at 10 min. No secretory response was detected after peppermint essential oil inhalation both at 5 and 10 min. The data obtained in the present work demonstrate a chemically induced airway secretion. The availability of a pharmacological magnetic resonance imaging approach opens new perspectives to test the action of volatile compounds on the airway.

Conductance changes associated with the secretory potential in the cockroach salivary gland.

PubMed

Ginsborg, B L; House, C R; Silinsky, E M

1974-02-01

1. Conductance changes in the acini of the cockroach salivary gland have been examined during nerve stimulation by means of two intracellular electrodes placed in the same acinus, the first electrode being used for recording membrane potential and the second for current injection.2. The transient hyperpolarization (secretory potential) in the acinus evoked by nerve stimuli is accompanied by a rise in membrane conductance. The conductance, however, remains high for a longer period than that of the response.3. Applying the analysis of Trautwein & Dudel (1958) to the secretory potentials recorded in the acinus (assumed to behave electrically like a single cell) gives estimates of the ;transmitter equilibrium potential'. The values indicate that the neurotransmitter increases the membrane potassium conductance.4. The hyperpolarization of the acinus evoked by 10(-6)M dopamine in the bathing fluid is also associated with an increase in membrane potassium conductance.

Evaluation of whole mount in situ hybridization as a tool for pathway-based toxicological research in early-life stage fathead minnows (poster)

EPA Science Inventory

Early-life stage fish can be more sensitive to chemical exposure than mature, adult fish. Therefore, defining adverse outcome pathways (AOPs) relevant to early-life stages is critical for linking perturbations of key events during fish development to potential adverse outcomes of...

N-Terminal Acetylation Inhibits Protein Targeting to the Endoplasmic Reticulum

PubMed Central

Forte, Gabriella M. A.; Pool, Martin R.; Stirling, Colin J.

2011-01-01

Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%–80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species. PMID:21655302

In Candida albicans hyphae, Sec2p is physically associated with SEC2 mRNA on secretory vesicles.

PubMed

Caballero-Lima, David; Hautbergue, Guillaume M; Wilson, Stuart A; Sudbery, Peter E

2014-11-01

Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans-Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Shared elements of host-targeting pathways among apicomplexan parasites of differing lifestyles.

PubMed

Pellé, Karell G; Jiang, Rays H Y; Mantel, Pierre-Yves; Xiao, Yu-Ping; Hjelmqvist, Daisy; Gallego-Lopez, Gina M; O T Lau, Audrey; Kang, Byung-Ho; Allred, David R; Marti, Matthias

2015-11-01

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM. © 2015 John Wiley & Sons Ltd.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

PubMed

Valkova, Christina; Albrizio, Marina; Röder, Ira V; Schwake, Michael; Betto, Romeo; Rudolf, Rüdiger; Kaether, Christoph

2011-01-11

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

MyRIP interaction with MyoVa on secretory granules is controlled by the cAMP-PKA pathway.

PubMed

Brozzi, Flora; Lajus, Sophie; Diraison, Frederique; Rajatileka, Shavanthi; Hayward, Katy; Regazzi, Romano; Molnár, Elek; Váradi, Anikó

2012-11-01

Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)-anchoring family, is implicated in hormone secretion. However, its mechanism of action is not fully elucidated. Here we investigate the role of MyRIP in myosin Va (MyoVa)-dependent secretory granule (SG) transport and secretion in pancreatic beta cells. These cells solely express the brain isoform of MyoVa (BR-MyoVa), which is a key motor protein in SG transport. In vitro pull-down, coimmunoprecipitation, and colocalization studies revealed that MyRIP does not interact with BR-MyoVa in glucose-stimulated pancreatic beta cells, suggesting that, contrary to previous notions, MyRIP does not link this motor protein to SGs. Glucose-stimulated insulin secretion is augmented by incretin hormones, which increase cAMP levels and leads to MyRIP phosphorylation, its interaction with BR-MyoVa, and phosphorylation of the BR-MyoVa receptor rabphilin-3A (Rph-3A). Rph-3A phosphorylation on Ser-234 was inhibited by small interfering RNA knockdown of MyRIP, which also reduced cAMP-mediated hormone secretion. Demonstrating the importance of this phosphorylation, nonphosphorylatable and phosphomimic Rph-3A mutants significantly altered hormone release when PKA was activated. These data suggest that MyRIP only forms a functional protein complex with BR-MyoVa on SGs when cAMP is elevated and under this condition facilitates phosphorylation of SG-associated proteins, which in turn can enhance secretion.

Early deprivation disruption of associative learning is a developmental pathway to depression and social problems.

PubMed

Sheridan, Margaret A; McLaughlin, Katie A; Winter, Warren; Fox, Nathan; Zeanah, Charles; Nelson, Charles A

2018-06-07

Exposure to psychosocial deprivation is associated with elevations in numerous forms of impairment throughout the life-course. Disruptions in associative learning may be a key mechanism through which adversity, particularly psychosocial deprivation, increases risk for impairment. Existing data consistent with this claim come entirely from correlational studies. Here, we present the first experimental evidence relating psychosocial deprivation and disruptions in multiple forms of associative learning. Using data from the Bucharest Early Intervention Project, we demonstrate that randomized placement into a family caregiving environment during the infant/toddler period as compared to prolonged institutional care normalizes two forms of associative learning in early adolescence: reward responsivity and implicit motor learning. These forms of associative learning significantly mediate the effect of institutional rearing on depressive symptoms and peer relationships. In sum, we provide evidence for a novel pathway linking early experience to psychopathology and peer relationships through basic associative learning mechanisms.

Non Secretory Multiple Myeloma With Extensive Extramedullary Plasmacytoma: A Diagnostic Dilemma

PubMed Central

Low, Soo Fin; Mohd Tap, Nor Hanani; Kew, Thean Yean; Ngiu, Chai Soon; Sridharan, Radhika

2015-01-01

Multiple myeloma (MM) is characterized by progressive proliferation of malignant plasma cells, usually initiating in the bone marrow. MM can affect any organ; a total of 7 - 18% of patients with MM demonstrate extramedullary involvement at diagnosis. Non-secretory multiple myeloma (NSMM) is a rare variant that accounts for 1 - 5% of all cases of multiple myeloma. The disease is characterized by the absence of monoclonal gammopathy in serum and urine electrophoresis. Our case report highlights the diagnostic challenge of a case of NSMM with extensive extramedullary involvement in a young female patient who initially presented with right shoulder pain and bilateral breasts lumps. Skeletal survey showed multiple lytic bony lesions. The initial diagnosis was primary breast carcinoma with osseous metastases. No monoclonal gammopathy was found in the serum or urine electrophoresis. Bone marrow and breast biopsies revealed marked plasmacytosis. The diagnosis was delayed for a month in view of the lack of clinical suspicion of multiple myeloma in a young patient and scant biochemical expression of non-secretory type of multiple myeloma. PMID:26528383

Secretory expression of Lentinula edodes intracellular laccase by yeast high-cell-density system: sub-milligram production of difficult-to-express secretory protein.

PubMed

Kurose, Takeshi; Saito, Yuta; Kimata, Koichi; Nakagawa, Yuko; Yano, Akira; Ito, Keisuke; Kawarasaki, Yasuaki

2014-06-01

While a number of heterologous expression systems have been reported for extracellular laccases, there are few for the intracellular counterparts. The Lentinula edodes intracellular laccase Lcc4 is an industrially potential enzyme with its unique substrate specificity. The heterologous production of the intracellular laccase, however, had been difficult because of its expression-dependent toxicity. We previously demonstrated that recombinant yeast cells synthesized and, interestingly, secreted Lcc4 only when they were suspended to an inducing medium in a high cell-density (J. Biosci. Bioeng., 113, 154-159, 2012). The high cell-density system was versatile and applicable to other difficult-to-express secretory proteins. Nevertheless, the system's great dependence on aeration, which was a practical obstacle to scale-up production of the enzyme and some other proteins, left the secretion pathway and enzymatic properties of the Lcc4 uncharacterized. In this report, we demonstrate a successful production of Lcc4 by applying a jar-fermentor to the high cell-density system. The elevated yield (0.6 mg L(-1)) due to the sufficient aeration allowed us to prepare and purify the enzyme to homogeneity. The enzyme had been secreted as a hyper-glycosylated protein, resulting in smear band-formations in SDS-PAGE. The amino acid sequencing analysis suggested that the N-terminal 17 residues had been recognized as a secretion signal. The recombinant enzyme showed similar enzymatic properties to the naturally occurring Lcc4. The characteristics of the scale-upped expression system, which includes helpful information for the potential users, have also been described. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Differentiation of osmotic and secretory diarrhoea by stool carbohydrate and osmolar gap measurements

PubMed Central

Castro-Rodriguez, J. A.; Salazar-Lindo, E.; Leon-Barua, R.

1997-01-01



 Clinical features and laboratory tests that determine carbohydrate in faeces were evaluated to determine which was best able to distinguish between osmotic and secretory diarrhoea in infants and children. For this purpose 80 boys aged 3 to 24 months, with acute watery diarrhoea, were studied prospectively. The faecal osmolar gap (FOG) was calculated as: serum osmolarity − [2 × (faecal sodium + potassium concentration)]. Fifty eight patients were classified as having predominantly osmotic diarrhoea (FOG >100 mosmol/l), and 22 as having predominantly secretory diarrhoea (FOG ⩽100 mosmol/l). The two groups were comparable in their clinical features on admission, in the results of blood and urine tests, and in the evolution of their diarrhoeal illness. Evidence of steatorrhoea (by positive Sudan III test) and of acid faecal pH on admission were significantly more frequent in patients with osmotic diarrhoea. Mean (SD) faecal osmolarity was not significantly different between the two groups (319 (80) mosmol/l in secretory diarrhoea v 361 (123) mosmol/l in osmotic diarrhoea). Tests for reducing substances in faeces such as Benedict's test—with and without hydrolysis—and glucose strip, all showed a positive and significant association with osmotic diarrhoea (p <0.05, <0.025, <0.05, respectively). The presence of excess reducing substances (Benedict's test with hydrolysis >++) on admission was the most sensitive and specific test with the best predictive value for differentiating between the two types of watery diarrhoea.

 PMID:9370895

Decreased PECAM1-mediated TGF-β1 expression in the mid-secretory endometrium in women with recurrent implantation failure.

PubMed

Guo, Feng; Si, Chenchen; Zhou, Mingjuan; Wang, Jingwen; Zhang, Dan; Leung, Peter C K; Xu, Bufang; Zhang, Aijun

2018-05-01

Is recurrent implantation failure (RIF) associated with decreased expression of platelet and endothelial cell adhesion molecule 1 (PECAM1) and transforming growth factor β1 (TGF-β1) in the endometrium during the implantation window? The present study demonstrates that the expression of PECAM1 and TGF-β1 is significantly decreased in the mid-secretory endometrium in women with RIF, which may account for embryo implantation failure. RIF has become a bottleneck issue that hampers the improvement of pregnancy rates in IVF-embryo transfer (IVF-ET). The causes of RIF are complex and may involve the dysregulation of various growth factors, metabolites, and inflammatory cytokines. At present, the precise pathogenesis of RIF has not been elucidated. This was a prospective case-control study. Endometrial tissue samples were obtained from January 2014 to December 2016 from two groups of women who had undergone IVF (RIF group, 22 women who underwent ≥3 ETs including a total of ≥4 good-quality embryos without pregnancy, control group, 18 women who conceived in their first treatment cycle). At the same time, samples were obtained from 18 women with infertility secondary to tubal factor in the early proliferative, late proliferative and mid-secretory phases of the menstrual cycle (n = 6 per group). Samples used for isolation of primary human endometrial epithelial cells and stromal cells (HEECs and HESCs) were collected in December 2017 from six women with infertility secondary to tubal factor. We investigated gene expression using integrative whole genome expression microarray analysis, including differentially expressed gene screening, principal component analysis, and functional enrichment analysis. RT-qPCR, western blotting, immunohistochemistry, immunofluorescence co-localization analysis and short hairpin RNA (shRNA) plasmid transfection in Ishikawa cell line, HEECs and HESCs were used to investigate the expression of PECAM1 and TGF-β1. Integrative data mining of

The Prohormone VGF Regulates β Cell Function via Insulin Secretory Granule Biogenesis.

PubMed

Stephens, Samuel B; Edwards, Robert J; Sadahiro, Masato; Lin, Wei-Jye; Jiang, Cheng; Salton, Stephen R; Newgard, Christopher B

2017-09-05

The prohormone VGF is expressed in neuroendocrine and endocrine tissues and regulates nutrient and energy status both centrally and peripherally. We and others have shown that VGF-derived peptides have direct action on the islet β cell as secretagogues and cytoprotective agents; however, the endogenous function of VGF in the β cell has not been described. Here, we demonstrate that VGF regulates secretory granule formation. VGF loss-of-function studies in both isolated islets and conditional knockout mice reveal a profound decrease in stimulus-coupled insulin secretion. Moreover, VGF is necessary to facilitate efficient exit of granule cargo from the trans-Golgi network and proinsulin processing. It also functions to replenish insulin granule stores following nutrient stimulation. Our data support a model in which VGF operates at a critical node of granule biogenesis in the islet β cell to coordinate insulin biosynthesis with β cell secretory capacity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Transcriptome Profiles of the Protoscoleces of Echinococcus granulosus Reveal that Excretory-Secretory Products Are Essential to Metabolic Adaptation

PubMed Central

Pan, Wei; Shen, Yujuan; Han, Xiuming; Wang, Ying; Liu, Hua; Jiang, Yanyan; Zhang, Yumei; Wang, Yanjuan; Xu, Yuxin; Cao, Jianping

2014-01-01

Background Cystic hydatid disease (CHD) is caused by the larval stages of the cestode and affects humans and domestic animals worldwide. Protoscoleces (PSCs) are one component of the larval stages that can interact with both definitive and intermediate hosts. Previous genomic and transcriptomic data have provided an overall snapshot of the genomics of the growth and development of this parasite. However, our understanding of how PSCs subvert the immune response of hosts and maintains metabolic adaptation remains unclear. In this study, we used Roche 454 sequencing technology and in silico secretome analysis to explore the transcriptome profiles of the PSCs from E. granulosus and elucidate the potential functions of the excretory-secretory proteins (ESPs) released by the parasite. Methodology/Principal Findings A large number of nonredundant sequences as unigenes were generated (26,514), of which 22,910 (86.4%) were mapped to the newly published E. granulosus genome and 17,705 (66.8%) were distributed within the coding sequence (CDS) regions. Of the 2,280 ESPs predicted from the transcriptome, 138 ESPs were inferred to be involved in the metabolism of carbohydrates, while 124 ESPs were inferred to be involved in the metabolism of protein. Eleven ESPs were identified as intracellular enzymes that regulate glycolysis/gluconeogenesis (GL/GN) pathways, while a further 44 antigenic proteins, 25 molecular chaperones and four proteases were highly represented. Many proteins were also found to be significantly enriched in development-related signaling pathways, such as the TGF-β receptor pathways and insulin pathways. Conclusions/Significance This study provides valuable information on the metabolic adaptation of parasites to their hosts that can be used to aid the development of novel intervention targets for hydatid treatment and control. PMID:25500817

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane[OPEN

PubMed Central

Karnik, Rucha; Zhang, Ben; Waghmare, Sakharam; Aderhold, Christin; Grefen, Christopher; Blatt, Michael R.

2015-01-01

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners. PMID:25747882

Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

PubMed

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

Correlative Scanning-Transmission Electron Microscopy Reveals that a Chimeric Flavivirus Is Released as Individual Particles in Secretory Vesicles

PubMed Central

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations. PMID:24681578

Effects of ozone on the cholinergic secretory responsiveness of ferret tracheal glands

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

McBride, R.K.; Oberdoerster, G.; Marin, M.G.

1991-06-01

Oxidant air pollutants exacerbate several pulmonary diseases. Inhalation of ozone has been shown to induce airway smooth muscle hyperresponsiveness. Oxidant injury could also affect airway secretory mechanisms. The authors postulated that oxidant exposure would alter the glycoconjugate secretory function of airway submucosal glands. To test this hypothesis they examined the effects of in vivo ozone exposure on the in vitro secretory responsiveness of ferret tracheal glands. Ferrets were exposed to 1 ppm ozone, 24 hr/day for 3 or 7 days. Following exposure, glandular explants, denuded of surface epithelial cells, were prepared and incubated in medium containing 3H-glucosamine for 18 hr.more » Basal secretion of labeled glycoconjugates was significantly increased 31% following 3 days of ozone exposure (P less than or equal to 0.05) and remained elevated 11% after 7 days of exposure compared to the air-exposed group. After 3 or 7 days of exposure to ozone, tracheal gland responsiveness to carbachol was increased as indicated by significantly lower EC50 values (log molar concentration) of -6.43 {plus minus} 0.04 (n = 6) and -6.50 {plus minus} 0.11 (n = 5), respectively; compared to -6.20 {plus minus} 0.08 (n = 6) for the air-exposed group. There was no difference in carbachol EC50 values for air and 7-day ozone-exposed animals treated with dexamethasone. Dexamethasone did not attenuate the ozone-induced increase in basal secretion. Tracheal gland responsiveness to {alpha}- or {beta}-adrenergic agonists was not changed by oxidant exposure. These experiments suggest that oxidant injury not only increases basal secretion of respiratory glycoconjugates but also increases tracheal gland sensitivity to a cholinergic agonist.« less

Foamy Monocytes Are Enriched in cis-7-Hexadecenoic Fatty Acid (16:1n-9), a Possible Biomarker for Early Detection of Cardiovascular Disease.

PubMed

Guijas, Carlos; Meana, Clara; Astudillo, Alma M; Balboa, María A; Balsinde, Jesús

2016-06-23

Human monocytes respond to arachidonic acid, a secretory product of endothelial cells, by activating the de novo pathway of fatty acid biosynthesis, resulting in the acquisition of a foamy phenotype due to accumulation of cytoplasmic lipid droplets. Recruitment of foamy monocytes to endothelium is a key step in the formation of atherosclerotic plaques. Here we describe that lipid droplets of foamy monocytes are enriched in a rather uncommon fatty acid, cis-7-hexadecenoic acid (16:1n-9), a positional isomer of palmitoleic acid. 16:1n-9 was found to possess an anti-inflammatory activity both in vitro and in vivo that is comparable with that of omega-3 fatty acids and clearly distinguishable from the effects of palmitoleic acid. Selective accumulation in neutral lipids of phagocytic cells of an uncommon fatty acid reveals an early phenotypic change that may provide a biomarker of proatherogenicity, and a potential target for intervention in the early stages of cardiovascular disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway

PubMed Central

Lim, Sanghee; Kwak, Minhye; Gray, Christy D.; Xu, Michael; Choi, Jun H.; Junn, Sue; Kim, Jieun; Xu, Jing; Schaefer, Michele; Johns, Roger A.; Song, Hongjun; Ming, Guo-Li; Mintz, C. David

2017-01-01

Clinical and preclinical studies indicate that early postnatal exposure to anesthetics can lead to lasting deficits in learning and other cognitive processes. The mechanism underlying this phenomenon has not been clarified and there is no treatment currently available. Recent evidence suggests that anesthetics might cause persistent deficits in cognitive function by disrupting key events in brain development. The hippocampus, a brain region that is critical for learning and memory, contains a large number of neurons that develop in the early postnatal period, which are thus vulnerable to perturbation by anesthetic exposure. Using an in vivo mouse model we demonstrate abnormal development of dendrite arbors and dendritic spines in newly generated dentate gyrus granule cell neurons of the hippocampus after a clinically relevant isoflurane anesthesia exposure conducted at an early postnatal age. Furthermore, we find that isoflurane causes a sustained increase in activity in the mechanistic target of rapamycin pathway, and that inhibition of this pathway with rapamycin not only reverses the observed changes in neuronal development, but also substantially improves performance on behavioral tasks of spatial learning and memory that are impaired by isoflurane exposure. We conclude that isoflurane disrupts the development of hippocampal neurons generated in the early postnatal period by activating a well-defined neurodevelopmental disease pathway and that this phenotype can be reversed by pharmacologic inhibition. PMID:28683067

Developmental Pathways from Childhood Aggression-Disruptiveness, Chronic Peer Rejection and Deviant Friendships to Early-Adolescent Rule Breaking

PubMed Central

Ettekal, Idean; Ladd, Gary W.

2015-01-01

Childhood aggression-disruptiveness, chronic peer rejection, and deviant friendships were examined as predictors of early-adolescent rule breaking behaviors. Using a sample of 383 children (193 girls and 190 boys) who were followed from ages 6 to 14, peer rejection trajectories were identified and incorporated into a series of alternative models to assess how chronic peer rejection and deviant friendships mediate the association between stable childhood aggression-disruptiveness and early-adolescent rule breaking. There were multiple mediated pathways to rule breaking that included both behavioral and relational risk factors and findings were consistent for boys and girls. Results have implications for better understanding the influence of multiple social processes in the continuity of antisocial behaviors from middle childhood to early adolescence. PMID:25403544

Human Plasma-derived Polymeric IgA and IgM Antibodies Associate with Secretory Component to Yield Biologically Active Secretory-like Antibodies*

PubMed Central

Longet, Stéphanie; Miled, Sarah; Lötscher, Marius; Miescher, Sylvia M.; Zuercher, Adrian W.; Corthésy, Blaise

2013-01-01

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins. PMID:23250751

A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation1[OPEN

PubMed Central

Wang, Hao; Zhuang, Xiaohong; Wang, Xiangfeng; Law, Angus Ho Yin; Zhao, Teng; Du, Shengwang; Loy, Michael M.T.; Jiang, Liwen

2016-01-01

Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGN-independent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles (GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1 follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGN-PM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1 for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent. PMID:27531442

An early colonisation pathway into northwest Australia 70-60,000 years ago

NASA Astrophysics Data System (ADS)

Norman, Kasih; Inglis, Josha; Clarkson, Chris; Faith, J. Tyler; Shulmeister, James; Harris, Daniel

2018-01-01

Colonisation of Sahul 70-60 thousand years ago (kya) represents the first great maritime migration undertaken by anatomically modern humans in one of the final phases of the Out of Africa dispersal. Visual connectivity network analyses, agent-based simulations and ocean current modelling reveal that modern humans could follow numerous northern and southern migration pathways into Sahul. Our results support a southern route out of Africa through South Asia with entry into ISEA through the Banda Arc, culminating in an early colonisation of Sahul on the northwest shelf. Our results show multiple colonisation events through other entry points were also probable, and raise interesting possibilities for complex regional migration and population histories.

G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua.

PubMed

Kolkova, Z; Noskova, V; Ehinger, A; Hansson, S; Casslén, B

2010-10-01

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERβ mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

Inhibition of untransformed prostaglandin H(2) production and stretch-induced contraction of rabbit pulmonary arteries by indoxam, a selective secretory phospholipase A(2) inhibitor.

PubMed

Tanabe, Yoshiyuki; Saito, Maki; Morikawa, Yuki; Kamataki, Akihisa; Sawai, Takashi; Hirose, Masamichi; Nakayama, Koichi

2011-01-01

Involvement of secretory phospholipase A(2) (sPLA(2)) in the stretch-induced production of untransformed prostaglandin H(2) (PGH(2)) in the endothelium of rabbit pulmonary arteries was investigated. The stretch-induced contraction was significantly inhibited by indoxam, a selective inhibitor for sPLA(2), and NS-398, a selective inhibitor for cyclooxygenase-2 (COX-2). Indoxam inhibited the RGD-sensitive-integrin-independent production of untransformed PGH(2), but did not affect the RGD-sensitive-integrin-dependent production of thromboxane A(2) (TXA(2)). These results suggest that the stretch-induced contraction and untransformed PGH(2) production was mediated by sPLA(2)-COX-2 pathway, making it a new possible target for pharmacological intervention of pulmonary artery contractility.

Glucose and Insulin Secretory Response Patterns Following Diet and Tolazamide Therapy in Diabetes

PubMed Central

Turtle, J. R.

1970-01-01

Glucose and insulin secretory response patterns during glucose tolerance tests were determined in 28 maturity-onset diabetics, and the sequential effects of diet and a sulphonylurea, tolazamide, were assessed. Untreated diabetics showed hyperglycaemia, increased serum immunoreactive insulin response patterns, delayed insulin release, and relative insulin deficiency. Diet alone partially corrected the hyperglycaemia and serum immunoreactive insulin response but had no effect on the delayed insulin release or relative insulin deficiency. Tolazamide plus diet restored all values towards normal. The net effect of maintenance tolazamide therapy was to (1) restore the insulin secretory response pattern to normal, (2) reduce total pancreatic insulin output, and (3) improve the efficiency of insulin secretion. The results suggest that there is a rational basis for the use of sulphonylurea in all maturity-onset diabetics, including patients with mild carbohydrate intolerance and those who are apparently controlled by diet alone. PMID:5470087

De novo epidermal regeneration using human eccrine sweat gland cells: higher competence of secretory over absorptive cells.

PubMed

Pontiggia, Luca; Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Oliveira, Carol; Braziulis, Erik; Klar, Agnieszka S; Meuli-Simmen, Claudia; Meuli, Martin; Reichmann, Ernst

2014-06-01

In our previous work, we showed that human sweat gland-derived epithelial cells represent an alternative source of keratinocytes to grow a near normal autologous epidermis. The role of subtypes of sweat gland cells in epidermal regeneration and maintenance remained unclear. In this study, we compare the regenerative potential of both secretory and absorptive sweat gland cell subpopulations. We demonstrate the superiority of secretory over absorptive cells in forming a new epidermis on two levels: first, the proliferative and colony-forming efficiencies in vitro are significantly higher for secretory cells (SCs), and second, SCs show a higher frequency of successful epidermis formation as well as an increase in the thickness of the formed epidermis in the in vitro and in vivo functional analyses using a 3D dermo-epidermal skin model. However, the ability of forming functional skin substitutes is not limited to SCs, which supports the hypothesis that multiple subtypes of sweat gland epithelial cells hold regenerative properties, while the existence and exact localization of a keratinocyte stem cell population in the human eccrine sweat gland remain elusive.

Secretory cavities and volatiles of Myrrhinium atropurpureum Schott var. atropurpureum (Myrtaceae): an endemic species collected in the restingas of Rio de Janeiro, Brazil.

PubMed

Victório, Cristiane Pimentel; Moreira, Claudio B; Souza, Marcelo da Costa; Sato, Alice; Arruda, Rosani do Carmo de Oliveira

2011-07-01

In this study, we investigated the leaf anatomy and the composition of volatiles in Myrrhinium atropurpureum var. atropurpureum endemic to Rio de Janeiro restingas. Particularly, leaf secretory structures were described using light microscopy, and histochemical tests were performed from fresh leaves to localize the secondary metabolites. To observe secretory cavities, fixed leaf samples were free-hand sectioned. To evaluate lipophilic compounds and terpenoids the following reagents were employed: Sudans III and IV, Red oil O and Nile blue. Leaf volatiles were characterized by gas chromatography after hydrodistillation (HD) or simultaneous distillation-extraction (SDE). Leaf analysis showed several cavities in mesophyll that are the main sites of lipophilic and terpenoid production. Monoterpenes, which represented more than 80% of the major volatiles, were characterized mainly by alpha- and beta-pinene and 1,8-cineole. In order to provide tools for M. atropurpureum identification, the following distinguishing characteristics were revealed by the following data: 1) adaxial face clear and densely punctuated by the presence of round or ellipsoidal secretory cavities randomly distributed in the mesophyll; 2) the presence of cells overlying the upper neck cells of secretory cavities; 3) the presence of numerous paracytic stomata distributed on the abaxial leaf surface, but absent in vein regions and leaf margin; and 4) non-glandular trichomes on both leaf surfaces. Our study of the compounds produced by the secretory cavities of M. atropurpureum led us to conclude that volatile terpenoid class are the main secretory compounds and that they consist of a high concentration of monoterpenes, which may indicate the phytotherapeutic importance of this plant.

Conformational study of the proline rich peptide from bovine neurohypophysis secretory granules

NASA Astrophysics Data System (ADS)

Alieva, Irada; Velieva, Lala; Aliev, Dshavanchir; Gojayev, Niftali; Demukhamedova, Svetlana

2004-01-01

The spatial organization and conformational properties of the Proline Rich Peptide (PRP) from bovine neurohypophysis secretory granules have been established by the methods of molecular mechanics and molecular dynamics simulations in water solution. Conformational studies showed the peptide with limited conformational flexibility. Two β-type III turns are observed in PRP spatial organization.

Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest.

PubMed

Diederen, J H; Vullings, H G

1995-03-01

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

Leptin secretory dynamics and associated disordered eating psychopathology across the weight spectrum

PubMed Central

Baskaran, Charumathi; Eddy, Kamryn T.; Miller, Karen K.; Meenaghan, Erinne; Misra, Madhusmita; Lawson, Elizabeth A.

2016-01-01

Leptin secretory dynamics across the weight spectrum and their relationship with disordered eating psychopathology have not been studied. Our objective was to compare leptin secretory dynamics in 13 anorexia nervosa (AN), 12 overweight/obese (OB) and 12 normal-weight women using deconvolution analysis. Methods In this cross-sectional study conducted at a tertiary referral center, serum leptin levels were obtained every 20 minutes from 2000-0800h. Dual energy X-ray absorptiometry was used to measure %body fat. Disordered eating psychopathology was assessed by the Eating Disorders Examination-Questionnaire (EDE-Q) and Eating Disorders Inventory-2 (EDI-2). Results The groups differed for basal leptin secretion (BASAL) (p=0.02). Mean leptin pulse amplitude, pulse mass, total pulsatile secretion (TPS) and area under the curve (AUC) were significantly different between groups before and after adjustment for BASAL (p<0.0001 for all). Leptin AUC correlated strongly with TPS (r=0.97, p<0.0001) and less with BASAL (r=0.35, p=0.03). On multivariate analysis, only TPS was a significant predictor of leptin AUC (p<0.0001). TPS was inversely associated with most EDE-Q and EDI-2 parameters and the associations remained significant for EDE-Q eating concern (p=0.01), and EDI-2 asceticism, ineffectiveness and social insecurity (p<0.05) after adjusting for BASAL. These relationships were not significant when controlled for %body fat. Conclusion Secretory dynamics of leptin differ across weight spectrum, with mean pulse amplitude, mean pulse mass and TPS being low in AN and high in OB. Pulsatile, rather than basal secretion, is the major contributor to leptin AUC. Decreased pulsatile leptin is associated with disordered eating psychopathology, possibly reflecting low %body fat in AN. PMID:26903591

Secretory immunoglobulin purification from whey by chromatographic techniques.

PubMed

Matlschweiger, Alexander; Engelmaier, Hannah; Himmler, Gottfried; Hahn, Rainer

2017-08-15

Secretory immunoglobulins (SIg) are a major fraction of the mucosal immune system and represent potential drug candidates. So far, platform technologies for their purification do not exist. SIg from animal whey was used as a model to develop a simple, efficient and potentially generic chromatographic purification process. Several chromatographic stationary phases were tested. A combination of two anion-exchange steps resulted in the highest purity. The key step was the use of a small-porous anion exchanger operated in flow-through mode. Diffusion of SIg into the resin particles was significantly hindered, while the main impurities, IgG and serum albumin, were bound. In this step, initial purity was increased from 66% to 89% with a step yield of 88%. In a second anion-exchange step using giga-porous material, SIg was captured and purified by step or linear gradient elution to obtain fractions with purities >95%. For the step gradient elution step yield of highly pure SIg was 54%. Elution of SIgA and SIgM with a linear gradient resulted in a step yield of 56% and 35%, respectively. Overall yields for both anion exchange steps were 43% for the combination of flow-through and step elution mode. Combination of flow-through and linear gradient elution mode resulted in a yield of 44% for SIgA and 39% for SIgM. The proposed process allows the purification of biologically active SIg from animal whey in preparative scale. For future applications, the process can easily be adopted for purification of recombinant secretory immunoglobulin species. Copyright © 2017 Elsevier B.V. All rights reserved.

Group IIA secretory phospholipase A2 (GIIA) mediates apoptotic death during NMDA receptor activation in rat primary cortical neurons.

PubMed

Chiricozzi, Elena; Fernandez-Fernandez, Seila; Nardicchi, Vincenza; Almeida, Angeles; Bolaños, Juan Pedro; Goracci, Gianfrancesco

2010-03-01

Phospholipases A(2) (PLA(2)) participate in neuronal death signalling pathways because of their ability to release lipid mediators, although the contribution of each isoform and mechanism of neurotoxicity are still elusive. Using a novel fluorogenic method to assess changes in a PLA(2) activity by flow cytometry, here we show that the group IIA secretory phospholipase A(2) isoform (GIIA) was specifically activated in cortical neurons following stimulation of N-methyl-d-aspartate glutamate receptor subtype (NMDAR). For activation, GIIA required Ca(2+) and reactive oxygen/nitrogen species, and inhibition of its activity fully prevented NMDAR-mediated neuronal apoptotic death. Superoxide, nitric oxide or peroxynitrite donors stimulated GIIA activity, which mediated neuronal death. Intriguingly, we also found that GIIA activity induced mitochondrial superoxide production after NMDAR stimulation. These results reveal a novel role for GIIA in excitotoxicity both as target and producer of superoxide in a positive-loop of activation that may contribute to the propagation of neurodegeneration.

Pathways of Prion Spread during Early Chronic Wasting Disease in Deer.

PubMed

Hoover, Clare E; Davenport, Kristen A; Henderson, Davin M; Denkers, Nathaniel D; Mathiason, Candace K; Soto, Claudio; Zabel, Mark D; Hoover, Edward A

2017-05-15

accumulation and pathways of prion spread during early CWD infection remain unknown. To investigate the chronological events of early prion pathogenesis, we exposed deer to CWD prions and monitored the tissue distribution of PrP CWD over the first 4 months of infection. We show CWD uptake occurs in the oropharynx with initial prion replication in the draining oropharyngeal lymphoid tissues, rapidly followed by dissemination to systemic lymphoid tissues without evidence of neuroinvasion. These data highlight the two phases of CWD infection: a robust prion amplification in systemic lymphoid tissues prior to neuroinvasion and establishment of a carrier state. Copyright © 2017 American Society for Microbiology.

Pathways of Prion Spread during Early Chronic Wasting Disease in Deer

PubMed Central

Hoover, Clare E.; Davenport, Kristen A.; Henderson, Davin M.; Denkers, Nathaniel D.; Mathiason, Candace K.; Soto, Claudio; Zabel, Mark D.

2017-01-01

accumulation and pathways of prion spread during early CWD infection remain unknown. To investigate the chronological events of early prion pathogenesis, we exposed deer to CWD prions and monitored the tissue distribution of PrPCWD over the first 4 months of infection. We show CWD uptake occurs in the oropharynx with initial prion replication in the draining oropharyngeal lymphoid tissues, rapidly followed by dissemination to systemic lymphoid tissues without evidence of neuroinvasion. These data highlight the two phases of CWD infection: a robust prion amplification in systemic lymphoid tissues prior to neuroinvasion and establishment of a carrier state. PMID:28250130

A miR-335/COX-2/PTEN axis regulates the secretory phenotype of senescent cancer-associated fibroblasts

PubMed Central

Kabir, Tasnuva D.; Leigh, Ross J.; Tasena, Hataitip; Mellone, Massimiliano; Coletta, Ricardo D.; Parkinson, Eric K.; Prime, Stephen S.; Thomas, Gareth J.; Paterson, Ian C.; Zhou, Donghui; McCall, John; Speight, Paul M.; Lambert, Daniel W.

2016-01-01

Senescent cancer-associated fibroblasts (CAF) develop a senescence-associated secretory phenotype (SASP) that is believed to contribute to cancer progression. The mechanisms underlying SASP development are, however, poorly understood. Here we examined the functional role of microRNA in the development of the SASP in normal fibroblasts and CAF. We identified a microRNA, miR-335, up-regulated in the senescent normal fibroblasts and CAF and able to modulate the secretion of SASP factors and induce cancer cell motility in co-cultures, at least in part by suppressing the expression of phosphatase and tensin homologue (PTEN). Additionally, elevated levels of cyclo-oxygenase 2 (PTGS2; COX-2) and prostaglandin E2 (PGE2) secretion were observed in senescent fibroblasts, and inhibition of COX-2 by celecoxib reduced the expression of miR-335, restored PTEN expression and decreased the pro-tumourigenic effects of the SASP. Collectively these data demonstrate the existence of a novel miRNA/PTEN-regulated pathway modulating the inflammasome in senescent fibroblasts. PMID:27385366

Resveratrol Interferes with an Early Step in the Fibrillization Pathway of Human Lysozyme and Modulates it towards Less-Toxic, Off-Pathway Aggregates.

PubMed

Kamal Zaidi, Fatima; Bhat, Rajiv

2018-01-18

The effect of resveratrol, a polyphenol in red wine, on the amyloid fibril formation of human lysozyme (HuL) was investigated, towards elucidating the mechanism of resveratrol action and probing its role as a possible modulator of lysozyme aggregation and toxicity. By using a number of biophysical tools, resveratrol was observed to alter the fibrillization kinetics of HuL and inhibit its fibrillization by binding with weak to moderate affinity to the conformations populated at the early stages of the pathway with concomitant stabilization of these initial conformations. The marginal decrease in the lifetime of HuL in the presence of resveratrol by time-resolved fluorescence measurements indicated the involvement of a static quenching mechanism in the interaction between HuL and resveratrol. Docking studies predicted the binding of resveratrol to aggregation-prone regions in HuL, and structure and activity analyses demonstrated the retention of much of the α-helical structure and activity of HuL in the presence of resveratrol. Resveratrol modulated the fibrillization pathway towards less-hydrophobic, less-toxic, off-pathway aggregates. These results demonstrate that binding of resveratrol to HuL could protect against the formation of pathogenic, cytotoxic aggregates formed in amyloidogenic disorders, such as systemic amyloidosis; thus suggesting its potential as a plausible therapeutic agent against lysozyme amyloidosis. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A pH-Regulated Quality Control Cycle for Surveillance of Secretory Protein Assembly

PubMed Central

Vavassori, Stefano; Cortini, Margherita; Masui, Shoji; Sannino, Sara; Anelli, Tiziana; Caserta, Imma R.; Fagioli, Claudio; Mossuto, Maria F.; Fornili, Arianna; van Anken, Eelco; Degano, Massimo; Inaba, Kenji; Sitia, Roberto

2013-01-01

Summary To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44’s active cysteine, simultaneously unmask the substrate binding site and −RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles. PMID:23685074

Triple DMARD treatment in early rheumatoid arthritis modulates synovial T cell activation and plasmablast/plasma cell differentiation pathways

PubMed Central

Wechalekar, Mihir D.; Guo, Yanxia; Yin, Xuefeng; Weedon, Helen; Proudman, Susanna M.; Smith, Malcolm D.; Nagpal, Sunil

2017-01-01

Objectives This study sought to investigate the genome-wide transcriptional effects of a combination of disease modifying anti-rheumatic drugs (tDMARD; methotrexate, sulfasalazine and hydroxychloroquine) in synovial tissues obtained from early rheumatoid arthritis (RA) patients. While combination DMARD strategies have been investigated for clinical efficacy, very little data exists on the potential molecular mechanism of action. We hypothesized that tDMARD would impact multiple biological pathways, but the specific pathways were unknown. Methods Paired synovial biopsy samples from early RA patients before and after 6 months of tDMARD therapy were collected by arthroscopy (n = 19). These biopsies as well as those from subjects with normal synovium (n = 28) were profiled by total RNA sequencing. Results Large differences in gene expression between RA and control biopsies (over 5000 genes) were identified. Despite clinical efficacy, the expression of a restricted set of less than 300 genes was reversed after 6 months of treatment. Many genes remained elevated, even in patients who achieved low disease activity. Interestingly, tDMARD downregulated genes included those involved in T cell activation and signaling and plasmablast/plasma cell differentiation and function. Conclusions We have identified transcriptomic signatures that characterize synovial tissue from RA patients with early disease. Analysis after 6 months of tDMARD treatment highlight consistent alterations in expression of genes related to T cell activation and plasmablast/plasma cell differentiation. These results provide novel insight into the biology of early RA and the mechanism of tDMARD action and may help identify novel drug targets to improve rates of treatment-induced disease remission. PMID:28863153

Triple DMARD treatment in early rheumatoid arthritis modulates synovial T cell activation and plasmablast/plasma cell differentiation pathways.

PubMed

Walsh, Alice M; Wechalekar, Mihir D; Guo, Yanxia; Yin, Xuefeng; Weedon, Helen; Proudman, Susanna M; Smith, Malcolm D; Nagpal, Sunil

2017-01-01

This study sought to investigate the genome-wide transcriptional effects of a combination of disease modifying anti-rheumatic drugs (tDMARD; methotrexate, sulfasalazine and hydroxychloroquine) in synovial tissues obtained from early rheumatoid arthritis (RA) patients. While combination DMARD strategies have been investigated for clinical efficacy, very little data exists on the potential molecular mechanism of action. We hypothesized that tDMARD would impact multiple biological pathways, but the specific pathways were unknown. Paired synovial biopsy samples from early RA patients before and after 6 months of tDMARD therapy were collected by arthroscopy (n = 19). These biopsies as well as those from subjects with normal synovium (n = 28) were profiled by total RNA sequencing. Large differences in gene expression between RA and control biopsies (over 5000 genes) were identified. Despite clinical efficacy, the expression of a restricted set of less than 300 genes was reversed after 6 months of treatment. Many genes remained elevated, even in patients who achieved low disease activity. Interestingly, tDMARD downregulated genes included those involved in T cell activation and signaling and plasmablast/plasma cell differentiation and function. We have identified transcriptomic signatures that characterize synovial tissue from RA patients with early disease. Analysis after 6 months of tDMARD treatment highlight consistent alterations in expression of genes related to T cell activation and plasmablast/plasma cell differentiation. These results provide novel insight into the biology of early RA and the mechanism of tDMARD action and may help identify novel drug targets to improve rates of treatment-induced disease remission.

Cortisol and Secretory Immunoglobulin A Response to Stress in German Shepherd Dogs

PubMed Central

Svobodová, Ivona; Chaloupková, Helena; Končel, Roman; Bartoš, Luděk; Hradecká, Lenka; Jebavý, Lukáš

2014-01-01

The aim of the study was to determine whether cortisol and secretory Immunoglobulin A (sIgA) could be used as an indicator of acute stress in both young and adult dogs. Seventeen German shepherd puppies were exposed to the Puppy test (challenge test) at the age of seven weeks. This test has been routinely used to assess the future working ability of potential police dogs. In addition, ten adult females were subjected to 4 minutes of defense training under stressful conditions. Saliva was collected from the puppies and adult females before testing and 20 minutes after the start of testing, using a cotton swab held for 1–2 minutes in each dog's mouth. Cortisol concentrations increased after the test compared to the control sample both in puppies and the adult females. However adult females showed a significant decrease in sIgA after defense training while puppies showed a tendency of increase in sIgA. We propose that salivary cortisol could be used as an indicator of stress in puppies during early ontogeny. It is not yet clear whether sIgA could be used as a useful indicator of short-term stress in dogs. PMID:24637917

Lysosomal Exoglycosidase Profile and Secretory Function in the Salivary Glands of Rats with Streptozotocin-Induced Diabetes

PubMed Central

Kossakowska, Agnieszka; Szulimowska, Julita; Klimiuk, Anna; Knaś, Małgorzata; Car, Halina; Niklińska, Wiesława; Ładny, Jerzy Robert; Chabowski, Adrian

2017-01-01

Before this study, there had been no research evaluating the relationship between a lysosomal exoglycosidase profile and secretory function in the salivary glands of rats with streptozotocin- (STZ-) induced type 1 diabetes. In our work, rats were divided into 4 groups of 8 animals each: control groups (C2, C4) and diabetic groups (STZ2, STZ4). The secretory function of salivary glands—nonstimulated and stimulated salivary flow, α-amylase, total protein—and salivary exoglycosidase activities—N-acetyl-β-hexosaminidase (HEX, HEX A, and HEX B), β-glucuronidase, α-fucosidase, β-galactosidase, and α-mannosidase—was estimated both in the parotid and submandibular glands of STZ-diabetic and control rats. The study has demonstrated that the activity of most salivary exoglycosidases is significantly higher in the parotid and submandibular glands of STZ-diabetic rats as compared to the healthy controls and that it increases as the disease progresses. Reduced secretory function of diabetic salivary glands was also observed. A significant inverse correlation between HEX B, α-amylase activity, and stimulated salivary flow in diabetic parotid gland has also been shown. Summarizing, STZ-induced diabetes leads to a change in the lysosomal exoglycosidase profile and reduced function of the salivary glands. PMID:29464184

Lysosomal Exoglycosidase Profile and Secretory Function in the Salivary Glands of Rats with Streptozotocin-Induced Diabetes.

PubMed

Maciejczyk, Mateusz; Kossakowska, Agnieszka; Szulimowska, Julita; Klimiuk, Anna; Knaś, Małgorzata; Car, Halina; Niklińska, Wiesława; Ładny, Jerzy Robert; Chabowski, Adrian; Zalewska, Anna

2017-01-01

Before this study, there had been no research evaluating the relationship between a lysosomal exoglycosidase profile and secretory function in the salivary glands of rats with streptozotocin- (STZ-) induced type 1 diabetes. In our work, rats were divided into 4 groups of 8 animals each: control groups (C2, C4) and diabetic groups (STZ2, STZ4). The secretory function of salivary glands-nonstimulated and stimulated salivary flow, α -amylase, total protein-and salivary exoglycosidase activities-N-acetyl- β -hexosaminidase (HEX, HEX A, and HEX B), β -glucuronidase, α -fucosidase, β -galactosidase, and α -mannosidase-was estimated both in the parotid and submandibular glands of STZ-diabetic and control rats. The study has demonstrated that the activity of most salivary exoglycosidases is significantly higher in the parotid and submandibular glands of STZ-diabetic rats as compared to the healthy controls and that it increases as the disease progresses. Reduced secretory function of diabetic salivary glands was also observed. A significant inverse correlation between HEX B, α -amylase activity, and stimulated salivary flow in diabetic parotid gland has also been shown. Summarizing, STZ-induced diabetes leads to a change in the lysosomal exoglycosidase profile and reduced function of the salivary glands.

Expression and secretory profile of buffalo fetal fibroblasts and Wharton's jelly feeder layers.

PubMed

Parmar, Mehtab S; Mishra, Smruti Ranjan; Somal, Anjali; Pandey, Sriti; Kumar, G Sai; Sarkar, Mihir; Chandra, Vikash; Sharma, G Taru

2017-05-01

The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Wharton's jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules viz. bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs. Copyright © 2017 Elsevier B.V. All rights reserved.

Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

PubMed

Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

2017-01-01

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Therapeutic potential of larval excretory/secretory proteins of the pig whipworm Trichuris suis in allergic disease.

PubMed

Ebner, F; Hepworth, M R; Rausch, S; Janek, K; Niewienda, A; Kühl, A; Henklein, P; Lucius, R; Hamelmann, E; Hartmann, S

2014-11-01

Gastrointestinal nematodes are currently being evaluated as a novel therapeutic in the treatment of chronic human inflammatory disorders, due to their unique ability to induce immunoregulatory pathways in their hosts. In particular, administration of ova from the pig whipworm Trichuris suis (T. suis; TSO) has been proposed for the treatment of allergic, inflammatory and autoimmune disorders. Despite these advances, the biological pathways through which TSO therapy modulates the host immune system in the context of human disease remain undefined. We characterized the dominant proteins present in the excretory/secretory (E/S) products of first-stage (L1) T. suis larvae (Ts E/S) using LC-MS/MS analysis and examined the immunosuppressive properties of whole larval Ts E/S in vitro and in a murine model of allergic airway disease. Administration of larval Ts E/S proteins in vivo during the allergen sensitization phase was sufficient to suppress airway hyperreactivity, bronchiolar inflammatory infiltrate and allergen-specific IgE production. Three proteins in larval Ts E/S were unambiguously identified. The immunomodulatory function of larval Ts E/S was found to be partially dependent on the immunoregulatory cytokine IL-10. Taken together, these data demonstrate that the released proteins of larval T. suis have significant immunomodulatory capacities and efficiently dampen allergic airway hyperreactivity. Thus, the therapeutic potential of defined larval E/S proteins should be exploited for the treatment of human allergic disorders. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The pathway to earthquake early warning in the US

NASA Astrophysics Data System (ADS)

Allen, R. M.; Given, D. D.; Heaton, T. H.; Vidale, J. E.; West Coast Earthquake Early Warning Development Team

2013-05-01

The development of earthquake early warning capabilities in the United States is now accelerating and expanding as the technical capability to provide warning is demonstrated and additional funding resources are making it possible to expand the current testing region to the entire west coast (California, Oregon and Washington). Over the course of the next two years we plan to build a prototype system that will provide a blueprint for a full public system in the US. California currently has a demonstrations warning system, ShakeAlert, that provides alerts to a group of test users from the public and private sector. These include biotech companies, technology companies, the entertainment industry, the transportation sector, and the emergency planning and response community. Most groups are currently in an evaluation mode, receiving the alerts and developing protocols for future response. The Bay Area Rapid Transit (BART) system is the one group who has now implemented an automated response to the warning system. BART now stops trains when an earthquake of sufficient size is detected. Research and development also continues to develop improved early warning algorithms to better predict the distribution of shaking in large earthquakes when the finiteness of the source becomes important. The algorithms under development include the use of both seismic and GPS instrumentation and integration with existing point source algorithms. At the same time, initial testing and development of algorithms in and for the Pacific Northwest is underway. In this presentation we will review the current status of the systems, highlight the new research developments, and lay out a pathway to a full public system for the US west coast. The research and development described is ongoing at Caltech, UC Berkeley, University of Washington, ETH Zurich, Southern California Earthquake Center, and the US Geological Survey, and is funded by the Gordon and Betty Moore Foundation and the US Geological

Mammalian TRAPPIII Complex positively modulates the recruitment of Sec13/31 onto COPII vesicles

PubMed Central

Zhao, Shan; Li, Chun Man; Luo, Xiao Min; Siu, Gavin Ka Yu; Gan, Wen Jia; Zhang, Lin; Wu, William K. K.; Chan, Hsiao Chang; Yu, Sidney

2017-01-01

The Transport protein particle (TRAPP) complex is a tethering factor for COPII vesicle. Of three forms of TRAPP (TRAPPI, II and III) complexes identified so far, TRAPPIII has been largely considered to play a role in autophagy. While depletion of TRAPPIII specific subunits caused defects in the early secretory pathway and TRAPPIII might interact with components of the COPII vesicle coat, its exact role remains to be determined. In this study, we studied the function of TRAPPIII in early secretory pathway using a TRAPPIII-specific subunit, TRAPPC12, as starting point. We found that TRAPPC12 was localized to the ER exit sites and ERGIC. In cells deleted with TRAPPC12, ERGIC and to a lesser extent, the Golgi became dispersed. ER-to-Golgi transport was also delayed. TRAPPC12, but not TRAPPC8, bound to Sec13/Sec31A tetramer but each Sec protein alone could not interact with TRAPPC12. TRAPPIII positively modulated the assembly of COPII outer layer during COPII vesicle formation. These results identified a novel function of TRAPPIII as a positive modulator of the outer layer of the COPII coat. PMID:28240221

Ionic and secretory response of pancreatic islet cells to minoxidil sulfate

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

Antoine, M.H.; Hermann, M.; Herchuelz, A.

Minoxidil sulfate is an antihypertensive agent belonging to the new class of vasodilators, the K+ channel openers. The present study was undertaken to characterize the effects of minoxidil sulfate on ionic and secretory events in rat pancreatic islets. The drug unexpectedly provoked a concentration-dependent decrease in 86Rb outflow. This inhibitory effect was reduced in a concentration-dependent manner by glucose and tolbutamide. Minoxidil sulfate did not affect 45Ca outflow from islets perfused in the presence of extracellular Ca++ and absence or presence of glucose. However, in islets exposed to a medium deprived of extracellular Ca++, the drug provoked a rise inmore » 45Ca outflow. Whether in the absence or presence of extracellular Ca++, minoxidil sulfate increased the cytosolic free Ca++ concentration of islet cells. Lastly, minoxidil sulfate increased the release of insulin from glucose-stimulated pancreatic islets. These results suggest that minoxidil sulfate reduces the activity of the ATP-sensitive K+ channels and promotes an intracellular translocation of Ca++. The latter change might account for the effect of the drug on the insulin-releasing process. However, the secretory response to minoxidil sulfate could also be mediated, at least in part, by a modest Ca++ entry.« less

Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells.

PubMed

Bahk, Young Yil; Pak, Jhang Ho

2016-10-01

Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.

Women's Work Pathways Across the Life Course.

PubMed

Damaske, Sarah; Frech, Adrianne

2016-04-01

Despite numerous changes in women's employment in the latter half of the twentieth century, women's employment continues to be uneven and stalled. Drawing from data on women's weekly work hours in the National Longitudinal Survey of Youth (NLSY79), we identify significant inequality in women's labor force experiences across adulthood. We find two pathways of stable full-time work for women, three pathways of part-time employment, and a pathway of unpaid labor. A majority of women follow one of the two full-time work pathways, while fewer than 10% follow a pathway of unpaid labor. Our findings provide evidence of the lasting influence of work-family conflict and early socioeconomic advantages and disadvantages on women's work pathways. Indeed, race, poverty, educational attainment, and early family characteristics significantly shaped women's work careers. Work-family opportunities and constraints also were related to women's work hours, as were a woman's gendered beliefs and expectations. We conclude that women's employment pathways are a product of both their resources and changing social environment as well as individual agency. Significantly, we point to social stratification, gender ideologies, and work-family constraints, all working in concert, as key explanations for how women are "tracked" onto work pathways from an early age.

Productive HIV-1 Infection of Human Cervical Tissue Ex Vivo is Associated with the Secretory Phase of the Menstrual Cycle

PubMed Central

Saba, Elisa; Origoni, Massimo; Taccagni, Gianluca; Ferrari, Davide; Doglioni, Claudio; Nava, Alice; Lisco, Andrea; Grivel, Jean-Charles; Margolis, Leonid; Poli, Guido

2013-01-01

Cervical tissue explants (CTE) from 22 HIV-1 seronegative women were exposed to R5 HIV-1 ex vivo. Eight CTE were productively infected in terms of HIV-1 p24Gag release in culture supernatants whereas 14 were not. Nonetheless, both accumulation of HIV-1gag DNA and of p24Gag+ CD4+ T cells and macrophages occurred in both productive and, at lower levels, in nonproductive CTE. Nonproductive CTE differed from productive CTE for higher secretion of CCL3 and CCL5. A post-hoc analysis revealed that all productive CTE were established from women in their secretory phase of the menstrual cycle, whereas nonproductive CTE derived from women either in their secretory (28%) or proliferative (36%) menstrual cycle phases or with an atrophic endometrium (36%). Thus, our results support the epidemiological observation that sexual HIV-1 transmission from males to women as well as from women to men is more efficient during their secretory phase of the menstrual cycle. PMID:23385427

Secretory Overexpression of Bacillus thermocatenulatus Lipase in Saccharomyces cerevisiae Using Combinatorial Library Strategy.

PubMed

Kajiwara, Shota; Yamada, Ryosuke; Ogino, Hiroyasu

2018-04-10

Simple and cost-effective lipase expression host microorganisms are highly desirable. A combinatorial library strategy is used to improve the secretory expression of lipase from Bacillus thermocatenulatus (BTL2) in the culture supernatant of Saccharomyces cerevisiae. A plasmid library including expression cassettes composed of sequences encoding one of each 15 promoters, 15 secretion signals, and 15 terminators derived from yeast species, S. cerevisiae, Pichia pastoris, and Hansenula polymorpha, is constructed. The S. cerevisiae transformant YPH499/D4, comprising H. polymorpha GAP promoter, S. cerevisiae SAG1 secretion signal, and P. pastoris AOX1 terminator, is selected by high-throughput screening. This transformant expresses BTL2 extra-cellularly with a 130-fold higher than the control strain, comprising S. cerevisiae PGK1 promoter, S. cerevisiae α-factor secretion signal, and S. cerevisiae PGK1 terminator, after cultivation for 72 h. This combinatorial library strategy holds promising potential for application in the optimization of the secretory expression of proteins in yeast. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sex-Specific Pathways to Early Puberty, Sexual Debut, and Sexual Risk Taking: Tests of an Integrated Evolutionary-Developmental Model

ERIC Educational Resources Information Center

James, Jenee; Ellis, Bruce J.; Schlomer, Gabriel L.; Garber, Judy

2012-01-01

The current study tested sex-specific pathways to early puberty, sexual debut, and sexual risk taking, as specified by an integrated evolutionary-developmental model of adolescent sexual development and behavior. In a prospective study of 238 adolescents (n = 129 girls and n = 109 boys) followed from approximately 12-18 years of age, we tested for…

Spatial and structural interrelationships between secretory cells of the subcommissural organ and blood vessels. An immunocytochemical study.

PubMed

Rodríguez, E M; Oksche, A; Hein, S; Rodríguez, S; Yulis, R

1984-01-01

In 76 specimens (amphibians, reptilians, mammals) belonging to 25 different vertebrate species, the region of the subcommissural organ (SCO) was investigated with the use of a primary antiserum raised against an extract of bovine Reissner's fiber + the immunoperoxidase procedure according to Sternberger et al. (1970). In the SCO of a toad (Bufo arenarum) and several species of reptiles (lacertilians, ophidians, crocodilians), the ependymal cells were the only type of secretory cell displaying vascular contacts, whereas in mammals ependymal and hypendymal cells established intimate spatial contacts with blood vessels. In Bufo arenarum, but especially in the reptilian species examined, the ependymo-vascular relationship was exerted by a population of ependymal cells having a rather constant location within the SCO and projecting to capillaries that showed a remarkably constant pattern of anatomical distribution. In the SCO of mammals the modality and degree of the structural relationships between secretory cells and blood vessels varied greatly from species to species. In the SCO of the armadillo and dog the secretory tissue was organized as a thick, highly vascularized layer with most of the cells oriented toward the capillaries. A rather opposite situation was found in the SCO of New- and Old-World monkeys, where vascular contacts were restricted to a few ependymal cells.

Reconstructing impairment of secretory ameloblast function in porcine teeth by analysis of morphological alterations in dental enamel

PubMed Central

Witzel, Carsten; Kierdorf, Uwe; Dobney, Keith; Ervynck, Anton; Vanpoucke, Sofie; Kierdorf, Horst

2006-01-01

We studied the relationship between the macroscopic appearance of hypoplastic defects in the dental enamel of wild boar and domestic pigs, and microstructural enamel changes, at both the light and the scanning electron microscopic levels. Deviations from normal enamel microstructure were used to reconstruct the functional and related morphological changes of the secretory ameloblasts caused by the action of stress factors during amelogenesis. The deduced reaction pattern of the secretory ameloblasts can be grouped in a sequence of increasingly severe impairments of cell function. The reactions ranged from a slight enhancement of the periodicity of enamel matrix secretion, over a temporary reduction in the amount of secreted enamel matrix, with reduction of the distal portion of the Tomes' process, to either a temporary or a definite cessation of matrix formation. The results demonstrate that analysis of structural changes in dental enamel allows a detailed reconstruction of the reaction of secretory ameloblasts to stress events, enabling an assessment of duration and intensity of these events. Analysing the deviations from normal enamel microstructure provides a deeper insight into the cellular changes underlying the formation of hypoplastic enamel defects than can be achieved by mere inspection of tooth surface characteristics alone. PMID:16822273

Activity-induced convergence of APP and BACE-1 in acidic microdomains via an endocytosis-dependent pathway

PubMed Central

Das, Utpal; Scott, David; Ganguly, Archan; Koo, Edward H.; Tang, Yong; Roy, Subhojit

2013-01-01

The convergence of APP (substrate) and BACE-1 (enzyme) is a rate-limiting, obligatory event triggering the amyloidogenic pathway – a key step in Alzheimer’s disease (AD) pathology. However, as both APP/BACE-1 are highly expressed in brain, mechanisms precluding their unabated convergence are unclear. Exploring dynamic localization of APP/BACE-1 in cultured hippocampal neurons, we found that after synthesis via the secretory-pathway, dendritic APP/BACE-1-containing vesicles are largely segregated in physiologic states. While BACE-1 is largely sorted into acidic recycling endosomes, APP is conveyed in Golgi-derived vesicles. However upon activity-induction – a known trigger of the amyloidogenic pathway – APP is routed into BACE-1-positive recycling endosomes via a clathrin-dependent mechanism. A partitioning/convergence of APP/BACE-1 vesicles is also apparent in control/AD brains respectively. Considering BACE-1 is optimally active in an acidic environment, our experiments suggest that neurons have evolved trafficking strategies that normally limit APP/BACE-1 proximity; and also uncover a pathway routing APP into BACE-1-containing organelles – triggering amyloidogenesis. PMID:23931995

Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

PubMed Central

Fuentes, Lida Q.; Reyes, Carlos E.; Sarmiento, José M.; Villanueva, Carolina I.; Figueroa, Carlos D.; Navarro, Javier; González, Carlos B.

2008-01-01

Activation of V1a receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. Here we found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V1a receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and β-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways. PMID:18571897

Lactobacillus frumenti Facilitates Intestinal Epithelial Barrier Function Maintenance in Early-Weaned Piglets

PubMed Central

Hu, Jun; Chen, Lingli; Zheng, Wenyong; Shi, Min; Liu, Liu; Xie, Chunlin; Wang, Xinkai; Niu, Yaorong; Hou, Qiliang; Xu, Xiaofan; Xu, Baoyang; Tang, Yimei; Zhou, Shuyi; Yan, Yiqin; Yang, Tao; Ma, Libao; Yan, Xianghua

2018-01-01

Increased intestinal epithelial barrier function damages caused by early weaning stress have adverse effects on swine health and feed utilization efficiency. Probiotics have emerged as the promising antibiotic alternatives used for intestinal barrier function damage prevention. Our previous data showed that Lactobacillus frumenti was identified as a predominant Lactobacillus in the intestinal microbiota of weaned piglets. However, whether the intestinal epithelial barrier function in piglets was regulated by L. frumenti is still unclear. Here, piglets received a PBS vehicle or PBS suspension (2 ml, 108 CFU/ml) containing the L. frumenti by oral gavage once a day during the period of 6–20 days of age prior to early weaning. Our data demonstrated that oral administration of L. frumenti significantly improved the intestinal mucosal integrity and decreased the serum endotoxin and D-lactic acid levels in early-weaned piglets (26 days of age). The intestinal tight junction proteins (including ZO-1, Occludin, and Claudin-1) were significantly up-regulated by L. frumenti administration. The serum immunoglobulin G (IgG) levels, intestinal secretory immunoglobulin A (sIgA) levels, and interferon-γ (IFN-γ) levels were significantly increased by L. frumenti administration. Furthermore, our data revealed that oral administration of L. frumenti significantly increased the relative abundances of health-promoting microbes (including L. frumenti, Lactobacillus gasseri LA39, Parabacteroides distasonis, and Kazachstania telluris) and decreased the relative abundances of opportunistic pathogens (including Desulfovibrio desulfuricans and Candida humilis). Functional alteration of the intestinal bacterial community by L. frumenti administration was characterized by the significantly increased fatty acids and protein metabolism and decreased diseases-associated metabolic pathways. These findings suggest that L. frumenti facilitates intestinal epithelial barrier function maintenance

Electromagnetic pulse activated brain microglia via the p38 MAPK pathway.

PubMed

Yang, Long-Long; Zhou, Yan; Tian, Wei-Dong; Li, Hai-Juan; Kang-Chu-Li; Miao, Xia; An, Guang-Zhou; Wang, Xiao-Wu; Guo, Guo-Zhen; Ding, Gui-Rong

2016-01-01

Previously, we found that electromagnetic pulses (EMP) induced an increase in blood brain barrier permeability and the leakage of albumin from blood into brain tissue. Albumin is known to activate microglia cells. Thus, we hypothesised that microglia activation could occur in the brain after EMP exposure. To test this hypothesis, the morphology and secretory function of microglia cells, including the expression of OX-42 (a marker of microglia activation), and levels of TNF-α, IL-10, IL-1β, and NO were determined in the rat cerebral cortex after EMP exposure. In addition, to examine the signalling pathway of EMP-induced microglia activation, protein and phosphorylated protein levels of p38, JNK and ERK were determined. It was found that the expression of OX-42increased significantly at 1, 6 and 12h (p<0.05) and recovered to the sham group level at 24h after EMP exposure. Levels of NO, TNF-α and IL-10 also changed significantly in vivo and in vitro after EMP exposure. The protein level of p38 and phosphorylated p38 increased significantly after EMP exposure (p<0.05) and recovered to sham levels at 12 and 24h, respectively. Protein and phosphorylated protein levels of ERK and JNK did not change. SB203580 (p38 inhibitor) partly prevented the change in NO, IL-10, IL-1β, TNF-α levels induced by EMP exposure. Taken together, these results suggested that EMP exposure (200kV/m, 200 pulses) could activate microglia in rat brain and affect its secretory function both in vivo and in vitro, and the p38 pathway is involved in this process. Copyright © 2015 Elsevier Inc. All rights reserved.

Pathways from maternal distress and child problem behavior to adolescent depressive symptoms: a prospective examination from early childhood to adolescence.

PubMed

Nilsen, Wendy; Gustavson, Kristin; Røysamb, Espen; Kjeldsen, Anne; Karevold, Evalill

2013-06-01

The main aim of this study was to identify the pathways from maternal distress and child problem behaviors (i.e., internalizing and externalizing problems) across childhood and their impact on depressive symptoms during adolescence among girls and boys. Data from families of 921 Norwegian children in a 15-year longitudinal community sample were used. Using structural equation modeling, the authors explored the interplay between maternal-reported distress and child problem behaviors measured at 5 time points from early (ages 1.5, 2.5, and 4.5 years) and middle (age 8.5 years) childhood to early adolescence (age 12.5 years), and their prediction of self-reported depressive symptoms during adolescence (ages 14.5 and 16.5 years). The findings revealed paths from internalizing and externalizing problems throughout the development for corresponding problems (homotypic paths) and paths from early externalizing to subsequent internalizing problems (heterotypic paths). The findings suggest 2 pathways linking maternal-rated risk factors to self-reported adolescent depressive symptoms. There was a direct path from early externalizing problems to depressive symptoms. There was an indirect path from early maternal distress going through child problem behavior to depressive symptoms. In general, girls and boys were similar, but some gender-specific effects appeared. Problem behaviors in middle childhood had heterotypic paths to subsequent problems only for girls. The findings highlight the developmental importance of child externalizing problems, as well as the impact of maternal distress as early as age 1.5 years for the development of adolescent depressive symptoms. Findings also indicate a certain vulnerable period in middle childhood for girls. NOTE: See Supplemental Digital Content 1, at http://links.lww.com/JDBP/A45, for a video introduction to this article.

Evaluation of the relationship between passive smoking and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in young children.

PubMed

Avşar, Aysun; Darka, Ozge; Bodrumlu, Ebru Hazar; Bek, Yüksel

2009-05-01

To evaluate the relationship between passive smoking as determined by salivary cotinine levels and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in children. Saliva was collected from 90 passive smoker (PS) subjects (the study group) and 90 healthy age-matched children (the control group). The study group was divided into three subgroups according the number of cigarettes smoked. Socio-economic status, dental and dietary habits were recorded by questionnaire. Stimulated salivary calcium (Ca), phosphate (P), sodium (Na), potassium (P), total protein, amylase activity, sialic acid level, secretory IgA concentration and cotinine level were analysed. All data were analysed using SPSS, version 13.0. Socio-economic status, dental and dietary habits were similar between the two groups. The salivary electrolytes concentrations did not reveal significant difference between the two groups (p>0.05). The mean cotinine levels of PS children were 1.58+/-4.3 ng/mL. The salivary concentrations of protein were similar between the two groups (p>0.05). The salivary secretory IgA concentration was significantly lower in the PS group than controls. The sialic acid level and amylase activity in PS group were found significantly higher compared with the controls (p<0.05). No difference was observed for all these parameters with sex (p>0.05). When saliva samples were analysed for output, the sialic acid level and amylase activity increased significantly in PS subjects (p<0.05). Further, the output of secretory IgA concentration was found significantly lower compared with the controls (p<0.05). In conclusion, we show that passive smoking was associated with a decrease in secretory IgA concentration, whereas with increase in amylase activity and sialic acid level of stimulated whole saliva in young children.

Structural insights into a secretory abundant heat-soluble protein from an anhydrobiotic tardigrade, Ramazzottius varieornatus.

PubMed

Fukuda, Yohta; Miura, Yoshimasa; Mizohata, Eiichi; Inoue, Tsuyoshi

2017-08-01

Upon stopping metabolic processes, some tardigrades can undergo anhydrobiosis. Secretory abundant heat-soluble (SAHS) proteins have been reported as candidates for anhydrobiosis-related proteins in tardigrades, which seem to protect extracellular components and/or secretory organelles. We determined structures of a SAHS protein from Ramazzottius varieornatus (RvSAHS1), which is one of the toughest tardigrades. RvSAHS1 shows a β-barrel structure similar to fatty acid-binding proteins (FABPs), in which hydrophilic residues form peculiar hydrogen bond networks, which would provide RvSAHS1 with better tolerance against dehydration. We identified two putative ligand-binding sites: one that superimposes on those of some FABPs and the other, unique to and conserved in SAHS proteins. These results indicate that SAHS proteins constitute a new FABP family. © 2017 Federation of European Biochemical Societies.

Huntingtons Disease Mice Infected with Toxoplasma gondii Demonstrate Early Kynurenine Pathway Activation, Altered CD8+ T-Cell Responses, and Premature Mortality.

PubMed

Donley, David W; Olson, Andrew R; Raisbeck, Merl F; Fox, Jonathan H; Gigley, Jason P

2016-01-01

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-repeat expansion in the huntingtin protein. Activation of the kynurenine pathway of tryptophan degradation is implicated in the pathogenesis of HD. Indoleamine-2,3-dioxygenase (IDO) catalyzes the oxidation of tryptophan to kynurenine, the first step in this pathway. The prevalent, neuroinvasive protozoal pathogen Toxoplasma gondii (T. gondii) results in clinically silent life-long infection in immune-competent individuals. T. gondii infection results in activation of IDO which provides some protection against the parasite by depleting tryptophan which the parasite cannot synthesize. The kynurenine pathway may therefore represent a point of synergism between HD and T. gondii infection. We show here that IDO activity is elevated at least four-fold in frontal cortex and striata of non-infected N171-82Q HD mice at 14-weeks corresponding to early-advanced HD. T. gondii infection at 5 weeks resulted in elevation of cortical IDO activity in HD mice. HD-infected mice died significantly earlier than wild-type infected and HD control mice. Prior to death, infected HD mice demonstrated decreased CD8+ T-lymphocyte proliferation in brain and spleen compared to wild-type infected mice. We demonstrate for the first time that HD mice have an altered response to an infectious agent that is characterized by premature mortality, altered immune responses and early activation of IDO. Findings are relevant to understanding how T. gondii infection may interact with pathways mediating neurodegeneration in HD.

Primary secretory otitis media in Cavalier King Charles spaniels.

PubMed

Cole, Lynette K

2012-11-01

Primary secretory otitis media (PSOM) is a disease that has been described in the Cavalier King Charles spaniel (CKCS). A large, bulging pars flaccida identified on otoscopic examination confirms the diagnosis. However, in many CKCS with PSOM the pars flaccida is flat, and radiographic imaging is needed to confirm the diagnosis. Current treatment for PSOM includes performing a myringotomy into the caudal-ventral quadrant of the pars tensa with subsequent flushing of the mucus out of the bulla using a video otoscope. Repeat myringotomies and flushing of the middle ear are necessary to keep the middle ear free of mucus. Copyright © 2012 Elsevier Inc. All rights reserved.

Entry and Exit Mechanisms at the cis-Face of the Golgi Complex

PubMed Central

Lorente-Rodríguez, Andrés; Barlowe, Charles

2011-01-01

Vesicular transport of protein and lipid cargo from the endoplasmic reticulum (ER) to cis-Golgi compartments depends on coat protein complexes, Rab GTPases, tethering factors, and membrane fusion catalysts. ER-derived vesicles deliver cargo to an ER-Golgi intermediate compartment (ERGIC) that then fuses with and/or matures into cis-Golgi compartments. The forward transport pathway to cis-Golgi compartments is balanced by a retrograde directed pathway that recycles transport machinery back to the ER. How trafficking through the ERGIC and cis-Golgi is coordinated to maintain organelle structure and function is poorly understood and highlights central questions regarding trafficking routes and organization of the early secretory pathway. PMID:21482742

Expression of CLAVATA3 fusions indicates rapid intracellular processing and a role of ERAD.

PubMed

De Marchis, Francesca; Colanero, Sara; Klein, Eva M; Mainieri, Davide; Prota, Viviana M; Bellucci, Michele; Pagliuca, Giampiero; Zironi, Elisa; Gazzotti, Teresa; Vitale, Alessandro; Pompa, Andrea

2018-06-01

The 12 amino acid peptide derived from the Arabidopsis soluble secretory protein CLAVATA3 (CLV3) acts at the cell surface in a signalling system that regulates the size of apical meristems. The subcellular pathway involved in releasing the peptide from its precursor is unknown. We show that a CLV3-GFP fusion expressed in transfected tobacco protoplasts or transgenic tobacco plants has very short intracellular half-life that cannot be extended by the secretory traffic inhibitors brefeldin A and wortmannin. The fusion is biologically active, since the incubation medium of protoplasts from CLV3-GFP-expressing tobacco contains the CLV3 peptide and inhibits root growth. The rapid disappearance of intact CLV3-GFP requires the signal peptide and is inhibited by the proteasome inhibitor MG132 or coexpression with a mutated CDC48 that inhibits endoplasmic reticulum-associated protein degradation (ERAD). The synthesis of CLV3-GFP is specifically supported by the endoplasmic reticulum chaperone endoplasmin in an in vivo assay. Our results indicate that processing of CLV3 starts intracellularly in an early compartment of the secretory pathway and that ERAD could play a regulatory or direct role in the active peptide synthesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster

PubMed Central

Rosenbaum, Erica E.; Vasiljevic, Eva; Brehm, Kimberley S.; Colley, Nansi Jo

2014-01-01

As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the

Cathepsin H Functions as an Aminopeptidase in Secretory Vesicles for Production of Enkephalin and Galanin Peptide Neurotransmitters

PubMed Central

Lu, W. Douglas; Funkelstein, Lydiane; Toneff, Thomas; Reinheckel, Thomas; Peters, Christoph; Hook, Vivian

2012-01-01

Peptide neurotransmitters function as key intercellular signaling molecules in the nervous system. These peptides are generated in secretory vesicles from proneuropeptides by proteolytic processing at dibasic residues, followed by removal of N- and/or C-terminal basic residues to form active peptides. Enkephalin biosynthesis from proenkephalin utilizes the cysteine protease cathepsin L and the subtilisin-like prohormone convertase 2 (PC2). Cathepsin L generates peptide intermediates with N-terminal basic residue extensions, which must be removed by an aminopeptidase. In this study, we identified cathepsin H as an aminopeptidase in secretory vesicles that produces (Met)enkephalin (ME) by sequential removal of basic residues from KR-ME and KK-ME, supported by in vivo knockout of the cathepsin H gene. Localization of cathepsin H in secretory vesicles was demonstrated by immunoelectron microscopy and confocal immunofluorescence microscopy. Purified human cathepsin H sequentially removes N-terminal basic residues to generate ME, with peptide products characterized by nano-LC-MS/MS tandem mass spectrometry. Cathepsin H shows highest activities for cleaving N-terminal basic residues (Arg and Lys) among amino acid fluorogenic substrates. Notably, knockout of the cathepsin H gene results in reduction of ME in mouse brain. Cathepsin H deficient mice also show a substantial decrease in galanin peptide neurotransmitter levels in brain. These results illustrate a role for cathepsin H as an aminopeptidase for enkephalin and galanin peptide neurotransmitter production. PMID:22582844

Crofelemer, a novel agent for treatment of secretory diarrhea.

PubMed

Crutchley, Rustin D; Miller, Jennifer; Garey, Kevin W

2010-05-01

To review the chemistry, pharmacology, pharmacokinetics, efficacy, and safety of crofelemer. A literature search using the terms SP-303, Provir, and crofelemer was performed with PubMed (up to April 2010), Google Scholar, and selected Ovid bibliography searches. Additional references from the bibliographies of articles included in the search, as well as company and Food and Drug Administration Web sites, were also assessed. English-language in vitro and clinical studies associated with the safety and efficacy of crofelemer were included. Crofelemer is a first-in-class agent that may be useful for different types of secretory diarrhea, since it prevents chloride and fluid secretion into the bowel by directly inhibiting 2 distinct intestinal chloride channels. Crofelemer significantly brought about faster symptom resolution in patients with traveler's diarrhea, along with lower rates of treatment failure compared to placebo-treated patients. In a post hoc analysis, crofelemer compared to placebo also appears to have reduced abnormal stool weight and frequency in patients with AIDS-associated diarrhea. In a third trial, crofelemer did not offer a significant benefit in improving stool consistency after 12 weeks of treatment in patients with diarrhea-predominant irritable bowel syndrome. However, a significant increase in pain-free days was noted in female patients. Preliminary studies also show that crofelemer may reduce watery stool output in patients with infectious diarrhea such as cholera. Oral crofelemer seemed to be well tolerated in clinical trials, with adverse effect profiles comparable to those with placebo. Crofelemer possesses a novel mechanism of action that shows promise in treating secretory diarrhea of several etiologies. However, results from further Phase 3 clinical trials are still needed in order to fully evaluate the efficacy and safety of this agent.

In vitro production of Toxocara canis excretory-secretory (TES) antigen.

PubMed

Thomas, Divyamol; Jeyathilakan, N; Abdul Basith, S; Senthilkumar, T M A

2016-09-01

Toxocara canis is a widespread gastrointestinal nematode parasite of dogs and cause Toxocara larva migrans, an important zoonotic disease in humans on ingestion of infective eggs. Toxocarosis is one of the few human parasitic diseases whose serodiagnosis uses a standardized antigen, T. canis excretory secretory antigen (TES). The present study describes collection of T. canis adult worm, collection and embryonation of T. canis eggs, hatching and separation of T. canis larvae, in vitro maintenance of T. canis second stage larvae for production of TES, concentration of culture fluid TES and yield of TES in correlation with various methods cited in literature.

Early evolution of radial glial cells in Bilateria

PubMed Central

Karl, Anett; Beckers, Patrick; Kaul-Strehlow, Sabrina; Ulbricht, Elke; Kourtesis, Ioannis; Kuhrt, Heidrun; Hausen, Harald; Reichenbach, Andreas; Bleidorn, Christoph

2017-01-01

Bilaterians usually possess a central nervous system, composed of neurons and supportive cells called glial cells. Whereas neuronal cells are highly comparable in all these animals, glial cells apparently differ, and in deuterostomes, radial glial cells are found. These particular secretory glial cells may represent the archetype of all (macro) glial cells and have not been reported from protostomes so far. This has caused controversial discussions of whether glial cells represent a homologous bilaterian characteristic or whether they (and thus, centralized nervous systems) evolved convergently in the two main clades of bilaterians. By using histology, transmission electron microscopy, immunolabelling and whole-mount in situ hybridization, we show here that protostomes also possess radial glia-like cells, which are very likely to be homologous to those of deuterostomes. Moreover, our antibody staining indicates that the secretory character of radial glial cells is maintained throughout their various evolutionary adaptations. This implies an early evolution of radial glial cells in the last common ancestor of Protostomia and Deuterostomia. Furthermore, it suggests that an intraepidermal nervous system—composed of sensory cells, neurons and radial glial cells—was probably the plesiomorphic condition in the bilaterian ancestor. PMID:28724733

Phyllosilicate weathering pathways in chlorite-talc bearing soil parent materials, D.R. Congo: early findings.

NASA Astrophysics Data System (ADS)

Dumon, Mathijs; Oostermeyer, Fran; Timmermans, Els; De Meulemeester, Aschwin; Mees, Florias; Van Driessche, Isabel; Erens, Hans; Bazirake Mujinya, Basile; Van Ranst, Eric

2015-04-01

The study of the formation and transformation of clay minerals is of the upmost importance to understand soil formation and to adjust land-use management to the land surface conditions. These clay minerals determine to a large extent the soil physical and chemical properties. It is commonly observed that over time the mineralogy of any parent material is transformed to a simple assemblage composed mostly of Al and Fe oxides and low-activity clays, e.g. kaolinite. This is especially obvious in the humid tropics, which have been protected from glacial erosion, allowing deep, highly weathered soils to form. Despite the abundant presence of kaolinite in these soils, its formation pathways are still under debate: either neoformation by dissolution-crystallisation reactions or solid-state transformation of 2:1 phyllosilicates. To elucidate this, weathering sequences in a unique 40 m core taken below a termite mound, reaching a talc-chlorite bearing substrate in the Lubumbashi area, Katanga, DR Congo are being investigated in detail using a.o. quantitative X-ray diffraction analysis, chemical characterization, micromorphology and µXRF-scanning with the main objective to improve the understanding of the formation pathways of kaolinite subgroup minerals in humid tropical environments. Based on an initial characterization of the core, two zones of interest were selected for more detailed analysis, for which the early findings will be presented. The first zone extends from ca. 9 m to 11 m below the surface is dominated by kaolinite but shows early traces of primary talc and micas. The second zone extends from 34 to 36 m below the surface and contains large amounts of chlorite, with smaller amounts of talc, micas and kaolinite.

Secretory IgM Exacerbates Tumor Progression by Inducing Accumulations of MDSCs in Mice.

PubMed

Tang, Chih-Hang Anthony; Chang, Shiun; Hashimoto, Ayumi; Chen, Yi-Ju; Kang, Chang Won; Mato, Anthony R; Del Valle, Juan R; Gabrilovich, Dmitry I; Hu, Chih-Chi Andrew

2018-06-01

Chronic lymphocytic leukemia (CLL) cells can secrete immunoglobulin M. However, it is not clear whether secretory IgM (sIgM) plays a role in disease progression. We crossed the Eμ-TCL1 mouse model of CLL, in which the expression of human TCL1 oncogene was driven by the V(H) promoter-Ig(H)-Eμ enhancer, with MD4 mice whose B cells produced B-cell receptor (membrane-bound IgM) and sIgM with specificity for hen egg lysozyme (HEL). CLL cells that developed in these MD4/Eμ-TCL1 mice reactivated a parental Ig gene allele and secreted IgM, and did not recognize HEL. The MD4/Eμ-TCL1 mice had reduced survival, increased myeloid-derived suppressor cells (MDSC), and decreased numbers of T cells. We tested whether sIgM could contribute to the accumulation of MDSCs by crossing μS -/- mice, which could not produce sIgM, with Eμ-TCL1 mice. The μS -/- /Eμ-TCL1 mice survived longer than Eμ-TCL1 mice and developed decreased numbers of MDSCs which were less able to suppress proliferation of T cells. We targeted the synthesis of sIgM by deleting the function of XBP-1s and showed that targeting XBP-1s genetically or pharmacologically could lead to decreased sIgM, accompanied by decreased numbers and reduced functions of MDSCs in MD4/Eμ-TCL1 mice. Additionally, MDSCs from μS -/- mice grafted with Lewis lung carcinoma were inefficient suppressors of T cells, resulting in slower tumor growth. These results demonstrate that sIgM produced by B cells can upregulate the functions of MDSCs in tumor-bearing mice to aggravate cancer progression. In a mouse model of CLL, production of secretory IgM led to more MDSCs, fewer T cells, and shorter survival times for the mice. Thus, secretory IgM may aggravate the progression of this cancer. Cancer Immunol Res; 6(6); 696-710. ©2018 AACR . ©2018 American Association for Cancer Research.

Regulation of traffic and organelle architecture of the ER-Golgi interface by signal transduction.

PubMed

Tillmann, Kerstin D; Millarte, Valentina; Farhan, Hesso

2013-09-01

The components that control trafficking between organelles of the secretory pathway as well as their architecture were uncovered to a reasonable extent in the past decades. However, only recently did we begin to explore the regulation of the secretory pathway by cellular signaling. In the current review, we focus on trafficking between the endoplasmic reticulum and the Golgi apparatus. We highlight recent advances that have been made toward a better understanding of how the secretory pathway is regulated by signaling and discuss how this knowledge is important to obtain an integrative view of secretion in the context of other homeostatic processes such as growth and proliferation.

The Developmental Pathway From Pubertal Timing to Delinquency and Sexual Activity From Early to Late Adolescence

PubMed Central

Negriff, Sonya; Elizabeth, J. Susman; Trickett, Penelope K.

2013-01-01

There is strong evidence that early pubertal timing is associated with adolescent problem behaviors. However, there has been limited investigation of the mechanisms or developmental relationships. The present study examined longitudinal models incorporating pubertal timing, delinquency, and sexual activity in a sample of 454 adolescents (9–13 years old at enrollment; 47% females). Participants were seen for three assessments approximately 1 year apart. Characteristics of friendship networks (older friends, male friends, older male friends) were examined as mediators. Structural equation modeling was used to test these associations as well as temporal relationships between sexual activity and delinquency. Results showed that early pubertal timing at Time 1 was related to more sexual activity at Time 2, which was related to higher delinquency at Time 3, a trend mediation effect. None of the friendship variables mediated these associations. Gender or maltreatment status did not moderate the meditational pathways. The results also supported the temporal sequence of sexual activity preceding increases in delinquency. These findings reveal that early maturing adolescents may actively seek out opportunities to engage in sexual activity which appears to be risk for subsequent delinquency. PMID:21191640

The developmental pathway from pubertal timing to delinquency and sexual activity from early to late adolescence.

PubMed

Negriff, Sonya; Susman, Elizabeth J; Trickett, Penelope K

2011-10-01

There is strong evidence that early pubertal timing is associated with adolescent problem behaviors. However, there has been limited investigation of the mechanisms or developmental relationships. The present study examined longitudinal models incorporating pubertal timing, delinquency, and sexual activity in a sample of 454 adolescents (9-13 years old at enrollment; 47% females). Participants were seen for three assessments approximately 1 year apart. Characteristics of friendship networks (older friends, male friends, older male friends) were examined as mediators. Structural equation modeling was used to test these associations as well as temporal relationships between sexual activity and delinquency. Results showed that early pubertal timing at Time 1 was related to more sexual activity at Time 2, which was related to higher delinquency at Time 3, a trend mediation effect. None of the friendship variables mediated these associations. Gender or maltreatment status did not moderate the meditational pathways. The results also supported the temporal sequence of sexual activity preceding increases in delinquency. These findings reveal that early maturing adolescents may actively seek out opportunities to engage in sexual activity which appears to be risk for subsequent delinquency.

Wide distribution of cysteine-rich secretory proteins in snake venoms: isolation and cloning of novel snake venom cysteine-rich secretory proteins.

PubMed

Yamazaki, Yasuo; Hyodo, Fumiko; Morita, Takashi

2003-04-01

Cysteine-rich secretory proteins (CRISPs) are found in epididymis and granules of mammals, and they are thought to function in sperm maturation and in the immune system. Recently, we isolated and obtained clones for novel snake venom proteins that are classified as CRISP family proteins. To elucidate the distribution of snake venom CRISP family proteins, we evaluated a wide range of venoms for immuno-cross-reactivity. Then we isolated, characterized, and cloned genes for three novel CRISP family proteins (piscivorin, ophanin, and catrin) from the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus), king cobra (Ophiophagus hannah), and western diamondback rattlesnake (Crotalus atrox). Our results show the wide distribution of snake venom CRISP family proteins among Viperidae and Elapidae from different continents, indicating that CRISP family proteins compose a new group of snake venom proteins.

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM).

PubMed

Yamazawa, Toshiko; Nakamura, Naotoshi; Sato, Mari; Sato, Chikara

2016-12-01

Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland-related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2- to 3-µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in-solution ASEM for histology and to study vesicle secretion systems. Further, the high-throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine-related diseases. © 2016 Wiley Periodicals, Inc.

Aspiration cytology of mammary analogue secretory carcinoma of the salivary gland.

PubMed

Jung, Min Jung; Kim, Sang Yoon; Nam, Soon Yuhl; Roh, Jong-Lyel; Choi, Seung-Ho; Lee, Jeong Hyun; Baek, Jung Hwan; Cho, Kyung-Ja

2015-04-01

Aspiration cytologic findings of mammary analogue secretory carcinoma (MASC), a newly established salivary gland neoplasm defined by a t(12;15)(p13;q25) ETV6-NTRK3 translocation, are not fully characterized to date. We report cytologic descriptions of nine cases of molecularly confirmed MASC, including two with unusual findings. Aspiration smears from nine MASCs of the salivary glands were retrospectively reviewed and analyzed according to the cellular and structural features of the corresponding surgical specimens. Aspiration smears of MASC generally reflected the histologic diversity of the tumors. Among usual histologic findings, a micropapillary pattern was associated with a predominance of vacuolated individual cells on aspiration smears, a papillary-cystic pattern with a predominance of thin branching papillary structures, and a microcystic pattern with a predominance of irregular sheets of eosinophilic cells. There were two unusual cases, one with three-dimensional groups of high-grade atypical cells, and one with epithelial clusters floating in a notably mucinous background. These cases represented MASC with high-grade transformation and MASC with cystadenocarcinoma-like features, respectively. The secretory activity of MASC was not prominent in the aspiration specimens. Although unusual cases were present, most MASC cases showed characteristic cytologic findings, which could aid the cytologic diagnosis of MASC. And knowledge of the histologic spectrum of MASC, including high-grade transformation, could be valuable for cytological differential diagnoses of salivary gland tumors, and the management of patients with MASC. © 2014 Wiley Periodicals, Inc.

Aspiration biopsy of mammary analogue secretory carcinoma of accessory parotid gland: another diagnostic dilemma in matrix-containing tumors of the salivary glands.

PubMed

Levine, Pascale; Fried, Karen; Krevitt, Lane D; Wang, Beverly; Wenig, Bruce M

2014-01-01

Mammary analogue secretory carcinoma (MASC) is a newly described rare salivary gland tumor, which shares morphologic features with acinic cell carcinoma, low-grade cystadenocarcinoma, and secretory carcinoma of the breast. This is the first reported case of MASC of an accessory parotid gland detected by aspiration biopsy with radiologic and histologic correlation in a 34-year-old patient. Sonographically-guided aspiration biopsy showed cytologic features mimicking those of low-grade mucoepidermoid carcinoma, including sheets of bland epithelial cells, dissociated histiocytoid cells with intracytoplasmic mucinous material, and spindle cells lying in a web-like matrix. Histologic sections showed a circumscribed tumor with microcystic spaces lined by bland uniform epithelial cells and containing secretory material. The tumor cells expressed mammaglobin and BRST-2. The cytologic features, differential diagnosis, and pitfalls are discussed. The pathologic stage was pT1N0. The patient showed no evidence of disease at 1 year follow-up. Copyright © 2012 Wiley Periodicals, Inc.

Early Intervention of Didang Decoction on MLCK Signaling Pathways in Vascular Endothelial Cells of Type 2 Diabetic Rats

PubMed Central

Song, Zhenqiang; Li, Jing; Li, Chunshen

2016-01-01

In the study, type 2 diabetic rat model was established using streptozotocin (STZ) combined with a high-fat diet, and the rats were divided into control and diabetic groups. Diabetic groups were further divided into nonintervening, simvastatin, Didang Decoction (DDD) early-phase intervening, DDD mid-phase intervening, and DDD late-phase intervening groups. The expression level of MLCK was detected using Western Blot analysis, and the levels of cyclic adenosine monophosphate (cAMP), protein kinase C (PKC), and protein kinase A (PKA) were examined using Real Time PCR. Under the electron microscope, the cells in the early-DDD-intervention group and the simvastatin group were significantly more continuous and compact than those in the diabetic group. Compared with the control group, the expression of cAMP-1 and PKA was decreased in all diabetic groups, whereas the expression of MLCK and PKC was increased in early- and mid-phase DDD-intervening groups (P < 0.05); compared with the late-phase DDD-intervening group, the expression of cAMP-1 and PKA was higher, but the level of MLCK and PKC was lower in early-phase DDD-intervening group (P < 0.05). In conclusion, the early use of DDD improves the permeability of vascular endothelial cells by regulating the MLCK signaling pathway. PMID:27703477

Chromaffin granules in the rat adrenal medulla release their secretory content in a particulate fashion.

PubMed

Crivellato, Enrico; Belloni, Anna; Nico, Beatrice; Nussdorfer, Gastone G; Ribatti, Domenico

2004-03-01

Exocytosis is considered the main route of granule discharge in chromaffin cells. We recently provided ultrastructural evidence suggesting that piecemeal degranulation (PMD) occurs in mouse adrenal chromaffin cells. In the present study, we processed rat adrenal glands for transmission electron microscopy (TEM), and examined chromaffin cells for changes characteristic of PMD. Both adrenaline (A)- and noradrenaline (NA)-storing cells express ultrastructural features suggestive of a slow and particulate mode of granule discharge. In adrenaline-containing cells, some granules present enlarged dimensions accompanied by eroded or dissolved matrices. Likewise, a number of granules in NA-releasing cells show content reduction with variably expanded granule chambers. Dilated, empty granule containers are recognizable in the cytoplasm of both cell types. Characteristically, altered granules and empty containers are seen intermingled with normal, resting granules. In addition, chromaffin granules often show irregular profiles, with budding or tail-like projections of their limiting membranes. Thirty 150-nm-diameter membrane-bound vesicles with a moderately electron-dense or -lucent internal structure are observable in the cytoplasm of both cell types. These vesicles are seen among the granules and some of them are fused with the perigranule membranes in the process of attachment to or budding from the granules. These data add further support to the concept that PMD may be an alternative secretory pathway in adrenal chromaffin cells. Copyright 2004 Wiley-Liss, Inc.

A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway

PubMed Central

Erdmann, Kai S.; Mao, Yuxin; McCrea, Heather J.; Zoncu, Roberto; Lee, Sangyoon; Paradise, Summer; Modregger, Jan; Biemesderfer, Daniel; Toomre, Derek; De Camilli, Pietro

2007-01-01

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, whose manifestations include mental retardation and renal Fanconi syndrome. OCRL has been implicated in membrane traffic, but disease mechanisms remain unclear. We show that OCRL visits late stage endocytic clathrin coated pits and binds the Rab5 effector APPL1 on peripheral early endosomes. The interaction with APPL1, which is mediated by the ASH-RhoGAP-like domains of OCRL and is abolished by disease mutations, provides a link to protein networks implicated in the reabsorptive function of kidney and in traffic and signaling of growth factor receptors in brain. Crystallographic studies reveal a role of the ASH-RhoGAP-like domains in positioning the phosphatase domain at the membrane interface and a clathrin box protruding from the RhoGAP-like domain. Our results support a role of OCRL in the early endocytic pathway consistent with the predominant localization of its preferred substrates, PI(4,5)P2 and PI(3,4,5)P3, at the cell surface. PMID:17765681

The tumor secretory factor ZAG promotes white adipose tissue browning and energy wasting.

PubMed

Elattar, Sawsan; Dimri, Manali; Satyanarayana, Ande

2018-03-23

Cachexia is a complex tissue-wasting syndrome characterized by inflammation, hypermetabolism, increased energy expenditure, and anorexia. Browning of white adipose tissue (WAT) is one of the significant factors that contribute to energy wasting in cachexia. By utilizing a cell implantation model, we demonstrate here that the lipid mobilizing factor zinc-α 2 -glycoprotein (ZAG) induces WAT browning in mice. Increased circulating levels of ZAG not only induced lipolysis in adipose tissues but also caused robust browning in WAT. Stimulating WAT progenitors with ZAG recombinant protein or expression of ZAG in mouse embryonic fibroblasts (MEFs) strongly enhanced brown-like differentiation. At the molecular level, ZAG stimulated peroxisome proliferator-activated receptor γ (PPARγ) and early B cell factor 2 expression and promoted their recruitment to the PR/SET domain 16 (Prdm16) promoter, leading to enhanced expression of Prdm16, which determines brown cell fate. In brown adipose tissue, ZAG stimulated the expression of PPARγ and PPARγ coactivator 1α and promoted recruitment of PPARγ to the uncoupling protein 1 (Ucp1) promoter, leading to increased expression of Ucp1. Overall, our results reveal a novel function of ZAG in WAT browning and highlight the targeting of ZAG as a potential therapeutic application in humans with cachexia.-Elattar, S., Dimri, M., Satyanarayana, A. The tumor secretory factor ZAG promotes white adipose tissue browning and energy wasting.

Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections

PubMed Central

Armitage, Charles W; O’Meara, Connor P; Harvie, Marina C G; Timms, Peter; Wijburg, Odilia L; Beagley, Kenneth W

2014-01-01

Immunoglobulin A is an important mucosal antibody that can neutralize mucosal pathogens by either preventing attachment to epithelia (immune exclusion) or alternatively inhibit intra-epithelial replication following transcytosis by the polymeric immunoglobulin receptor (pIgR). Chlamydia trachomatis is a major human pathogen that initially targets the endocervical or urethral epithelium in women and men, respectively. As both tissues contain abundant secretory IgA (SIgA) we assessed the protection afforded by IgA targeting different chlamydial antigens expressed during the extra- and intra-epithelial stages of infection. We developed an in vitro model using polarizing cells expressing the murine pIgR together with antigen-specific mouse IgA, and an in vivo model using pIgR−/− mice. Secretory IgA targeting the extra-epithelial chlamydial antigen, the major outer membrane protein, significantly reduced infection in vitro by 24% and in vivo by 44%. Conversely, pIgR-mediated delivery of IgA targeting the intra-epithelial inclusion membrane protein A bound to the inclusion but did not reduce infection in vitro or in vivo. Similarly, intra-epithelial IgA targeting the secreted protease Chlamydia protease-like activity factor also failed to reduce infection. Together, these data suggest the importance of pIgR-mediated delivery of IgA targeting extra-epithelial, but not intra-epithelial, chlamydial antigens for protection against a genital tract infection. PMID:24827556

MUM ENHANCERS are important for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana

PubMed Central

Arsovski, Andrej A.; Villota, Maria M.; Rowland, Owen; Subramaniam, Rajagopal; Western, Tamara L.

2009-01-01

Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis. To identify additional genes acting in mucilage synthesis and secretion, a screen for enhancers of the mum4 phenotype was performed. Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61). Our results show that, in a mum4 background, mutations in MEN1, MEN4, and MEN5 lead to further reductions in mucilage compared to mum4 single mutants, suggesting that they are involved in mucilage synthesis or secretion. Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity. With the exception of men4, whose single mutant exhibits reduced mucilage, none of these genes have a single mutant phenotype, suggesting that they would not have been identified outside the compromised mum4 background. PMID:19401413

Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

PubMed

Harthoorn, L F; Diederen, J H; Oudejans, R C; Verstegen, M M; Vullings, H G; Van der Horst, D J

2000-01-01

The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

[Curcumin alleviates early brain injury following subarachnoid hemorrhage in rats by inhibiting JNK/c-Jun signal pathway].

PubMed

Li, Xia; Zhu, Ji

2018-03-01

Objective To investigate the inhibitory effect of curcumin on early brain injury following subarachnoid hemorrhage (SAH) by inhibiting JNK/ c-Jun signal pathway. Methods Sixty adult male SD rats were randomly divided into four groups: sham operation group (sham group), SAH group, SAH group treated with 100 mg/(kg.d) curcumin and SAH group treated with 200 mg/(kg.d) curcumin, with 15 rats in each group. Endovascular puncture was used to induce SAH model. Nissl staining was used to test whether neurons were broken. TUNEL staining was used to detect apoptosis. Immunohistochemistry was used to investigate the expression of caspase-3. Western blot analysis was used to detect the expressions of p-JNK, JNK, p-c-Jun, c-Jun, and caspase-3. Results Nissl staining indicated the decrease of Nissl bodies in SAH group, but increase of Nissl bodies in SAH group treated with curcumin. TUNEL staining showed that there were more apoptotic neurons in SAH group compared with sham group, while apoptotic neurons decreased after the treatment with curcumin, more obviously in the group treated with 200 mg/(kg.d) curcumin. The expressions of p-JNK, JNK, p-c-Jun, c-Jun, and caspase-3 were up-regulated in SAH group compared with sham group. However, the expressions of those proteins were down-regulated after the treatment with curcumin, especially by higher-dose curcumin treatment. Conclusion Curcumin might suppress early brain injury after SAH by inhibiting JNK/c-Jun signal pathway and neuron apoptosis.

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin.

PubMed

Kim, Young-Sun; Jin, Hyeon-Ok; Hong, Sung-Eun; Song, Jie-Young; Hwang, Chang-Sun; Park, In-Chul

2018-01-08

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Secretory expression of a heterologous nattokinase in Lactococcus lactis.

PubMed

Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

2007-05-01

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

Sperm-storage defects and live birth in Drosophila females lacking spermathecal secretory cells.

PubMed

Schnakenberg, Sandra L; Matias, Wilfredo R; Siegal, Mark L

2011-11-01

Male Drosophila flies secrete seminal-fluid proteins that mediate proper sperm storage and fertilization, and that induce changes in female behavior. Females also produce reproductive-tract secretions, yet their contributions to postmating physiology are poorly understood. Large secretory cells line the female's spermathecae, a pair of sperm-storage organs. We identified the regulatory regions controlling transcription of two genes exclusively expressed in these spermathecal secretory cells (SSC): Spermathecal endopeptidase 1 (Send1), which is expressed in both unmated and mated females, and Spermathecal endopeptidase 2 (Send2), which is induced by mating. We used these regulatory sequences to perform precise genetic ablations of the SSC at distinct time points relative to mating. We show that the SSC are required for recruiting sperm to the spermathecae, but not for retaining sperm there. The SSC also act at a distance in the reproductive tract, in that their ablation: (1) reduces sperm motility in the female's other sperm-storage organ, the seminal receptacle; and (2) causes ovoviviparity--the retention and internal development of fertilized eggs. These results establish the reproductive functions of the SSC, shed light on the evolution of live birth, and open new avenues for studying and manipulating female fertility in insects.

The serrated neoplasia pathway of colorectal tumors: Identification of MUC5AC hypomethylation as an early marker of polyps with malignant potential.

PubMed

Renaud, Florence; Mariette, Christophe; Vincent, Audrey; Wacrenier, Agnès; Maunoury, Vincent; Leclerc, Julie; Coppin, Lucie; Crépin, Michel; Van Seuningen, Isabelle; Leteurtre, Emmanuelle; Buisine, Marie-Pierre

2016-03-15

The serrated neoplasia pathway accounts for 20-30% of colorectal cancers (CRC), which are characterized by extensive methylation (CpG island methylation phenotype, CIMP), frequent BRAF mutation and high microsatellite instability (MSI). We recently identified MUC5AC mucin gene hypomethylation as a specific marker of MSI CRC. The early identification of preneoplastic lesions among serrated polyps is currently challenging. Here, we performed a detailed pathological and molecular analysis of a large series of colorectal serrated polyps and evaluated the usefulness of mucin genes MUC2 and MUC5AC to differentiate serrated polyps and to identify lesions with malignant potential. A series of 330 colorectal polyps including 218 serrated polyps [42 goblet cell-rich hyperplastic polyps (GCHP), 68 microvesicular hyperplastic polyps (MVHP), 100 sessile serrated adenoma (SSA) and eight traditional serrated adenoma (TSA)] and 112 conventional adenomas was analyzed for BRAF/KRAS mutations, MSI, CIMP, MLH1 and MGMT methylation, and MUC2 and MUC5AC expression and methylation. We show that MUC5AC hypomethylation is an early event in the serrated neoplasia pathway, and specifically detects MVHP and SSA, arguing for a filiation between MVHP, SSA and CIMP-H/MSI CRC, whereas GCHP and TSA arise from a distinct pathway. Moreover, MUC5AC hypomethylation specifically identified serrated lesions with BRAF mutation, CIMP-H or MSI, suggesting that it may be useful to identify serrated neoplasia pathway-related precursor lesions. Our data suggest that MVHP should be recognized among HP and require particular attention. © 2015 UICC.

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

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

Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215; Weller, Peter F.

Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombreromore » Vesicles – EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. - Highlights: • Application of EM to understand the complex secretory pathway in human eosinophils. • EM techniques reveal an active vesicular system associated with secretory granules. • Tubular vesicles are involved in the transport of granule-derived immune mediators.« less

Loss of ascl1a prevents secretory cell differentiation within the zebrafish intestinal epithelium resulting in a loss of distal intestinal motility

PubMed Central

Roach, Gillian; Wallace, Rachel Heath; Cameron, Amy; Ozel, Rifat Emrah; Hongay, Cintia F.; Baral, Reshica; Andreescu, Silvana; Wallace, Kenneth N.

2013-01-01

The vertebrate intestinal epithelium is renewed continuously from stem cells at the base of the crypt in mammals or base of the fold in fish over the life of the organism. As stem cells divide, newly formed epithelial cells make an initial choice between a secretory or enterocyte fate. This choice has previously been demonstrated to involve Notch signaling as well as Atonal and Her transcription factors in both embryogenesis and adults. Here, we demonstrate that in contrast to the atoh1 in mammals, ascl1a is responsible for formation of secretory cells in zebrafish. ascl1a−/− embryos lack all intestinal epithelial secretory cells and instead differentiate into enterocytes. ascl1a−/− embryos also fail to induce intestinal epithelial expression of deltaD suggesting that ascl1a plays a role in initiation of Notch signaling. Inhibition of Notch signaling increases the number of ascl1a and deltaD expressing intestinal epithelial cells as well as the number of developing secretory cells during two specific time periods: between 30 and 34 hpf and again between 64 and 74 hpf. Loss of enteroendocrine products results in loss of anterograde motility in ascl1a−/− embryos. 5HT produced by enterochromaffin cells is critical in motility and secretion within the intestine. We find that addition of exogenous 5HT to ascl1a−/− embryos at near physiological levels (measured by differential pulse voltammetry) induce anterograde motility at similar levels to wild type velocity, distance, and frequency. Removal or doubling the concentration of 5HT in WT embryos does not significantly affect anterograde motility, suggesting that the loss of additional enteroendocrine products in ascl1a−/− embryos also contributes to intestinal motility. Thus, zebrafish intestinal epithelial cells appear to have a common secretory progenitor from which all subtypes form. Loss of enteroendocrine cells reveals the critical need for enteroendocrine products in maintenance of normal

The Role of the Polymeric Immunoglobulin Receptor and Secretory Immunoglobulins during Mucosal Infection and Immunity.

PubMed

Turula, Holly; Wobus, Christiane E

2018-05-03

The gastrointestinal tract houses millions of microbes, and thus has evolved several host defense mechanisms to keep them at bay, and prevent their entry into the host. One such mucosal surface defense is the secretion of secretory immunoglobulins (SIg). Secretion of SIg depends on the polymeric immunoglobulin receptor (pIgR), which transports polymeric Ig (IgA or IgM) from the basolateral surface of the epithelium to the apical side. Upon reaching the luminal side, a portion of pIgR, called secretory component (SC) is cleaved off to release Ig, forming SIg. Through antigen-specific and non-specific binding, SIg can modulate microbial communities and pathogenic microbes via several mechanisms: agglutination and exclusion from the epithelial surface, neutralization, or via host immunity and complement activation. Given the crucial role of SIg as a microbial scavenger, some pathogens also evolved ways to modulate and utilize pIgR and SIg to facilitate infection. This review will cover the regulation of the pIgR/SIg cycle, mechanisms of SIg-mediated mucosal protection as well as pathogen utilization of SIg.

Distorted secretory granule composition in mast cells with multiple protease deficiency.

PubMed

Grujic, Mirjana; Calounova, Gabriela; Eriksson, Inger; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Tchougounova, Elena; Kjellén, Lena; Pejler, Gunnar

2013-10-01

Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

Women’s Work Pathways Across the Life Course1

PubMed Central

Damaske, Sarah; Frech, Adrianne

2016-01-01

Despite numerous changes in women’s employment in the latter half of the 20th century, women’s employment continues to be uneven and stalled. Drawing from data on women’s weekly work hours in the National Longitudinal Survey of Youth (NLSY79), we identify significant inequality in women’s labor force experiences across adulthood. We find two pathways of stable fulltime work for women, three pathways of part-time employment, and a pathway of unpaid labor. A majority of women follow one of the two fulltime work pathways, while fewer than 10 percent follow a pathway of unpaid labor. Our findings provide evidence of the lasting influence of work-family conflict and early socio-economic advantages and disadvantages on women’s work pathways. Indeed, race, poverty, educational attainment, and early family characteristics significantly shaped women’s work careers. Work-family opportunities and constraints also were related to women’s work hours, as were a woman’s gendered beliefs and expectations. We conclude that women’s employment pathways are a product of both their resources and changing social environment as well as individual agency. Significantly, we point to social stratification, gender ideologies, and work-family constraints, working in concert, as key explanations for how women are “tracked” onto work pathways from an early age. PMID:27001314

[Unilateral exophthalmos as the debut of a non-secretory multiple myeloma].

PubMed

Castro-Rebollo, M; Cañones-Zafra, R; Vleming-Pinilla, E N; Drake-Rodríguez-Casanova, P; Pérez-Rico, C

2009-12-01

A 56 year-old male presented blurred vision and diplopia for 2 months, left unilateral exophthalmos, restricted ocular motility and papilledema. The imaging proofs showed osteolytic lesions in the left sphenoid bone, fourth rib and fourth dorsal vertebral body with associated masses of soft tissues. Biopsy was performed and the diagnosis of plasma cell neoplasm was established. The diagnosis of non-secretory multiple myeloma was made by analytical criteria and bone marrow biopsy. Local radiotherapy and polychemotherapy was prescribed. The ophthalmologist can play an important role in the diagnosis of systemic neoplasms that require the intervention of a multidisciplinary team.

Adult Sexual Experiences as a Mediator Between Child Abuse and Current Secretory Immunoglobulin A Levels.

PubMed

Waldron, Jonathan C; Scarpa, Angela; Kim-Spoon, Jungmeen; Coe, Christopher L

2016-03-01

The current study investigated whether a history of child abuse is a predictor of adult immune status, with unwanted adult sexual experiences as a proximal mediator. Participants included 89 young adult women (M(age) = 19.24) who were classified as having experienced no child abuse, child physical abuse, or child sexual abuse, based upon self-reported victimization history before 14 years of age. Participants also reported on unwanted sexual experiences in young adulthood and provided four saliva samples, which were collected over two consecutive days to determine secretory immunoglobulin A (sIgA). Age and negative life events were considered as covariates. The results indicated that adult sexual victimization partially mediated the relationship between child abuse (physical and sexual) and sIgA. Specifically, child abuse experiences predicted more adult sexual victimization experiences, which in turn predicted lower sIgA levels. These findings support long-term health effects of victimization, and suggest that the influence of child abuse on sIgA may be perpetuated through adult victimization. Prevention efforts should aim to empower child maltreatment survivors with skills to prevent adult re-victimization. By thwarting future unwanted sexual experiences in adulthood, individuals will be better protected from the health impairments associated with early abuse experiences. © The Author(s) 2014.

A Family of Secretory Proteins Is Associated with Different Morphotypes in Cryptococcus neoformans.

PubMed

Gyawali, Rachana; Upadhyay, Srijana; Way, Joshua; Lin, Xiaorong

2017-03-01

Cryptococcus neoformans , an opportunistic human fungal pathogen, can undergo a yeast-to-hypha transition in response to environmental cues. This morphological transition is associated with changes in the expression of cell surface proteins. The Cryptococcus cell surface and secreted protein Cfl1 was the first identified adhesin in the Basidiomycota. Cfl1 has been shown to regulate morphology, biofilm formation, and intercellular communication. Four additional homologs of CFL1 are harbored by the Cryptococcus genome: DHA1 , DHA2 , CPL1 , and CFL105 The common features of this gene family are the conserved C-terminal SIGC domain and the presence of an N-terminal signal peptide. We found that all these Cfl1 homolog proteins are indeed secreted extracellularly. Interestingly, some of these secretory proteins display cell type-specific expression patterns: Cfl1 is hypha specific, Dha2 is yeast specific, and Dha1 (delayed hypersensitivity antigen 1) is expressed in all cell types but is particularly enriched at basidia. Interestingly, Dha1 is induced by copper limitation and suppressed by excessive copper in the medium. This study further attests to the physiological heterogeneity of the Cryptococcus mating colony, which is composed of cells with heterogeneous morphotypes. The differential expression of these secretory proteins contributes to heterogeneity, which is beneficial for the fungus to adapt to changing environments. IMPORTANCE Heterogeneity in physiology and morphology is an important bet-hedging strategy for nonmobile microbes such as fungi to adapt to unpredictable environmental changes. Cryptococcus neoformans , a ubiquitous basidiomycetous fungus, is known to switch from the yeast form to the hypha form during sexual development. However, in a mating colony, only a subset of yeast cells switch to hyphae, and only a fraction of the hyphal subpopulation will develop into fruiting bodies, where meiosis and sporulation occur. Here, we investigated a

Secretory IgA in complex with Lactobacillus rhamnosus potentiates mucosal dendritic cell-mediated Treg cell differentiation via TLR regulatory proteins, RALDH2 and secretion of IL-10 and TGF-β

PubMed Central

Mikulic, Josip; Longet, Stéphanie; Favre, Laurent; Benyacoub, Jalil; Corthesy, Blaise

2017-01-01

The importance of secretory IgA in controlling the microbiota is well known, yet how the antibody affects the perception of the commensals by the local immune system is still poorly defined. We have previously shown that the transport of secretory IgA in complex with bacteria across intestinal microfold cells results in an association with dendritic cells in Peyer’s patches. However, the consequences of such an interaction on dendritic cell conditioning have not been elucidated. In this study, we analyzed the impact of the commensal Lactobacillus rhamnosus, alone or associated with secretory IgA, on the responsiveness of dendritic cells freshly recovered from mouse Peyer’s patches, mesenteric lymph nodes, and spleen. Lactobacillus rhamnosus-conditioned mucosal dendritic cells are characterized by increased expression of Toll-like receptor regulatory proteins [including single immunoglobulin interleukin-1 receptor-related molecule, suppressor of cytokine signaling 1, and Toll-interacting molecule] and retinaldehyde dehydrogenase 2, low surface expression of co-stimulatory markers, high anti- versus pro-inflammatory cytokine production ratios, and induction of T regulatory cells with suppressive function. Association with secretory IgA enhanced the anti-inflammatory/regulatory Lactobacillus rhamnosus-induced conditioning of mucosal dendritic cells, particularly in Peyer’s patches. At the systemic level, activation of splenic dendritic cells exposed to Lactobacillus rhamnosus was partially dampened upon association with secretory IgA. These data suggest that secretory IgA, through coating of commensal bacteria, contributes to the conditioning of mucosal dendritic cells toward tolerogenic profiles essential for the maintenance of intestinal homeostasis. PMID:26972771

Immediate Early Genes Anchor a Biological Pathway of Proteins Required for Memory Formation, Long-Term Depression and Risk for Schizophrenia

PubMed Central

Marballi, Ketan K.; Gallitano, Amelia L.

2018-01-01

While the causes of myriad medical and infectious illnesses have been identified, the etiologies of neuropsychiatric illnesses remain elusive. This is due to two major obstacles. First, the risk for neuropsychiatric disorders, such as schizophrenia, is determined by both genetic and environmental factors. Second, numerous genes influence susceptibility for these illnesses. Genome-wide association studies have identified at least 108 genomic loci for schizophrenia, and more are expected to be published shortly. In addition, numerous biological processes contribute to the neuropathology underlying schizophrenia. These include immune dysfunction, synaptic and myelination deficits, vascular abnormalities, growth factor disruption, and N-methyl-D-aspartate receptor (NMDAR) hypofunction. However, the field of psychiatric genetics lacks a unifying model to explain how environment may interact with numerous genes to influence these various biological processes and cause schizophrenia. Here we describe a biological cascade of proteins that are activated in response to environmental stimuli such as stress, a schizophrenia risk factor. The central proteins in this pathway are critical mediators of memory formation and a particular form of hippocampal synaptic plasticity, long-term depression (LTD). Each of these proteins is also implicated in schizophrenia risk. In fact, the pathway includes four genes that map to the 108 loci associated with schizophrenia: GRIN2A, nuclear factor of activated T-cells (NFATc3), early growth response 1 (EGR1) and NGFI-A Binding Protein 2 (NAB2); each of which contains the “Index single nucleotide polymorphism (SNP)” (most SNP) at its respective locus. Environmental stimuli activate this biological pathway in neurons, resulting in induction of EGR immediate early genes: EGR1, EGR3 and NAB2. We hypothesize that dysfunction in any of the genes in this pathway disrupts the normal activation of Egrs in response to stress. This may result in

Traffic to the malaria parasite food vacuole: a novel pathway involving a phosphatidylinositol 3-phosphate-binding protein.

PubMed

McIntosh, Michael T; Vaid, Ankush; Hosgood, H Dean; Vijay, Justin; Bhattacharya, Anindita; Sahani, Mayurbhai H; Baevova, Pavlina; Joiner, Keith A; Sharma, Pushkar

2007-04-13

Phosphatidylinositol 3-phosphate (PI3P) is a key ligand for recruitment of endosomal regulatory proteins in higher eukaryotes. Subsets of these endosomal proteins possess a highly selective PI3P binding zinc finger motif belonging to the FYVE domain family. We have identified a single FYVE domain-containing protein in Plasmodium falciparum which we term FCP. Expression and mutagenesis studies demonstrate that key residues are involved in specific binding to PI3P. In contrast to FYVE proteins in other organisms, endogenous FCP localizes to a lysosomal compartment, the malaria parasite food vacuole (FV), rather than to cytoplasmic endocytic organelles. Transfections of deletion mutants further indicate that FCP is essential for trophozoite and FV maturation and that it traffics to the FV via a novel constitutive cytoplasmic to vacuole targeting pathway. This newly discovered pathway excludes the secretory pathway and is directed by a C-terminal 44-amino acid peptide domain. We conclude that an FYVE protein that might be expected to participate in vesicle targeting in the parasite cytosol instead has a vital and functional role in the malaria parasite FV.

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles

PubMed Central

Woo, Sang Su; James, Declan J.; Martin, Thomas F. J.

2017-01-01

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. PMID:28100639

Ubiquitylation and the Fanconi Anemia Pathway

PubMed Central

Garner, Elizabeth; Smogorzewska, Agata

2012-01-01

The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic stability. PMID:21605559

EARLY LIFE RISKS, ANTISOCIAL TENDENCIES, AND PRETEEN DELINQUENCY.

PubMed

Staff, Jeremy; Whichard, Corey; Siennick, Sonja; Maggs, Jennifer

2015-11-01

Early age-of-onset delinquency and substance use confer a major risk for continued criminality, alcohol and drug abuse, and other serious difficulties throughout the life course. Our objective is to examine the developmental roots of preteen delinquency and substance use. Using nationally representative longitudinal data from the UK Millennium Cohort Study ( n = 13,221), we examine the influence of early childhood developmental and family risks on latent pathways of antisocial tendencies from ages 3 to 7, and the influence of those pathways on property crime and substance use by age 11. We identified a normative, non-antisocial pathway; a pathway marked by oppositional behavior and fighting; a pathway marked by impulsivity and inattention; and a rare pathway characterized by a wide range of antisocial tendencies. Children with developmental and family risks that emerged by age 3-specifically difficult infant temperament, low cognitive ability, weak parental closeness, and disadvantaged family background-face increased odds of antisocial tendencies. There is minimal overlap between the risk factors for early antisocial tendencies and those for preteen delinquency. Children on an antisocial pathway are more likely to engage in preteen delinquency and substance use by age 11, even after accounting for early life risk factors.

EARLY LIFE RISKS, ANTISOCIAL TENDENCIES, AND PRETEEN DELINQUENCY*

PubMed Central

Staff, Jeremy; Whichard, Corey; Siennick, Sonja; Maggs, Jennifer

2015-01-01

Early age-of-onset delinquency and substance use confer a major risk for continued criminality, alcohol and drug abuse, and other serious difficulties throughout the life course. Our objective is to examine the developmental roots of preteen delinquency and substance use. Using nationally representative longitudinal data from the UK Millennium Cohort Study (n = 13,221), we examine the influence of early childhood developmental and family risks on latent pathways of antisocial tendencies from ages 3 to 7, and the influence of those pathways on property crime and substance use by age 11. We identified a normative, non-antisocial pathway; a pathway marked by oppositional behavior and fighting; a pathway marked by impulsivity and inattention; and a rare pathway characterized by a wide range of antisocial tendencies. Children with developmental and family risks that emerged by age 3—specifically difficult infant temperament, low cognitive ability, weak parental closeness, and disadvantaged family background—face increased odds of antisocial tendencies. There is minimal overlap between the risk factors for early antisocial tendencies and those for preteen delinquency. Children on an antisocial pathway are more likely to engage in preteen delinquency and substance use by age 11, even after accounting for early life risk factors. PMID:26900167

Early storage lesions in apheresis platelets are induced by the activation of the integrin αIIbβ₃ and focal adhesion signaling pathways.

PubMed

Thiele, Thomas; Iuga, Cristina; Janetzky, Susann; Schwertz, Hansjorg; Gesell Salazar, Manuela; Fürll, Birgit; Völker, Uwe; Greinacher, Andreas; Steil, Leif

2012-12-05

Production and storage of platelet concentrates (PC) induce protein changes in platelets leading to impaired platelet function. This study aimed to identify signaling pathways involved in the development of early platelet storage lesions in apheresis-PCs stored in plasma or additive solution (PAS). Apheresis-PCs from four donors were stored in plasma or in PAS at 22°C (n=4 each). Platelets were analyzed at day 0 (production day) and after 1, 6 and 9 days of storage. Platelet response to agonists (TRAP, collagen, ADP) and to hypotonic shock decreased, CD62P expression increased in both storage media over time. Using DIGE 1550 protein spots were monitored and compared to baseline values at day 0. Platelets in plasma displayed changes in 352 spots (166/day 1, 263/day 6 and 201/day 9); in PAS 325 spots changed (202/day 1, 221/day 6, 200/day 9). LC-ESI-MS/MS analysis of 405 platelet proteins revealed 32 proteins changed during storage in plasma (9/day 1, 15/day 6 and 26/day 9) and 28 in PAS (5/day 1, 20/day 6, 26/day 9). Ingenuity pathway analysis found integrin-αII(b)β(3) and focal adhesion signaling pathways involved in early alterations, being confirmed by Western blotting. Corresponding mRNAs in platelets were identified by next generation sequencing for 84 changed proteins. Integrin-αII(b)β(3) and focal adhesion signaling cause irreversible early storage lesions in apheresis platelets. This article is part of a Special Issue entitled: Integrated omics. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of threonine on secretory immune system using a chicken intestinal ex vivo model with lipopolysaccharide challenge

USDA-ARS?s Scientific Manuscript database

Secretory IgA (sIgA) and its transcytosis receptor, polymeric immunoglobulin receptor (pIgR), along with mucus, form the first lines of intestinal defense. Threonine (Thr) is a major constituent component of intestinal mucins and IgA, which are highly secreted under lipopolysaccharide (LPS) induced ...

THE LONG REACH OF EDUCATION: EARLY RETIREMENT.

PubMed

Venti, Steven; Wise, David A

2015-12-01

The goal of this paper is to draw attention to the long lasting effect of education on economic outcomes. We use the relationship between education and two routes to early retirement - the receipt of Social Security Disability Insurance (DI) and the early claiming of Social Security retirement benefits - to illustrate the long-lasting influence of education. We find that for both men and women with less than a high school degree the median DI participation rate is 6.6 times the participation rate for those with a college degree or more. Similarly, men and women with less than a high school education are over 25 percentage points more likely to claim Social Security benefits early than those with a college degree or more. We focus on four critical "pathways" through which education may indirectly influence early retirement - health, employment, earnings, and the accumulation of assets. We find that for women health is the dominant pathway through which education influences DI participation. For men, the health, earnings, and wealth pathways are of roughly equal magnitude. For both men and women the principal channel through which education influences early Social Security claiming decisions is the earnings pathway. We also consider the direct effect of education that does not operate through these pathways. The direct effect of education is much greater for early claiming of Social Security benefits than for DI participation, accounting for 72 percent of the effect of education for men and 67 percent for women. For women the direct effect of education on DI participation is not statistically significant, suggesting that the total effect may be through the four pathways.

Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells.

PubMed Central

Ortega, E; Schweitzer-Stenner, R; Pecht, I

1988-01-01

Three biologically active monoclonal antibodies (mAbs) specific for the monovalent, high-affinity membrane receptor for IgE (Fc epsilon R) were employed in analysing the secretory response of mast cells of the RBL-2H3 line to crosslinking of their Fc epsilon R. All three mAbs (designated F4, H10 and J17) compete with each other and with IgE for binding to the Fc epsilon R. Their stoichiometry of binding is 1 Fab:1 Fc epsilon R, hence, the intact mAbs can aggregate the Fc epsilon Rs to dimers only. Since all three mAbs induce secretion, we conclude that Fc epsilon R dimers constitute a sufficient 'signal element' for secretion of mediators for RBL-2H3 cells. The secretory dose-response of the cells to these three mAbs are, however, markedly different: F4 caused rather high secretion, reaching almost 80% of the cells' content, while J17 and H10 induced release of only 30-40% mediators content. Both the intrinsic affinities and equilibrium constants for the receptor dimerization were derived from analysis of binding data of the Fab fragments and intact mAbs. These parameters were used to compute the extent of Fc epsilon R dimerization caused by each of the antibodies. However, the different secretory responses to the three mAbs could not be rationalized simply in terms of the extent of Fc epsilon R dimerization which they produce. This suggests that it is not only the number of crosslinked Fc epsilon Rs which determines the magnitude of secretion-causing signal, but rather other constraints imposed by each individual mAb are also important.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2977332

Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C

PubMed Central

Müller, Thomas; Rasool, Insha; Heinz-Erian, Peter; Mildenberger, Eva; Hülstrunk, Christian; Müller, Andreas; Michaud, Laurent; Koot, Bart G P; Ballauff, Antje; Vodopiutz, Julia; Rosipal, Stefan; Petersen, Britt-Sabina; Franke, Andre; Fuchs, Irene; Witt, Heiko; Zoller, Heinz; Janecke, Andreas R; Visweswariah, Sandhya S

2016-01-01

Objective Congenital sodium diarrhoea (CSD) refers to a form of secretory diarrhoea with intrauterine onset and high faecal losses of sodium without congenital malformations. The molecular basis for CSD remains unknown. We clinically characterised a cohort of infants with CSD and set out to identify disease-causing mutations by genome-wide genetic testing. Design We performed whole-exome sequencing and chromosomal microarray analyses in 4 unrelated patients, followed by confirmatory Sanger sequencing of the likely disease-causing mutations in patients and in their family members, followed by functional studies. Results We identified novel de novo missense mutations in GUCY2C, the gene encoding receptor guanylate cyclase C (GC-C) in 4 patients with CSD. One patient developed severe, early-onset IBD and chronic arthritis at 4 years of age. GC-C is an intestinal brush border membrane-bound guanylate cyclase, which functions as receptor for guanylin, uroguanylin and Escherichia coli heat-stable enterotoxin. Mutations in GUCY2C were present in different intracellular domains of GC-C, and were activating mutations that enhanced intracellular cyclic guanosine monophosphate accumulation in a ligand-independent and ligand-stimulated manner, following heterologous expression in HEK293T cells. Conclusions Dominant gain-of-function GUCY2C mutations lead to elevated intracellular cyclic guanosine monophosphate levels and could explain the chronic diarrhoea as a result of decreased intestinal sodium and water absorption and increased chloride secretion. Thus, mutations in GUCY2C indicate a role for this receptor in the pathogenesis of sporadic CSD. PMID:25994218

Assessing the secretory capacity of pancreatic acinar cells.

PubMed

Geron, Erez; Schejter, Eyal D; Shilo, Ben-Zion

2014-08-28

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex vivo culture will represent the in vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

Pathways to fraction learning: Numerical abilities mediate the relation between early cognitive competencies and later fraction knowledge.

PubMed

Ye, Ai; Resnick, Ilyse; Hansen, Nicole; Rodrigues, Jessica; Rinne, Luke; Jordan, Nancy C

2016-12-01

The current study investigated the mediating role of number-related skills in the developmental relationship between early cognitive competencies and later fraction knowledge using structural equation modeling. Fifth-grade numerical skills (i.e., whole number line estimation, non-symbolic proportional reasoning, multiplication, and long division skills) mapped onto two distinct factors: magnitude reasoning and calculation. Controlling for participants' (N=536) demographic characteristics, these two factors fully mediated relationships between third-grade general cognitive competencies (attentive behavior, verbal and nonverbal intellectual abilities, and working memory) and sixth-grade fraction knowledge (concepts and procedures combined). However, specific developmental pathways differed by type of fraction knowledge. Magnitude reasoning ability fully mediated paths from all four cognitive competencies to knowledge of fraction concepts, whereas calculation ability fully mediated paths from attentive behavior and verbal ability to knowledge of fraction procedures (all with medium to large effect sizes). These findings suggest that there are partly overlapping, yet distinct, developmental pathways from cognitive competencies to general fraction knowledge, fraction concepts, and fraction procedures. Copyright © 2016 Elsevier Inc. All rights reserved.

Secretory production of tetrameric native full-length streptavidin with thermostability using Streptomyces lividans as a host.

PubMed

Noda, Shuhei; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

2015-01-13

Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin system have been widely studied. Streptavidin is generally produced using protein expression in Escherichia coli. In the present study, the secretory production of streptavidin was carried out using Streptomyces lividans as a host. In this study, we used the gene encoding native full-length streptavidin, whereas the core region is generally used for streptavidin production in E. coli. Tetrameric streptavidin composed of native full-length streptavidin monomers was successfully secreted in the culture supernatant of S. lividans transformants, and had specific biotin binding affinity as strong as streptavidin produced by E. coli. The amount of Sav using S. lividans was about 9 times higher than using E. coli. Surprisingly, streptavidin produced by S. lividans exhibited affinity to biotin after boiling, despite the fact that tetrameric streptavidin is known to lose its biotin binding ability after brief boiling. We successfully produced a large amount of tetrameric streptavidin as a secretory-form protein with unique thermotolerance.

Host-derived apolipoproteins play comparable roles with viral secretory proteins Erns and NS1 in the infectious particle formation of Flaviviridae

PubMed Central

Ono, Chikako; Shiokawa, Mai; Mori, Hiroyuki; Uemura, Kentaro; Yamamoto, Satomi; Okamoto, Toru; Suzuki, Ryosuke; Yoshii, Kentaro; Kurosu, Takeshi; Igarashi, Manabu; Aoki, Hiroshi; Sakoda, Yoshihiro

2017-01-01

Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein Erns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of Erns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded Erns and NS1 in the formation of infectious particles. We examined whether Erns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and non-hepatic 293T cells. We found that exogenous expression of either Erns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an Erns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of Erns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while Erns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene. PMID:28644867

Early activation of deleterious molecular pathways in the kidney in experimental heart failure with atrial remodeling.

PubMed

Ichiki, Tomoko; Huntley, Brenda K; Harty, Gail J; Sangaralingham, S Jeson; Burnett, John C

2017-05-01

Heart failure (HF) is a major health problem with worsening outcomes when renal impairment is present. Therapeutics for early phase HF may be effective for cardiorenal protection, however the detailed characteristics of the kidney in early-stage HF (ES-HF), and therefore treatment for potential renal protection, are poorly defined. We sought to determine the gene and protein expression profiles of specific maladaptive pathways of ES-HF in the kidney and heart. Experimental canine ES-HF, characterized by de-novo HF with atrial remodeling but not ventricular fibrosis, was induced by right ventricular pacing for 10 days. Kidney cortex (KC), medulla (KM), left ventricle (LV), and left atrial (LA) tissues from ES-HF versus normal canines ( n  = 4 of each) were analyzed using RT-PCR microarrays and protein assays to assess genes and proteins related to inflammation, renal injury, apoptosis, and fibrosis. ES-HF was characterized by increased circulating natriuretic peptides and components of the renin-angiotensin-aldosterone system and decreased sodium and water excretion with mild renal injury and up-regulation of CNP and renin genes in the kidney. Compared to normals, widespread genes, especially genes of the inflammatory pathways, were up-regulated in KC similar to increases seen in LA Protein expressions related to inflammatory cytokines were also augmented in the KC Gene and protein changes were less prominent in the LV and KM The ES-HF displayed mild renal injury with widespread gene changes and increased inflammatory cytokines. These changes may provide important clues into the pathophysiology of ES-HF and for therapeutic molecular targets in the kidney of ES-HF. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Axonal damage and loss of connectivity in nigrostriatal and mesolimbic dopamine pathways in early Parkinson's disease.

PubMed

Caminiti, Silvia Paola; Presotto, Luca; Baroncini, Damiano; Garibotto, Valentina; Moresco, Rosa Maria; Gianolli, Luigi; Volonté, Maria Antonietta; Antonini, Angelo; Perani, Daniela

2017-01-01

A progressive loss of dopamine neurons in the substantia nigra (SN) is considered the main feature of idiopathic Parkinson's disease (PD). Recent neuropathological evidence however suggests that the axons of the nigrostriatal dopaminergic system are the earliest target of α-synuclein accumulation in PD, thus the principal site for vulnerability. Whether this applies to in vivo PD, and also to the mesolimbic system has not been investigated yet. We used [ 11 C]FeCIT PET to measure presynaptic dopamine transporter (DAT) activity in both nigrostriatal and mesolimbic systems, in 36 early PD patients (mean disease duration in months ± SD 21.8 ± 10.7) and 14 healthy controls similar for age. We also performed anatomically-driven partial correlation analysis to evaluate possible changes in the connectivity within both the dopamine networks at an early clinical phase. In the nigrostriatal system, we found a severe DAT reduction in the afferents to the dorsal putamen (DPU) (η 2  = 0.84), whereas the SN was the less affected region (η 2  = 0.31). DAT activity in the ventral tegmental area (VTA) and the ventral striatum (VST) were also reduced in the patient group, but to a lesser degree (VST η 2  = 0.71 and VTA η 2  = 0.31). In the PD patients compared to the controls, there was a marked decrease in dopamine network connectivity between SN and DPU nodes, supporting the significant derangement in the nigrostriatal pathway. These results suggest that neurodegeneration in the dopamine pathways is initially more prominent in the afferent axons and more severe in the nigrostriatal system. Considering PD as a disconnection syndrome starting from the axons, it would justify neuroprotective interventions even if patients have already manifested clinical symptoms.

How do secretory products cross the plant cell wall to be released? A new hypothesis involving cyclic mechanical actions of the protoplast

PubMed Central

Paiva, Elder Antônio Sousa

2016-01-01

Background In plants, the products of secretory activity leave the protoplast and cross the plasma membrane by means of transporters, fusion with membranous vesicles or, less commonly, as result of disintegration of the cell. These mechanisms do not address an intriguing question: How do secretory products cross the cell wall? Furthermore, how do these substances reach the external surface of the plant body? Such diverse substances as oils, polysaccharides or nectar are forced to cross the cell wall and, in fact, do so. How are chemical materials that are repelled by the cell wall or that are sufficiently viscous to not cross passively released from plant cells? Scope and Conclusions I propose a cell-cycle model developed based on observations of different secreting systems, some unpublished results and an extensive literature review, aiming to understand the processes involved in both the secretory process and the release of secretion products. In the absence of facilitated diffusion, a mechanical action of the protoplast is necessary to ensure that some substances can cross the cell wall. The mechanical action of the protoplast, in the form of successive cycles of contraction and expansion, causes the material accumulated in the periplasmic space to cross the cell wall and the cuticle. This action is particularly relevant for the release of lipids, resins and highly viscous hydrophilic secretions. The proposed cell-cycle model and the statements regarding exudate release will also apply to secretory glands not elaborated upon here. Continuous secretion of several days, as observed in extrafloral nectaries, salt glands and some mucilage-producing glands, is only possible because the process is cyclical. PMID:26929201

Community Structure Analysis of Transcriptional Networks Reveals Distinct Molecular Pathways for Early- and Late-Onset Temporal Lobe Epilepsy with Childhood Febrile Seizures

PubMed Central

Moreira-Filho, Carlos Alberto; Bando, Silvia Yumi; Bertonha, Fernanda Bernardi; Iamashita, Priscila; Silva, Filipi Nascimento; Costa, Luciano da Fontoura; Silva, Alexandre Valotta; Castro, Luiz Henrique Martins; Wen, Hung-Tzu

2015-01-01

Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolonged febrile seizures in early childhood (FS) constitute an initial precipitating insult (IPI) commonly associated with mesial temporal lobe epilepsy (MTLE). FS-MTLE patients may have early disease onset, i.e. just after the IPI, in early childhood, or late-onset, ranging from mid-adolescence to early adult life. The mechanisms governing early (E) or late (L) disease onset are largely unknown. In order to unveil the molecular pathways underlying E and L subtypes of FS-MTLE we investigated global gene expression in hippocampal CA3 explants of FS-MTLE patients submitted to hippocampectomy. Gene coexpression networks (GCNs) were obtained for the E and L patient groups. A network-based approach for GCN analysis was employed allowing: i) the visualization and analysis of differentially expressed (DE) and complete (CO) - all valid GO annotated transcripts - GCNs for the E and L groups; ii) the study of interactions between all the system’s constituents based on community detection and coarse-grained community structure methods. We found that the E-DE communities with strongest connection weights harbor highly connected genes mainly related to neural excitability and febrile seizures, whereas in L-DE communities these genes are not only involved in network excitability but also playing roles in other epilepsy-related processes. Inversely, in E-CO the strongly connected communities are related to compensatory pathways (seizure inhibition, neuronal survival and responses to stress conditions) while in L-CO these communities harbor several genes related to pro-epileptic effects, seizure-related mechanisms and vulnerability to epilepsy. These results fit the concept, based on fMRI and behavioral studies, that early onset epilepsies, although impacting more severely the hippocampus, are associated to compensatory mechanisms, while in late MTLE development the brain is less able to

Clonorchis sinensis excretory-secretory products promote the migration and invasion of cholangiocarcinoma cells by activating the integrin β4-FAK/Src signaling pathway.

PubMed

Pak, Jhang Ho; Bashir, Qudsia; Kim, In Ki; Hong, Sung-Jong; Maeng, Sejung; Bahk, Young Yil; Kim, Tong-Soo

2017-06-01

Cholangiocarcinoma (CCA) is a slow-growing but highly metastatic cancer. Its metastatic potential largely explains its high mortality rate. A recognized risk factor for CCA development is infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis. We previously reported that the excretory-secretory products (ESPs) of C. sinensis promoted the three-dimensional aggregation and invasion of CCA cells. In the present study, a quantitative real-time PCR array of extracellular matrix (ECM) and adhesion molecules was used to examine the regulatory mechanism of ESP-mediated CCA cell migration and invasion. In particular, the expression levels of integrin α isoforms and β4 were upregulated in response to ESPs. Increased expression of integrin β4 was probably correlated with activation of focal adhesion kinase (FAK) and the steroid receptor coactivator (Src) family kinase and the subsequent activation of two downstream focal adhesion molecules, paxillin and vinculin. Moreover, inhibition of FAK/Src activation reduced paxillin and vinculin phosphorylation and attenuated ESP-induced CCA cell migration and invasion. These findings suggest that the integrin β4-FAK/Src signaling axis may play a crucial role in clonorchiasis-associated CCA metastasis during tumor progression. Copyright © 2017 Elsevier B.V. All rights reserved.

Origin and evolution of group XI secretory phospholipase A2 from flax (Linum usitatissimum) based on phylogenetic analysis of conserved domains.

PubMed

Gupta, Payal; Saini, Raman; Dash, Prasanta K

2017-07-01

Phospholipase A 2 (PLA 2 ) belongs to class of lipolytic enzymes (EC 3.1.1.4). Lysophosphatidic acid (LPA) and free fatty acids (FFAs) are the products of PLA 2 catalyzed hydrolysis of phosphoglycerides at sn-2 position. LPA and FFA that act as second mediators involved in the development and maturation of plants and animals. Mining of flax genome identified two phospholipase A 2 encoding genes, viz., LusPLA 2 I and LusPLA 2 II (Linum usitatissimum secretory phospholipase A 2 ). Molecular simulation of LusPLA 2 s with already characterized plant sPLA 2 s revealed the presence of conserved motifs and signature domains necessary to classify them as secretory phospholipase A 2 . Phylogenetic analysis of flax sPLA 2 with representative sPLA 2 s from other organisms revealed that they evolved rapidly via gene duplication/deletion events and shares a common ancestor. Our study is the first report of detailed phylogenetic analysis for secretory phospholipase A 2 in flax. Comparative genomic analysis of two LusPLA 2 s with earlier reported plant sPLA 2 s, based on their gene architectures, sequence similarities, and domain structures are presented elucidating the uniqueness of flax sPLA 2 .

The Akt signaling pathway

PubMed Central

Madhunapantula, SubbaRao V; Mosca, Paul J

2011-01-01

Studies using cultured melanoma cells and patient tumor biopsies have demonstrated deregulated PI3 kinase-Akt3 pathway activity in ∼70% of melanomas. Furthermore, targeting Akt3 and downstream PRAS40 has been shown to inhibit melanoma tumor development in mice. Although these preclinical studies and several other reports using small interfering RNAs and pharmacological agents targeting key members of this pathway have been shown to retard melanoma development, analysis of early Phase I and Phase II clinical trials using pharmacological agents to target this pathway demonstrate the need for (1) selection of patients whose tumors have PI3 kinase-Akt pathway deregulation, (2) further optimization of therapeutic agents for increased potency and reduced toxicity, (3) the identification of additional targets in the same pathway or in other signaling cascades that synergistically inhibit the growth and progression of melanoma, and (4) better methods for targeted delivery of pharmaceutical agents inhibiting this pathway. In this review we discuss key potential targets in PI3K-Akt3 signaling, the status of pharmacological agents targeting these proteins, drugs under clinical development, and strategies to improve the efficacy of therapeutic agents targeting this pathway. PMID:22157148

Distinct roles of prolactin, epidermal growth factor, and glucocorticoids in β-casein secretion pathway in lactating mammary epithelial cells.

PubMed

Kobayashi, Ken; Oyama, Shoko; Kuki, Chinatsu; Tsugami, Yusaku; Matsunaga, Kota; Suzuki, Takahiro; Nishimura, Takanori

2017-01-15

Beta-casein is a secretory protein contained in milk. Mammary epithelial cells (MECs) synthesize and secrete β-casein during lactation. However, it remains unclear how the β-casein secretion pathway is developed after parturition. In this study, we focused on prolactin (PRL), epidermal growth factor (EGF), and glucocorticoids, which increase in blood plasma and milk around parturition. MECs cultured with PRL, EGF and dexamethasone (DEX: glucocorticoid analog) developed the β-casein secretion pathway. In the absence of PRL, MECs hardly expressed β-casein. EGF enhanced the expression and secretion of β-casein in the presence of PRL and DEX. DEX treatment rapidly increased secreted β-casein concurrent with enhancing β-casein expression. DEX also up-regulated the expression of SNARE proteins, such as SNAP-23, VAMP-8 and Syntaxin-12. Furthermore, PRL and DEX regulated the expression ratio of α s1 -, β- and κ-casein. These results indicate that PRL, EGF and glucocorticoids have distinct roles in the establishment of β-casein secretion pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Investigating dysregulated pathways in Staphylococcus aureus (SA) exposed macrophages based on pathway interaction network.

PubMed

Zhou, Wei; Zhang, Yan; Li, Yue-Hua; Wang, Shuang; Zhang, Jing-Jing; Zhang, Cui-Xia; Zhang, Zhi-Sheng

2017-02-01

This work aimed to identify dysregulated pathways for Staphylococcus aureus (SA) exposed macrophages based on pathway interaction network (PIN). The inference of dysregulated pathways was comprised of four steps: preparing gene expression data, protein-protein interaction (PPI) data and pathway data; constructing a PIN dependent on the data and Pearson correlation coefficient (PCC); selecting seed pathway from PIN by computing activity score for each pathway according to principal component analysis (PCA) method; and investigating dysregulated pathways in a minimum set of pathways (MSP) utilizing seed pathway and the area under the receiver operating characteristics curve (AUC) index implemented in support vector machines (SVM) model. A total of 20,545 genes, 449,833 interactions and 1189 pathways were obtained in the gene expression data, PPI data and pathway data, respectively. The PIN was consisted of 8388 interactions and 1189 nodes, and Respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins was identified as the seed pathway. Finally, 15 dysregulated pathways in MSP (AUC=0.999) were obtained for SA infected samples, such as Respiratory electron transport and DNA Replication. We have identified 15 dysregulated pathways for SA infected macrophages based on PIN. The findings might provide potential biomarkers for early detection and therapy of SA infection, and give insights to reveal the molecular mechanism underlying SA infections. However, how these dysregulated pathways worked together still needs to be studied. Copyright © 2016 Elsevier Ltd. All rights reserved.

Predicting pathway cross-talks in ankylosing spondylitis through investigating the interactions among pathways.

PubMed

Gu, Xiang; Liu, Cong-Jian; Wei, Jian-Jie

2017-11-13

Given that the pathogenesis of ankylosing spondylitis (AS) remains unclear, the aim of this study was to detect the potentially functional pathway cross-talk in AS to further reveal the pathogenesis of this disease. Using microarray profile of AS and biological pathways as study objects, Monte Carlo cross-validation method was used to identify the significant pathway cross-talks. In the process of Monte Carlo cross-validation, all steps were iterated 50 times. For each run, detection of differentially expressed genes (DEGs) between two groups was conducted. The extraction of the potential disrupted pathways enriched by DEGs was then implemented. Subsequently, we established a discriminating score (DS) for each pathway pair according to the distribution of gene expression levels. After that, we utilized random forest (RF) classification model to screen out the top 10 paired pathways with the highest area under the curve (AUCs), which was computed using 10-fold cross-validation approach. After 50 bootstrap, the best pairs of pathways were identified. According to their AUC values, the pair of pathways, antigen presentation pathway and fMLP signaling in neutrophils, achieved the best AUC value of 1.000, which indicated that this pathway cross-talk could distinguish AS patients from normal subjects. Moreover, the paired pathways of SAPK/JNK signaling and mitochondrial dysfunction were involved in 5 bootstraps. Two paired pathways (antigen presentation pathway and fMLP signaling in neutrophil, as well as SAPK/JNK signaling and mitochondrial dysfunction) can accurately distinguish AS and control samples. These paired pathways may be helpful to identify patients with AS for early intervention.

αS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form

PubMed Central

2010-01-01

Background Caseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known. Results In this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues. The majority of both αS1- and β-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature β-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature αS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of αS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of αS1-casein with membranes. Conclusions These experiments reveal for the first time the existence of a membrane-associated form of αS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that αS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway. PMID:20704729

Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter.

PubMed

Hong, Seung-Pyo; Seip, John; Walters-Pollak, Dana; Rupert, Ross; Jackson, Raymond; Xue, Zhixiong; Zhu, Quinn

2012-02-01

Oleaginous yeast Yarrowia lipolytica is an important host for the production of lipid-derived compounds or heterologous proteins. Selection of strong promoters and effective expression systems is critical for heterologous protein secretion. To search for a strong promoter in Y. lipolytica, activities of FBA1, TDH1 and GPM1 promoters were compared to that of TEF1 promoter by constructing GUS reporter fusions. The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively. The FBA1IN promoter (FBA1 sequence of -826 to +169) containing an intron (+64 to +165) showed five-fold higher expression than the FBA1 promoter (-831 to -1). The transcriptional enhancement by the 5'-region within the FBA1 gene was confirmed by GPM1::FBA1 chimeric promoter construction. Using the strong FBA1IN promoter, four different S. cerevisiae SUC2 expression cassettes were tested for the SUC+ phenotype in Y. lipolytica. Functional invertase secretion was facilitated by the Xpr2 prepro-region with an additional 13 amino acids of mature Xpr2, or by the native Suc2 signal sequence. However, these two secretory signals in tandem, or the mature Suc2 with no secretory signal, did not direct secretion of functional invertase. Unlike previously reported Y. lipolytica SUC+ strains, our engineered stains secreted most of invertase into the medium. Copyright © 2011 John Wiley & Sons, Ltd.

Effects of sexually dimorphic growth hormone secretory patterns on arachidonic acid metabolizing enzymes in rodent heart

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

Zhang, Furong; Yu, Xuming; He, Chunyan

The arachidonic acid (AA) metabolizing enzymes are the potential therapeutic targets of cardiovascular diseases (CVDs). As sex differences have been shown in the risk and outcome of CVDs, we investigated the regulation of heart AA metabolizing enzymes (COXs, LOXs, and CYPs) by sex-dependent growth hormone (GH) secretory patterns. The pulsatile (masculine) GH secretion at a physiological concentration decreased CYP1A1 and CYP2J3 mRNA levels more efficiently in the H9c2 cells compared with the constant (feminine) GH secretion; however, CYP1B1 mRNA levels were higher following the pulsatile GH secretion. Sex differences in CYP1A1, CYP1B1, and CYP2J11 mRNA levels were observed in bothmore » the wild-type and GHR deficient mice. No sex differences in the mRNA levels of COXs, LOXs, or CYP2E1 were observed in the wild-type mice. The constant GH infusion induced heart CYP1A1 and CYP2J11, and decreased CYP1B1 in the male C57/B6 mice constantly infused with GH (0.4 μg/h, 7 days). The activity of rat Cyp2j3 promoter was inhibited by the STAT5B protein, but was activated by C/EBPα (CEBPA). Compared with the constant GH administration, the levels of the nuclear phosphorylated STAT5B protein and its binding to the rat Cyp2j3 promoter were higher following the pulsatile GH administration. The constant GH infusion decreased the binding of the nuclear phosphorylated STAT5B protein to the mouse Cyp2j11 promoter. The data suggest the sexually dimorphic transcription of heart AA metabolizing enzymes, which might alter the risk and outcome of CVDs. GHR-STAT5B signal transduction pathway may be involved in the sex difference in heart CYP2J levels. - Highlights: • The transcription of heart Cyp1a1, Cyp1b1 and Cyp2j genes is sexually dimorphic. • There are no sex differences in the mRNA levels of heart COXs, LOXs, or CYP2E1. • GHR-STAT5B pathway is involved in sexually dimorphic transcription of heart Cpy2j genes. • Heart CYPs-mediated metabolism pathway of arachidonic acid

Oxygen and hydrogen peroxide in the early evolution of life on earth: in silico comparative analysis of biochemical pathways.

PubMed

Slesak, Ireneusz; Slesak, Halina; Kruk, Jerzy

2012-08-01

In the Universe, oxygen is the third most widespread element, while on Earth it is the most abundant one. Moreover, oxygen is a major constituent of all biopolymers fundamental to living organisms. Besides O(2), reactive oxygen species (ROS), among them hydrogen peroxide (H(2)O(2)), are also important reactants in the present aerobic metabolism. According to a widely accepted hypothesis, aerobic metabolism and many other reactions/pathways involving O(2) appeared after the evolution of oxygenic photosynthesis. In this study, the hypothesis was formulated that the Last Universal Common Ancestor (LUCA) was at least able to tolerate O(2) and detoxify ROS in a primordial environment. A comparative analysis was carried out of a number of the O(2)-and H(2)O(2)-involving metabolic reactions that occur in strict anaerobes, facultative anaerobes, and aerobes. The results indicate that the most likely LUCA possessed O(2)-and H(2)O(2)-involving pathways, mainly reactions to remove ROS, and had, at least in part, the components of aerobic respiration. Based on this, the presence of a low, but significant, quantity of H(2)O(2) and O(2) should be taken into account in theoretical models of the early Archean atmosphere and oceans and the evolution of life. It is suggested that the early metabolism involving O(2)/H(2)O(2) was a key adaptation of LUCA to already existing weakly oxic zones in Earth's primordial environment.

THE LONG REACH OF EDUCATION: EARLY RETIREMENT

PubMed Central

Wise, David A.

2015-01-01

The goal of this paper is to draw attention to the long lasting effect of education on economic outcomes. We use the relationship between education and two routes to early retirement – the receipt of Social Security Disability Insurance (DI) and the early claiming of Social Security retirement benefits – to illustrate the long-lasting influence of education. We find that for both men and women with less than a high school degree the median DI participation rate is 6.6 times the participation rate for those with a college degree or more. Similarly, men and women with less than a high school education are over 25 percentage points more likely to claim Social Security benefits early than those with a college degree or more. We focus on four critical “pathways” through which education may indirectly influence early retirement – health, employment, earnings, and the accumulation of assets. We find that for women health is the dominant pathway through which education influences DI participation. For men, the health, earnings, and wealth pathways are of roughly equal magnitude. For both men and women the principal channel through which education influences early Social Security claiming decisions is the earnings pathway. We also consider the direct effect of education that does not operate through these pathways. The direct effect of education is much greater for early claiming of Social Security benefits than for DI participation, accounting for 72 percent of the effect of education for men and 67 percent for women. For women the direct effect of education on DI participation is not statistically significant, suggesting that the total effect may be through the four pathways. PMID:26664822

The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis.

PubMed

Durfee, Tim; Roe, Judith L; Sessions, R Allen; Inouye, Carla; Serikawa, Kyle; Feldmann, Kenneth A; Weigel, Detlef; Zambryski, Patricia C

2003-07-08

The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens. The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1. In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development. This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity. However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development. In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants. These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity.

Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation.

PubMed

Chen, Kevin G; Mallon, Barbara S; Johnson, Kory R; Hamilton, Rebecca S; McKay, Ronald D G; Robey, Pamela G

2014-05-01

Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We further highlight core signaling pathways that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyze emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Furthermore, based on the influence of cell-cell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids, which are pivotal steps needed for future clinical applications. Published by Elsevier B.V.

Horizontal Transfers and Gene Losses in the Phospholipid Pathway of Bartonella Reveal Clues about Early Ecological Niches

PubMed Central

Zhu, Qiyun; Kosoy, Michael; Olival, Kevin J.; Dittmar, Katharina

2014-01-01

Bartonellae are mammalian pathogens vectored by blood-feeding arthropods. Although of increasing medical importance, little is known about their ecological past, and host associations are underexplored. Previous studies suggest an influence of horizontal gene transfers in ecological niche colonization by acquisition of host pathogenicity genes. We here expand these analyses to metabolic pathways of 28 Bartonella genomes, and experimentally explore the distribution of bartonellae in 21 species of blood-feeding arthropods. Across genomes, repeated gene losses and horizontal gains in the phospholipid pathway were found. The evolutionary timing of these patterns suggests functional consequences likely leading to an early intracellular lifestyle for stem bartonellae. Comparative phylogenomic analyses discover three independent lineage-specific reacquisitions of a core metabolic gene—NAD(P)H-dependent glycerol-3-phosphate dehydrogenase (gpsA)—from Gammaproteobacteria and Epsilonproteobacteria. Transferred genes are significantly closely related to invertebrate Arsenophonus-, and Serratia-like endosymbionts, and mammalian Helicobacter-like pathogens, supporting a cellular association with arthropods and mammals at the base of extant Bartonella spp. Our studies suggest that the horizontal reacquisitions had a key impact on bartonellae lineage specific ecological and functional evolution. PMID:25106622

Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

PubMed Central

Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

2016-01-01

In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals’ endocrine system. PMID:27775083

Epithelial–mesenchymal transition-associated secretory phenotype predicts survival in lung cancer patients

PubMed Central

Reka, Ajaya Kumar; Chen, Guoan; Keshamouni, Venkateshwar G.

2014-01-01

In cancer cells, the process of epithelial–mesenchymal transition (EMT) confers migratory and invasive capacity, resistance to apoptosis, drug resistance, evasion of host immune surveillance and tumor stem cell traits. Cells undergoing EMT may represent tumor cells with metastatic potential. Characterizing the EMT secretome may identify biomarkers to monitor EMT in tumor progression and provide a prognostic signature to predict patient survival. Utilizing a transforming growth factor-β-induced cell culture model of EMT, we quantitatively profiled differentially secreted proteins, by GeLC-tandem mass spectrometry. Integrating with the corresponding transcriptome, we derived an EMT-associated secretory phenotype (EASP) comprising of proteins that were differentially upregulated both at protein and mRNA levels. Four independent primary tumor-derived gene expression data sets of lung cancers were used for survival analysis by the random survival forests (RSF) method. Analysis of 97-gene EASP expression in human lung adenocarcinoma tumors revealed strong positive correlations with lymph node metastasis, advanced tumor stage and histological grade. RSF analysis built on a training set (n = 442), including age, sex and stage as variables, stratified three independent lung cancer data sets into low-, medium- and high-risk groups with significant differences in overall survival. We further refined EASP to a 20 gene signature (rEASP) based on variable importance scores from RSF analysis. Similar to EASP, rEASP predicted survival of both adenocarcinoma and squamous carcinoma patients. More importantly, it predicted survival in the early-stage cancers. These results demonstrate that integrative analysis of the critical biological process of EMT provides mechanism-based and clinically relevant biomarkers with significant prognostic value. PMID:24510113

Proteomics of the human endometrial glandular epithelium and stroma from the proliferative and secretory phases of the menstrual cycle.

PubMed

Hood, Brian L; Liu, Baoquan; Alkhas, Addie; Shoji, Yutaka; Challa, Rusheeswar; Wang, Guisong; Ferguson, Susan; Oliver, Julie; Mitchell, Dave; Bateman, Nicholas W; Zahn, Christopher M; Hamilton, Chad A; Payson, Mark; Lessey, Bruce; Fazleabas, Asgerally T; Maxwell, G Larry; Conrads, Thomas P; Risinger, John I

2015-04-01

Despite its importance in reproductive biology and women's health, a detailed molecular-level understanding of the human endometrium is lacking. Indeed, no comprehensive studies have been undertaken to elucidate the important protein expression differences between the endometrial glandular epithelium and surrounding stroma during the proliferative and midsecretory phases of the menstrual cycle. We utilized laser microdissection to harvest epithelial cells and stromal compartments from proliferative and secretory premenopausal endometrial tissue and performed a global, quantitative mass spectrometry-based proteomics analysis. This analysis identified 1224 total proteins from epithelial cells, among which 318 were differentially abundant between the proliferative and secretory phases (q < 0.05), and 1005 proteins from the stromal compartments, 19 of which were differentially abundant between the phases (q < 0.05). Several proteins were chosen for validation by immunohistochemistry in an independent set of uterine tissues, including carboxypeptidase M, tenascin C, neprilysin, and ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3). ENPP3, which was elevated in epithelial glandular cells in the secretory phase, was confirmed to be elevated in midsecretory-phase baboon uterine lavage samples and also observed to have an N-linked glycosylated form that was not observed in the proliferative phase. This study provides a detailed view into the global proteomic alterations of the epithelial cells and stromal compartments of the cycling premenopausal endometrium. These proteomic alterations during endometrial remodeling provide a basis for numerous follow-up investigations on the function of these differentially regulated proteins and their role in reproductive biology and endometrial pathologies. © 2015 by the Society for the Study of Reproduction, Inc.

Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV-2 Microglial Cells.

PubMed

Lee, Jinyoung; Kang, Jung-Mi; Kim, Tae Im; Kim, Jong-Hyun; Sohn, Hae-Jin; Na, Byoung-Kuk; Shin, Ho-Joon

2017-03-01

Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV-2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV-2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL-1α and TNF-α. NfESP-induced IL-1α and TNF-α expression in BV-2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP-induced IL-1α and TNF-α production in BV-2 cells were effectively downregulated by inhibition of NF-kB and AP-1. These results collectively suggest that NfESP stimulates BV-2 cells to release IL-1α and TNF-α via NF-kB- and AP-1-dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

Monitoring the Secretory Behavior of the Rat Adrenal Medulla by High-Performance Liquid Chromatography-Based Catecholamine Assay from Slice Supernatants

PubMed Central

De Nardi, Frédéric; Lefort, Claudie; Bréard, Dimitri; Richomme, Pascal; Legros, Christian; Guérineau, Nathalie C.

2017-01-01

Catecholamine (CA) secretion from the adrenal medullary tissue is a key step of the adaptive response triggered by an organism to cope with stress. Whereas molecular and cellular secretory processes have been extensively studied at the single chromaffin cell level, data available for the whole gland level are much scarcer. We tackled this issue in rat by developing an easy to implement experimental strategy combining the adrenal acute slice supernatant collection with a high-performance liquid chromatography-based epinephrine and norepinephrine (NE) assay. This technique affords a convenient method for measuring basal and stimulated CA release from single acute slices, allowing thus to individually address the secretory function of the left and right glands. Our data point that the two glands are equally competent to secrete epinephrine and NE, exhibiting an equivalent epinephrine:NE ratio, both at rest and in response to a cholinergic stimulation. Nicotine is, however, more efficient than acetylcholine to evoke NE release. A pharmacological challenge with hexamethonium, an α3-containing nicotinic acetylcholine receptor antagonist, disclosed that epinephrine- and NE-secreting chromaffin cells distinctly expressed α3 nicotinic receptors, with a dominant contribution in NE cells. As such, beyond the novelty of CA assays from acute slice supernatants, our study contributes at refining the secretory behavior of the rat adrenal medullary tissue, and opens new perspectives for monitoring the release of other hormones and transmitters, especially those involved in the stress response. PMID:28993760

Linking Family Economic Hardship to Early Childhood Health: An Investigation of Mediating Pathways.

PubMed

Hsu, Hui-Chin; Wickrama, Kandauda A S

2015-12-01

The underlying mechanisms through which family economic adversity influences child health are less understood. Taking a process-oriented approach, this study examined maternal mental health and investment in children, child health insurance, and child healthcare as mediators linking family economic hardship (FEH) to child health. A structural equation modeling was applied to test the hypothesized mediating model. After adjustment for sociodemographic risk factors, results revealed: (1) a significant direct path linking FEH to poor child health (effect size = .372), and (2) six significant mediating pathways (total effect size = .089). In two mediating pathways, exposures to FEH undermined mothers' mental health: in the first pathway poor maternal mental health led to decreased parental investment, which, in turn, contributed to poor child health, whereas in the second pathway the adverse effect of poor maternal mental health was cascaded through child unmet healthcare need, which resulted in poor child health. One pathway involved child insurance status, where the effect of FEH increased the likelihood to be uninsured, which led to unmet healthcare need, and, in turn, to poor health. Three pathways involved preventive care: in one pathway FEH contributed to poor preventive care, which led to unmet healthcare need and then to poor health; in the other two pathways where poor preventive care respectively gave rise to decreased investment in children or poor maternal mental health, which further contributed to poor child health. Results suggest that the association between FEH and children's health is mediated by multiple pathways.

Longitudinal pathways from early maternal depression to children's dysregulated representations: a moderated mediation analysis of harsh parenting and gender.

PubMed

Martoccio, Tiffany L; Brophy-Herb, Holly E; Maupin, Angela N; Robinson, Joann L

2016-01-01

There is some evidence linking maternal depression, harsh parenting, and children's internal representations of attachment, yet, longitudinal examinations of these relationships and differences in the developmental pathways between boys and girls are lacking. Moderated mediation growth curves were employed to examine harsh parenting as a mechanism underlying the link between maternal depression and children's dysregulated representations using a nationally-representative, economically-vulnerable sample of mothers and their children (n = 575; 49% boys, 51% girls). Dysregulation representations were measured using the MacArthur Story Stem Battery at five years of age (M = 5.14, SD = 0.29). Harsh parenting mediated the association between early maternal depression and dysregulated representations for girls. Though initial harsh parenting was a significant mediator for boys, a stronger direct effect of maternal depression to dysregulated representations emerged over time. Results are discussed in terms of their implications for intervention efforts aimed at promoting early supportive parenting.

Plasma secretory phospholipase A2-IIa as a potential biomarker for lung cancer in patients with solitary pulmonary nodules

PubMed Central

2011-01-01

Background Five-year survival for lung cancer has remained at 16% over last several decades largely due to the fact that over 50% of patients are diagnosed with locally-advanced or metastatic disease. Diagnosis at an earlier and potentially curable stage is crucial. Solitary pulmonary nodules (SPNs) are common, but the difficulty lies in the determination of which SPN is malignant. Currently, there is no convenient and reliable biomarker effective for early diagnosis. Secretory phospholipase A2-IIa (sPLA2-IIa) is secreted into the circulation by cancer cells and may allow for an early detection of lung cancer. Methods Plasma samples from healthy donors, patients with only benign SPN, and patients with lung cancer were analyzed. Expression of sPLA2-IIa protein in lung cancer tissues was also determined. Results We found that the levels of plasma sPLA2-IIa were significantly elevated in lung cancer patients. The receiver operating characteristic curve analysis, comparing lung cancer patients to patients with benign nodules, revealed an optimum cutoff value for plasma sPLA2-IIa of 2.4 ng/ml to predict an early stage cancer with 48% sensitivity and 86% specificity and up to 67% sensitivity for T2 stage lung cancer. Combined sPLA2-IIa, CEA, and Cyfra21.1 tests increased the sensitivity for lung cancer prediction. High level of plasma sPLA2-IIa was associated with a decreased overall cancer survival. sPLA2-IIa was overexpressed in almost all non-small cell lung cancer and in the majority of small cell lung cancer by immunohistochemistry analysis. Conclusion Our finding strongly suggests that plasma sPLA2-IIa is a potential lung biomarker to distinguish benign nodules from lung cancer and to aid lung cancer diagnosis in patients with SPNs. PMID:22151235

A genetic screen in Drosophila reveals novel cytoprotective functions of the autophagy-lysosome pathway.

PubMed

Arsham, Andrew M; Neufeld, Thomas P

2009-06-29

The highly conserved autophagy-lysosome pathway is the primary mechanism for breakdown and recycling of macromolecular and organellar cargo in the eukaryotic cell. Autophagy has recently been implicated in protection against cancer, neurodegeneration, and infection, and interest is increasing in additional roles of autophagy in human health, disease, and aging. To search for novel cytoprotective features of this pathway, we carried out a genetic mosaic screen for mutations causing increased lysosomal and/or autophagic activity in the Drosophila melanogaster larval fat body. By combining Drosophila genetics with live-cell imaging of the fluorescent dye LysoTracker Red and fixed-cell imaging of autophagy-specific fluorescent protein markers, the screen was designed to identify essential metazoan genes whose disruption causes increased flux through the autophagy-lysosome pathway. The screen identified a large number of genes associated with the protein synthesis and ER-secretory pathways (e.g. aminoacyl tRNA synthetases, Oligosaccharyl transferase, Sec61alpha), and with mitochondrial function and dynamics (e.g. Rieske iron-sulfur protein, Dynamin-related protein 1). We also observed that increased lysosomal and autophagic activity were consistently associated with decreased cell size. Our work demonstrates that disruption of the synthesis, transport, folding, or glycosylation of ER-targeted proteins at any of multiple steps leads to autophagy induction. In addition to illuminating cytoprotective features of autophagy in response to cellular damage, this screen establishes a genetic methodology for investigating cell biological phenotypes in live cells, in the context of viable wild type organisms.

Hyphae-specific genes HGC1, ALS3, HWP1, and ECE1 and relevant signaling pathways in Candida albicans.

PubMed

Fan, Yan; He, Hong; Dong, Yan; Pan, Hengbiao

2013-12-01

Fungal virulence mechanisms include adhesion to epithelia, morphogenesis, production of secretory hydrolytic enzymes, and phenotype switching, all of which contribute to the process of pathogenesis. A striking feature of the biology of Candida albicans is its ability to grow in yeast, pseudohyphal, and hyphal forms. The hyphal form plays an important role in causing disease, by invading epithelial cells and causing tissue damage. In this review, we illustrate some of the main hyphae-specific genes, namely HGC1, UME6, ALS3, HWP1, and ECE1, and their relevant and reversed signal transduction pathways in reactions stimulated by environmental factors, including pH, CO2, and serum.

Modulation of neuronal pentraxin 1 expression in rat pancreatic β-cells submitted to chronic glucotoxic stress.

PubMed

Schvartz, Domitille; Couté, Yohann; Brunner, Yannick; Wollheim, Claes B; Sanchez, Jean-Charles

2012-08-01

Insulin secretory granules are β-cell vesicles dedicated to insulin processing, storage, and release. The secretion of insulin secretory granule content in response to an acute increase of glucose concentration is a highly regulated process allowing normal glycemic homeostasis. Type 2 diabetes is a metabolic disease characterized by chronic hyperglycemia. The consequent prolonged glucose exposure is known to exert deleterious effects on the function of various organs, notably impairment of insulin secretion by pancreatic β-cells and induction of apoptosis. It has also been described as modifying gene and protein expression in β-cells. Therefore, we hypothesized that a modulation of insulin secretory granule protein expression induced by chronic hyperglycemia may partially explain β-cell dysfunction. To identify the potential early molecular mechanisms underlying β-cell dysfunction during chronic hyperglycemia, we performed SILAC and mass spectrometry experiments to monitor changes in the insulin secretory granule proteome from INS-1E rat insulinoma β-cells cultivated either with 11 or 30 mm of glucose for 24 h. Fourteen proteins were found to be differentially expressed between these two conditions, and several of these proteins were not described before to be present in β-cells. Among them, neuronal pentraxin 1 was only described in neurons so far. Here we investigated its expression and intracellular localization in INS-1E cells. Furthermore, its overexpression in glucotoxic conditions was confirmed at the mRNA and protein levels. According to its role in hypoxia-ischemia-induced apoptosis described in neurons, this suggests that neuronal pentraxin 1 might be a new β-cell mediator in the AKT/GSK3 apoptotic pathway. In conclusion, the modification of specific β-cell pathways such as apoptosis and oxidative stress may partially explain the impairment of insulin secretion and β-cell failure, observed after prolonged exposure to high glucose concentrations.

The Y-organ secretory activity fluctuates in relation to seasons of molt and reproduction in the brachyuran crab, Metopograpsus messor (Grapsidae): Ultrastructural and immunohistochemical study.

PubMed

Shyamal, Sharmishtha; Sudha, K; Gayathri, N; Anilkumar, G

2014-01-15

This paper presents a first-time report on the localization, structure and seasonal secretory activity of the Y-organ of a grapsid brachyuran crab (Metopograpsus messor). Having exhibited discrete seasonality with reference to the programming of molt and reproduction, this brachyuran crab has offered us an excellent model to obtain a clear picture of the fluctuating secretory nature of the Yorgan, all the way through the reproductive (August-December) as well as the molt-reproduction active (January-May) and inactive (June-July) seasons. Ultrastructural studies revealed that the secretion of the Y-organ was at its peak in premolt crabs during molt-reproduction season (January-May). Interestingly, the Y-organs of the intermolt females that engaged in breeding activity showed higher levels of secretion than those of the molt-reproduction inactive season (June-July), implicating the gland's involvement in reproduction. Immunohistochemical studies using the antiserum raised against 2-succinyl conjugate of ecdysone have demonstrated the ecdysteroid nature of the secretion from the Y-organ, and results of the quantitative assay of ecdysteroids (through radioimmunoassay) revealed that the hormone titer fluctuates in consonance with the Y-organ's secretory activity during seasons of molt and reproduction. Pertinently, not only that the paper gives us a comprehensive understanding on the secretory activity of the Y-organ in a season-dependent fashion, it also allows us to have a better insight into the gland's function related to molting and reproduction (for the first time) in a grapsid brachyuran crab. Copyright © 2013 Elsevier Inc. All rights reserved.

Toxoplasma gondii Syntaxin 6 Is Required for Vesicular Transport Between Endosomal-Like Compartments and the Golgi Complex

PubMed Central

Jackson, Allison J; Clucas, Caroline; Mamczur, Nicola J; Ferguson, David J; Meissner, Markus

2013-01-01

Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post-Golgi and it has been speculated that they pass through endosomal-like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well-established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant-like secretory pathway. PMID:23962112

Working Together for Children: Strengthening Transition Pathways

ERIC Educational Resources Information Center

Collie, Louise; Willis, Felicity; Paine, Crystal; Windsor, Corina

2007-01-01

The "Working Together for Children: Strengthening Transition Pathways" Team has identified that there is a gap between the Early Childhood settings and schools within the Dubbo area. Through meetings with early childhood professionals within Dubbo the authors have identified that the current landscape of care and education within their community…

Building Adaptive Capacity of Pathways in Technology Early College High School Stakeholders: A Multiple-Case Study on the Influence of Performance, Leadership, and Organizational Learning

ERIC Educational Resources Information Center

Michaud-Wells, Amy

2016-01-01

The purpose of this qualitative study was to explore the perceptions and beliefs of Pathways in Technology Early College High School (P-TECH) leaders and stakeholders regarding the personal and professional experiences that contributed to the development of adaptive capacity. This embedded multiple-case study was anchored by the interrelated…

Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

PubMed Central

1992-01-01

Mutant V.24.1, a member of the End4 complementation group of temperature-sensitive CHO cells, is defective in secretion at the restrictive temperature (Wang, R.-H., P. A. Colbaugh, C.-Y. Kao, E. A. Rutledge, and R. K. Draper. 1990. J. Biol. Chem. 265:20179-20187; Presley, J. F., R. K. Draper, and D. T. Brown. 1991. J. Virol. 65:1332- 1339). We have further investigated the secretory lesion and report three main findings. First, the block in secretion is not due to aberrant folding or oligomerization of secretory proteins in the endoplasmic reticulum because the hemagglutinin of influenza virus folded and oligomerized at the same rate in mutant and parental cells at the restrictive temperature. Second, secretory proteins accumulated in a compartment intermediate between the ER and the Golgi. Several lines of evidence support this conclusion, the most direct being the colocalization by immunofluorescence microscopy of influenza virus hemagglutinin with a 58-kD protein that is known to reside in an intermediate compartment. Third, at the resolution of fluorescence microscopy, the Golgi complex in the mutant cells vanished at the restrictive temperature. PMID:1577851

Early Components of the Complement Classical Activation Pathway in Human Systemic Autoimmune Diseases

PubMed Central

Lintner, Katherine E.; Wu, Yee Ling; Yang, Yan; Spencer, Charles H.; Hauptmann, Georges; Hebert, Lee A.; Atkinson, John P.; Yu, C. Yung

2016-01-01

The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases. PMID:26913032

A pilot project using evidence-based clinical pathways and payment reform in China's rural hospitals shows early success.

PubMed

Cheng, Tsung-Mei

2013-05-01

Reforming China's public hospitals to curb widespread overtreatment and improve the quality and affordability of care has been the most challenging aspect of that nation's ambitious health reform, which began in 2009. This article describes a pilot project under way in several of China's provinces that combines payment reform with the implementation of evidence-based clinical pathways at a few hospitals serving rural areas. Results to date include reduced length-of-stay and prescription drug use and higher patient and provider satisfaction. These early results suggest that the pilot may be achieving its goals, which may have far-reaching and positive implications for China's ongoing reform.

Detailed educational pathways among females after very early sexual intercourse.

PubMed

Steward, Nicole R; Farkas, George; Bingenheimer, Jeffrey B

2009-12-01

Although studies have shown that early sex is negatively associated with high school graduation and college enrollment, no recent study has examined the extent to which early sex affects educational attainment, and the mediating mechanisms have not been established. Data from 4,613 female participants in the National Educational Longitudinal Study were used to examine the relationship between early sexual intercourse (i.e., first sex prior to age 15) and educational attainment. Logistic regression analyses that controlled for background characteristics, school performance, behavioral factors and academic aspirations were conducted to assess this association, as well as to explore the potential mediating role of early marriage and childbearing. Young women who had had early sex had reduced odds of having graduated from high school (odds ratio, 0.4) and from college (0.5). Postsecondary enrollment was positively associated with expectations of postsecondary education (1.5) and levels of academic achievement (1.4-1.5), but not with early sex. Respondents who had married before their expected high school graduation date had reduced odds of having graduated from high school (0.1), enrolled in postsecondary school (0.4) or completed college (0.1); those who had a child before their expected high school graduation date had reduced odds of having graduated from high school (0.3) or college (0.1). Programs that target early parenthood and marriage, and that provide hope for future educational opportunities, could lessen the impact of early sex for young women.

Insect haptoelectrical stimulation of Venus flytrap triggers exocytosis in gland cells

PubMed Central

Scherzer, Sönke; Shabala, Lana; Hedrich, Benjamin; Fromm, Jörg; Bauer, Hubert; Munz, Eberhard; Jakob, Peter; Al-Rascheid, Khaled A. S.; Kreuzer, Ines; Becker, Dirk; Eiblmeier, Monika; Rennenberg, Heinz; Shabala, Sergey; Neher, Erwin; Hedrich, Rainer

2017-01-01

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin’s pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H+ and Cl− fuse with the plasma membrane, hyperacidifying the “green stomach”-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal. PMID:28416693

Insect haptoelectrical stimulation of Venus flytrap triggers exocytosis in gland cells.

PubMed

Scherzer, Sönke; Shabala, Lana; Hedrich, Benjamin; Fromm, Jörg; Bauer, Hubert; Munz, Eberhard; Jakob, Peter; Al-Rascheid, Khaled A S; Kreuzer, Ines; Becker, Dirk; Eiblmeier, Monika; Rennenberg, Heinz; Shabala, Sergey; Bennett, Malcolm; Neher, Erwin; Hedrich, Rainer

2017-05-02

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin's pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H + and Cl - fuse with the plasma membrane, hyperacidifying the "green stomach"-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal.

Thyroid hormone affects secretory activity and uncoupling protein-3 expression in rat harderian gland.

PubMed

Chieffi Baccari, Gabriella; Monteforte, Rossella; de Lange, Pieter; Raucci, Franca; Farina, Paola; Lanni, Antonia

2004-07-01

The effects of T(3) administration on the rat Harderian gland were examined at morphological, biochemical, and molecular levels. T(3) induced hypertrophy of the two cell types (A and B) present in the glandular epithelium. In type A cells, the hypertrophy was mainly due to an increase in the size of the lipid compartment. The acinar lumina were filled with lipoproteic substances, and the cells often showed an olocrine secretory pattern. In type B cells, the hypertrophy largely consisted of a marked proliferation of mitochondria endowed with tightly packed cristae, the mitochondrial number being nearly doubled (from 62 to 101/100 microm(2)). Although the average area of individual mitochondria decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 microm(2)). This could be ascribed to T(3)-induced mitochondrial proliferation. The morphological and morphometric data correlated well with our biochemical results, which indicated that mitochondrial respiratory activity is increased in hyperthyroid rats. T(3), by influencing the metabolic function of the mitochondrial compartment, induces lipogenesis and the release of secretory product by type A cells. Mitochondrial uncoupling proteins 2 and 3 were expressed at both mRNA and protein levels in the euthyroid rat Harderian gland. T(3) treatment increased the mRNA levels of both uncoupling protein 2 (UCP2) and UCP3, but the protein level only of UCP3. A possible role for these proteins in the Harderian gland is discussed.

Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic beta-cells.

PubMed

Hatanaka, Masayuki; Tanabe, Katsuya; Yanai, Akie; Ohta, Yasuharu; Kondo, Manabu; Akiyama, Masaru; Shinoda, Koh; Oka, Yoshitomo; Tanizawa, Yukio

2011-04-01

Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.

MiR-338 controls BPA-triggered pancreatic islet insulin secretory dysfunction from compensation to decompensation by targeting Pdx-1.

PubMed

Wei, Jie; Ding, Dongxiao; Wang, Tao; Liu, Qiong; Lin, Yi

2017-12-01

Bisphenol A (BPA) can disrupt glucose homeostasis and impair pancreatic islet function; however, the mechanisms behind these effects are poorly understood. Male mice (4 wk old) were treated with BPA (50 or 500 μg/kg/d) for 8 wk. Whole-body glucose homeostasis, pancreatic islet morphology and function, and miR-338-mediated molecular signal transduction analyses were examined. We showed that BPA treatment led to a disruption of glucose tolerance and a compensatory increase of pancreatic islets insulin secretion and pancreatic and duodenal homeobox 1 ( Pdx1 ) expression in mice. Inhibition of Pdx1 reduced glucose-stimulated insulin secretion and ATP production in the islets of BPA-exposed mice. Based on primary pancreatic islets, we also confirmed that miR-338 regulated Pdx1 and thus contributed to BPA-induced insulin secretory dysfunction from compensation to decompensation. Short-term BPA exposure downregulated miR-338 through activation of G-protein-coupled estrogen receptor 1 (Gpr30), whereas long-term BPA exposure upregulated miR-338 through suppression of glucagon-like peptide 1 receptor (Glp1r). Taken together, our results reveal a molecular mechanism, whereby BPA regulates Gpr30/Glp1r to mediate the expression of miR-338, which acts to control Pdx1-dependent insulin secretion. The Gpr30/Glp1r-miR-338-Pdx1 axis should be represented as a novel mechanism by which BPA induces insulin secretory dysfunction in pancreatic islets.-Wei, J., Ding, D., Wang, T., Liu, Q., Lin, Y. MiR-338 controls BPA-triggered pancreatic islet insulin secretory dysfunction from compensation to decompensation by targeting Pdx-1. © FASEB.

Immunohistochemical localization of cystic fibrosis transmembrane regulator and clara cell secretory protein in taste receptor cells of rat circumvallate papillae.

PubMed

Merigo, Flavia; Benati, Donatella; Galiè, Mirco; Crescimanno, Caterina; Osculati, Francesco; Sbarbati, Andrea

2008-03-01

Taste receptor cells (TRCs) are the sensory cells of taste transduction and are organized into taste buds embedded in the epithelium of the tongue, palate, pharynx, and larynx. Several studies have demonstrated that TRCs involved in sweet as well as bitter and umami responses express alpha-gustducin, an alpha-subunit of the G-protein complex. It has been further demonstrated that this typical taste protein is a potent marker of chemosensory cells located in several tissues, including gastric and pancreatic mucosa and the respiratory apparatus. We recently observed that alpha-gustducin and phospholipase C beta 2-immunoreactive cells were colocalized in the airways with cystic fibrosis transmembrane regulator (CFTR) and Clara cell-specific secretory protein of 10 (CC10) and 26 kDa (CC26). This finding suggests that TRCs might themselves express secretory markers. To test this hypothesis, we investigated the expression of CFTR, CC10, and CC26 in rat circumvallate papillae using reverse transcriptase-polymerase chain reaction analysis, immunohistochemistry, and confocal laser microscopy. The results showed that secretory markers such as CFTR, CC10, and CC26 are present in taste cells of rat circumvallate papillae, and their immunoreactivity is expressed, to a different extent, in subsets of taste cells that express alpha-gustducin. The presence of CFTR, CC10, and CC26 in taste bud cells and their coexpression pattern with alpha-gustducin confirms and extends our previous findings in airway epithelium, lending further credence to the notion that chemoreception and secretion may be related processes.

Myrip couples the capture of secretory granules by the actin-rich cell cortex and their attachment to the plasma membrane.