Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion.

PubMed

Ortega-Sáenz, Patricia; Macías, David; Levitsky, Konstantin L; Rodríguez-Gómez, José A; González-Rodríguez, Patricia; Bonilla-Henao, Victoria; Arias-Mayenco, Ignacio; López-Barneo, José

2016-12-15

Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin-deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. Biotin is a water-soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin-deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin-deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin-deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin-deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation.

PubMed

Kasai, Kazuo; Ohara-Imaizumi, Mica; Takahashi, Noriko; Mizutani, Shin; Zhao, Shengli; Kikuta, Toshiteru; Kasai, Haruo; Nagamatsu, Shinya; Gomi, Hiroshi; Izumi, Tetsuro

2005-02-01

The monomeric small GTPase Rab27a is specifically localized on both secretory granules and lysosome-related organelles. Although natural mutations of the Rab27a gene in human Griscelli syndrome and in ashen mice cause partial albinism and immunodeficiency reflecting the dysfunction of lysosome-related organelles, phenotypes resulting from the defective exocytosis of secretory granules have not been reported. To explore the roles of Rab27a in secretory granules, we analyzed insulin secretion profiles in ashen mice. Ashen mice showed glucose intolerance after a glucose load without signs of insulin resistance in peripheral tissues or insulin deficiency in the pancreas. Insulin secretion from isolated islets was decreased specifically in response to high glucose concentrations but not other nonphysiological secretagogues such as high K+ concentrations, forskolin, or phorbol ester. Neither the intracellular Ca2+ concentration nor the dynamics of fusion pore opening after glucose stimulation were altered. There were, however, marked reductions in the exocytosis from insulin granules predocked on the plasma membrane and in the replenishment of docked granules during glucose stimulation. These results provide the first genetic evidence to our knowledge for the role of Rab27a in the exocytosis of secretory granules and suggest that the Rab27a/effector system mediates glucose-specific signals for the exocytosis of insulin granules in pancreatic beta cells.

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.

Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

PubMed

Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

2014-03-01

Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

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.

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

Missing secretory granules, dilated endoplasmic reticulum, and nuclear dislocation in the thyroid gland of rdw rats with hereditary dwarfism.

PubMed

Sakai, Y; Yamashina, S; Furudate, S I

2000-05-01

Previous studies on the rdw rat have suggested that its dwarfism is caused primarily by dysfunction of the thyroid gland. In this study, rat thyroid glands were analyzed endocrinologically and morphologically to clarify the primary cause of dwarfism in the rdw rat. The rdw rat showed lowered thyroid hormone (T4 and T3) levels but elevated TSH in serum. The rdw thyroid gland was almost proportional in size and it was not goiter in gross inspection. Our histological investigation produced three results that may lend important evidence in understanding the problem in the thyroid gland of rdw rats. First of all, secretory granules could not be detected in the follicular epithelial cells of the rdw. Secondly, thyroglobulin was found at very low levels in the follicular lumen by immunohistochemical analysis. In contrast, it could be detected in a substantial quantity inside the dilated rER and in the huge vacuoles that are formed by swelling of the rough endoplasmic reticulum (rER) at the basal side of the follicular epithelial cells. Additionally, the nucleus of the follicular epithelial cells was pressed to the luminal side by the enlarged rER. These morphological changes would indicate that the transport of thyroglobulin is stopped at or before the formation of the secretory granules and thyroglobulin is not secreted into the follicular lumen. The rdw characterization strongly supports that rdw dwarfism is induced by hypothyroidism due to some defect(s) in the thyroid gland. Copyright 2000 Wiley-Liss, Inc.

Autoantibodies against Muscarinic Type 3 Receptor in Sjögren's Syndrome Inhibit Aquaporin 5 Trafficking

PubMed Central

Lee, Byung Ha; Gauna, Adrienne E.; Perez, Geidys; Park, Yun-jong; Pauley, Kaleb M.; Kawai, Toshihisa; Cha, Seunghee

2013-01-01

Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS. PMID:23382834

Changes in the oviducal epithelium during the estrous cycle in the marsupial Monodelphis domestica

PubMed Central

Kress, Annetrudi; Morson, Gianni

2007-01-01

The Monodelphis oviduct can be divided into four anatomical segments: preampulla (comprising fimbriae and infundibulum), ampulla, isthmus with crypts and uterotubal junction. Ovaries are enclosed in a periovarial sac, the bursa, and in some specimens tubules of an epoophoron could be identified. In both structures non-ciliated cells develop small translucent vesicles, which accumulate in the cell apices and presumably produce fluid as often seen in the bursa and in the tubules of the epooophoron. These vesicles do not stain with Alcian blue or PAS. The same applies also to the non-ciliated cells of the fimbriae. The oviducal epithelium of ampulla and the surface epithelium of the isthmus consisting of ciliated and non-ciliated, secretory cells undergo considerable changes during the estrous cycle. Proestrus shows low numbers of ciliated cells, some are in the process of neo-ciliogenesis, non-ciliated cells carry solitary cilia and few remnant secretory granules from the previous cycle may be found. At estrus the amount of ciliated cells in ampulla and isthmus has increased, most non-cililated cells lost the solitary cilia, developed longer microvilli and formed numerous secretory granules in their cell apices. At postestrus secretory products, often surrounded by membranes, are extruded into the oviducal lumen and contribute towards egg coat formation. First signs of deciliation processes are apparent. Solitary cilia reappear. At metestrus only few secretory cells are left with some secretory material. The lumen is often filled with shed cilia and cell apices. Proliferation of basal bodies within non-secretory cells indicate the formation of new ciliated cells. The non-ciliated epithelial cells of the isthmic crypts form no secretory granules but accumulate a great number of translucent vesicles, which in contrast to the secretory granules do not stain with Alcian blue or PAS. PMID:17883438

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

Specificity and disease in the ubiquitin system

PubMed Central

Chaugule, Viduth K.; Walden, Helen

2016-01-01

Post-translational modification (PTM) of proteins by ubiquitination is an essential cellular regulatory process. Such regulation drives the cell cycle and cell division, signalling and secretory pathways, DNA replication and repair processes and protein quality control and degradation pathways. A huge range of ubiquitin signals can be generated depending on the specificity and catalytic activity of the enzymes required for attachment of ubiquitin to a given target. As a consequence of its importance to eukaryotic life, dysfunction in the ubiquitin system leads to many disease states, including cancers and neurodegeneration. This review takes a retrospective look at our progress in understanding the molecular mechanisms that govern the specificity of ubiquitin conjugation. PMID:26862208

Elovl6 Deficiency Improves Glycemic Control in Diabetic db/db Mice by Expanding β-Cell Mass and Increasing Insulin Secretory Capacity.

PubMed

Zhao, Hui; Matsuzaka, Takashi; Nakano, Yuta; Motomura, Kaori; Tang, Nie; Yokoo, Tomotaka; Okajima, Yuka; Han, Song-Iee; Takeuchi, Yoshinori; Aita, Yuichi; Iwasaki, Hitoshi; Yatoh, Shigeru; Suzuki, Hiroaki; Sekiya, Motohiro; Yahagi, Naoya; Nakagawa, Yoshimi; Sone, Hirohito; Yamada, Nobuhiro; Shimano, Hitoshi

2017-07-01

Dysfunctional fatty acid (FA) metabolism plays an important role in the pathogenesis of β-cell dysfunction and loss of β-cell mass in type 2 diabetes (T2D). Elovl6 is a microsomal enzyme that is responsible for converting C16 saturated and monounsaturated FAs into C18 species. We previously showed that Elovl6 played a critical role in the development of obesity-induced insulin resistance by modifying FA composition. To further define its role in T2D development, we assessed the effects of Elovl6 deletion in leptin receptor-deficient C57BL/KsJ db / db mice, a model of T2D. The db / db ; Elovl6 -/- mice had a markedly increased β-cell mass with increased proliferation and decreased apoptosis, an adaptive increase in insulin, and improved glycemic control. db / db islets were characterized by a prominent elevation of oleate (C18:1n-9), cell stress, and inflammation, which was completely suppressed by Elovl6 deletion. As a mechanistic ex vivo experiment, isolated islets from Elovl6 -/- mice exhibited reduced susceptibility to palmitate-induced inflammation, endoplasmic reticulum stress, and β-cell apoptosis. In contrast, oleate-treated islets resulted in impaired glucose-stimulated insulin secretion with suppressed related genes irrespective of the Elovl6 gene. Taken together, Elovl6 is a fundamental factor linking dysregulated lipid metabolism to β-cell dysfunction, islet inflammation, and β-cell apoptosis in T2D, highlighting oleate as the potential culprit of β-cell lipotoxicity. © 2017 by the American Diabetes Association.

Normal and abnormal secretion by haemopoietic cells

PubMed Central

STINCHCOMBE, JANE C; GRIFFITHS, GILLIAN M

2001-01-01

The secretory lysosomes found in haemopoietic cells provide a very efficient mechanism for delivering the effector proteins of many immune cells in response to antigen recognition. Although secretion shows some similarities to the secretion of specialized granules in other secretory cell types, some aspects of secretory lysosome release appear to be unique to melanocytes and cells of the haemopoietic lineage. Mast cells and platelets have provided excellent models for studying secretion, but recent advances in characterizing the immunological synapse allow a very fine dissection of the secretory process in T lymphocytes. These studies show that secretory lysosomes are secreted from the centre of the talin ring at the synapse. Proper secretion requires a series of Rab and cytoskeletal elements which play critical roles in the specialized secretion of lysosomes in haemopoietic cells. PMID:11380687

A Sustained Activation of Pancreatic NMDARs Is a Novel Factor of β-Cell Apoptosis and Dysfunction.

PubMed

Huang, Xiao-Ting; Yue, Shao-Jie; Li, Chen; Huang, Yan-Hong; Cheng, Qing-Mei; Li, Xiao-Hong; Hao, Cai-Xia; Wang, Ling-Zhi; Xu, Jian-Ping; Ji, Ming; Chen, Chen; Feng, Dan-Dan; Luo, Zi-Qiang

2017-11-01

Type 2 diabetes, which features β-cell failure, is caused by the decrease of β-cell mass and insulin secretory function. Current treatments fail to halt the decrease of functional β-cell mass. Strategies to prevent β-cell apoptosis and dysfunction are highly desirable. Recently, our group and others have reported that blockade of N-methyl-d-aspartate receptors (NMDARs) in the islets has been proposed to prevent the progress of type 2 diabetes through improving β-cell function. It suggests that a sustained activation of the NMDARs may exhibit deleterious effect on β-cells. However, the exact functional impact and mechanism of the sustained NMDAR stimulation on islet β-cells remains unclear. Here, we identify a sustained activation of pancreatic NMDARs as a novel factor of apoptotic β-cell death and function. The sustained treatment with NMDA results in an increase of intracellular [Ca2+] and reactive oxygen species, subsequently induces mitochondrial membrane potential depolarization and a decrease of oxidative phosphorylation expression, and then impairs the mitochondrial function of β-cells. NMDA specifically induces the mitochondrial-dependent pathway of apoptosis in β-cells through upregulation of the proapoptotic Bim and Bax, and downregulation of antiapoptotic Bcl-2. Furthermore, a sustained stimulation of NMDARs impairs β-cell insulin secretion through decrease of pancreatic duodenal homeobox-1 (Pdx-1) and adenosine triphosphate synthesis. The activation of nuclear factor-κB partly contributes to the reduction of Pdx-1 expression induced by overstimulation of NMDARs. In conclusion, we show that the sustained stimulation of NMDARs is a novel mediator of apoptotic signaling and β-cell dysfunction, providing a mechanistic insight into the pathological role of NMDARs activation in diabetes. Copyright © 2017 Endocrine Society.

The functional basis for hemophagocytic lymphohistiocytosis in a patient with co-inherited missense mutations in the perforin (PFN1) gene.

PubMed

Voskoboinik, Ilia; Thia, Marie-Claude; De Bono, Annette; Browne, Kylie; Cretney, Erika; Jackson, Jacob T; Darcy, Phillip K; Jane, Stephen M; Smyth, Mark J; Trapani, Joseph A

2004-09-20

About 30% of cases of the autosomal recessive immunodeficiency disorder hemophagocytic lymphohistiocytosis are believed to be caused by inactivating mutations of the perforin gene. We expressed perforin in rat basophil leukemia cells to define the basis of perforin dysfunction associated with two mutations, R225W and G429E, inherited by a compound heterozygote patient. Whereas RBL cells expressing wild-type perforin (67 kD) efficiently killed Jurkat target cells to which they were conjugated, the substitution to tryptophan at position 225 resulted in expression of a truncated ( approximately 45 kD) form of the protein, complete loss of cytotoxicity, and failure to traffic to rat basophil leukemia secretory granules. By contrast, G429E perforin was correctly processed, stored, and released, but the rat basophil leukemia cells possessed reduced cytotoxicity. The defective function of G429E perforin mapped downstream of exocytosis and was due to its reduced ability to bind lipid membranes in a calcium-dependent manner. This study elucidates the cellular basis for perforin dysfunctions in hemophagocytic lymphohistiocytosis and provides the means for studying structure-function relationships for lymphocyte perforin.

Older Subjects with β-cell Dysfunction have an Accentuated Incretin Release.

PubMed

Garduno-Garcia, José de Jesús; Gastaldelli, Amalia; DeFronzo, Ralph A; Lertwattanarak, Raweewan; Holst, Jens J; Musi, Nicolas

2018-04-16

Insulin secretion declines with age and this contributes to the increased risk of developing impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM) in older subjects. Insulin secretion is regulated by the incretin hormones glucagon-like peptide (GLP) 1 and glucose-dependent insulinotropic peptide (GIP). Here we tested the hypotheses that incretin release is reduced in older subjects, and that this decline is associated with β-cell dysfunction. 40 young (25±3 y) and 53 older (74±7 y) lean non-diabetic subjects underwent a 2 h oral glucose tolerance test (OGTT). Based on the OGTT, subjects were divided in 3 groups: young normal glucose tolerant (Y-NGT, n=40), older with NGT (O-NGT, n=32), and older with IGT (O-IGT, n=21). Plasma insulin, C-peptide, GLP-1, and GIP concentrations were measured every 15-30 min. We quantitated insulin sensitivity (Matsuda index) and insulin secretory rate (ISR) by deconvolution of C-peptide with the calculation of β-cell glucose sensitivity. Matsuda index, early phase ISR (0-30min) and parameters of β-cell function were reduced in O-IGT vs. Y-NGT, but not in O-NGT. GLP-1 concentrations were elevated in both older groups [GLP-1_AUC0-120 was 2.8±0.1 in Y-NGT, 3.8±0.5 in O-NGT, and 3.7±0.4 nmol/l∙120 min in O-IGT (P<0.05)] while GIP secretion was elevated in O-NGT vs. Y-NGT [GIP_AUC0-120 was 4.7±0.3 in Y-NGT, 6.0±0.4 in O-NGT, and 4.8±0.3 nmol/l∙120 min in O-IGT (P<0.05)]. Aging is associated with an exaggerated GLP-1 secretory response. However, this was not sufficient to increase insulin first phase release in O-IGT and overcome insulin resistance.

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.

Changes in pituitary growth hormone cells prepared from rats flown on Spacelab 3

NASA Technical Reports Server (NTRS)

Grindeland, R.; Hymer, W. C.; Farrington, M.; Fast, T.; Hayes, C.; Motter, K.; Patil, L.; Vasques, M.

1987-01-01

The effect of exposure to microgravity on pituitary gland was investigated by examining cells isolated from anterior pituitaries of rats flown on the 7-day Spacelab 3 mission and, subsequently, cultured for 6 days. Compared with ground controls, flight cells contained more intracellular growth hormone (GH); however, the flight cells released less GH over the 6-day culture period and after implantation into hypophysectomized rats than did the control cells. Compared with control rats, glands from large rats (400 g) contained more somatotrophs (44 percent compared with 37 percent in control rats); small rats (200 g) showed no difference. No major differences were found in the somatotroph ultrastructure (by TEM) or in the pattern of the immunoactive GH variants. However, high-performance liquid chromatography fractionation of culture media indicated that flight cells released much less of a biologically active high-molecular weight GH variant, suggesting that space flight may lead to secretory dysfunction.

Molecular characterization of the equine testis-specific protein 1 (TPX1) and acidic epididymal glycoprotein 2 (AEG2) genes encoding members of the cysteine-rich secretory protein (CRISP) family.

PubMed

Giese, Alexander; Jude, Rony; Kuiper, Heidi; Raudsepp, Terje; Piumi, Francois; Schambony, Alexandra; Guérin, Gérard; Chowdhary, Bhanu P; Distl, Ottmar; Töpfer-Petersen, Edda; Leeb, Tosso

2002-10-16

The cysteine-rich secretory protein (CRISP) family consists of three members called acidic epididymal glycoprotein 1 (AEG1), AEG2, and testis-specific protein 1 (TPX1), which share 16 conserved cysteine residues at their C-termini. The CRISP proteins are primarily expressed in different sections of the male genital tract and are thought to mediate cell-cell interactions of male germ cells with other cells during sperm maturation or during fertilization. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 137 kb of horse genomic DNA from equine chromosome 20q22 containing the closely linked equine TPX1 and AEG2 genes are described. The equine TPX1 gene consists of ten exons spanning 18 kb while the AEG2 gene consists of eight exons that are spread over 24 kb. The expression of these two genes was investigated in several tissues by reverse transcription polymerase chain reaction analysis and Western blotting. Comparative genome analysis between horse, human, and mouse indicates that all three CRISP genes are clustered on one chromosomal location, which shows conserved synteny between these species.

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, and only 20-45% increase in porosome diameter is demonstrated following the docking and fusion of 0.2-1.2 μm in diameter secretory vesicles, it is concluded that secretory vesicles ``transiently'' dock and fuse, rather than completely merge at the base of the porosome complex to release their contents to the outside. In agreement, it has been demonstrated that ``secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells''; that ``single synaptic vesicles fuse transiently and successively without loss of identity''; and that``zymogen granule (the secretory vesicle in exocrine pancreas) exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity.'' In this presentation, the discovery of the porosome, resulting in a paradigm shift in our understanding of cell secretion will be briefly discussed.

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.

Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease

PubMed Central

Tsedensodnom, Orkhontuya; Vacaru, Ana M.; Howarth, Deanna L.; Yin, Chunyue; Sadler, Kirsten C.

2013-01-01

SUMMARY Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 μmol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1), the major enzymes that metabolize ethanol, prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus, we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol, whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly, these treatments also blocked ethanol-induced steatosis and reduced UPR activation, whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively, these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD, and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes. PMID:23798569

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.

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.

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.

Endoplasmic Reticulum Stress and Type 2 Diabetes

PubMed Central

Back, Sung Hoon; Kaufman, Randal J.

2013-01-01

Given the functional importance of the endoplasmic reticulum (ER), an organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, the maintenance of ER homeostasis in insulin-secreting β-cells is very important. When ER homeostasis is disrupted, the ER generates adaptive signaling pathways, called the unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis fails to be restored, the ER initiates death signaling pathways. New observations suggest that both chronic hyperglycemia and hyperlipidemia, known as important causative factors of type 2 diabetes (T2D), disrupt ER homeostasis to induce unresolvable UPR activation and β-cell death. This review examines how the UPR pathways, induced by high glucose and free fatty acids (FFAs), interact to disrupt ER function and cause β-cell dysfunction and death. PMID:22443930

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.

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

Regulation of Insulin Synthesis and Secretion and Pancreatic Beta-Cell Dysfunction in Diabetes

PubMed Central

Fu, Zhuo; Gilbert, Elizabeth R.; Liu, Dongmin

2014-01-01

Pancreatic β-cell dysfunction plays an important role in the pathogenesis of both type 1 and type 2 diabetes. Insulin, which is produced in β-cells, is a critical regulator of metabolism. Insulin is synthesized as preproinsulin and processed to proinsulin. Proinsulin is then converted to insulin and C-peptide and stored in secretary granules awaiting release on demand. Insulin synthesis is regulated at both the transcriptional and translational level. The cis-acting sequences within the 5′ flanking region and trans-activators including paired box gene 6 (PAX6), pancreatic and duodenal homeobox-1(PDX-1), MafA, and B-2/Neurogenic differentiation 1 (NeuroD1) regulate insulin transcription, while the stability of preproinsulin mRNA and its untranslated regions control protein translation. Insulin secretion involves a sequence of events in β-cells that lead to fusion of secretory granules with the plasma membrane. Insulin is secreted primarily in response to glucose, while other nutrients such as free fatty acids and amino acids can augment glucose-induced insulin secretion. In addition, various hormones, such as melatonin, estrogen, leptin, growth hormone, and glucagon like peptide-1 also regulate insulin secretion. Thus, the β-cell is a metabolic hub in the body, connecting nutrient metabolism and the endocrine system. Although an increase in intracellular [Ca2+] is the primary insulin secretary signal, cAMP signaling-dependent mechanisms are also critical in the regulation of insulin secretion. This article reviews current knowledge on how β-cells synthesize and secrete insulin. In addition, this review presents evidence that genetic and environmental factors can lead to hyperglycemia, dyslipidemia, inflammation, and autoimmunity, resulting in β-cell dysfunction, thereby triggering the pathogenesis of diabetes. PMID:22974359

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

Exo- and endocytotic trafficking of SCAMP2.

PubMed

Toyooka, Kiminori; Matsuoka, Ken

2009-12-01

Exo- and endocytotic membrane trafficking is an essential process for transport of secretory proteins, extracellular glycans, transporters and lipids in plant cells. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco BY-2 cells as a model system, we recently demonstrated that SCAMP2 positive structures containing secretory materials are transported from the Golgi apparatus to the plasma membrane (PM) and/or cell plate. This structure is consisted with clustered vesicles and was thus named the secretory vesicle cluster (SVC). Here, we have utilized the reversible photoswitching fluorescent protein Dronpa1 to trace the movement of SCAMP2 on the PM and cell plate. Activated SCAMP2-Dronpa fluorescence on the PM and cell plate moved into the BY-2 cells within several minutes, but did not spread around PM. This is consistent with recycling of SCAMP2 among endomembrane compartments such as the TGN, PM and cell plate. The relationship between SVC-mediated trafficking and exo- and endocytosis of plant cells is discussed taking into account this new data and knowledge provided by recent reports.

Endoplasmic Reticulum Stress in Beta Cells and Development of Diabetes

PubMed Central

Fonseca, Sonya G.; Burcin, Mark; Gromada, Jesper; Urano, Fumihiko

2009-01-01

The endoplasmic reticulum (ER) is a cellular compartment responsible for multiple important cellular functions including the biosynthesis and folding of newly synthesized proteins destined for secretion, such as insulin. A myriad of pathological and physiological factors perturb ER function and cause dysregulation of ER homeostasis, leading to ER stress. ER stress elicits a signaling cascade to mitigate stress, the Unfolded Protein Response (UPR). As long as the UPR can relieve stress, cells can produce the proper amount of proteins and maintain ER homeostasis. If the UPR, however, fails to maintain ER homeostasis, cells will undergo apoptosis. Activation of the UPR is critical to the survival of insulin-producing pancreatic β-cells with high secretory protein production. Any disruption of ER homeostasis in β-cells can lead to cell death and contribute to the pathogenesis of diabetes. There are several models of ER stress-mediated diabetes. In this review, we outline the underlying molecular mechanisms of ER stress-mediated β-cell dysfunction and death during the progression of diabetes. PMID:19665428

PIGN prevents protein aggregation in the endoplasmic reticulum independently of its function in the GPI synthesis.

PubMed

Ihara, Shinji; Nakayama, Sohei; Murakami, Yoshiko; Suzuki, Emiko; Asakawa, Masayo; Kinoshita, Taroh; Sawa, Hitoshi

2017-02-01

Quality control of proteins in the endoplasmic reticulum (ER) is essential for ensuring the integrity of secretory proteins before their release into the extracellular space. Secretory proteins that fail to pass quality control form aggregates. Here we show the PIGN-1/PIGN is required for quality control in Caenorhabditis elegans and in mammalian cells. In C. elegans pign-1 mutants, several proteins fail to be secreted and instead form abnormal aggregation. PIGN-knockout HEK293 cells also showed similar protein aggregation. Although PIGN-1/PIGN is responsible for glycosylphosphatidylinositol (GPI)-anchor biosynthesis in the ER, certain mutations in C. elegans pign-1 caused protein aggregation in the ER without affecting GPI-anchor biosynthesis. These results show that PIGN-1/PIGN has a conserved and non-canonical function to prevent deleterious protein aggregation in the ER independently of the GPI-anchor biosynthesis. PIGN is a causative gene for some human diseases including multiple congenital seizure-related syndrome (MCAHS1). Two pign-1 mutations created by CRISPR/Cas9 that correspond to MCAHS1 also cause protein aggregation in the ER, implying that the dysfunction of the PIGN non-canonical function might affect symptoms of MCAHS1 and potentially those of other diseases. © 2017. Published by The Company of Biologists Ltd.

Ultrastructure of posterior sternal glands of Macrotermes annandalei (Silvestri): new members of the sexual glandular set found in termites (Insecta).

PubMed

Quennedey, André; Peppuy, Alexis; Courrent, Annie; Robert, Alain; Everaerts, Claude; Bordereau, Christian

2004-12-01

In female alates of Macrotermes annandalei, two types of abdominal glands are involved in the secretion of sex pheromone. Tergal glands are found at the anterior margin of tergites 6-10 and posterior sternal glands (PSGs) are located at the anterior margin of sternites 6-7. The cytological features of both types of glands are quite similar. The fine structural organization of PSGs is studied more precisely and described for the first time. The glandular cuticle is pitted with narrow apertures corresponding to the openings of numerous subcuticular pouches. Several Class 3 glandular units open in each pouch. One canal cell and one secretory cell make an individual glandular unit. The canal cell is enlarged apically and is connected with the other canal cells to form a common pouch. Based on the structural features found in these glands, we propose a common secretory process for PSGs and tergal glands. During the physiological maturation of alates inside the nest, secretory vesicles amass in the cytoplasm of secretory cells, while large intercellular spaces collapse the cuticular pouches. At the time of dispersal flight, pouches are filled with the content of secretory vesicles while intercellular spaces are sharply reduced. After calling behavior, no secretion remains in the glands and pouches collapse again, while secretory cells are drastically reduced in size. The structure and the secretory processes of PSGs and tergal glands are compared to those of abdominal sexual glands known in termites.

Spontaneous water secretion in T84 cells: effects of STa enterotoxin, bumetanide, VIP, forskolin, and A-23187.

PubMed

Toriano, R; Kierbel, A; Ramirez, M A; Malnic, G; Parisi, M

2001-09-01

The regulated Cl(-) secretory apparatus of T84 cells responds to several pharmacological agents via different second messengers (Ca(2+), cAMP, cGMP). However, information about water movements in T84 cells has not been available. In the absence of osmotic or chemical gradient, we observed a net secretory transepithelial volume flux (J(w) = -0.16 +/- 0.02 microl.min(-1).cm(-2)) in parallel with moderate short-circuit current values (I(sc) = 1.55 +/- 0.23 microA/cm(2)). The secretory J(w) reversibly reverted to an absorptive value when A-23187 was added to the serosal bath. Vasoactive intestinal polypeptide increased I(sc), but, unexpectedly, J(w) was not affected. Bumetanide, an inhibitor of basolateral Na(+)-K(+)-2Cl(-) cotransporter, completely blocked secretory J(w) with no change in I(sc). Conversely, serosal forskolin increased I(sc), but J(w) switched from secretory to absorptive values. Escherichia coli heat-stable enterotoxin increased secretory J(w) and I(sc). No difference between the absorptive and secretory unidirectional Cl(-) fluxes was observed in basal conditions, but after STa stimulation, a significant net secretory Cl(-) flux developed. We conclude that, under these conditions, the presence of secretory or absorptive J(w) values cannot be shown by I(sc) and ion flux studies. Furthermore, RT-PCR experiments indicate that aquaporins were not expressed in T84 cells. The molecular pathway for water secretion appears to be transcellular, moving through the lipid bilayer or, as recently proposed, through water-solute cotransporters.

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.

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

Antral G-cell in gastrin and gastrin-cholecystokinin knockout animals.

PubMed

Friis-Hansen, Lennart; Wierup, Nils; Rehfeld, Jens F; Sundler, Frank

2005-07-01

The antral hormone gastrin is the key regulator of gastric acid secretion, mucosal growth and differentiation. Gastrin is synthesized in the endocrine G-cells in the antroduodenal mucosa. We have now examined the way in which the loss of gastrin alone or gastrin plus cholecystokinin (CCK) affects the antral G-cell. Immunohistochemistry, radioimmunoassay and quantitative real-time polymerase chain reaction techniques were employed to examine the expression of genes belonging to the G-cell secretory pathway in gastrin and gastrin-CCK knockout mice. Transmission electron microscopy was used to examine the ultrastructure of the G-cells. The number of G-cells increased but the secretory granules were few and abnormally small in the G-cells of both mouse models compared with wildtypes. Thus, gastrin is not necessary for the formation of G-cells as such but the lack of gastrin reduces the number and size of their secretory granules suggesting that gastrin is vital for the formation and/or maintenance of secretory granules in G-cells.

Neurotrophin Signaling Is Required for Glucose-Induced Insulin Secretion.

PubMed

Houtz, Jessica; Borden, Philip; Ceasrine, Alexis; Minichiello, Liliana; Kuruvilla, Rejji

2016-11-07

Insulin secretion by pancreatic islet β cells is critical for glucose homeostasis, and a blunted β cell secretory response is an early deficit in type 2 diabetes. Here, we uncover a regulatory mechanism by which glucose recruits vascular-derived neurotrophins to control insulin secretion. Nerve growth factor (NGF), a classical trophic factor for nerve cells, is expressed in pancreatic vasculature while its TrkA receptor is localized to islet β cells. High glucose rapidly enhances NGF secretion and increases TrkA phosphorylation in mouse and human islets. Tissue-specific deletion of NGF or TrkA, or acute disruption of TrkA signaling, impairs glucose tolerance and insulin secretion in mice. We show that internalized TrkA receptors promote insulin granule exocytosis via F-actin reorganization. Furthermore, NGF treatment augments glucose-induced insulin secretion in human islets. These findings reveal a non-neuronal role for neurotrophins and identify a new regulatory pathway in insulin secretion that can be targeted to ameliorate β cell dysfunction. Copyright © 2016 Elsevier Inc. All rights reserved.

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 droplets, it had 10.4 ± 2.1 secretory cells of oil droplets per mm2. All of Zingiberaceae's root and leaves did not have secretory cells of protein. Zingiberaceae's rhizomes had amylum grain, protein granules, and oil droplets. Jahe merah's rhizomes had the greatest density of amylum grain, it had 198.3 ± 21.1 cells of amylum grain per mm2. Jahe emprit's rhizomes had the greatest density of protein granules, it had254.0 ± 90.0 cells of protein granules per mm². Kunyit putih's rhizomes had the greatest density of oil droplets, it had 254.0 ± 90.0 cells of oil droplets per mm².

Multiple roles for the actin cytoskeleton during regulated exocytosis

PubMed Central

Porat-Shliom, Natalie; Milberg, Oleg; Masedunskas, Andrius; Weigert, Roberto

2014-01-01

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules. PMID:22986507

The role of the parenchyma sheath and PCD during the development of oil cavities in Pterodon pubescens (Leguminosae-Papilionoideae).

PubMed

Rodrigues, Tatiane Maria; Santos, Daniela Carvalho Dos; Machado, Silvia Rodrigues

2011-07-01

Pterodon pubescens cavities are constituted by lumen and uniseriated epithelium surrounded by multiseriate parenchyma sheath. We studied the development of secretory cavities, including the role of parenchyma sheath, using light and transmission electron microscopy. A Tunel assay was performed to verify whether programmed cell death (PCD) occurs during the process. The lumen is formed by schizogeny and lysigeny occur in later developmental stages of the secretory cavities. Ultrastructurally, epithelial cells in later developmental stages become dark and with sinuous walls; the protoplast becomes retracted and the cytoplasm shows low organelle definition. Degenerated cells are released toward the lumen. Our results showed that PCD occurs during later developmental stages of cavities and plays a critical role in functioning of these glands. New cells originated from the parenchyma sheath differentiate into secretory cells and replace those degenerated ones. This fact associated to PCD guarantees epithelium renovation during the secretory cycle and the maintenance of secretory activity of cavities. Copyright © 2011 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Dacomitinib-induced diarrhea: Targeting chloride secretion with crofelemer.

PubMed

Van Sebille, Ysabella Z A; Gibson, Rachel J; Wardill, Hannah R; Ball, Imogen A; Keefe, Dorothy M K; Bowen, Joanne M

2018-01-15

Dacomitinib, an irreversible small-molecule pan-ErbB TKI, has a high incidence of diarrhea, which has been suggested to be due to chloride secretory mechanisms. Based on this hypothesis, crofelemer, an antisecretory agent may be an effective intervention. T84 monolayers were treated with 1 µM dacomitinib and 10 µM crofelemer, and mounted into Ussing chambers for electrogenic ion analysis. Crofelemer attenuated increases in chloride secretion in cells treated with dacomitinib. Albino Wistar rats (n = 48) were treated with 7.5 mg/kg dacomitinib and/or 25 mg/kg crofelemer via oral gavage for 21 days. Crofelemer significantly worsened dacomitinib-induced diarrhea (p = 0.0003), and did not attenuate weight loss (p < 0.0001). Sections of the ileum and colon were mounted into Ussing chambers, and secretory processes analyzed. This indicated that crofelemer lost its anti-secretory action in the presence of dacomitinib in this model. Mass spectrometry revealed that crofelemer did not change serum concentration of dacomitinib. Serum FITC dextran levels indicated that crofelemer was unable to attenuate dacomitinib-induced barrier dysfunction. Tight junction proteins were visualized with immunofluorescence. Qualitative analysis showed dacomitinib induced proteolysis of ZO-1 and occludin, and internalization of claudin-1, which was not attenuated by crofelemer. Detailed histopathological analysis showed that crofelemer was unable to attenuate dacomitinib-induced ileal damage. Crofelemer worsened dacomitinib-induced diarrhea, suggesting that antisecretory drug therapy may be ineffective in this setting. © 2017 UICC.

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.

Morphological and histochemical observations on the crural gland-spur apparatus of the echidna (Tachyglossus aculeatus) together with comparative observations on the femoral gland-spur apparatus of the duckbilled platypus (Ornithorhyncus anatinus).

PubMed

Krause, William J

2010-01-01

The echidna and platypus have a crural/femoral gland that is linked by a large duct to a canalized, keratinous spur located on the medial side of the ankle. The echidna crural gland, like the femoral gland of the platypus, exhibits cyclic activity, being prominent in both monotremes when they are sexually active. In the present study, we compared the structure and histochemistry of these glands. During the active phase, the secretory epithelium forming the respective glands of both species increased in height and became packed with secretory granules that differed markedly in structure. Secretory granules of the echidna crural gland were electron dense and characterized by cores or areas of increased electron density. Those of the platypus were initially electron dense, but then became less dense and coalesced into irregular complexes of secretory material. Large cytoplasmic blebs extended from epithelial cell apices and appeared to be shed into the lumen, resulting in an apocrine mode of secretion. Exocytosis was also observed. A similar form of release of secretory product was not observed in the echidna. Secretory granules of both species were periodic acid-Schiff positive and stained for protein, suggesting that much of the secretory product was glycoprotein. Myoepithelial cells enveloped the secretory tubules of the platypus femoral gland, whereas they were not observed surrounding tubules comprising the echidna crural gland. During the quiescent phase, the epithelial cells of both species lost their secretory granules and decreased in height. As a result, the secretory tubules became smaller, intralobular connective tissue increased and the glands decreased in overall size.

Capitate glandular trichomes of Helianthus annuus (Asteraceae): ultrastructure and cytological development.

PubMed

Amrehn, Evelyn; Heller, Annerose; Spring, Otmar

2014-01-01

Previous studies have shown that capitate glandular trichomes (CGT) of the common sunflower, Helianthus annuus, produce sesquiterpene lactones (STL) and flavonoids, which are sequestered and accumulated between the apical cuticle and the wall of the tip cells. To explore the cellular structures required and putatively involved in the STL biosynthesis and secretion, the present study was focused on the development of CGT and the comparison of the ultrastructure of its different cell types. Gradual maturation of flowers in the capitulum of the sunflower provided the possibility to study the simultaneous differentiation from the primordial to the secretory stage of CGT located by light microscopy (bright field, differential interference contrast and fluorescence) as well as transmission electron microscopy. It was shown that the CGT of sunflower anthers had a biseriate structure with up to 14 cell pairs. In mature trichomes, the apical cells called secretory cells were covered entirely by a large cuticle globe, which enclosed the resinous terpenoids and was specialised in thickness and structure. The secretory cells lacked chloroplasts and contained mainly smooth endoplasmic reticulum (sER). Conspicuous cell wall protuberances and an accumulation of mitochondria nearby occurred in the horizontally oriented cell walls. The cytological differences between stalk cells and secretory cells indicate a different function. The dominance of sER suggests its involvement in STL biosynthesis and cell wall protuberances enlarge the surface of the plasmamembrane of secretory cells and may be involved in the secretion processes of STL into the subcuticular space.

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.

Actin cytoskeleton and exocytosis in rat melanotrophs.

PubMed

Chowdhury, Helana H; Popoff, Michel R; Zorec, Robert

2000-01-01

We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (C m ). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1μM [Ca 2+ ] i . Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.

Actin cytoskeleton and exocytosis in rat melanotrophs.

PubMed

Chowdhury, H H; Popoff, M R; Zorec, R

2000-01-01

We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (Cm). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1 microM [Ca2+]i. Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.

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.

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

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.

The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus.

PubMed

Gerber, Philipp A; Rutter, Guy A

2017-04-01

Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene-environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn 2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501-518.

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

Baconnais, S.; Delavoie, F.; Zahm, J.M.

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections.more » We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.« less

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

Three-dimensional cell shapes and arrangements in human sweat glands as revealed by whole-mount immunostaining

PubMed Central

Kurata, Ryuichiro; Futaki, Sugiko; Nakano, Itsuko; Fujita, Fumitaka; Tanemura, Atsushi; Murota, Hiroyuki; Katayama, Ichiro; Okada, Fumihiro

2017-01-01

Because sweat secretion is facilitated by mechanical contraction of sweat gland structures, understanding their structure-function relationship could lead to more effective treatments for patients with sweat gland disorders such as heat stroke. Conventional histological studies have shown that sweat glands are three-dimensionally coiled tubular structures consisting of ducts and secretory portions, although their detailed structural anatomy remains unclear. To better understand the details of the three-dimensional (3D) coiled structures of sweat glands, a whole-mount staining method was employed to visualize 3D coiled gland structures with sweat gland markers for ductal luminal, ductal basal, secretory luminal, and myoepithelial cells. Imaging the 3D coiled gland structures demonstrated that the ducts and secretory portions were comprised of distinct tubular structures. Ductal tubules were occasionally bent, while secretory tubules were frequently bent and formed a self-entangled coiled structure. Whole-mount staining of complex coiled gland structures also revealed the detailed 3D cellular arrangements in the individual sweat gland compartments. Ducts were composed of regularly arranged cuboidal shaped cells, while secretory portions were surrounded by myoepithelial cells longitudinally elongated along entangled secretory tubules. Whole-mount staining was also used to visualize the spatial arrangement of blood vessels and nerve fibers, both of which facilitate sweat secretion. The blood vessels ran longitudinally parallel to the sweat gland tubules, while nerve fibers wrapped around secretory tubules, but not ductal tubules. Taken together, whole-mount staining of sweat glands revealed the 3D cell shapes and arrangements of complex coiled gland structures and provides insights into the mechanical contraction of coiled gland structures during sweat secretion. PMID:28636607

PAR-2 receptor-induced effects on human eccrine sweat gland cells.

PubMed

L Bovell, Douglas; Kofler, Barbara; Lang, Roland

2009-01-01

Serine proteases can induce cell signaling by stimulating G-protein-coupled receptors, called proteinase-activated receptors (PAR's) on a variety of epithelial cells. While PAR-2, one such receptor, activates cell signaling in a secretory cell line derived from human sweat glands, there was no information on their presence and effects on intact sweat glands. PAR-2 presence and activation of eccrine sweat glands isolated from human skin samples was investigated using Western blot analysis, immunohistochemistry, electron microscopy (EM) and Ca(2+) imaging. Anti-human PAR-2 antibody demonstrated the presence of these receptors in eccrine sweat glands. EM showed that PAR-2 activation resulted in degranulation of secretory cells. Ca(2+) imaging using PAR-2 activators demonstrated a two phase increase in [Ca(2+)](i) which was dependent on extracellular Ca(2+) for the second phase, and that the response could be blocked by prior incubation with xestospongin, the IP(3) receptor blocker. The results demonstrated that PAR-2 receptors are present in human sweat gland secretory cells and that these receptors are functionally active and can induce changes associated with secretory events in eccrine glands.

Localization of sesquiterpene lactone biosynthesis in cells of capitate glandular trichomes of Helianthus annuus (Asteraceae).

PubMed

Amrehn, Evelyn; Aschenbrenner, Anna-Katharina; Heller, Annerose; Spring, Otmar

2016-03-01

Capitate glandular trichomes (CGT) of sunflower, Helianthus annuus, synthesize bioactive sesquiterpene lactones (STLs) within a short period of only a few days during trichome development. In the current project, the subcellular localization of H. annuus germacrene A monooxygenase (HaGAO), a key enzyme of the STL biosynthesis in sunflower CGT, was investigated. A polyclonal antibody raised against this enzyme was used for immunolabelling. HaGAO was found in secretory and stalk cells of CGT. This correlated with the appearance of smooth endoplasmic reticulum in both cell types. Stalk cells and secretory cells differed in form, size and types of plastids, but both had structures necessary for secretion. No HaGAO-specific immunoreaction was found in sunflower leaf tissue outside of CGT or in developing CGT before the secretory phase had started. Our results indicated that not only secretory cells but also nearly all cells of the CGT were involved in the biosynthesis of STL and that this process was not linked to the presence or absence of a specific type of plastid.

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.

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.

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).

Vascular repair strategies in type 2 diabetes: novel insights

PubMed Central

Kuschnerus, Kira; Landmesser, Ulf

2015-01-01

Impaired functions of vascular cells are responsible for the majority of complications in patients with type 2 diabetes (T2D). Recently a better understanding of mechanisms contributing to development of vascular dysfunction and the role of systemic inflammatory activation and functional alterations of several secretory organs, of which adipose tissue has more recently been investigated, has been achieved. Notably, the progression of vascular disease within the context of T2D appears to be driven by a multitude of incremental signaling shifts. Hence, successful therapies need to target several mechanisms in parallel, and over a long time period. This review will summarize the latest molecular strategies and translational developments of cardiovascular therapy in patients with T2D. PMID:26543824

Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes

PubMed Central

Patwardhan, Anand; Bardin, Sabine; Miserey-Lenkei, Stéphanie; Larue, Lionel; Goud, Bruno; Raposo, Graça; Delevoye, Cédric

2017-01-01

Exocytic carriers convey neo-synthesized components from the Golgi apparatus to the cell surface. While the release and anterograde movement of Golgi-derived vesicles require the small GTPase RAB6, its effector ELKS promotes the targeting and docking of secretory vesicles to particular areas of the plasma membrane. Here, we show that specialized cell types exploit and divert the secretory pathway towards lysosome related organelles. In cultured melanocytes, the secretory route relies on RAB6 and ELKS to directly transport and dock Golgi-derived carriers to melanosomes. By delivering specific cargos, such as MART-1 and TYRP2/ DCT, the RAB6/ELKS-dependent secretory pathway controls the formation and maturation of melanosomes but also pigment synthesis. In addition, pigmentation defects are observed in RAB6 KO mice. Our data together reveal for the first time that the secretory pathway can be directed towards intracellular organelles of endosomal origin to ensure their biogenesis and function. PMID:28607494

[Microscopic structure of the epithelium of the oviducts in cows during the estrus cycle].

PubMed

Uhrín, V

1983-03-01

The mucous membrane of a cow is covered with ciliary and secretory cells. The so-called basal cells occur at the basal membrane. The counts of ciliary cells vary during the sexual cycle: they reach the maximum (up to 68%) during oestrus. About 13% of cells lose cilia during metoestrus and at the beginning of dioestrus. Reciliation occurs during pro-oestrus. Light and dark ciliary cells can be discerned by the staining of cytoplasm and by the density of nuclei. A higher variability was found in the secretory cells. There are light and dark cells, cells with a wedge shape and rod-shaped cells. Their frequency and function are discussed. Mitoses of epithelium were found in rare cases. The relative volume of epithelium and the mucous membrane of connective tissues change during the sexual cycle. The volume of secretory cells increases during metoestrus and dioestrus and the volume of ciliary cells increases during pro-oestrus and heat. The volume of nuclei decreases in metoestrus and mainly in dioestrus. PAS positive granules occur in the cytoplasm of secretory cells, mainly during metoestrus, in the apical regions. Ptyalin-resistant polysaccharides, besides glycogen, were detected in the cells. The occurrence rate of lipids varies just slightly during the oestrous cycle.

Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei.

PubMed

Ooi, Cher-Pheng; Smith, Terry K; Gluenz, Eva; Wand, Nadina Vasileva; Vaughan, Sue; Rudenko, Gloria

2018-06-01

The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre-cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post-mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post-mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol-anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans-face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi. © 2018 The Authors. Traffic published by John Wiley & Sons Ltd.

Fine structure of the middle ear epithelium in the chicken (Gallus gallus).

PubMed Central

Giannessi, F; Ruffoli, R

1993-01-01

The epithelium lining the tympanic cavity of the chicken possesses distinct morphological characteristics. Its ultrastructure was studied using 2 preparative techniques. (1) After fixation in Karnovsky's solution, postfixation in osmium tetroxide and embedding in Epon, the epithelium was observed to contain 2 kinds of cell: secretory and basal. The secretory cells (which we refer to as mixed granulated cells) showed numerous secretory vesicles that varied in appearance, some containing paracrystalline formations. The basal cells, located close to the basement membrane, showed no evidence of secretory activity. (2) Other specimens were immersed in Karnovsky fixative and subsequently in a mixture of glutaraldehyde and tannic acid. They were then osmicated and embedded in polar Epon mix. With this method, the epithelium was seen to be covered by electron-dense material made up of thin intertwined tubules. In addition, the secretory cells contained vesicles with concentrically arranged lamellae; such vesicles resembled the multilamellar bodies of mammalian type II pneumocytes. The hypothesis is advanced that tubules and lamellar vesicles are related to the presence of surfactant substances. Images Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 13 Fig. 14 Fig. 15 Fig. 16 Fig. 17 Fig. 18 Fig. 19 PMID:8270465

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 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

Down-regulation of zinc transporter 8 (SLC30A8) in pancreatic beta-cells promotes cell survival

USDA-ARS?s Scientific Manuscript database

The pancreatic islet contains high levels of zinc in granular vesicles of beta-cells where insulin is matured, crystallized, and stored before secretion. Zinc is an essential co-factor for insulin crystallization forming dense core in secretory granules. In insulin-containing secretory granules, zin...

Down-regulation of zinc transporter 8 (SLC30A8) in pancreatic beta-cells promotes cell survival.

USDA-ARS?s Scientific Manuscript database

The pancreatic islet contains high levels of zinc in granular vesicles of ß-cells where insulin is matured, crystallized, and stored before secretion. Zinc is an essential co-factor for insulin crystallization forming dense cores in secretory granules. In insulin-containing secretory granules, zinc ...

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.

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

Ageing induced vascular smooth muscle cell senescence in atherosclerosis.

PubMed

Uryga, Anna K; Bennett, Martin R

2016-04-15

Atherosclerosis is a disease of ageing in that its incidence and prevalence increase with age. However, atherosclerosis is also associated with biological ageing, manifest by a number of typical hallmarks of ageing in the atherosclerotic plaque. Thus, accelerated biological ageing may be superimposed on the effects of chronological ageing in atherosclerosis. Tissue ageing is seen in all cells that comprise the plaque, but particularly in vascular smooth muscle cells (VSMCs). Hallmarks of ageing include evidence of cell senescence, DNA damage (including telomere attrition), mitochondrial dysfunction, a pro-inflammatory secretory phenotype, defects in proteostasis, epigenetic changes, deregulated nutrient sensing, and exhaustion of progenitor cells. In this model, initial damage to DNA (genomic, telomeric, mitochondrial and epigenetic changes) results in a number of cellular responses (cellular senescence, deregulated nutrient sensing and defects in proteostasis). Ultimately, ongoing damage and attempts at repair by continued proliferation overwhelm reparative capacity, causing loss of specialised cell functions, cell death and inflammation. This review summarises the evidence for accelerated biological ageing in atherosclerosis, the functional consequences of cell ageing on cells comprising the plaque, and the causal role that VSMC senescence plays in atherogenesis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Scanning and transmission electron microscopic analysis of ampullary segment of oviduct during estrous cycle in caprines.

PubMed

Sharma, R K; Singh, R; Bhardwaj, J K

2015-01-01

The ampullary segment of the mammalian oviduct provides suitable milieu for fertilization and development of zygote before implantation into uterus. It is, therefore, in the present study, the cyclic changes in the morphology of ampullary segment of goat oviduct were studied during follicular and luteal phases using scanning and transmission electron microscopy techniques. Topographical analysis revealed the presence of uniformly ciliated ampullary epithelia, concealing apical processes of non-ciliated cells along with bulbous secretory cells during follicular phase. The luteal phase was marked with decline in number of ciliated cells with increased occurrence of secretory cells. The ultrastructure analysis has demonstrated the presence of indented nuclear membrane, supranuclear cytoplasm, secretory granules, rough endoplasmic reticulum, large lipid droplets, apically located glycogen masses, oval shaped mitochondria in the secretory cells. The ciliated cells were characterized by the presence of elongated nuclei, abundant smooth endoplasmic reticulum, oval or spherical shaped mitochondria with crecentric cristae during follicular phase. However, in the luteal phase, secretory cells were possessing highly indented nucleus with diffused electron dense chromatin, hyaline nucleosol, increased number of lipid droplets. The ciliated cells had numerous fibrous granules and basal bodies. The parallel use of scanning and transmission electron microscopy techniques has enabled us to examine the cyclic and hormone dependent changes occurring in the topography and fine structure of epithelium of ampullary segment and its cells during different reproductive phases that will be great help in understanding major bottle neck that limits success rate in vitro fertilization and embryo transfer technology. © Wiley Periodicals, Inc.

Leaf blade structure of Verbesina macrophylla (Cass.) F. S. Blake (Asteraceae): ontogeny, duct secretion mechanism and essential oil composition.

PubMed

Bezerra, L D A; Mangabeira, P A O; de Oliveira, R A; Costa, L C D B; Da Cunha, M

2018-05-01

Secretory structures are common in Asteraceae, where they exhibit a high degree of morphological diversity. The species Verbesina macrophylla, popularly known as assa-peixe, is native to Brazil where it is widely used for medicinal purposes. Despite its potential medical importance, there have been no studies of the anatomy of this species, especially its secretory structures and secreted compounds. This study examined leaves of V. macrophylla with emphasis on secretory structures and secreted secondary metabolites. Development of secretory ducts and the mechanism of secretion production are described for V. macrophylla using ultrastructure, yield and chemical composition of its essential oils. Verbesina macrophylla has a hypostomatic leaf blade with dorsiventral mesophyll and secretory ducts associated with vascular bundles of schizogenous origin. Histochemistry identified the presence of lipids, terpenes, alkaloids and mucopolysaccharides. Ultrastructure suggests that the secretion released into the duct lumen is produced in plastids of transfer cells, parenchymal sheath cells and stored in vacuoles in these cells and duct epithelial cells. The essential oil content was 0.8%, and its major components were germacrene D, germacrene D-4-ol, β-caryophyllene, bicyclogermacrene and α-cadinol. Secretory ducts of V. macrophylla are squizogenous. Substances identified in tissues suggest that both secretions stored in the ducts and in adjacent parenchyma cells are involved in chemical defence. The essential oil is rich in sesquiterpenes, with germacrene D and its derivatives being notable components. © 2018 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

Quantification of β-cell insulin secretory function using a graded glucose infusion with C-peptide deconvolution in dysmetabolic, and diabetic cynomolgus monkeys.

PubMed

Wang, Xiaoli; Hansen, Barbara C; Shi, Da; Fang, Yupeng; Du, Fenglai; Wang, Bingdi; Chen, Yaxiong Michael; Gregoire, Francine M; Wang, Yi-Xin Jim

2013-07-25

Quantitation of β-cell function is critical in better understanding of the dynamic interactions of insulin secretion, clearance and action at different phases in the progression of diabetes. The present study aimed to quantify β-cell secretory function independently of insulin sensitivity in the context of differential metabolic clearance rates of insulin (MCRI) in nonhuman primates (NHPs). Insulin secretion rate (ISR) was derived from deconvolution of serial C-peptide concentrations measured during a 5 stage graded glucose infusion (GGI) in 12 nondiabetic (N), 8 prediabetic or dysmetabolic (DYS) and 4 overtly diabetic (DM) cynomolgus monkeys. The characterization of the monkeys was based on the fasting glucose and insulin concentrations, glucose clearance rate measured by intravenous glucose tolerance test, and insulin resistance indices measured in separate experiments. The molar ratio of C-peptide/insulin (C/I) was used as a surrogate index of hepatic MCRI. Compared to the N monkeys, the DYS with normal glycemia and hyperinsulinemia had significantly higher basal and GGI-induced elevation of insulin and C-peptide concentrations and lower C/I, however, each unit of glucose-stimulated ISR increment was not significantly different from that in the N monkeys. In contrast, the DM monkeys with β-cell failure and hyperglycemia had a depressed GGI-stimulated ISR response and elevated C/I. The present data demonstrated that in addition to β-cell hypersecretion of insulin, reduced hepatic MCRI may also contribute to the development of hyperinsulinemia in the DYS monkeys. On the other hand, hyperinsulinemia may cause the saturation of hepatic insulin extraction capacity, which in turn reduced MCRI in the DYS monkeys. The differential contribution of ISR and MCRI in causing hyperinsulinemia provides a new insight into the trajectory of β-cell dysfunction in the development of diabetes. The present study was the first to use the GGI and C-peptide deconvolution method to quantify the β-cell function in NHPs.

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.

The morphology and ultrastructure of salivary glands of Zoraptera (Insecta).

PubMed

Dallai, R; Mercati, D; Mashimo, Y; Machida, R; Beutel, R G

2017-07-01

The salivary glands of two species of Zoraptera, Zorotypus caudelli and Zorotypus hubbardi, were examined and documented mainly using transmission electron microscopy (TEM). The results obtained for males and females of the two species are compared and functional aspects related to ultrastructural features are discussed. The salivary glands are divided into two regions: the secretory cell region and the long efferent duct, the latter with its distal end opening in the salivarium below the hypopharyngeal base. The secretory region consists of a complex of secretory cells provided with microvillated cavities connected by short ectodermal ducts to large ones, which are connected with the long efferent duct. The secretory cell cytoplasm contains a large system of rough endoplasmic reticulum and Golgi apparatus producing numerous dense secretions. The cells of the efferent duct, characterized by reduced cytoplasm and the presence of long membrane infoldings associated with mitochondria, are possibly involved in fluid uptaking from the duct lumen. Copyright © 2017 Elsevier Ltd. All rights reserved.

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy.

PubMed

Melo, Rossana C N; Weller, Peter F

2016-10-01

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 Sombrero 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. Copyright © 2016 Elsevier Inc. All rights reserved.

Placental membrane aging and HMGB1 signaling associated with human parturition.

PubMed

Menon, Ramkumar; Behnia, Faranak; Polettini, Jossimara; Saade, George R; Campisi, Judith; Velarde, Michael

2016-02-01

Aging is associated with the onset of several diseases in various organ systems; however, different tissues may age differently, rendering some of them dysfunctional sooner than others. Placental membranes (fetal amniochorionic membranes) protect the fetus throughout pregnancy, but their longevity is limited to the duration of pregnancy. The age-associated dysfunction of these membranes is postulated to trigger parturition. Here, we investigated whether cellular senescence-the loss of cell division potential as a consequence of stress-is involved in placental membrane function at term. We show telomere reduction, p38 MAPK activation, increase in p21 expression, loss of lamin B1 loss, increase in SA-β-galactosidase , and senescence-associated secretory phenotype (SASP) gene expression in placental membranes after labor and delivery (term labor [TL]) compared to membranes prior to labor at term (term, not-in-labor [TNIL]). Exposing TNIL placental membranes to cigarette smoke extract, an oxidative stress inducer, also induced markers of cellular senescence similar to those in TL placental membranes. Bioinformatics analysis of differentially expressed SASP genes revealed HMGB1 signaling among the top pathways involved in labor. Further, we show that recombinant HMGB1 upregulates the expression of genes associated with parturition in myometrial cells. These data suggest that the natural physiologic aging of placental tissues is associated with cellular senescence and human parturition.

Role of estrogen receptors alpha, beta and GPER1/GPR30 in pancreatic beta-cells.

PubMed

Nadal, Angel; Alonso-Magdalena, Paloma; Soriano, Sergi; Ripoll, Cristina; Fuentes, Esther; Quesada, Ivan; Ropero, Ana Belen

2011-01-01

Estrogen receptors (ER) are emerging as important molecules involved in the adaptation of beta-cells to insulin resistance. The onset of type 2 diabetes is marked by insulin secretory dysfunction and decreased beta-cell mass. During pregnancy, puberty and obesity there is increased metabolic demand and insulin resistance is developed. This metabolic state increases the demand on beta-cells to augment insulin biosynthesis and release. In this respect, ERalpha is directly implicated in the E2-regulation of insulin content and secretion, while ERbeta is in the E2-potentiation of glucose-induced insulin release. Both receptors develop their actions within the physiological range of E2. In addition, the G protein-coupled estrogen receptor (GPER1/GPR30) seems to be implicated in the E2-regulation of stimulus-secretion coupling in the three cell types of the islet. The increased demand of insulin production for long time may lead to beta-cell stress and apoptosis. ERalpha, ERbeta and GPER1/GPR30 are involved in preventing beta-cell apoptosis, impeding the loss of critical beta-cell mass. Therefore, estrogen receptors may play an essential role in the adaptation of the pancreas to insulin resistant periods.

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.

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

Dong, Hu, E-mail: austhudong@126.com; Medical Inspection Center, Anhui University of Science and Technology, Huainan; Jing, Wu, E-mail: wujing8008@126.com

In recent years, increasing studies have found that pathogenic Mycobacterium tuberculosis (Mtb) inhibits autophagy, which mediates the anti-mycobacterial response, but the mechanism is not clear. We previously reported that secretory acid phosphatase (SapM) of Mtb can negatively regulate autophagy flux. Recently, another virulence factor of Mtb, early secretory antigenic target 6 (ESAT6), has been found to be involved in inhibiting autophagy, but the mechanism remains unclear. In this study, we show that ESAT6 hampers autophagy flux to boost bacillus Calmette-Guerin (BCG) proliferation and reveals a mechanism by which ESAT6 blocks autophagosome-lysosome fusion in a mammalian target of rapamycin (MTOR)-dependent manner.more » In both Raw264.7 cells and primary macrophages derived from the murine abdominal cavity (ACM), ESAT6 repressed autophagy flux by interfering with the autophagosome-lysosome fusion, which resulted in an increased load of BCG. Impaired degradation of LC3Ⅱ and SQSTM1 by ESAT6 was related to the upregulated activity of MTOR. Contrarily, inhibiting MTOR with Torin1 removed the ESAT6-induced autophagy block and lysosome dysfunction. Furthermore, in both Raw264.7 and ACM cells, MTOR inhibition significantly suppressed the survival of BCG. In conclusion, our study highlights how ESAT6 blocks autophagy and promotes BCG survival in a way that activates MTOR. - Highlights: • A mechanism for disruping autophagy flux induced by ESAT6. • ESAT6-inhibited autophagy is MTOR-dependent. • ESAT6-boosted BCG is MTOR-dependent.« less

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.

Susceptibility to fatty acid-induced β-cell dysfunction is enhanced in prediabetic diabetes-prone biobreeding rats: a potential link between β-cell lipotoxicity and islet inflammation.

PubMed

Tang, Christine; Naassan, Anthony E; Chamson-Reig, Astrid; Koulajian, Khajag; Goh, Tracy T; Yoon, Frederick; Oprescu, Andrei I; Ghanim, Husam; Lewis, Gary F; Dandona, Paresh; Donath, Marc Y; Ehses, Jan A; Arany, Edith; Giacca, Adria

2013-01-01

β-Cell lipotoxicity is thought to play an important role in the development of type 2 diabetes. However, no study has examined its role in type 1 diabetes, which could be clinically relevant for slow-onset type 1 diabetes. Reports of enhanced cytokine toxicity in fat-laden islets are consistent with the hypothesis that lipid and cytokine toxicity may be synergistic. Thus, β-cell lipotoxicity could be enhanced in models of autoimmune diabetes. To determine this, we examined the effects of prolonged free fatty acids elevation on β-cell secretory function in the prediabetic diabetes-prone BioBreeding (dp-BB) rat, its diabetes-resistant BioBreeding (dr-BB) control, and normal Wistar-Furth (WF) rats. Rats received a 48-h iv infusion of saline or Intralipid plus heparin (IH) (to elevate free fatty acid levels ~2-fold) followed by hyperglycemic clamp or islet secretion studies ex vivo. IH significantly decreased β-cell function, assessed both by the disposition index (insulin secretion corrected for IH-induced insulin resistance) and in isolated islets, in dp-BB, but not in dr-BB or WF, rats, and the effect of IH was inhibited by the antioxidant N-acetylcysteine. Furthermore, IH significantly increased islet cytokine mRNA and plasma cytokine levels (monocyte chemoattractant protein-1 and IL-10) in dp-BB, but not in dr-BB or WF, rats. All dp-BB rats had mononuclear infiltration of islets, which was absent in dr-BB and WF rats. In conclusion, the presence of insulitis was permissive for IH-induced β-cell dysfunction in the BB rat, which suggests a link between β-cell lipotoxicity and islet inflammation.

The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus

PubMed Central

Gerber, Philipp A.

2017-01-01

Abstract Significance: Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Critical Issues: Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene–environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Future Directions: Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501–518. PMID:27225690

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

Ultrastructure of the platypus and echidna mandibular glands.

PubMed

Krause, W J

2011-10-01

The secretory units of the platypus and echidna mandibular glands consist of a single serous cell type. Secretory granules within the cells of the platypus mandibular gland stained intensely with the periodic acid-Schiff staining procedure but failed to stain with Alcian Blue, suggesting the granules contained neutral glycoproteins. Secretory granules within the mandibular glands of the echidna failed to stain with the methods used indicating little if any glycoprotein was associated with the secretory granules. Ultrastructurally, secretory granules of the platypus mandibular gland were electron dense with a central core of less electron-dense material and were membrane bound. In contrast, those of the echidna presented a lamellated appearance and also were limited by a membrane. These secretory granules appeared to form as a result of concentric layering of lamellae within cisternae of the Golgi membranes. The intralobular ductal system of the platypus was more extensively developed than that of the echidna. The striated ducts of both species were characterized by elaborate infoldings of the basolateral plasmalemma and an abundance of associated mitochondria. © 2011 Blackwell Verlag GmbH.

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 intestinal motility. PMID:23353550

[Changes of right atrial myoendocrine cells during hypertension and after arterial pressure decrease].

PubMed

Maksimov, V F; Korostyshevskaia, I M; Kurganov, S A; Markel', A L; Rudenko, N S; Iakobson, G S

2014-01-01

It is well known now that atrial cardiomyocytes carry out both contractile and endocrine activities--they synthesize, accumulate in specific secretory granules and release the natriuretic peptides. The main physiological effects of natriuretic peptides are antagonistic to the renin-angiotensin-aldostrol system, but their role in the development of hypertension is still disputable. The aim of this investigation is to estimate using electron microscopy the secretory activities of atrial myoendocrine cells in rats with inherited stress-induced arterial hypertension (ISIAH stain). It has been shown that myoendocrine cells in the ISIAH rats with arterial pressure about 180 mm Hg reveal morphological features of increased synthesis, extra accumulation and release of natriuretic peptides compared with normotensive control rats. In the ISIAH rats treated with losartan (angiotensin II receptor blocker) and therefore having a sustained decrease in arterial pressure to 140 mm Hg, changes in granular pool composition, reduction of the number and diameter of the secretory granules, reduction of Golgi complexes, and increased intracellular degradation of secretory stores were found in the myoendocrine cells. At the same time the marked capillary hyperemia and interstitial edema in the myocardium were observed. Thus, in rats with severe inherited hypertension, the secretory activity of heart myoendocrine cells is sharply increased and directly depends on the arterial blood pressure level. This proves that natriuretic peptides actively participate in the regulation of hemodynamics during with cardiovascular pathology.

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…

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.

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

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

[An immunocytochemical study of the C-cell function of the thyroid in rats exposed on the Kosmos-2044 biosatellite].

PubMed

Loginov, V I

1993-01-01

Immunocytochemical analysis of thyroid gland C-cells of the rats exposed to a 14-day space flight revealed a decrease in the number of C-cells, volume of their nuclei and a declined percentage of active secretory C-cells, which point to a decline of calcitonin proactive and calcitonin secretory hypofunction of the thyroid C-cells system in flown rats. Tail suspension as a microgravity model caused similar changes in C-cells.

["Light" epithelial cells of swine and bovine oviducts].

PubMed

Suuroia, T; Aunapuu, M; Arend, A; Sépp, E

2002-01-01

The ultrastructure of oviduct epithelium of clinically healthy cows and 15 sows was investigated using scanning and transmission electron microscopy. In all parts of the oviduct, ciliated and non-ciliated epithelial cells are present, but their number varies in both the investigated animals in different regions of the oviduct, depending on the phase of the estrous cycle. In addition to ciliated cells with numerous cilia on their luminal surface, so-called pale ciliary cells were found in all parts of the oviduct of cows and sows. The cytoplasm of these cells is electron-lucent, their luminal surface carries few cilia and short microvilli. The apical cytoplasm contains species specific secretory granules, which means that these cells have features characteristic of both secretory and ciliated cells. It is suggested that the pale ciliated and non-ciliated secretory cells are functional stages of the same tubar epithelium cell, and that the transformation between these two cell types is regulated by functional requirements of the organ in different phases of the estrous cycle.

Pharmacotherapy for Irritable Bowel Syndrome

PubMed Central

Camilleri, Michael

2017-01-01

Irritable bowel syndrome (IBS) is a disorder of the brain-gut axis; the pathophysiological mechanisms include altered colonic motility, bile acid metabolism, neurohormonal regulation, immune dysfunction, alterations in the epithelial barrier and secretory properties of the gut. This article reviews the mechanisms, efficacy, and safety of current pharmacotherapy, and medications that are in phase III trials for the treatment of IBS. There remains a significant unmet need for effective treatments—particularly for the pain component of IBS—although the introduction of drugs directed at secretion, motility and a non-absorbable antibiotic provide options for the bowel dysfunction in IBS. PMID:29077050

Life sciences, biotechnology, and microgravity

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Hayes, C.; Grindeland, R.; Lanhan, J. W.; Morrison, D.

1987-01-01

Growth hormone (GH) studies on rats flown aboard Spacelab 3 are discussed, and evidence for the direct effect of microgravity on cell function is reviewed. SL-3 rat GH cells were found to experience a secretory lesion (they contained more hormone per cell, but released less per cell relative to controls). Pituitary cell culture experiments on the STS-8 mission showed that GH cells did not subsequently release as much hormone as did control cells, indicating a secretory lesion. Changes in bone and muscle noted in SL-3 rats are related to GH cell findings.

Loss of proteostasis induced by amyloid beta peptide in brain endothelial cells.

PubMed

Fonseca, Ana Catarina; Oliveira, Catarina R; Pereira, Cláudia F; Cardoso, Sandra M

2014-06-01

Abnormal accumulation of amyloid-β (Aβ) peptide in the brain is a pathological hallmark of Alzheimer's disease (AD). In addition to neurotoxic effects, Aβ also damages brain endothelial cells (ECs) and may thus contribute to the degeneration of cerebral vasculature, which has been proposed as an early pathogenic event in the course of AD and is able to trigger and/or potentiate the neurodegenerative process and cognitive decline. However, the mechanisms underlying Aβ-induced endothelial dysfunction are not completely understood. Here we hypothesized that Aβ impairs protein quality control mechanisms both in the secretory pathway and in the cytosol in brain ECs, leading cells to death. In rat brain RBE4 cells, we demonstrated that Aβ1-40 induces the failure of the ER stress-adaptive unfolded protein response (UPR), deregulates the ubiquitin-proteasome system (UPS) decreasing overall proteasome activity with accumulation of ubiquitinated proteins and impairs the autophagic protein degradation pathway due to failure in the autophagic flux, which culminates in cell demise. In conclusion, Aβ deregulates proteostasis in brain ECs and, as a consequence, these cells die by apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Changes in endometrial natural killer cell expression of CD94, CD158a and CD158b are associated with infertility.

PubMed

McGrath, Emma; Ryan, Elizabeth J; Lynch, Lydia; Golden-Mason, Lucy; Mooney, Eoghan; Eogan, Maeve; O'Herlihy, Colm; O'Farrelly, Cliona

2009-04-01

Cycle-dependent fluctuations in natural killer (NK) cell populations in endometrium and circulation may differ, contributing to unexplained infertility. NK cell phenotypes were determined by flow cytometry in endometrial biopsies and matched blood samples. While circulating and endometrial T cell populations remained constant throughout the menstrual cycle in fertile and infertile women, circulating NK cells in infertile women increased during the secretory phase. However, increased expression of CD94, CD158b (secretory phase), and CD158a (proliferative phase) by endometrial NK cells from infertile women was observed. These changes were not reflected in the circulation. In infertile women, changes in circulating NK cell percentages are found exclusively during the secretory phase and not in endometrium; cycle-related changes in NK receptor expression are observed only in infertile endometrium. While having exciting implications for understanding NK cell function in fertility, our data emphasize the difficulty in attaching diagnostic or prognostic significance to NK cell analyses in individual patients.

THE OPPORTUNISTIC PATHOGEN TOXOPLASMA GONDII DEPLOYS A DIVERSE LEGION OF INVASION AND SURVIVAL PROTEINS

PubMed Central

Zhou, Xing W.; Kafsack, Björn F. C.; Cole, Robert N.; Beckett, Phil; Shen, Rong F.; Carruthers, Vern B.

2006-01-01

Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins using two complementary methodologies, 2-DE/MS and LC/ESI-MS-MS (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by 2-DE revealed ∼100 spots, most of which were successfully identified by protein microsequencing or MALDI-MS analysis. Many proteins were present in multiple species suggesting they are subjected to substantial posttranslational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. The findings provide a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection. PMID:16002397

Global deficits in development, function, and gene expression in the endocrine pancreas in a deletion mouse model of Prader-Willi syndrome.

PubMed

Stefan, Mihaela; Simmons, Rebecca A; Bertera, Suzanne; Trucco, Massimo; Esni, Farzad; Drain, Peter; Nicholls, Robert D

2011-05-01

Prader-Willi syndrome (PWS) is a multisystem disorder caused by genetic loss of function of a cluster of imprinted, paternally expressed genes. Neonatal failure to thrive in PWS is followed by childhood-onset hyperphagia and obesity among other endocrine and behavioral abnormalities. PWS is typically assumed to be caused by an unknown hypothalamic-pituitary dysfunction, but the underlying pathogenesis remains unknown. A transgenic deletion mouse model (TgPWS) has severe failure to thrive, with very low levels of plasma insulin and glucagon in fetal and neonatal life prior to and following onset of progressive hypoglycemia. In this study, we tested the hypothesis that primary deficits in pancreatic islet development or function may play a fundamental role in the TgPWS neonatal phenotype. Major pancreatic islet hormones (insulin, glucagon) were decreased in TgPWS mice, consistent with plasma levels. Immunohistochemical analysis of the pancreas demonstrated disrupted morphology of TgPWS islets, with reduced α- and β-cell mass arising from an increase in apoptosis. Furthermore, in vivo and in vitro studies show that the rate of insulin secretion is significantly impaired in TgPWS β-cells. In TgPWS pancreas, mRNA levels for genes encoding all pancreatic hormones, other secretory factors, and the ISL1 transcription factor are upregulated by either a compensatory response to plasma hormone deficiencies or a primary effect of a deleted gene. Our findings identify a cluster of imprinted genes required for the development, survival, coordinate regulation of genes encoding hormones, and secretory function of pancreatic endocrine cells, which may underlie the neonatal phenotype of the TgPWS mouse model.

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

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

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.

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

The ultrastructure of the sweat glands of the ox, sheep and goat during sweating and recovery.

PubMed Central

Jenkinson, D M; Montgomery, I; Elder, H Y

1979-01-01

The ultrastructure of the sweat glands of cattle, sheep and goats was studied before, during, and after, exposure of the animals to controlled warm environments. In cattle, sweating induced little ultrastructural change in the gland, although fluid-filled spaces appeared between the myo- and secretory epithelial layers. The mechanism appears to be one of fluid transport and exocytosis of secretory vesicles, which in this species seem to be derived from the Golgi apparatus and/or mitochondria. The glands of the sheep and goat also displayed signs of vesicle exocytosis and of fluid transport during sweating. The sweating 'fatigue' in these species was apparently due to failure of the secretory cells, some of which ruptured and were extruded into the lumen. The evidence during subsequent recovery indicates that neighbouring cells spread to make contact, encase remnants of atretic cells between them and the underlying myoepithelium, and engulf them. Sweat in these species appears to be formed (a) by secretion and (b) from cells which can no longer meet the demands of stimulation. The role in sweating of cell replacement, and of undifferentiated cells found between the myo- and secretory epithelia, is discussed. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 PMID:511758

Modelling the spatio-temporal cell dynamics reveals novel insights on cell differentiation and proliferation in the small intestinal crypt.

PubMed

Pin, Carmen; Watson, Alastair J M; Carding, Simon R

2012-01-01

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through Wnt and Notch signals, the strength of which depends on the local cell composition. The highest level of Wnt activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of Wnt signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high Wnt signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4-5 days with 10-12 days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered Wnt and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days.

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.

Comparative morphology and ultrastructure of the prosomal salivary glands in the unfed larvae Leptotrombidium orientale (Acariformes, Trombiculidae), a possible vector of tsutsugamushi disease agent.

PubMed

Shatrov, Andrew B

2015-07-01

The prosomal salivary glands of the unfed larvae Leptotrombidium orientale (Schluger) were investigated using transmission electron microscopy. In total, four pairs of the prosomal glands were identified--three pairs, the lateral, the medial and the anterior, belong to the podocephalic system, and one pair, the posterior, is separate having an own excretory duct. All glands are simple alveolar/acinous with prismatic cells arranged around a relatively small intra-alveolar lumen with the duct base. The cells of all glands besides the lateral ones contain practically mature electron-dense secretory granules ready to be discharged from the cells. The secretory granules in the lateral glands undergo formation and maturation due to the Golgi body activity. The cells of all gland types contain a large basally located nucleus and variously expressed rough endoplasmic reticulum. Specialized duct-forming cells filled with numerous freely scattered microtubules are situated in the middle zone of each gland's acinus and form the intra-alveolar lumen and the duct base. Both the acinar (secretory) and the duct-forming cells contact each other via gap junctions and septate desmosomes. Axons of nerve cells come close to the basal extensions of the duct-forming cells where they form the bulb-shaped synaptic terminations. The process of secretion is under the control of the nerve system that provides contraction of the duct-forming cells and discharge of secretion from the secretory cells into the intra-alveolar lumen and further to the exterior. Unfed larvae of L. orientale, the potential vector of tsutsugamushi disease agents, contain the most simply organized salivary secretory granules among known trombiculid larvae, and this secretion, besides the lateral glands, does not undergo significant additional maturation. Thus, the larvae are apparently ready to feed on the appropriate host just nearly after hatching.

Identification of Human Islet Amyloid Polypeptide as a BACE2 Substrate

PubMed Central

Rulifson, Ingrid C.; Cao, Ping; Miao, Li; Kopecky, David; Huang, Linda; White, Ryan D.; Samayoa, Kim; Gardner, Jonitha; Wu, Xiaosu; Chen, Kui; Tsuruda, Trace; Homann, Oliver; Baribault, Helene; Yamane, Harvey; Carlson, Tim; Wiltzius, Jed; Li, Yang

2016-01-01

Pancreatic amyloid formation by islet amyloid polypeptide (IAPP) is a hallmark pathological feature of type 2 diabetes. IAPP is stored in the secretory granules of pancreatic beta-cells and co-secreted with insulin to maintain glucose homeostasis. IAPP is innocuous under homeostatic conditions but imbalances in production or processing of IAPP may result in homodimer formation leading to the rapid production of cytotoxic oligomers and amyloid fibrils. The consequence is beta-cell dysfunction and the accumulation of proteinaceous plaques in and around pancreatic islets. Beta-site APP-cleaving enzyme 2, BACE2, is an aspartyl protease commonly associated with BACE1, a related homolog responsible for amyloid processing in the brain and strongly implicated in Alzheimer’s disease. Herein, we identify two distinct sites of the mature human IAPP sequence that are susceptible to BACE2-mediated proteolytic activity. The result of proteolysis is modulation of human IAPP fibrillation and human IAPP protein degradation. These results suggest a potential therapeutic role for BACE2 in type 2 diabetes-associated hyperamylinaemia. PMID:26840340

Cytokine markers of B lymphocytes in minor salivary gland infiltrates in Sjögren's syndrome.

PubMed

Navarro-Mendoza, Erika P; Aguirre-Valencia, David; Posso-Osorio, Iván; Correa-Forero, Shirley Vanessa; Torres-Cutiva, Daniel-Felipe; Loaiza, Diana; Tobón, Gabriel J

2018-05-03

Sjögren's syndrome (SS) is a chronic autoimmune disorder characterised by the clinical presence of sicca syndrome. SS compromises the dysfunction of exocrine glands due to the presence of focal, mononuclear cell infiltrates that surround the ducts and replace the secretory units. Abnormal expression of different cytokines and chemokines such as B-cell activating factor, CXC Motif Chemokine Ligand 13, interleukin 6 (IL-6), IL-22, and FMS-like tyrosine kinase 3 ligand as well as that of their corresponding receptors has been implicated in the inflammatory process. The severity of glandular infiltration has been suggested to be associated with the presence of extra-glandular systemic manifestations, contributing to a clinical spectrum of the most severe disease. This review describes several cytokines and chemokines associated with B lymphocytes expressed in the minor salivary gland, their chemical structures, and their roles in SS as possible early predictors of lymphoma development and disease progression. Copyright © 2018. Published by Elsevier B.V.

Ultrastructural aspects of feeding and secretion-excretion by the equine parasite Strongylus vulgaris.

PubMed

Mobarak, M S; Ryan, M F

1999-06-01

Light, scanning, and transmission electron microscopy were employed to provide further data on the putative origins of the immunogenic secretory-excretory product (ESP) of Strongylus vulgaris (Looss 1900). The sharply delineated but superficial attachment to the equine caecum by the mouth leaves behind an oval area devoid of epithelial cells. Attachment does not extend deeply enough to reach the muscularis mucosa layer of the equine intestine. The progressive digestion of the ingested plug of tissue (epithelial cells, blood cells and mucous) was visualized. The coelomocytes, floating cells and membranous structures located in the pseudocoelom and intimately associated with the digestive, excretory and reproductive systems, and with the somatic muscles are described. The secretory-excretory system comprises two, ventrally-located, secretory-excretory glands connected to tubular elements. These glands synthesize granules of various sizes and densities that are delineated.

Placental membrane aging and HMGB1 signaling associated with human parturition

PubMed Central

Menon, Ramkumar; Behnia, Faranak; Polettini, Jossimara; Saade, George R; Campisi, Judith; Velarde, Michael

2016-01-01

Aging is associated with the onset of several diseases in various organ systems; however, different tissues may age differently, rendering some of them dysfunctional sooner than others. Placental membranes (fetal amniochorionic membranes) protect the fetus throughout pregnancy, but their longevity is limited to the duration of pregnancy. The age-associated dysfunction of these membranes is postulated to trigger parturition. Here, we investigated whether cellular senescence—the loss of cell division potential as a consequence of stress—is involved in placental membrane function at term. We show telomere reduction, p38 MAPK activation, increase in p21 expression, loss of lamin B1 loss, increase in SA-β-galactosidase, and senescence-associated secretory phenotype (SASP) gene expression in placental membranes after labor and delivery (term labor [TL]) compared to membranes prior to labor at term (term, not-in-labor [TNIL]). Exposing TNIL placental membranes to cigarette smoke extract, an oxidative stress inducer, also induced markers of cellular senescence similar to those in TL placental membranes. Bioinformatics analysis of differentially expressed SASP genes revealed HMGB1 signaling among the top pathways involved in labor. Further, we show that recombinant HMGB1 upregulates the expression of genes associated with parturition in myometrial cells. These data suggest that the natural physiologic aging of placental tissues is associated with cellular senescence and human parturition. PMID:26851389

Effect of steroid hormones on Bufo arenarum oviduct. Ultrastructural study.

PubMed

Medina, Marcela Fátima; Crespo, Claudia Alejandra; Ramos, Inés; Cisint, Susana Beatriz; Fernández, Silvia Nélida

2007-06-01

The endocrine regulation of the mucosa of the oviductal pars convoluta was analyzed by ultrastructural studies demonstrating that ovariectomy, together with a decrease in ovarian steroids circulating levels, caused a marked regression in this portion of Bufo arenarum oviduct. Twenty-five days after ovariectomy, a decrease in the depth of the epithelial and glandular layers was observed due to the notable loss of secretory cells, whose number was clearly smaller than in nonovariectomized females. The remaining secretory cells showed involution signs, with few secretory granules in their cytoplasm, little endoplasmic reticulum near poorly developed Golgi complexes and a large amount of lipid droplets. Cells in an advanced autolysis state were found in the lumen. These characteristics evidence a nonfunctional state of the pars convoluta. Treatment with 5alpha-dihydrotestosterone (DHT) completely reversed the ovariectomy effect, inducing pars convoluta growths and restoring the characteristics of epithelial and glandular secretory cells in the whole pars convoluta, with micrographs similar to the control. These same effects were observed after treatment with estradiol-17beta (E2), progesterone (P) o E(2)+P in the glandular layer of the whole pars convoluta, but only in the epithelial layer of the most anterior region of this duct. In the secretory cells of other segments these treatments induced the formation of granules of high electron density and homogeneous aspect. Each steroid had a particular effect on the pars convoluta. Although E2 and DHT induced the development of the organoids involved in the proteins biosynthesis, P and DHT acted as secretagogues. (c) 2007 Wiley-Liss, Inc.

Ultrastructure and post-floral secretion of the pericarpial nectaries of Erythrina speciosa (Fabaceae)

PubMed Central

Paiva, Elder Antônio Sousa

2009-01-01

Background and Aims The occurrence of nectaries in fruits is restricted to a minority of plant families and consistent reports of their occurrence are not found associated with Fabaceae, mainly showing cellular details. The present study aims to describe the anatomical organization and ultrastructure of the pericarpial nectaries (PNs) in Erythrina speciosa, a bird-pollinated species, discussing functional aspects of these unusual structures. Methods Samples of floral buds, ovaries of flowers at anthesis and fruits at several developmental stages were fixed and processed by the usual methods for studies using light, and scanning and transmission electron microscopy. Nectar samples collected by filter paper wicks were subjected to chemical analysis using thin-layer chromatography. Key Results The PNs are distributed in isolation on the exocarp. Each PN is represented by a single hyaline trichome that consists of a basal cell at epidermal level, stalk cell(s) and a small secretory multicellular head. The apical stalk cell shows inner periclinal and anticlinal walls impregnated by lipids and lignin and has dense cytoplasm with a prevalence of mitochondria and endoplasmic reticulum. The secretory cells show voluminous nuclei and dense cytoplasm, which predominantly has dictyosomes, rough endoplasmic reticulum, plastids, mitochondria and free ribosomes. At the secretory stage the periplasmic space is prominent and contains secretion residues. Tests for sugar indicate the presence of non-reducing sugars in the secretory cells. Nectar samples from PNs contained sucrose, glucose and fructose. Conclusions The secretory stage of these PNs extends until fruit maturation and evidence suggests that the energetic source of nectar production is based on pericarp photosynthesis. Patrolling ants were seen foraging on fruits during all stages of fruit development, which suggests that the PNs mediate a symbiotic relationship between ants and plant, similar to the common role of many extrafloral nectaries. PMID:19617593

Ultrastructure and post-floral secretion of the pericarpial nectaries of Erythrina speciosa (Fabaceae).

PubMed

Paiva, Elder Antônio Sousa

2009-10-01

The occurrence of nectaries in fruits is restricted to a minority of plant families and consistent reports of their occurrence are not found associated with Fabaceae, mainly showing cellular details. The present study aims to describe the anatomical organization and ultrastructure of the pericarpial nectaries (PNs) in Erythrina speciosa, a bird-pollinated species, discussing functional aspects of these unusual structures. Samples of floral buds, ovaries of flowers at anthesis and fruits at several developmental stages were fixed and processed by the usual methods for studies using light, and scanning and transmission electron microscopy. Nectar samples collected by filter paper wicks were subjected to chemical analysis using thin-layer chromatography. The PNs are distributed in isolation on the exocarp. Each PN is represented by a single hyaline trichome that consists of a basal cell at epidermal level, stalk cell(s) and a small secretory multicellular head. The apical stalk cell shows inner periclinal and anticlinal walls impregnated by lipids and lignin and has dense cytoplasm with a prevalence of mitochondria and endoplasmic reticulum. The secretory cells show voluminous nuclei and dense cytoplasm, which predominantly has dictyosomes, rough endoplasmic reticulum, plastids, mitochondria and free ribosomes. At the secretory stage the periplasmic space is prominent and contains secretion residues. Tests for sugar indicate the presence of non-reducing sugars in the secretory cells. Nectar samples from PNs contained sucrose, glucose and fructose. The secretory stage of these PNs extends until fruit maturation and evidence suggests that the energetic source of nectar production is based on pericarp photosynthesis. Patrolling ants were seen foraging on fruits during all stages of fruit development, which suggests that the PNs mediate a symbiotic relationship between ants and plant, similar to the common role of many extrafloral nectaries.

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

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

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.

The roles of supernatant of macrophage treated by excretory-secretory products from muscle larvae of Trichinella spiralis on the differentiation of C2C12 myoblasts

USDA-ARS?s Scientific Manuscript database

The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or...

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

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

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.

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.

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

Hexavalent chromium induces apoptosis in male somatic and spermatogonial stem cells via redox imbalance

PubMed Central

Das, Joydeep; Kang, Min-Hee; Kim, Eunsu; Kwon, Deug-Nam; Choi, Yun-Jung; Kim, Jin-Hoi

2015-01-01

Hexavalent chromium [Cr(VI)], an environmental toxicant, causes severe male reproductive abnormalities. However, the actual mechanisms of toxicity are not clearly understood and have not been studied in detail. The present in vitro study aimed to investigate the mechanism of reproductive toxicity of Cr(VI) in male somatic cells (mouse TM3 Leydig cells and TM4 Sertoli cells) and spermatogonial stem cells (SSCs) because damage to or dysfunction of these cells can directly affect spermatogenesis, resulting in male infertility. Cr(VI) by inducing oxidative stress was cytotoxic to both male somatic cells and SSCs in a dose-dependent manner, and induced mitochondria-dependent apoptosis. Although the mechanism of Cr(VI)-induced cytotoxicity was similar in both somatic cells, the differences in sensitivity of TM3 and TM4 cells to Cr(VI) could be attributed, at least in part, to cell-specific regulation of P-AKT1, P-ERK1/2, and P-P53 proteins. Cr(VI) affected the differentiation and self-renewal mechanisms of SSCs, disrupted steroidogenesis in TM3 cells, while in TM4 cells, the expression of tight junction signaling and cell receptor molecules was affected as well as the secretory functions were impaired. In conclusion, our results show that Cr(VI) is cytotoxic and impairs the physiological functions of male somatic cells and SSCs. PMID:26355036

Absorbed dose in target cell nuclei and dose conversion coefficient of radon progeny in the human lung.

PubMed

Nikezic, D; Lau, B M F; Stevanovic, N; Yu, K N

2006-01-01

To calculate the absorbed dose in the human lung due to inhaled radon progeny, ICRP focussed on the layers containing the target cells, i.e., the basal and secretory cells. Such an approach did not consider details of the sensitive cells in the layers. The present work uses the microdosimetric approach and determines the absorbed alpha-particle energy in non-spherical nuclei of target cells (basal and secretory cells). The absorbed energy for alpha particles emitted by radon progeny in the human respiratory tract was calculated in basal- and secretory-cell nuclei, assuming conical and ellipsoidal forms for these cells. Distributions of specific energy for different combinations of alpha-particle sources, energies and targets are calculated and shown. The dose conversion coefficient for radon progeny is reduced for about 2mSv/WLM when conical and ellipsoidal cell nuclei are considered instead of the layers. While changes in the geometry of secretory-cell nuclei do not have significant effects on their absorbed dose, changes from spherical to conical basal-cell nuclei have significantly reduced their absorbed dose from approximately 4 to approximately 3mGy/WLM. This is expected because basal cells are situated close to the end of the range of 6MeV alpha particles. This also underlines the significance of better and more precise information on targets in the T-B tree. A further change in the dose conversion coefficient can be achieved if a different weighting scheme is adopted for the doses for the cells. The results demonstrate the necessity for better information on the target cells for more accurate dosimetry for radon progeny.

Potent Insulin Secretagogue from Scoparia dulcis Linn of Nepalese Origin.

PubMed

Sharma, Khaga Raj; Adhikari, Achyut; Hafizur, Rahman M; Hameed, Abdul; Raza, Sayed Ali; Kalauni, Surya Kant; Miyazaki, Jun-Ichi; Choudhary, M Iqbal

2015-10-01

Ethno-botanical inspired isolation from plant Scoparia dulcis Linn. (Sweet Broomweed) yielded six compounds, coixol (1), glutinol (2), glutinone (3), friedelin (4), betulinic acid (5), and tetratriacontan-1-ol (6). There structures were identified using mass and 1D- and 2D-NMR spectroscopy techniques. Compounds 1-6 were evaluated for their insulin secretory activity on isolated mice islets and MIN-6 pancreatic β-cell line, and compounds 1 and 2 were found to be potent and mildly active, respectively. Compound 1 was further evaluated for insulin secretory activity on MIN-6 cells. Compound 1 was subjected to in vitro cytotoxicity assay against MIN-6, 3T3 cell lines, and islet cells, and in vivo acute toxicity test in mice that was found to be non-toxic. The insulin secretory activity of compounds 1 and 2 supported the ethno-botanic uses of S. dulcis as an anti-diabetic agent. Copyright © 2015 John Wiley & Sons, Ltd.

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.

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.

The fine structure of the stomach mucosa of the Llama (Llama guanacoe). II. The fundic region of the hind stomach.

PubMed

Luciano, L; Reale, E; von Engelhardt, W

1980-01-01

The epithelium of the fundic region mucosa of the hind stomach in the Llama guanacoe has been studied using morphological and histochemical methods. Morphology suggests that solute and water absorption may occur in the epithelium of the surface and of the foveolae, although this absorption can not be estimated because of the extensive secretion of the gastric glands. The same cells of the surface and foveolar epithelium show numerous secretory granules. The glands reveal neck cells, chief cells, a large number of oxyntic cells, four types of endocrine cells (A-like, ECL, D and EC), brush cells and wandering cells. PAS and Alcian blue reactions for light microscopy suggest a secretion of neutral and acidic mucosubstances in the surface and foveolar epithelium, of neutral mucosubstances only in the neck cells. Periodic acid-thiocarbohydrazide silver proteinate (PA-TCH-SP) reaction for electron microscopy confirms the presence of neutral mucosubstances within the secretory granules of the surface, foveolar and neck epithelial cells. In all these cells, the reaction product is also evident within sacculi and vesicles of the maturing surface of the Golgi apparatus. A positive PA-TCH-SP reaction also occurs on the membrane (and not on the contents) of the Golgi apparatus (maturing surface) and of the secretory granules of the chief cells as well as on the membrane of the Golgi apparatus and of apical vesicles and tubules of the oxyntic cells. In addition, silver granules slightly enhance the electron desity of the contents of the secretory granules in the endocrine cells. Morphological and histochemical findings are discussed and compared with results described by others for monogastric mammals.

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania*

PubMed Central

Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

2015-01-01

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. PMID:26499792

A case of unfulfilled expectations. Cytokines in idiopathic minimal lesion nephrotic syndrome.

PubMed

Araya, Carlos E; Wasserfall, Clive H; Brusko, Todd M; Mu, Wei; Segal, Mark S; Johnson, Richard J; Garin, Eduardo H

2006-05-01

Idiopathic minimal lesion nephrotic syndrome (IMLNS) was proposed to be a disorder of T-cell dysfunction by Shalhoub in 1974. The mechanisms by which T-cells increase glomerular permeability have remained elusive (and unproven). There is evidence that IMLNS may be due to a circulating factor released from activated T-cells. In recent years, efforts have been made to identify this pathogenetic cytokine as well as to understand the mechanism(s) for the increased release of this factor. This review attempts to critically analyze the available published data. Using different methodologies, investigators have focused on the production of cytokines in patients with IMLNS during relapse and remission. This has resulted in a plethora of data without definitive conclusions. The pathogenetic cytokine has not been identified, and it is questionable whether there is a Th2 dominance in IMLNS. The review of the available data illustrates the difficulties encountered when one is studying the cytokine secretory pattern in patients with IMLNS. Differences in patient population, type of cells studies, sample preservation, and methodology used to measure cytokines are some of the factors that could account for the disparity of observed results.

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...

A new type of exocrine gland and its function in mass recruitment in the ant Cylindromyrmex whymperi (Formicidae, Cerapachyinae)

NASA Astrophysics Data System (ADS)

Gobin, Bruno; Rüppell, Olav; Hartmann, Annegret; Jungnickel, Harald; Morgan, David; Billen, Johan

2001-08-01

Workers of the ant Cylindromyrmex whymperi display mass trail recruitment. Bioassays show that the trail pheromone originates from a unique gland between abdominal sternites 6 and 7. The gland has a hitherto unknown structural organization. Upon leaving the secretory cell, the duct cell widens to form a sclerotized pear-shaped reservoir chamber, lined with multiple duct cells. Each duct thus forms a miniature reservoir for the secretions of each single secretory cell, a novel structural arrangement in exocrine glands of social Hymenoptera.

The pelvic kidney of male Ambystoma maculatum (Amphibia, urodela, ambystomatidae) with special reference to the sexual collecting ducts.

PubMed

Siegel, Dustin S; Sever, David M; Aldridge, Robert D

2010-12-01

This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct, synthesizes neutral carbohydrates and proteins, and is also lined by apical ciliated cells intercalated between secretory cells. Although functional aspects associated with the morphological variation along the length of the proximal portions of the nephron have been investigated, the role of a highly secretory collecting duct has not. Historical data that implicated secretory activity concordant with mating activity, and similarity of structure and chemistry to sexual segments of the kidneys in other vertebrates, lead us to believe that the collecting duct functions as a secondary sexual organ in Ambystoma maculatum. © 2010 Wiley-Liss, Inc.

Electrophoretic separation of kidney and pituitary cells on STS-8

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Nachtwey, D. S.; Barlow, G. H.; Cleveland, C.; Lanham, J. W.; Farrington, M. A.; Hatfield, J. M.; Hymer, W. C.; Grindeland, R.; Lewis, M. L.

1984-01-01

Specific secretory cells were separated from suspensions of cultured primary human embryonic cells and rat pituitary cells in microgravity conditions, with an objective of isolating the subfractions of kidney cells that produce the largest amount of urakinase, and the subfractions of rat pituitary cells that secrete growth hormones (GH), prolactin (PRL), and other hormones. It is inferred from the experimental observations that the surface charge distributions of the GH-containing cells differ from those of the PRL-containing cells, which is explained by the presence of secretory products on the surface of pituitary cells. For kidney cells, the electrophoretic mobility distributions in flight experiments were spread more than the ground controls.

Induced trefoil factor family 1 expression by trans-differentiating Clara cells in a murine asthma model.

PubMed

Kouznetsova, Irina; Chwieralski, Caroline E; Bälder, Ralf; Hinz, Margitta; Braun, Armin; Krug, Norbert; Hoffmann, Werner

2007-03-01

Asthma is a chronic inflammatory disease of the airways that is accompanied by goblet cell metaplasia and mucus hypersecretion. Trefoil factor family (TFF) peptides represent major secretory products of the respiratory tract and are synthesized together with mucins. In the murine lung, TFF2 is mainly expressed, whereas TFF1 transcripts represent only a minor species. TFF peptides are well known for their motogenic and anti-apoptotic effects, and they modulate the inflammatory response of bronchial epithelial cells. Here, an established mouse model of asthma was investigated (i.e., exposure to Aspergillus fumigatus [AF] antigens). RT-PCR analysis of lung tissue showed elevated levels particularly of TFF1 transcripts in AF-sensitized/challenged animals. In contrast, transcripts encoding Clara cell secretory protein (CCSP/CC10) were strongly diminished in these animals. For comparison, the expression of the goblet cell secretory granule marker mCLCA3/Gob-5, the mucins Muc1-Muc6 and Muc19, and the secretoglobins ScgB3A1 and ScgB3A2, as well as the mammalian ependymin-related gene MERP2, were monitored. Immunohistochemistry localized TFF1 mainly in cells with a mixed phenotype (e.g., TFF1-positive cells stain with the lectin wheat germ agglutinin (WGA), which recognizes mucins characteristic of goblet cells). In addition, these cells express CCSP/CC10, a Clara cell marker. When compared with mucins or CCSP/CC10, TFF1 was stored in a different population of secretory granules localized at the more basolateral portion of these cells. Thus, the results presented indicate for the first time that allergen exposure leads to the trans-differentiation of Clara cells toward a TFF1-expressing mucous phenotype.

Structural associations between organelle membranes in nectary parenchyma cells.

PubMed

Machado, Silvia Rodrigues; Gregório, Elisa A; Rodrigues, Tatiane M

2018-05-01

The close association between membranes and organelles, and the intense chloroplast remodeling in parenchyma cells of extrafloral nectaries occurred only at the secretion time and suggest a relationship with the nectar secretion. Associations between membranes and organelles have been well documented in different tissues and cells of plants, but poorly explored in secretory cells. Here, we described the close physical juxtaposition between membranes and organelles, mainly with chloroplasts, in parenchyma cells of Citharexylum myrianthum (Verbenaeceae) extrafloral nectaries under transmission electron microscopy, using conventional and microwave fixation. At the time of nectar secretion, nectary parenchyma cells exhibit a multitude of different organelle and membrane associations as mitochondria-mitochondria, mitochondria-endoplasmic reticulum, mitochondria-chloroplast, chloroplast-nuclear envelope, mitochondria-nuclear envelope, chloroplast-plasmalemma, chloroplast-chloroplast, chloroplast-tonoplast, chloroplast-peroxisome, and mitochondria-peroxisome. These associations were visualized as amorphous electron-dense material, a network of dense fibrillar material and/or dense bridges. Chloroplasts exhibited protrusions variable in shape and extension, which bring them closer to each other and to plasmalemma, tonoplast, and nuclear envelope. Parenchyma cells in the pre- and post-secretory stages did not exhibit any association or juxtaposition of membranes and organelles, and chloroplast protrusions were absent. Chloroplasts had peripheral reticulum that was more developed in the secretory stage. We propose that such subcellular phenomena during the time of nectar secretion optimize the movement of signaling molecules and the exchange of metabolites. Our results open new avenues on the potential mechanisms of organelle contact in parenchyma nectary cells, and reveal new attributes of the secretory cells on the subcellular level.

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.

Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.

PubMed

Ohara-Imaizumi, Mica; Nakamichi, Yoko; Tanaka, Toshiaki; Katsuta, Hidenori; Ishida, Hitoshi; Nagamatsu, Shinya

2002-04-01

The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic beta-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH 7.4, but not at approximately pH 5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy (5 or 10 s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50 mM KCl and 22 mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin-pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

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.

Age-related alterations in IL-1beta, TNF-alpha, and IL-6 concentrations in parotid acinar cells from BALB/c and non-obese diabetic mice.

PubMed

Yamakawa, M; Weinstein, R; Tsuji, T; McBride, J; Wong, D T; Login, G R

2000-08-01

IL-1beta, TNF-alpha, and IL-6 have been implicated in the destruction of parotid gland acinar cells (but not duct cells) in autoimmune sialoadenitis. Here we report the temporal alterations of these cytokines in parotid acinar cells that may lead to this specificity in cell death in the non-obese diabetic (NOD) mouse model for Sjögren's syndrome. Immunohistochemistry on paraffin sections of parotid gland from 5- and 10-week-old BALB/c and NOD mice confirmed the presence of many peri-acinar lymphoid nodules but few T-cells and macrophages between acinar cells. RT-PCR on enzymatically dispersed mouse parotid acinar cells (MPACs) showed no bands for CD3varepsilon, CD20, or F4/80 regardless of mouse strain or age. By ELISA, MPACs from 10-week-old NODs showed a small but highly significant (p<0.003) increase in IL-1beta and a large significant decrease (p<0.008) in IL-6 compared to 5-week-old NODs. Norepinephrine-stimulated amylase release from MPACs was not different regardless of mouse strain or age. These data show that alterations in acinar cell production of IL-1beta and IL-6 in aging NODs precede periductal lymphoid aggregates and acinar cell secretory dysfunction. (J Histochem Cytochem 48:1033-1041,2000)

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

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

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.

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

Clear cell and endometrioid carcinomas: are their differences attributable to distinct cells of origin?

PubMed

Cochrane, Dawn R; Tessier-Cloutier, Basile; Lawrence, Katherine M; Nazeran, Tayyebeh; Karnezis, Anthony N; Salamanca, Clara; Cheng, Angela S; McAlpine, Jessica N; Hoang, Lien N; Gilks, C Blake; Huntsman, David G

2017-09-01

Endometrial epithelium is the presumed tissue of origin for both eutopic and endometriosis-derived clear cell and endometrioid carcinomas. We had previously hypothesized that the morphological, biological and clinical differences between these carcinomas are due to histotype-specific mutations. Although some mutations and genomic landscape features are more likely to be found in one of these histotypes, we were not able to identify a single class of mutations that was exclusively present in one histotype and not the other. This lack of genomic differences led us to an alternative hypothesis that these cancers could arise from distinct cells of origin within endometrial tissue, and that it is the cellular context that accounts for their differences. In a proteomic screen, we identified cystathionine γ-lyase (CTH) as a marker for clear cell carcinoma, as it is expressed at high levels in clear cell carcinomas of the ovary and endometrium. In the current study, we analysed normal Müllerian tissues, and found that CTH is expressed in ciliated cells of endometrium (both eutopic endometrium and endometriosis) and fallopian tubes. We then demonstrated that other ciliated cell markers are expressed in clear cell carcinomas, whereas endometrial secretory cell markers are expressed in endometrioid carcinomas. The same differential staining of secretory and ciliated cells was demonstrable in a three-dimensional organoid culture system, in which stem cells were stimulated to differentiate into an admixture of secretory and ciliated cells. These data suggest that endometrioid carcinomas are derived from cells of the secretory cell lineage, whereas clear cell carcinomas are derived from, or have similarities to, cells of the ciliated cell lineage. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses

PubMed Central

2011-01-01

Background The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications. Results The impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low Pi supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter. Conclusions Our study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins. PMID:22151908

Pseudomonas aeruginosa lipopolysaccharide induces CF-like alteration of protein secretion by human tracheal gland cells.

PubMed

Kammouni, W; Figarella, C; Baeza, N; Marchand, S; Merten, M D

1997-12-18

Human tracheal gland (HTG) serous cells are now believed to play a major role in the physiopathology of cystic fibrosis. Because of the persistent inflammation and the specific infection by Pseudomonas aeruginosa in the lung, we looked for the action of the lipopolysaccharide (LPS) of this bacteria on human tracheal gland cells in culture by studying the secretion of the secretory leukocyte proteinase inhibitor (SLPI) which is a specific serous secretory marker of these cells. Treatment with Pseudomonas aeruginosa LPS resulted in a significant dose-dependent increase in the basal production of SLPI (+ 250 +/- 25%) whilst the SLPI transcript mRNA levels remained unchanged. This LPS-induced increase in secretion was inhibited by glucocorticoides. Furthermore, LPS treatment of HTG cells induces a loss of responsiveness to carbachol and isoproterenol but not to adenosine triphosphate. These findings indicate that HTG cells treated by Pseudomonas aeruginosa LPS have the same behavior as those previously observed with CF-HTG cells. Exploration by using reverse transcriptase polymerase chain reaction amplification showed that LPS downregulated cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in HTG cells indicative of a link between CFTR function and consequent CF-like alteration in protein secretory process.

Leaf colleters in Tontelea micrantha (Celastraceae, Salacioideae): ecological, morphological and structural aspects.

PubMed

Mercadante-Simões, Maria Olívia; Paiva, Elder Antônio Sousa

2013-08-01

The colleter secretion can be useful to protect plants of Cerrado (Brazilian savanna) biome during the long and pronounced dry season. This study describes the presence of colleters in Tontelea micrantha and represents the first record of these structures in Celastraceae. To investigate colleter structure and their secretory processes, young leaves were collected, fixed, and processed according to conventional techniques for light, and electron microscopy. Colleters were observed at the marginal teeth on the leaf. They produce mucilaginous secretions that spread over the leaf surface. After secretory phase, colleters abscise. The secretory epithelium is uniseriate and composed of elongated cells whose dense cytoplasm is rich in organelles. The ultrastructure of the secretory cells is compatible with the pectin-rich secretion. Observations of the young leaves surface revealed the presence of superficial hydrophilic secretion films that appeared to have the function of maintaining the water status of those organs. Copyright © 2013 Académie des sciences. Published by Elsevier SAS. 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.

The defensive secretion of Carabus lefebvrei Dejean 1826 pupa (Coleoptera, Carabidae): gland ultrastructure and chemical identification.

PubMed

Giglio, Anita; Brandmayr, Pietro; Dalpozzo, Renato; Sindona, Giovanni; Tagarelli, Antonio; Talarico, Federica; Brandmayr, Tullia Zetto; Ferrero, Enrico A

2009-05-01

This study documents the defensive function of flavored humor secreted by the abdominal glands of Carabus lefebvrei pupae. The morphology and the ultrastructure of these glands were described and the volatile compounds of glands secretion were identified by gas chromatography/mass spectrometry. The ultrastructure analysis shows an acinose complex formed by about 50 clusters. Each cluster has 20 glandular units and the unit-composed of one secretory and one canal cell lying along a duct-belongs to the class 3 cell type of Quennedey (1998). In the cytoplasm, the secretory cell contains abundant rough endoplasmatic reticula, glycogen granules, numerous mitochondria, and many well-developed Golgi complexes producing electron-dense secretory granules. Mitochondria are large, elongated, and often adjoining electronlucent vesicles. The kind and the origin of secretory granules varying in size and density were discussed. The chemical analysis of the gland secretion revealed the presence of a mixture of low molecular weight terpenes, ketones, aldehydes, alcohols, esters, and carboxylic acids. Monoterpenes, especially linalool, were the major products. We supposed that ketones, aldehydes, alcohols, esters, and carboxylic acids have a deterrent function against the predators and monoterpenes provide a prophylaxis function against pathogens. (c) 2008 Wiley-Liss, Inc.

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.

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.

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency

PubMed Central

Tourlakis, Marina E.; Zhang, Siyi; Ball, Heather L.; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S.; Guidos, Cynthia J.; Durie, Peter R.; Rommens, Johanna M.

2015-01-01

Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15Ink4b and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to be p53-dependent. Our findings therefore point to cell/tissue-specific responses to p53-activation that include distinction between apoptosis and senescence pathways, in the context of translation disruption. PMID:26057580

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

PubMed

Tourlakis, Marina E; Zhang, Siyi; Ball, Heather L; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S; Guidos, Cynthia J; Durie, Peter R; Rommens, Johanna M

2015-06-01

Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to be p53-dependent. Our findings therefore point to cell/tissue-specific responses to p53-activation that include distinction between apoptosis and senescence pathways, in the context of translation disruption.

Cell-to-cell contact dependence and junctional protein content are correlated with in vivo maturation of pancreatic beta cells.

PubMed

Santos-Silva, Junia Carolina; Carvalho, Carolina Prado de França; de Oliveira, Ricardo Beltrame; Boschero, Antonio Carlos; Collares-Buzato, Carla Beatriz

2012-07-01

In this study, we investigated the cellular distribution of junctional proteins and the dependence on cell-cell contacts of pancreatic beta cells during animal development. Fetus and newborn rat islets, which display a relatively poor insulin secretory response to glucose, present an immature morphology and cytoarchitecture when compared with young and adult islets that are responsive to glucose. At the perinatal stage, beta cells display a low junctional content of neural cell adhesion molecule (N-CAM), α- and β-catenins, ZO-1, and F-actin, while a differential distribution of N-CAM and Pan-cadherin was seen in beta cells and nonbeta cells only from young and adult islets. In the absence of intercellular contacts, the glucose-stimulated insulin secretion was completely blocked in adult beta cells, but after reaggregation they partially reestablished the secretory response to glucose. By contrast, neonatal beta cells were poorly responsive to sugar, regardless of whether they were arranged as intact islets or as isolated cells. Interestingly, after 10 days of culturing, neonatal beta cells, known to display increased junctional protein content in vitro, became responsive to glucose and concomitantly dependent on cell-cell contacts. Therefore, our data suggest that the developmental acquisition of an adult-like insulin secretory pattern is paralleled by a dependence on direct cell-cell interactions.

Culture of prostate epithelial cells of the rhesus monkey on extracellular matrix substrate: influence of steroids and insulin-like growth factors.

PubMed

Udayakumar, T S; Jeyaraj, D A; Rajalakshmi, M; Sharma, R S

1999-09-01

Rhesus monkey prostate epithelial cells from the cranial lobe were isolated and cultured in flasks coated either with collagen IV or laminin. The effects of stromal cell medium, androgens and growth factors on cell number, thymidine incorporation and secretory activity were assessed. The results indicate that dihydrotestosterone (DHT) and androstenedione have stimulatory influences on cell proliferation and secretion in coated flasks. DHT was more effective in increasing cell number but the induction of secretory activity was similar with both steroids. The combination of IGF-I and -II resulted in inducing better cell proliferation and secretory activity than the individual IGFs but, of the two IGFs, IGF-I was more effective than IGF-II. DHT with IGFs was more potent in inducing proliferation, differentiation and secretion than androstenedione. Even in the absence of steroids or growth factors, colony formation and confluence occurred in coated flasks but cell differentiation and secretion only to a limited extent. In conclusion, we were able to establish an in vitro primary culture of prostate epithelial cells from rhesus monkey using extracellular matrix proteins, steroids and growth factors as additional supplements. This culture system may be useful to study prostate cell physiology and to identify drugs that can inhibit cell proliferation.

Brefeldin A Inhibits Cell-Free, De Novo Synthesis of Poliovirus

PubMed Central

Cuconati, Andrea; Molla, Akhteruzzaman; Wimmer, Eckard

1998-01-01

Brefeldin A (BFA), an inhibitor of intracellular vesicle-dependent secretory transport, is a potent inhibitor of poliovirus RNA replication in infected cells. We have determined that the unknown mechanism of BFA inhibition of replication is reproduced in the cell-free poliovirus translation, replication, and encapsidation system. Furthermore, we provide evidence suggesting that the cellular mechanism targeted by BFA, the GTP-dependent synthesis of secretory transport vesicles, may be involved in viral RNA replication in the system via a soluble cellular GTP-binding and -hydrolyzing activity. This activity is related to the ARF (ADP-ribosylation factor) family of GTP-binding proteins. ARFs are required for the formation of several classes of secretory vesicles, and some family members are indirectly inactivated by BFA. Peptides that function as competitive inhibitors of ARF activity in cell-free transport systems also inhibit poliovirus RNA replication, and this inhibitory effect can be countered by the addition of exogenous ARF. We suggest that BFA inhibition of replication is diagnostic of a requirement for ARF activity in the cell-free system. PMID:9658088

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.

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania.

PubMed

Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

2015-12-11

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Biosynthesis, Trafficking and Secretion of Pro-opiomelanocortin-derived peptides

PubMed Central

Cawley, Niamh X.; Li, Zhaojin; Loh, Y. Peng

2016-01-01

Pro-opiomelanocortin (POMC) is a prohormone that encodes multiple smaller peptide hormones within its structure. These peptide hormones can be generated by cleavage of POMC at basic-residue cleavage sites by prohormone converting enzymes in the regulated secretory pathway of POMC synthesizing endocrine cells and neurons. The peptides are stored inside the cells in dense core secretory granules until released in a stimulus dependent manner. The complexity of the regulation of the biosynthesis, trafficking and secretion of POMC and its peptides reflect an impressive level of control over many factors involved in the ultimate role of POMC expressing cells, i.e. to produce a range of different biologically active peptide hormones ready for action when signaled by the body. From the discovery of POMC as the precursor to ACTH and β-Lipotropin in the late 1970s to our current knowledge, the understanding of POMC physiology remains a monumental body of work that has provided insight into many aspects of molecular endocrinology. In this chapter, we describe the intracellular trafficking of POMC in endocrine cells, its sorting into dense core secretory granules and transport of these granules to the regulated secretory pathway. Additionally, we review the enzymes involved in the maturation of POMC to its various peptides and the mechanisms involved in the differential processing of POMC in different cell types. Finally, we highlight studies pertaining to the regulation of ACTH secretion in the anterior and intermediate pituitary and POMC neurons of the hypothalamus. PMID:26880796

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

Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function.

PubMed

Freedman, S D; Scheele, G A

1994-03-23

The role of acid-base interactions during coordinated acinar and duct cell secretion in the exocrine pancreas is described. The sequence of acid-base events may be summarized as follows: (1) Sorting of secretory proteins and membrane components into the regulated secretory pathway of pancreatic acinar cells is triggered by acid- and calcium-induced aggregation and association mechanisms located in the trans-Golgi network. (2) Cholecystokinin-stimulated exocytosis in acinar cells releases the acidic contents of secretory granules into the acinar lumen. (3) Secretin-stimulated bicarbonate secretion from duct and duct-like cells neutralizes the acidic pH of exocytic contents, which leads to dissociation of protein aggregates and solubilization of (pro)enzymes within the acinar lumen. (4) Stimulated fluid secretion transports solubilized enzymes through the ductal system. (5) Further alkalinization of acinar lumen pH accelerates the enzymatic cleavage of the glycosyl phosphatidyl-inositol anchor associated with GP2 and thus releases the GP2/proteoglycan matrix from lumenal membranes, a process that appears to be required for vesicular retrieval of granule membranes from the apical plasma membrane and their reuse in the secretory process. We conclude that the central function of bicarbonate secretion by centroacinar and duct cells in the pancreas is to neutralize and then alkalinize the pH of the acinar lumen, sequential process that are required for (a) solubilization of secreted proteins and (b) cellular retrieval of granule membranes, respectively.

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

Dietary toxins, endoplasmic reticulum (ER) stress and diabetes.

PubMed

Hettiarachchi, Kalindi D; Zimmet, Paul Z; Myers, Mark A

2008-05-01

The incidence of Type 1 diabetes has been increasing at a rate too rapid to be due to changes in genetic risk. Instead changes in environmental factors are the likely culprit. The endoplasmic reticulum (ER) plays an important role in the production of newly synthesized proteins and interference with these processes leads to ER stress. The insulin-producing beta cells are particularly prone to ER stress as a result of their heavy engagement in insulin production. Increasing evidence suggests ER stress is central to initiation and progression of Type 1 diabetes. An early environmental exposure, such as toxins and viral infections, can impart a significant physiological load on beta cells to initiate abnormal processing of proinsulin, ER stress and insulin secretory defects. Release of altered proinsulin from the beta cells early in life may trigger autoimmunity in those with genetic susceptibility leading to cytokine-induced nitric oxide production and so exacerbating ER stress in beta cells, ultimately leading to apoptosis of beta cells and diabetes. Here we suggest that ER stress is an inherent cause of beta cell dysfunction and environmental factors, in particular dietary toxins derived from Streptomyces in infected root vegetables, can impart additional stress that aggravates beta cell death and progression to diabetes. Furthermore, we propose that the increasing incidence of Type 1 diabetes may be accounted for by increased dietary exposure to ER-stress-inducing Streptomyces toxins.

Curcumin attenuates palmitate-induced apoptosis in MIN6 pancreatic β-cells through PI3K/Akt/FoxO1 and mitochondrial survival pathways.

PubMed

Hao, Feng; Kang, Jinsen; Cao, Yajun; Fan, Shengjun; Yang, Haopeng; An, Yu; Pan, Yan; Tie, Lu; Li, Xuejun

2015-11-01

Lipotoxicity plays a vital role in development and progression of type 2 diabetes. Prolonged elevation of free fatty acids especially the palmitate leads to pancreatic β-cell dysfunction and apoptosis. Curcumin (diferuloylmethane), a polyphenol from the curry spice turmeric, is considered to be a broadly cytoprotective agent. The present study was designed to determine the protective effect of curcumin on palmitate-induced apoptosis in β-cells and investigate underlying mechanisms. Our results showed that curcumin improved cell viability and enhanced glucose-induced insulin secretory function in MIN6 pancreatic β-cells. Palmitate incubation evoked chromatin condensation, DNA nick end labeling and activation of caspase-3 and -9. Curcumin treatment inhibited palmitate-induced apoptosis, relieved mitochondrial depolarization and up-regulated Bcl-2/Bax ratio. Palmitate induced the generation of reactive oxygen species and inhibited activities of antioxidant enzymes, which could be neutralized by curcumin treatment. Moreover, curcumin could promote rapid phosphorylation of Akt and nuclear exclusion of FoxO1 in MIN6 cells under lipotoxic condition. Phosphatidylinositol 3-kinase and Akt specific inhibitors abolished the anti-lipotoxic effect of curcumin and stimulated FoxO1 nuclear translocation. These findings suggested that curcumin protected MIN6 pancreatic β-Cells against apoptosis through activation of Akt, inhibition of nuclear translocation of FoxO1 and mitochondrial survival pathway.

Alpha-synuclein Toxicity in the Early Secretory Pathway: How It Drives Neurodegeneration in Parkinsons Disease

PubMed Central

Wang, Ting; Hay, Jesse C.

2015-01-01

Alpha-synuclein is a predominant player in the pathogenesis of Parkinson's Disease. However, despite extensive study for two decades, its physiological and pathological mechanisms remain poorly understood. Alpha-synuclein forms a perplexing web of interactions with lipids, trafficking machinery, and other regulatory factors. One emerging consensus is that synaptic vesicles are likely the functional site for alpha-synuclein, where it appears to facilitate vesicle docking and fusion. On the other hand, the dysfunctions of alpha-synuclein are more dispersed and numerous; when mutated or over-expressed, alpha-synuclein affects several membrane trafficking and stress pathways, including exocytosis, ER-to-Golgi transport, ER stress, Golgi homeostasis, endocytosis, autophagy, oxidative stress, and others. Here we examine recent developments in alpha-synuclein's toxicity in the early secretory pathway placed in the context of emerging themes from other affected pathways to help illuminate its underlying pathogenic mechanisms in neurodegeneration. PMID:26617485

Epithelial-stromal interaction via Notch signaling is essential for the full maturation of gut-associated lymphoid tissues.

PubMed

Obata, Yuuki; Kimura, Shunsuke; Nakato, Gaku; Iizuka, Keito; Miyagawa, Yurika; Nakamura, Yutaka; Furusawa, Yukihiro; Sugiyama, Machiko; Suzuki, Keiichiro; Ebisawa, Masashi; Fujimura, Yumiko; Yoshida, Hisahiro; Iwanaga, Toshihiko; Hase, Koji; Ohno, Hiroshi

2014-12-01

Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE. © 2014 The Authors.

How does the stimulus define exocytosis in adrenal chromaffin cells?

PubMed

Marengo, Fernando D; Cárdenas, Ana M

2018-01-01

The extent and type of hormones and active peptides secreted by the chromaffin cells of the adrenal medulla have to be adjusted to physiological requirements. The chromaffin cell secretory activity is controlled by the splanchnic nerve firing frequency, which goes from approximately 0.5 Hz in basal conditions to more than 15 Hz in stress. Thus, these neuroendocrine cells maintain a tonic release of catecholamines under resting conditions, massively discharge intravesicular transmitters in response to stress, or adequately respond to moderate stimuli. In order to adjust the secretory response to the stimulus, the adrenal chromaffin cells have an appropriate organization of Ca 2+ channels, secretory granules pools, and sets of proteins dedicated to selectively control different steps of the secretion process, such as the traffic, docking, priming and fusion of the chromaffin granules. Among the molecules implicated in such events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Ca 2+ sensors like Munc13 and synaptotagmin-1, chaperon proteins such as Munc18, and the actomyosin complex. In the present review, we discuss how these different actors contribute to the extent and maintenance of the stimulus-dependent exocytosis in the adrenal chromaffin cells.

Ultrastructure and Glycoconjugate Pattern of the Foot Epithelium of the Abalone Haliotis tuberculata (Linnaeus, 1758) (Gastropoda, Haliotidae)

PubMed Central

Bravo Portela, I.; Martinez-Zorzano, V. S.; Molist- Perez, I.; Molist García, P.

2012-01-01

The foot epithelium of the gastropod Haliotis tuberculata is studied by light and electron microscopy in order to contribute to the understanding of the anatomy and functional morphology of the mollusks integument. Study of the external surface by scanning electron microscopy reveals that the side foot epithelium is characterized by a microvillus border with a very scant presence of small ciliary tufts, but the sole foot epithelium bears a dense field of long cilia. Ultrastructural examination by transmission electron microscopy of the side epithelial cells shows deeply pigmented cells with high electron-dense granular content which are not observed in the epithelial sole cells. Along the pedal epithelium, seven types of secretory cells are present; furthermore, two types of subepithelial glands are located just in the sole foot. The presence and composition of glycoconjugates in the secretory cells and subepithelial glands are analyzed by conventional and lectin histochemistry. Subepithelial glands contain mainly N-glycoproteins rich in fucose and mannose whereas secretory cells present mostly acidic sulphated glycoconjugates such as glycosaminoglycans and mucins, which are rich in galactose, N-acetyl-galactosamine, and N-acetyl-glucosamine. No sialic acid is present in the foot epithelium. PMID:22645482

Diminished parathyroid gland responsiveness to hypocalcemia in diabetic patients with uremia.

PubMed

Heidbreder, E; Götz, R; Schafferhans, K; Heidland, A

1986-01-01

The parathyroid gland responsiveness to hypocalcemia induced by short-term calcium-free hemodialysis in patients with insulin-dependent diabetes mellitus was investigated in comparison with 10 nondiabetic uremic patients and compared with test results from the autonomic nervous system. Diabetic patients had lower C-terminal parathyroid hormone (cPTH) levels before hemodialysis than uremic control patients and showed a significantly smaller increase in cPTH during hypocalcemia. The neurological tests revealed severe disturbances of the autonomic functions in the diabetic group. In conclusion, the disturbances observed in the parathyroid secretory pattern are probably caused by gland dysfunction; it is hypothesized that the defective autonomic nervous system has an additional effect on the development of this hormonal dysfunction.

Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility.

PubMed

Bolnick, Alan D; Bolnick, Jay M; Kilburn, Brian A; Stewart, Tamika; Oakes, Jonathan; Rodriguez-Kovacs, Javier; Kohan-Ghadr, Hamid-Reza; Dai, Jing; Diamond, Michael P; Hirota, Yasushi; Drewlo, Sascha; Dey, Sudhansu K; Armant, D Randall

2016-09-01

Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility? MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples. Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility. MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and β-catenin in epithelial cells. MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases. MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and β-catenin in epithelial cell junctions in the mid- and late-secretory phases. Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model. This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation. This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A Role for Glutamate Transporters in the Regulation of Insulin Secretion

PubMed Central

Gammelsaeter, Runhild; Coppola, Thierry; Marcaggi, Païkan; Storm-Mathisen, Jon; Chaudhry, Farrukh A.; Attwell, David; Regazzi, Romano; Gundersen, Vidar

2011-01-01

In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules. PMID:21853059

Transcriptional and Functional Plasticity Induced by Chronic Insulin Exposure in a Mast Cell-Like Basophilic Leukemia Cell Model

PubMed Central

Jansen, Chad; Speck, Mark; Greineisen, William E; Maaetoft-Udsen, Kristina; Cordasco, Edward; Shimoda, Lori MN; Stokes, Alexander J; Turner, Helen

2018-01-01

Objective Secretory granules (SG) and lipid bodies (LB) are the primary organelles that mediate functional responses in mast cells. SG contains histamine and matrix-active proteases, while LB are reservoirs of arachidonic acid and its metabolites, precursors for rapid synthesis of eicosanoids such as LTC4. Both of these compartments can be dynamically or ontologically regulated, with metabolic and immunological stimuli altering lipid body content and granule numbers responding to contextual signals from tissue. We previously described that chronic in vitro or in vivo hyperinsulinemia expands the LB compartment with a concomitant loss of SG capacity, suggesting that this ratio is dynamically regulated. The objective of the current study is to determine if chronic insulin exposure initiates a transcriptional program that biases model mast cells towards a lipogenic state with accompanying loss of secretory granule biogenesis. Methods We used a basophilic leukemic cell line with mucosal mast cell-like features as a model system. We tested the hypothesis that chronic insulin exposure initiates a transcriptional program that biases these model mast cells towards a lipogenic state with accompanying loss of secretory granule biogenesis. Transcriptional arrays were used to map gene expression patterns. Biochemical, immunocytochemical and mediator release assays were used to evaluate organelle numbers and functional responses. Results In a mucosal mast cell model, the rat basophilic leukemia line RBL2H3, mast cell granularity and SG numbers are inversely correlated with LB numbers. Chronic insulin exposure appears to modulate gene networks involved in both lipid body biogenesis and secretory granule formation. Western blot analysis confirms upregulation of protein levels for LB proteins, and decreases in proteins that are markers for SG cargo. Conclusions The levels of insulin in the extracellular milieu may modify the phenotype of mast cell-like cells in vitro. PMID:29430572

Impairment of glucose-induced insulin secretion in human pancreatic islets transplanted to diabetic nude mice.

PubMed

Jansson, L; Eizirik, D L; Pipeleers, D G; Borg, L A; Hellerström, C; Andersson, A

1995-08-01

Hyperglycemia-induced beta-cell dysfunction may be an important component in the pathogenesis of non-insulin-dependent diabetes mellitus. However, most available data in this field were obtained from rodent islets. To investigate the relevance of this hypothesis for human beta-cells in vivo, human pancreatic islets were transplanted under the renal capsule of nude mice. Experimental groups were chosen so that grafted islets were exposed to either hyper- or normoglycemia or combinations of these for 4 or 6 wk. Grafts of normoglycemic recipients responded with an increased insulin release to a glucose stimulus during perfusion, whereas grafts of hyperglycemic recipients failed to respond to glucose. The insulin content of the grafts in the latter groups was only 10% of those observed in controls. Recipients initially hyperglycemic (4 wk), followed by 2 wk of normoglycemia regained a normal graft insulin content, but a decreased insulin response to glucose remained. No ultrastructural signs of beta-cell damage were observed, with the exception of increased glycogen deposits in animals hyperglycemic at the time of killing. It is concluded that prolonged exposure to a diabetic environment induces a long-term secretory defect in human beta-cells, which is not dependent on the size of the islet insulin stores.

Impairment of glucose-induced insulin secretion in human pancreatic islets transplanted to diabetic nude mice.

PubMed Central

Jansson, L; Eizirik, D L; Pipeleers, D G; Borg, L A; Hellerström, C; Andersson, A

1995-01-01

Hyperglycemia-induced beta-cell dysfunction may be an important component in the pathogenesis of non-insulin-dependent diabetes mellitus. However, most available data in this field were obtained from rodent islets. To investigate the relevance of this hypothesis for human beta-cells in vivo, human pancreatic islets were transplanted under the renal capsule of nude mice. Experimental groups were chosen so that grafted islets were exposed to either hyper- or normoglycemia or combinations of these for 4 or 6 wk. Grafts of normoglycemic recipients responded with an increased insulin release to a glucose stimulus during perfusion, whereas grafts of hyperglycemic recipients failed to respond to glucose. The insulin content of the grafts in the latter groups was only 10% of those observed in controls. Recipients initially hyperglycemic (4 wk), followed by 2 wk of normoglycemia regained a normal graft insulin content, but a decreased insulin response to glucose remained. No ultrastructural signs of beta-cell damage were observed, with the exception of increased glycogen deposits in animals hyperglycemic at the time of killing. It is concluded that prolonged exposure to a diabetic environment induces a long-term secretory defect in human beta-cells, which is not dependent on the size of the islet insulin stores. Images PMID:7635965

Preparation of the Secretory Recombinant ALV-J gp85 Protein Using Pichia pastoris and Its Immunoprotection as Vaccine Antigen Combining with CpG-ODN Adjuvant.

PubMed

Jing, Weifang; Zhou, Jinrun; Wang, Chunyang; Qiu, Jianhua; Guo, Huijun; Li, Hongmei

2018-04-26

This study focuses on preparing the secretory recombinant J subgroup of avian leukosis virus (ALV-J) gp85 protein using Pichia pastoris and evaluating its immunoprotection as vaccine antigen combining with CpG-ODN adjuvant. The secretory recombinant plasmid pPIC9-gp85 containing ALV-J gp85 gene was designed and was transfected into the genome of P. pastoris (GS115) cells. The recombinant plasmid was expressed under the induction of methanol. The expressed products in the medium of the cells were purified and identified with endoglycosidase digestion assay and western blot mediated with monoclonal antibody (MAb) JE9. The purified product combining with CpG-ODN adjuvant was inoculated intramuscularly into 7-day-old chickens and three booster inoculations were performed on 21 days post first inoculation (dpfi), 42, and 56 dpfi. The antibody responses and cellular immune responses were detected, and the protective effects were analyzed after challenge with ALV-J. The results showed that the secretory pPIC9-gp85 plasmid was successfully constructed and could be stably expressed in GS115 cells. The expressed products were N-acetylglucosylated and could specifically combine with MAb (JE9). The secreted gp85 protein combining with CpG-ODN adjuvant could induce higher antibody response and spleen lymphocyte proliferation response and IFN-γ-inducing response, and could protect all the inoculated chickens against the viremia and the immunosuppressive lesions caused by ALV-J challenge. The results of neutralizing test in vitro suggested that the antisera with some ALV-J antibody titers could neutralize ALV-J strain and inhibit the growth of virus in vitro. The result of IFA showed that IgG antibody in the antisera could specifically combine with ALV-J strain in cells. It can be concluded that the secretory recombinant gp85 protein, as a new acetylglucosylated gp85 protein, was successfully prepared and combining with CpG-ODN adjuvant could protect the inoculated chickens against ALV-J infection. This study first reported the methods on preparing the secretory recombinant ALV-J gp85 protein using P. pastoris and evaluated its immunoprotection.

Myb permits multilineage airway epithelial cell differentiation

PubMed Central

Pan, Jie-hong; Adair-Kirk, Tracy L.; Patel, Anand C.; Huang, Tao; Yozamp, Nicholas S.; Xu, Jian; Reddy, E. Premkumar; Byers, Derek E.; Pierce, Richard A.; Holtzman, Michael J.; Brody, Steven L.

2014-01-01

The epithelium of the pulmonary airway is specially differentiated to provide defense against environmental insults, but also subject to dysregulated differentiation that results in lung disease. The current paradigm for airway epithelial differentiation is a one-step program whereby a p63+ basal epithelial progenitor cell generates a ciliated or secretory cell lineage, but the cue for this transition and whether there are intermediate steps is poorly defined. Here we identify transcription factor Myb as a key regulator that permits early multilineage differentiation of airway epithelial cells. Myb+ cells were identified as p63− and therefore distinct from basal progenitor cells, but were still negative for markers of differentiation. Myb RNAi treatment of primary-culture airway epithelial cells and Myb gene deletion in mice resulted in a p63− population with failed maturation of Foxj1+ ciliated cells, as well as Scbg1a1+ and Muc5ac+ secretory cells. Consistent with these findings, analysis of whole genome expression of Myb-deficient cells identified Myb-dependent programs for ciliated and secretory cell differentiation. Myb+ cells were rare in human airways but were increased in regions of ciliated cells and mucous cell hyperplasia in samples from subjects with chronic obstructive pulmonary disease. Together, the results show that a p63− Myb+ population of airway epithelial cells represents a distinct intermediate stage of differentiation that is required under normal conditions and may be heightened in airway disease. PMID:25103188

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.

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.

Histological convergent evolution of the accessory submandibular glands in four species of frog-eating bats.

PubMed

Tandler, B; Phillips, C J; Nagato, T

1996-08-01

The accessory submandibular glands in four species of bats were examined by electron microscopy. These four species represent two independently evolved lineages. The fringe-lipped bat, Trachops cirrhosis, is a Neotropical phyllostomid species, whereas the false vampire bats of southeast Asia, Megaderma lyra and M. spasma, and the heart-nosed bat, Cardioderma cor, of East Africa are megadermatid species. These glands show extreme deviation from typical salivary gland histology: their secretory endpieces are in the form of follicles and their ducts lack the cytological details that permit identification of diverse duct segments. Despite their unusual histology, the secretory endpieces in M. lyra, M. spasma, and C. cor consist of secretory cells that conform to typical secretory cell morphology. In contrast, secretion by follicular cells in T. cirrhosis involves unusual cytoplasmic bodies, and their mitochondria frequently have intracristal crystalloids. Ducts in all four species consist of simple cuboidal to columnar epithelium without basal striations. Follicles and ducts in all four bats are surrounded by numerous myoepithelial cells and are heavily innervated by hypolemmal nerve terminals. Despite their widely separated geographical ranges, all four bat species consume frogs and other vertebrates. Frogs and toads often possess toxic cutaneous glands that provide a chemical defense against predation. It is postulated that the unusual accessory glands in the four frog-eating species secrete toxin-neutralizing salivary factors. The follicular form of the endpieces permits storage of preformed saliva and their coterie of myoepithelial cells and hypolemmal nerve terminals facilitates the sudden and rapid expulsion of saliva into the oral cavity during the consumption of noxious amphibians.

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.

Differentially regulated gene expression in quiescence versus senescence and identification of ARID5A as a quiescence associated marker.

PubMed

Anwar, Tarique; Sen, Bijoya; Aggarwal, Savera; Nath, Rhisita; Pathak, Niteen; Katoch, Ajay; Aiyaz, Mohamed; Trehanpati, Nirupma; Khosla, Sanjeev; Ramakrishna, Gayatri

2018-05-01

In multicellular organisms majority of the cells remain in a non-dividing states of either quiescence (reversible) or senescence (irreversible). In the present study, gene expression signatures unique to quiescence and senescence were identified using microarray in osteosarcoma cell line, U2OS. It was noted that certain genes and pathways like NOD pathway was shared by both the growth arrest conditions. A major highlight of the present study was increased expression of number of chemokines and cytokines in both quiescence and senescence. While senescence-associated secretory phenotype (SASP) is well known, the quiescence-associated secretory phenotype (QASP) is relatively unknown and appeared novel in this study. ARID5A, a subunit of SWI/SNF complex was identified as a quiescence associated gene. The endogenous expression of ARID5A increased during serum starved condition of quiescence. Overexpression of ARID5A resulted in more number of cells in G0/G1 phase of cell cycle. Further ARID5A overexpressing cells when subjected to serum starvation showed a pronounced secretory phenotype. Overall, the present work has identified gene expression signatures which can distinguish quiescence from senescence. © 2017 Wiley Periodicals, Inc.

Ultrastructural evidence of a vesicle-mediated mode of cell degranulation in chicken chromaffin cells during the late phase of embryonic development

PubMed Central

Crivellato, Enrico; Nico, Beatrice; Travan, Luciana; Isola, Miriam; Ribatti, Domenico

2009-01-01

In the present investigation, we attempted to determine whether ultrastructural features indicative of a vesicle-mediated mode of cell secretion were detectable in chick chromaffin cells during embryo development. The adrenal anlagen of domestic fowls were examined at embryonic days (E) 12, 15, 19 and 21 by electron microscopy quantitative analysis. Morphometric evaluation revealed a series of granule and cytoplasmic changes highly specific for piecemeal degranulation (PMD), a secretory process based on vesicular transport of cargoes from within granules for extracellular release. At E19 and E21 we found a significant peak in the percentage of granules exhibiting changes indicative of progressive release of secretory materials, i.e. granules with lucent areas in their cores, reduced electron density, disassembled matrices, residual cores and membrane empty containers. A dramatic raise in the density of 30–80-nm-diameter, membrane-bound, electron-dense and electron-lucent vesicles – which were located either next to granules or close to the plasma membrane – was recognizable at E19, that is, during the prehatching phase. The cytoplasmic burst of dense and clear vesicles was paralleled by the appearance of chromaffin granules showing outpouches or protrusions of their profiles (‘budding features’). These ultrastructural data are indicative of an augmented vesicle-mediated transport of chromaffin granule products for extracellular release in chick embryo chromaffin cells during the prehatching stage. In conclusion, this study provides new data on the fine structure of chromaffin cell organelles during organ development and suggests that PMD may be part of an adrenomedullary secretory response that occurs towards the end of chicken embryogenesis. From an evolutionary point of view, this study lends support to the concept that PMD is a secretory mechanism highly conserved throughout vertebrate classes. PMID:19245498

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

PubMed Central

Noe, BD; Baste, CA; Bauer, GE

1977-01-01

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level. PMID:328517

Labellar anatomy and secretion in Bulbophyllum Thouars (Orchidaceae: Bulbophyllinae) sect. Racemosae Benth. & Hook. f.

PubMed Central

Davies, Kevin L.; Stpiczyńska, Malgorzata

2014-01-01

Background and Aims Floral secretions are common in Bulbophyllum Thouars, and the labella of a number of Asian species are said to produce secretions rich in lipids that act as food rewards for insect pollinators. Although some of these reports are based on simple histochemical tests, a much greater number are anecdotal and, hitherto, neither the ultrastructure of the labellum nor the secretory process has been investigated in detail. Furthermore, sophisticated histochemical approaches have generally not been applied. Here, both the labellar structure and the secretory process are investigated for four species of Asian Bulbophyllum sect. Racemosae Benth. & Hook. f., namely Bulbophyllum careyanum (Hook.) Spreng., B. morphologorum Kraenzl., B. orientale Seidenf. and B. wangkaense Seidenf., and compared with those of unequivocal lipid-secreting orchids. Methods Labellar, secretory tissue was investigated using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. Key Results The adaxial median longitudinal groove of the labellum contained secretory tissue comprising palisade-like epidermal cells, similar to those of certain lipid-secreting Oncidiinae Benth. However, these cells and their secretions gave positive results mainly for protein and mucilage, and their organelle complement was consistent with that of cells involved in protein and mucilage synthesis. Sub-cuticular accumulation of secretion resulted in cuticular distension and blistering. The sub-epidermal layer of isodiametric parenchyma contained starch and, like the epidermal cells, ultrastructure consistent with mucilage synthesis. Lipids were mainly confined to the cuticle, and hardly any intracellular lipid droplets were observed. Conclusions It is proposed that mucilage is produced by dictyosomes present in the palisade-like epidermal cells. Mucilage precursors may also be produced by these same organelles in sub-epidermal cells and are thought to pass along the symplast via plasmodesmata into the adjoining palisade-like secretory cells, which contain abundant arrays of rough endoplasmic reticulum. Here, they become chemically modified and form a protein-rich, mucilaginous secretion that, following vesicle-mediated transport across the cytoplasm, traverses the cell wall and accumulates in blisters formed from the distended cuticle. Rupture of these blisters releases the secretion onto the labellar surface. However, in certain species, there is some evidence that the secretion may traverse the cuticle via cuticular pores, and micro-channels may permit the passage of fragrance. Hydrolysis of sub-epidermal starch probably generates the carbohydrate and, together with mitochondria, much of the energy required for the secretory process. This anatomical organization resembles that found in certain lipid-secreting, Neotropical species of Bulbophyllum and Oncidiinae, but since the chemical composition of their secretions is different, and these taxa occur on a separate continent and have different insect pollinators, parallelism of floral anatomy is likely. PMID:25122654

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…

Protein quality control in the early secretory pathway

PubMed Central

Anelli, Tiziana; Sitia, Roberto

2008-01-01

Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)–Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. ‘Public' and ‘private' chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion. PMID:18216874

Immunohistochemical evidence suggests intrinsic regulatory activity of human eccrine sweat glands

PubMed Central

ZANCANARO, CARLO; MERIGO, FLAVIA; CRESCIMANNO, CATERINA; ORLANDINI, SIMONETTA; OSCULATI, ANTONIO

1999-01-01

Immunohistochemistry of normal eccrine sweat glands was performed on paraffin sections of human skin. Immunoreactivity (ir) for neuron specific enolase, S100 protein (S100), regulatory peptides, nitric oxide synthase type I (NOS-I) and choline-acetyltransferase (ChAT) was found in small nerve bundles close to sweat glands. In the glands, secretory cells were labelled with anticytokeratin antibody. Using antibodies to S100, calcitonin gene-related peptide (CGRP) and substance P (SP) a specific distribution pattern was found in secretory cells. Granulated (dark) and parietal (clear) cells were immunopositive for CGRP, and S100 and SP, respectively. Immunoreactivity was diffuse in the cytoplasm for CGRP and S100, and peripheral for SP. Myoepithelial cells were not labelled. Electron microscopy revealed electron dense granules, probably containing peptide, in granulated cells. Using antibodies to NOS-I and ChAT, ir was exclusively found in myoepithelial cells. Immunoreactivity for the atrial natriuretic peptide was absent in sweat glands. These results provide evidence for the presence of both regulatory peptides involved in vasodilation and key enzymes for the synthesis of nitric oxide and acetylcholine in the secretory coil of human sweat glands. It is suggested that human sweat glands are capable of some intrinsic regulation in addition to that carried out by their nerve supply. PMID:10386780

Eccrine sweat gland development and sweat secretion

PubMed Central

Cui, Chang-Yi; Schlessinger, David

2017-01-01

Eccrine sweat glands help to maintain homoeostasis, primarily by stabilizing body temperature. Derived from embryonic ectoderm, millions of eccrine glands are distributed across human skin and secrete litres of sweat per day. Their easy accessibility has facilitated the start of analyses of their development and function. Mouse genetic models find sweat gland development regulated sequentially by Wnt, Eda and Shh pathways, although precise subpathways and additional regulators require further elucidation. Mature glands have two secretory cell types, clear and dark cells, whose comparative development and functional interactions remain largely unknown. Clear cells have long been known as the major secretory cells, but recent studies suggest that dark cells are also indispensable for sweat secretion. Dark cell-specific Foxa1 expression was shown to regulate a Ca2+-dependent Best2 anion channel that is the candidate driver for the required ion currents. Overall, it was shown that cholinergic impulses trigger sweat secretion in mature glands through second messengers – for example InsP3 and Ca2+ – and downstream ion channels/transporters in the framework of a Na+-K+-Cl− cotransporter model. Notably, the microenvironment surrounding secretory cells, including acid–base balance, was implicated to be important for proper sweat secretion, which requires further clarification. Furthermore, multiple ion channels have been shown to be expressed in clear and dark cells, but the degree to which various ion channels function redundantly or indispensably also remains to be determined. PMID:26014472

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

Dielectric properties of isolated adrenal chromaffin cells determined by microfluidic impedance spectroscopy.

PubMed

Sabuncu, A C; Stacey, M; Craviso, G L; Semenova, N; Vernier, P T; Leblanc, N; Chatterjee, I; Zaklit, J

2018-02-01

Knowledge of the dielectric properties of biological cells plays an important role in numerical models aimed at understanding how high intensity ultrashort nanosecond electric pulses affect the plasma membrane and the membranes of intracellular organelles. To this end, using electrical impedance spectroscopy, the dielectric properties of isolated, neuroendocrine adrenal chromaffin cells were obtained. Measured impedance data of the cell suspension, acquired between 1kHz and 20MHz, were fit into a combination of constant phase element and Cole-Cole models from which the effect of electrode polarization was extracted. The dielectric spectrum of each cell suspension was fit into a Maxwell-Wagner mixture model and the Clausius-Mossotti factor was obtained. Lastly, to extract the cellular dielectric parameters, the cell dielectric data were fit into a granular cell model representative of a chromaffin cell, which was based on the inclusion of secretory granules in the cytoplasm. Chromaffin cell parameters determined from this study were the cell and secretory granule membrane specific capacitance (1.22 and 7.10μF/cm 2 , respectively), the cytoplasmic conductivity, which excludes and includes the effect of intracellular membranous structures (1.14 and 0.49S/m, respectively), and the secretory granule milieu conductivity (0.35S/m). These measurements will be crucial for incorporating into numerical models aimed at understanding the differential poration effect of nanosecond electric pulses on chromaffin cell membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines.

PubMed

Adalsteinsson, Viktor A; Tahirova, Narmin; Tallapragada, Naren; Yao, Xiaosai; Campion, Liam; Angelini, Alessandro; Douce, Thomas B; Huang, Cindy; Bowman, Brittany; Williamson, Christina A; Kwon, Douglas S; Wittrup, K Dane; Love, J Christopher

2013-10-01

Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

Morphology of the femoral glands in the lizard Ameiva ameiva (teiidae) and their possible role in semiochemical dispersion.

PubMed

Imparato, Beatriz A; Antoniazzi, Marta M; Rodrigues, Miguel T; Jared, Carlos

2007-07-01

Many lizards have epidermal glands in the cloacal or femoral region with semiochemical function related to sexual behavior and/or territorial demarcation. Externally, these glands are recognized as a row of pores, opening individually in the center of a modified scale. In many species the pores are used as systematic characters. They form a glandular cord or, in some species, a row of glandular beads below the dermis, and are connected to the exterior through the ducts, which continuously liberate a solid secretion. Dead cells, desquamated from the secretory epithelium, constitute the secretion, known as "a secretion plug." The present work focuses on the morphology of the femoral glands of the teiid lizard Ameiva ameiva, correlating it to the way in which the secretion is deposited in the environment. The results here obtained are compared to those available for other lizards and amphisbaenians. We observed that the diameter of the glandular pores did not show significant differences between males and females. The glands comprise germinative and secretory cells, which pass through at least three stages of differentiation, during which an accumulation of cytoplasmic granules, with a glycoprotein content, occurs. The cells eventually die and desquamate from the secretory epithelium, forming a secretory plug mostly constituted by juxtaposed nonfragmented secretory cells. Because of the arrangement of the rosette-like scales surrounding the femoral pores, we suggest that when the animal is in a resting position, with its femoral regions touching the ground, these scales may be involved in the breakage of their respective plugs, depositing tiny portions on the substrate. In this manner, it seems that the method for signal dispersion in this species involves specifically adapted structures and does not simply involve the chance breakage of the plug, as the gland secretes it. Signal dispersion must also be intimately associated with the animal's movement within its territory.

Reciprocal links between metabolic and ionic events in islet cells. Their relevance to the rhythmics of insulin release.

PubMed

Malaisse, W J

1998-02-01

The notion of reciprocal links between metabolic and ionic events in islet cells and the rhythmics of insulin release is based on (i) the rhythmic pattern of hormonal release from isolated perfused rat pancreas, which supports the concept of an intrapancreatic pacemaker; (ii) the assumption that this phasic pattern is due to the integration of secretory activity in distinct functional units, e.g. distinct islets; and (iii) the fact that reciprocal coupling between metabolic and ionic events is operative in the secretory sequence.

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.

Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells.

PubMed

Vomund, Anthony N; Zinselmeyer, Bernd H; Hughes, Jing; Calderon, Boris; Valderrama, Carolina; Ferris, Stephen T; Wan, Xiaoxiao; Kanekura, Kohsuke; Carrero, Javier A; Urano, Fumihiko; Unanue, Emil R

2015-10-06

Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system.

Allergen valency, dose and FcERI occupancy set thresholds for secretory responses to Pen a 1 and motivate design of hypoallergens

USDA-ARS?s Scientific Manuscript database

Antigen-mediated cross-linking of IgE-Fc'RI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to recom...

The Unfolded Protein Response in Amelogenesis and Enamel Pathologies.

PubMed

Brookes, Steven J; Barron, Martin J; Dixon, Michael J; Kirkham, Jennifer

2017-01-01

During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other "professional" secretory cells, ameloblasts employ the unfolded protein response (UPR) to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum)/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI) and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed.

Is nectar reabsorption restricted by the stalk cells of floral and extrafloral nectary trichomes?

PubMed

Cardoso-Gustavson, P; Davis, A R

2015-01-01

Reabsorption is a phase of nectar dynamics that occurs concurrently with secretion; it has been described in floral nectaries that exude nectar through stomata or unicellular trichomes, but has not yet been recorded in extrafloral glands. Apparently, nectar reabsorption does not occur in multicellular secretory trichomes (MST) due to the presence of lipophilic impregnations - which resemble Casparian strips - in the anticlinal walls of the stalk cells. It has been assumed that these impregnations restrict solute movement within MST to occur unidirectionally and exclusively by the symplast, thereby preventing nectar reflux toward the underlying nectary tissues. We hypothesised that reabsorption is absent in nectaries possessing MST. The fluorochrome lucifer yellow (LYCH) was applied to standing nectar of two floral and extrafloral glands of distantly related species, and then emission spectra from nectary sections were systematically analysed using confocal microscopy. Passive uptake of LYCH via the stalk cells to the nectary tissues occurred in all MST examined. Moreover, we present evidence of nectar reabsorption in extrafloral nectaries, demonstrating that LYCH passed the stalk cells of MST, although it did not reach the deepest nectary tissues. Identical (control) experiments performed with neutral red (NR) demonstrated no uptake of this stain by actively secreting MST, whereas diffusion of NR did occur in plasmolysed MST of floral nectaries at the post-secretory phase, indicating that nectar reabsorption by MST is governed by stalk cell physiology. Interestingly, non-secretory trichomes failed to reabsorb nectar. The role of various nectary components is discussed in relation to the control of nectar reabsorption by secretory trichomes. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

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

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles.

PubMed

Woo, Sang Su; James, Declan J; Martin, Thomas F J

2017-03-15

Munc13-4 is a Ca 2+ -dependent SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca 2+ -evoked secretion in various secretory cells. Studies in mast cell-like RBL-2H3 cells provide direct evidence that Munc13-4 with its two Ca 2+ -binding C2 domains functions as a Ca 2+ sensor for SG exocytosis. Unexpectedly, Ca 2+ 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 Ca 2+ binding to Munc13-4. Munc13-4 promoted the Ca 2+ -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 Ca 2+ 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. © 2017 Woo et al. 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).

A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype.

PubMed

Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-11-24

DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance.

A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype

PubMed Central

Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-01-01

DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance. PMID:26503466

The Type 7 Serotonin Receptor, 5-HT7, Is Essential in the Mammary Gland for Regulation of Mammary Epithelial Structure and Function

PubMed Central

Pai, Vaibhav P.; Hernandez, Laura L.; Stull, Malinda A.; Horseman, Nelson D.

2015-01-01

Autocrine-paracrine activity of serotonin (5-hydroxytryptamine, 5-HT) is a crucial homeostatic parameter in mammary gland development during lactation and involution. Published studies suggested that the 5-HT7 receptor type was important for mediating several effects of 5-HT in the mammary epithelium. Here, using 5-HT7 receptor-null (HT7KO) mice we attempt to understand the role of this receptor in mediating 5-HT actions within the mammary gland. We demonstrate for the first time that HT7KO dams are inefficient at sustaining their pups. Histologically, the HT7KO mammary epithelium shows a significant deviation from the normal secretory epithelium in morphological architecture, reduced secretory vesicles, and numerous multinucleated epithelial cells with atypically displaced nuclei, during lactation. Mammary epithelial cells in HT7KO dams also display an inability to transition from lactation to involution as normally seen by transition from a columnar to a squamous cell configuration, along with alveolar cell apoptosis and cell shedding. Our results show that 5-HT7 is required for multiple actions of 5-HT in the mammary glands including core functions that contribute to changes in cell shape and cell turnover, as well as specialized secretory functions. Understanding these actions may provide new interventions to improve lactation performance and treat diseases such as mastitis and breast cancer. PMID:25664318

Fatty Acid-Binding Protein 4 (FABP4): Pathophysiological Insights and Potent Clinical Biomarker of Metabolic and Cardiovascular Diseases

PubMed Central

Furuhashi, Masato; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

2014-01-01

Over the past decade, evidences of an integration of metabolic and inflammatory pathways, referred to as metaflammation in several aspects of metabolic syndrome, have been accumulating. Fatty acid-binding protein 4 (FABP4), also known as adipocyte FABP (A-FABP) or aP2, is mainly expressed in adipocytes and macrophages and plays an important role in the development of insulin resistance and atherosclerosis in relation to metaflammation. Despite lack of a typical secretory signal peptide, FABP4 has been shown to be released from adipocytes in a non-classical pathway associated with lipolysis, possibly acting as an adipokine. Elevation of circulating FABP4 levels is associated with obesity, insulin resistance, diabetes mellitus, hypertension, cardiac dysfunction, atherosclerosis, and cardiovascular events. Furthermore, ectopic expression and function of FABP4 in several types of cells and tissues have been recently demonstrated. Here, we discuss both the significant role of FABP4 in pathophysiological insights and its usefulness as a biomarker of metabolic and cardiovascular diseases. PMID:25674026

Fatty Acid-Binding Protein 4 (FABP4): Pathophysiological Insights and Potent Clinical Biomarker of Metabolic and Cardiovascular Diseases.

PubMed

Furuhashi, Masato; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

2014-01-01

Over the past decade, evidences of an integration of metabolic and inflammatory pathways, referred to as metaflammation in several aspects of metabolic syndrome, have been accumulating. Fatty acid-binding protein 4 (FABP4), also known as adipocyte FABP (A-FABP) or aP2, is mainly expressed in adipocytes and macrophages and plays an important role in the development of insulin resistance and atherosclerosis in relation to metaflammation. Despite lack of a typical secretory signal peptide, FABP4 has been shown to be released from adipocytes in a non-classical pathway associated with lipolysis, possibly acting as an adipokine. Elevation of circulating FABP4 levels is associated with obesity, insulin resistance, diabetes mellitus, hypertension, cardiac dysfunction, atherosclerosis, and cardiovascular events. Furthermore, ectopic expression and function of FABP4 in several types of cells and tissues have been recently demonstrated. Here, we discuss both the significant role of FABP4 in pathophysiological insights and its usefulness as a biomarker of metabolic and cardiovascular diseases.

Biochemical nature of Russell Bodies

PubMed Central

Francesca Mossuto, Maria; Ami, Diletta; Anelli, Tiziana; Fagioli, Claudio; Maria Doglia, Silvia; Sitia, Roberto

2015-01-01

Professional secretory cells produce and release abundant proteins. Particularly in case of mutations and/or insufficient chaperoning, these can aggregate and become toxic within or amongst cells. Immunoglobulins (Ig) are no exception. In the extracellular space, certain Ig-L chains form fibrils causing systemic amyloidosis. On the other hand, Ig variants lacking the first constant domain condense in dilated cisternae of the early secretory compartment, called Russell Bodies (RB), frequently observed in plasma cell dyscrasias, autoimmune diseases and chronic infections. RB biogenesis can be recapitulated in lymphoid and non-lymphoid cells by expressing mutant Ig-μ, providing powerful models to investigate the pathophysiology of endoplasmic reticulum storage disorders. Here we analyze the aggregation propensity and the biochemical features of the intra- and extra-cellular Ig deposits in human cells, revealing β-aggregated features for RB. PMID:26223695

Biochemical nature of Russell Bodies.

PubMed

Mossuto, Maria Francesca; Ami, Diletta; Anelli, Tiziana; Fagioli, Claudio; Doglia, Silvia Maria; Sitia, Roberto

2015-07-30

Professional secretory cells produce and release abundant proteins. Particularly in case of mutations and/or insufficient chaperoning, these can aggregate and become toxic within or amongst cells. Immunoglobulins (Ig) are no exception. In the extracellular space, certain Ig-L chains form fibrils causing systemic amyloidosis. On the other hand, Ig variants lacking the first constant domain condense in dilated cisternae of the early secretory compartment, called Russell Bodies (RB), frequently observed in plasma cell dyscrasias, autoimmune diseases and chronic infections. RB biogenesis can be recapitulated in lymphoid and non-lymphoid cells by expressing mutant Ig-μ, providing powerful models to investigate the pathophysiology of endoplasmic reticulum storage disorders. Here we analyze the aggregation propensity and the biochemical features of the intra- and extra-cellular Ig deposits in human cells, revealing β-aggregated features for RB.

The integrity of the RRGDL sequence of the proprotein convertase PC1 is critical for its zymogen and C-terminal processing and for its cellular trafficking.

PubMed Central

Lusson, J; Benjannet, S; Hamelin, J; Savaria, D; Chrétien, M; Seidah, N G

1997-01-01

In order to define the functional importance of the conserved RRGDL motif in the P-domain of the mammalian proprotein convertases(PCs) we generated and cellularly expressed three mutant PC1 vaccinia-virus (VV) recombinants: ARGDL-PC1, RAGDL-PC1 and RRGEL-PC1. Functionally, these mutants caused a decreased level of processing of pro-opiomelanocortin (POMC) into beta-lipotropic pituitary hormone (beta-LPH), especially in the constitutively secreting BSC40 cells. Pulse-chase analyses demonstrated that, in part, this effect was due to both an increased degradation of the mutant PC1s within the endoplasmic reticulum and to a diminished level of zymogen processing in the same compartment. In addition, within cells containing secretory granules such as PC12 and GH4C1 cells, such mutations prevented the C-terminal auto-processing of PC1 into the fully mature 66 kDa form stored in the secretory granules of regulated cells. Since the 66 kDa PC1 is the most active form of the enzyme, it is proposed that the RRGDL sequence is critical for the generation of maximal intracellular PC1 activity. In regulated cells, co-expression of POMC with PC1 or its mutants together with the general PC inhibitor alpha1-antitrypsin Portland (alpha1-PDX), which acts primarily within the constitutive secretory pathway, demonstrated that the latter completely inhibited the formation of beta-LPH by PC1 mutants, whereas it only partially inhibited the ability of wild-type PC1 to process POMC. This suggests that RRGDL mutations prevent PC1 from entering secretory granules and hence the formation of the 66 kDa PC1, and result in the mis-sorting of PC1 mutants towards the constitutive secretory pathway. This conclusion was further supported by immunocytochemical data demonstrating that RRGDL mutants exhibit an intracellular localization pattern different from that of the granule-associated wild-type PC1,but similar to that of the Golgi-localized convertase PC5-B. PMID:9307023

Notch inhibition counteracts Paneth cell death in absence of caspase-8.

PubMed

Jeon, M K; Kaemmerer, E; Schneider, U; Schiffer, M; Klaus, C; Hennings, J; Clahsen, T; Ackerstaff, T; Niggemann, M; Schippers, A; Longerich, T; Sellge, G; Trautwein, C; Wagner, N; Liedtke, C; Gassler, N

2018-05-16

Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8 ∆int ) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8 ∆int mice. The secretory cell metaplasia in DBZ-treated casp8 ∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8 ∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

Investigation into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipase A2 using Cells Deficient in the Expression of Scramblase

PubMed Central

Nelson, Jennifer; Francom, Lyndee L.; Anderson, Lynn; Damm, Kelly; Baker, Ryan; Chen, Joseph; Franklin, Sarah; Hamaker, Amy; Izidoro, Izadora; Moss, Eric; Orton, Mikayla; Stevens, Evan; Yeung, Celestine; Judd, Allan M.; Bell, John D.

2012-01-01

Summary Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt’s lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. PMID:22266334

Specific storage of glycoconjugates with terminal α-galactosyl moieties in the exocrine pancreas of Fabry disease patients with blood group B.

PubMed

Rybová, Jitka; Kuchar, Ladislav; Hulková, Helena; Asfaw, Befekadu; Dobrovolný, Robert; Sikora, Jakub; Havlícek, Vladimír; Škultéty, Ludovít; Ledvinová, Jana

2018-06-01

Blood group B glycosphingolipids (B-GSLs) are substrates of the lysosomal alpha-galactosidase A (AGAL). Similar to its major substrate-globotriaosylceramide (Gb3Cer)-B-GSLs are not degraded and accumulate in the cells of patients affected by an inherited defect of AGAL activity (Fabry disease-FD).The pancreas is a secretory organ known to have high biosynthesis of blood group GSLs. Herein, we provide a comprehensive overview of the biochemical and structural abnormalities in pancreatic tissue from two male FD patients with blood group B. In both patients, we found major accumulation of a variety of complex B-GSLs carrying predominantly hexa- and hepta-saccharide structures. The subcellular pathology was dominated by deposits containing B-glycoconjugates and autofluorescent ceroid. The contribution of Gb3Cer to the storage was minor. This abnormal storage pattern was specific for the pancreatic acinar epithelial cells. Other pancreatic cell types including those of islets of Langerhans were affected much less or not at all.Altogether, we provide evidence for a key role of B-antigens in the biochemical and morphological pathology of the exocrine pancreas in FD patients with blood group B. We believe that our findings will trigger further studies aimed at assessing the potential pancreatic dysfunction in this disease.

Myrip couples the capture of secretory granules by the actin-rich cell cortex and their attachment to the plasma membrane.

PubMed

Huet, Sébastien; Fanget, Isabelle; Jouannot, Ouardane; Meireles, Patricia; Zeiske, Tim; Larochette, Nathanaël; Darchen, François; Desnos, Claire

2012-02-15

Exocytosis of secretory granules (SGs) requires their delivery to the actin-rich cell cortex followed by their attachment to the plasma membrane (PM). How these reactions are executed and coordinated is still unclear. Myrip, which is also known as Slac-2c, binds to the SG-associated GTPase Rab27 and is thought to promote the delivery of SGs to the PM by recruiting the molecular motor myosin Va. Myrip also interacts with actin and the exocyst complex, suggesting that it may exert multiple roles in the secretory process. By combining total internal reflection fluorescence microscopy, single-particle tracking, a photoconversion-based assay, and mathematical modeling, we show that, in human enterochromaffin cells, Myrip (1) inhibits a class of SG motion characterized by fast and directed movement, suggesting that it facilitates the dissociation of SGs from microtubules; (2) enhances their motion toward the PM and the probability of SG attachment to the PM; and (3) increases the characteristic time of immobilization at the PM, indicating that it is a component of the molecular machinery that tether SGs to the PM. Remarkably, while the first two effects of Myrip depend on its ability to recruit myosin Va on SGs, the third is myosin Va independent but relies on the C-terminal domain of Myrip. We conclude that Myrip couples the retention of SGs in the cell cortex, their transport to the PM, and their attachment to the PM, and thus promotes secretion. These three steps of the secretory process are thus intimately coordinated.

Endometrial proteins: a reappraisal.

PubMed

Seppälä, M; Julkunen, M; Riittinen, L; Koistinen, R

1992-06-01

Uterine factors influence reproduction at the macro-anatomy level, and the effects of hormonal steroids on endometrial morphology are well recognized in the histopathological diagnosis of dysfunctional bleeding and infertility. During the past decade, attention has been paid to endometrial protein synthesis and secretion with respect to endocrine stimuli and implantation, and to the paracrine/autocrine effects of endometrial peptide growth factors, their binding proteins and other factors. The emphasis of this presentation is on protein secretion of the secretory endometrium, in which progesterone plays a pivotal role. Insulin-like growth factors have receptors on the endometrium, and IGF-binding proteins, stimulated by progesterone, modulate the effects of IGFs locally. Also other protein products of the secretory endometrium have been reviewed in this communication, with special emphasis on studies of a progesterone-associated endometrial protein which has many names in the literature, such as PEP, PP14, alpha 2-PEG and AUP. Extensive studies are ongoing in many laboratories to elucidate the regulation, function, interplay at tissue and cellular levels, and clinical significance of these proteins.

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.

Tarsomere and distal tibial glands: structure and potential roles in termites (Isoptera: Rhinotermitidae, Termitidae).

PubMed

Costa-Leonardo, Ana Maria; Soares, Helena Xavier; Haifig, Ives; Laranjo, Lara Teixeira

2015-09-01

Social insects have numerous exocrine glands, but these organs are understudied in termites compared to hymenopterans. The tarsomere and distal tibial glands of the termites Heterotermes tenuis, Coptotermes gestroi and Silvestritermes euamignathus were investigated by scanning and transmission electron microscopy. Pore plates are visible in scanning micrographs on the distal tibial surfaces and on the ventral surface of the first and second tarsomeres of workers of H. tenuis and C. gestroi. In contrast, workers of S. euamignathus have isolated pores spread throughout the ventral surfaces of the first, second, and third tarsomeres and the distal tibia. In all three species each pore corresponds to the opening of a class-3 secretory unit, composed of one secretory and one canal cell. Clusters of class-3 glandular cells are arranged side by side underneath the cuticle. The main characteristics of these exocrine glands include their presence on all the legs and the electron-lucent secretion in the secretory cells. Possible functions of these glands are discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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.

Degranulation and shrinkage of dark cells in eccrine glands and elevated serum carcinoembryonic antigen in patients with acquired idiopathic generalized anhidrosis.

PubMed

Sano, K; Asahina, M; Uehara, T; Matsumoto, K; Araki, N; Okuyama, R

2017-12-01

Acquired idiopathic generalized anhidrosis (AIGA) is characterized by anhidrosis/hypohidrosis without other autonomic and neurological dysfunctions. Pathologically, AIGA is considered to usually present no significant morphological alterations in eccrine glands, the secretory portion which consists of clear cells, dark cells, and myoepithelial cells. AIGA patients recently have been reported to show high serum concentrations of carcinoembryonic antigen (CEA). Our aim is to reveal morphological abnormalities of dark cells and investigate their relationship with serum CEA. We performed comparative analysis of eccrine glands between sweat-preserved and non-sweating skin in four AIGA patients. Serum CEA concentrations in 22 cases with AIGA were measured with healthy volunteers. Furthermore, we semiquantitatively investigated dermcidin, FoxA1 and CEA expression in eccrine glands of 12 cases with AIGA and 5 cases with non-AIGA. Marked degranulation and shrinkage of dark cells consistently occurred in AIGA. Furthermore, high serum CEA concentrations were found in 14 of 22 AIGA patients (over 60%), but serum CEA levels were not correlated with CEA expression in eccrine glands. Dermcidin expression in dark cells apparently decreased in AIGA patients, severely in those with high serum CEA and moderately in those with low serum CEA, while well-preserved expression was found in non-AIGA subjects. Our study suggests morphological damage and molecular dysregulation of dark cells, leading to impairment of their functions in AIGA patients. Severely damaged dark cells correspond to high serum CEA. Accordingly, these pathological changes in eccrine dark cells may be involved in anhidrosis/hypohidrosis of AIGA. © 2017 European Academy of Dermatology and Venereology.

Labellar anatomy and secretion in Bulbophyllum Thouars (Orchidaceae: Bulbophyllinae) sect. Racemosae Benth. & Hook. f.

PubMed

Davies, Kevin L; Stpiczyńska, Malgorzata

2014-10-01

Floral secretions are common in Bulbophyllum Thouars, and the labella of a number of Asian species are said to produce secretions rich in lipids that act as food rewards for insect pollinators. Although some of these reports are based on simple histochemical tests, a much greater number are anecdotal and, hitherto, neither the ultrastructure of the labellum nor the secretory process has been investigated in detail. Furthermore, sophisticated histochemical approaches have generally not been applied. Here, both the labellar structure and the secretory process are investigated for four species of Asian Bulbophyllum sect. Racemosae Benth. & Hook. f., namely Bulbophyllum careyanum (Hook.) Spreng., B. morphologorum Kraenzl., B. orientale Seidenf. and B. wangkaense Seidenf., and compared with those of unequivocal lipid-secreting orchids. Labellar, secretory tissue was investigated using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. The adaxial median longitudinal groove of the labellum contained secretory tissue comprising palisade-like epidermal cells, similar to those of certain lipid-secreting Oncidiinae Benth. However, these cells and their secretions gave positive results mainly for protein and mucilage, and their organelle complement was consistent with that of cells involved in protein and mucilage synthesis. Sub-cuticular accumulation of secretion resulted in cuticular distension and blistering. The sub-epidermal layer of isodiametric parenchyma contained starch and, like the epidermal cells, ultrastructure consistent with mucilage synthesis. Lipids were mainly confined to the cuticle, and hardly any intracellular lipid droplets were observed. It is proposed that mucilage is produced by dictyosomes present in the palisade-like epidermal cells. Mucilage precursors may also be produced by these same organelles in sub-epidermal cells and are thought to pass along the symplast via plasmodesmata into the adjoining palisade-like secretory cells, which contain abundant arrays of rough endoplasmic reticulum. Here, they become chemically modified and form a protein-rich, mucilaginous secretion that, following vesicle-mediated transport across the cytoplasm, traverses the cell wall and accumulates in blisters formed from the distended cuticle. Rupture of these blisters releases the secretion onto the labellar surface. However, in certain species, there is some evidence that the secretion may traverse the cuticle via cuticular pores, and micro-channels may permit the passage of fragrance. Hydrolysis of sub-epidermal starch probably generates the carbohydrate and, together with mitochondria, much of the energy required for the secretory process. This anatomical organization resembles that found in certain lipid-secreting, Neotropical species of Bulbophyllum and Oncidiinae, but since the chemical composition of their secretions is different, and these taxa occur on a separate continent and have different insect pollinators, parallelism of floral anatomy is likely. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting

PubMed Central

Lindeman, Geoffrey J.; Dagnino, Lina; Gaubatz, Stefan; Xu, Yuhui; Bronson, Roderick T.; Warren, Henry B.; Livingston, David M.

1998-01-01

Homozygous E2F-5 knockout embryos and mice have been generated. Although embryonic development appeared normal, newborn mice developed nonobstructive hydrocephalus, suggesting excessive cerebrospinal fluid (CSF) production. Although the CSF-producing choroid plexus displayed normal cellular organization, it contained abundant electron-lucent epithelial cells, consistent with excessive CSF secretory activity. Moreover, E2F-5 CNS expression in normal animals was largely confined to the choroid plexus. Cell cycle kinetics were not perturbed in homozygous knockout embryo fibroblasts. Thus, E2F-5 is not essential for cell proliferation. Rather, it affects the secretory behavior of a differentiated neural tissue. PMID:9553039

The Ets transcription factor Elf5 specifies mammary alveolar cell fate

PubMed Central

Oakes, Samantha R.; Naylor, Matthew J.; Asselin-Labat, Marie-Liesse; Blazek, Katrina D.; Gardiner-Garden, Margaret; Hilton, Heidi N.; Kazlauskas, Michael; Pritchard, Melanie A.; Chodosh, Lewis A.; Pfeffer, Peter L.; Lindeman, Geoffrey J.; Visvader, Jane E.; Ormandy, Christopher J.

2008-01-01

Hormonal cues regulate mammary development, but the consequent transcriptional changes and cell fate decisions are largely undefined. We show that knockout of the prolactin-regulated Ets transcription factor Elf5 prevented formation of the secretory epithelium during pregnancy. Conversely, overexpression of Elf5 in an inducible transgenic model caused alveolar differentiation and milk secretion in virgin mice, disrupting ductal morphogenesis. CD61+ luminal progenitor cells accumulated in Elf5-deficient mammary glands and were diminished in glands with Elf5 overexpression. Thus Elf5 specifies the differentiation of CD61+ progenitors to establish the secretory alveolar lineage during pregnancy, providing a link between prolactin, transcriptional events, and alveolar development. PMID:18316476

Secretory vesicles of immune cells contain only a limited number of interleukin 6 molecules.

PubMed

Verboogen, Daniëlle R J; Ter Beest, Martin; Honigmann, Alf; van den Bogaart, Geert

2018-05-01

Immune cells communicate by releasing large quantities of cytokines. Although the mechanisms of cytokine secretion are increasingly understood, quantitative knowledge of the number of cytokines per vesicle is still lacking. Here, we measured with quantitative microscopy the release rate of vesicles potentially carrying interleukin-6 (IL-6) in human dendritic cells. By comparing this to the total secreted IL-6, we estimate that secretory vesicles contain about 0.5-3 IL-6 molecules, but with a large spread among cells/donors. Moreover, IL-6 did not accumulate within most cells, indicating that synthesis and not trafficking is the bottleneck for IL-6 production. IL-6 accumulated in the Golgi apparatus only in ~ 10% of the cells. Understanding how immune cells produce cytokines is important for designing new immunomodulatory drugs. © 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

PubMed Central

Morosky, Stefanie; Lennemann, Nicholas J.

2016-01-01

ABSTRACT Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCE Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression correlates with pronounced defects in the secretory pathway and greatly reduces the replication of CVB, PV, and EV71. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter enterovirus replication. PMID:26962226

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication.

PubMed

Morosky, Stefanie; Lennemann, Nicholas J; Coyne, Carolyn B

2016-05-15

Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression correlates with pronounced defects in the secretory pathway and greatly reduces the replication of CVB, PV, and EV71. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter enterovirus replication. Copyright © 2016, American Society for Microbiology. 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

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.

Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction.

PubMed

Ferreira de Oliveira, José Miguel P; van Passel, Mark W J; Schaap, Peter J; de Graaff, Leo H

2010-07-01

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.

Eccrine sweat gland development and sweat secretion.

PubMed

Cui, Chang-Yi; Schlessinger, David

2015-09-01

Eccrine sweat glands help to maintain homoeostasis, primarily by stabilizing body temperature. Derived from embryonic ectoderm, millions of eccrine glands are distributed across human skin and secrete litres of sweat per day. Their easy accessibility has facilitated the start of analyses of their development and function. Mouse genetic models find sweat gland development regulated sequentially by Wnt, Eda and Shh pathways, although precise subpathways and additional regulators require further elucidation. Mature glands have two secretory cell types, clear and dark cells, whose comparative development and functional interactions remain largely unknown. Clear cells have long been known as the major secretory cells, but recent studies suggest that dark cells are also indispensable for sweat secretion. Dark cell-specific Foxa1 expression was shown to regulate a Ca(2+) -dependent Best2 anion channel that is the candidate driver for the required ion currents. Overall, it was shown that cholinergic impulses trigger sweat secretion in mature glands through second messengers - for example InsP3 and Ca(2+) - and downstream ion channels/transporters in the framework of a Na(+) -K(+) -Cl(-) cotransporter model. Notably, the microenvironment surrounding secretory cells, including acid-base balance, was implicated to be important for proper sweat secretion, which requires further clarification. Furthermore, multiple ion channels have been shown to be expressed in clear and dark cells, but the degree to which various ion channels function redundantly or indispensably also remains to be determined. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

[The structure of the gastric mucosa of the llamas (Lama guanocoe and Lama lamae). I. Forestomach].

PubMed

Luciano, L; Voss-Wermbter, G; Behnke, M; von Engelhardt, W; Reale, E

1979-01-01

The mucous membrane of the first and second compartments (ventral regions) as well as of the third compartment of Lama guanacoe and Lama lamae stomach shows tubular glands opening into pits. Below the surface epithelium blood capillaries of the fenestrated type form a regular network, each mesh of which surrounds a gastric pit. From a morphological point of view (thin section and freeze-fracture replicas) the columnar cells of the surface epithelium and those of the pits closest to the capillaries are largely similar to the epithelial cells of the rabbit gallbladder. This similarity suggests that at the level of the columnar cells sodium-dependent water reabsorption occurs. This reabsorption has already been demonstrated in the abovementioned compartments by physiological methods. The surface and foveolae epithelial cells as well as some cells of the tubular glands have a secretory function. Their secretory granules contain mucosubstances, as indicated by light-(PAS- and Alcian blue reactions) and electron microscopic (PA-TCH-Ag-reaction) histochemistry. The secretory granules originate from the Golgi complex which shows a positive histochemical reaction in its innermost sacculi at the electron microscope level. Endocrine cells (s. second part of this investigation) are rare. The mucosal membrane of each muscular lip separating the glandular sacs in the first compartment shows a stratified, not keratinized, squamous epithelium.

VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion.

PubMed Central

Regazzi, R; Wollheim, C B; Lang, J; Theler, J M; Rossetto, O; Montecucco, C; Sadoul, K; Weller, U; Palmer, M; Thorens, B

1995-01-01

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta-cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP-2 and cellubrevin, but not VAMP-1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta-cells contain secretory granules that store and secrete insulin as well as synaptic-like microvesicles carrying gamma-aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP-2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP-2 with insulin-containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta-cells. Pretreatment of streptolysin-O permeabilized insulin-secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP-2 and cellubrevin and abolished Ca(2+)-induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S-stimulated insulin secretion. Taken together, our results show that pancreatic beta-cells express VAMP-2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)-mediated insulin secretion. Images PMID:7796801

Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells. [Pisum sativum

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

Camirand, A.; Brummell, D.; MacLachlan, G.

1987-07-01

Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-(/sup 14/C) fucose and UDP-(/sup 14/C)xylose to xyloglucan occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with (/sup 3/H)fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.

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

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

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

Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

PubMed

Sundaramoorthy, Vinod; Walker, Adam K; Tan, Vanessa; Fifita, Jennifer A; Mccann, Emily P; Williams, Kelly L; Blair, Ian P; Guillemin, Gilles J; Farg, Manal A; Atkin, Julie D

2015-07-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that optineurin-myosin VI dysfunction is a common feature of both sporadic and familial ALS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Augmented cell survival in eutopic endometrium from women with endometriosis: Expression of c-myc, TGF-beta1 and bax genes

PubMed Central

Johnson, M Cecilia; Torres, Marisa; Alves, Alessandra; Bacallao, Ketty; Fuentes, Ariel; Vega, Margarita; Boric, M Angélica

2005-01-01

Background Endometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside of the uterus. The fragments in normal menstruation are composed of necrotic and living cells, which do not survive in ectopic locations because of programmed cell death. The aim of this study was to evaluate if the balance between cell proliferation and apoptosis is changed in eutopic endometrium from women with endometriosis throughout the menstrual cycle by studying bax (pro-apoptotic), c-myc (regulator of cell cycle) and TGF-beta1 (involved in cell differentiation) genes. Methods Eutopic endometrium was obtained from: 30 women with endometriosis (32.8 +/- 5 years) and 34 fertile eumenorrheic women (36 +/- 5.3 years). We analyzed apoptosis (TUNEL: DNA fragmentation); cell proliferation (immunohistochemistry (IHC) for Ki67); c-myc, bax and TGF-beta1 mRNA abundance (RT-PCR) and TGF-beta1 protein (IHC) in endometrial explants. Results Cell proliferation strongly decreased from proliferative to late secretory phases in glands, but not in stroma, in both endometria. Positive staining in glands and stroma from proliferative endometrium with endometriosis was 1.9- and 2.2-fold higher than control endometrium, respectively (p < 0.05). Abundance of c-myc mRNA was 65% higher in proliferative endometrium from endometriosis than normal tissue (p < 0.05). TGF-beta1 (mRNA and protein) augmented during mid secretory phase in normal endometrium, effect not observed in endometrium with endometriosis. In normal endometrium, the percentage of apoptotic epithelial and stromal cells increased more than 30-fold during late secretory phase. In contrast, in endometrium from endometriosis, not only this increase was not observed, besides bax mRNA decreased 63% versus normal endometrium (p < 0.05). At once, in early secretory phase, apoptotic stromal cells increased 10-fold with a concomitant augment of bax mRNA abundance (42%) in endometria from endometriosis (p < 0.05). Conclusion An altered expression of c-myc, TGF-beta1 and bax was observed in eutopic endometrium from endometriosis, suggesting its participation in the regulation of cell survival in this disease. The augmented cell viability in eutopic endometrium from these patients as a consequence of a reduction in cell death by apoptosis, and also an increase in cell proliferation indicates that this condition may facilitate the invasive feature of the endometrium. PMID:16150151

Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis

PubMed Central

Tian, Hua; Biehs, Brian; Chiu, Cecilia; Siebel, Chris; Wu, Yan; Costa, Mike; de Sauvage, Frederic J.; Klein, Ophir D.

2015-01-01

Summary Proper organ homeostasis requires tight control of adult stem cells and differentiation through integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a de-repression of the Wnt signaling pathway, leading to mis-expression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology. PMID:25818302

Airway epithelial stem cells and the pathophysiology of chronic obstructive pulmonary disease.

PubMed

Randell, Scott H

2006-11-01

Characteristic pathologic changes in chronic obstructive pulmonary disease (COPD) include an increased fractional volume of bronchiolar epithelial cells, fibrous thickening of the airway wall, and luminal inflammatory mucus exudates, which are positively correlated with airflow limitation and disease severity. The mechanisms driving general epithelial expansion, mucous secretory cell hyperplasia, and mucus accumulation must relate to the effects of initial toxic exposures on patterns of epithelial stem and progenitor cell proliferation and differentiation, eventually resulting in a self-perpetuating, and difficult to reverse, cycle of injury and repair. In this review, current concepts in stem cell biology and progenitor-progeny relationships related to COPD are discussed, focusing on the factors, pathways, and mechanisms leading to mucous secretory cell hyperplasia and mucus accumulation in the airways. A better understanding of alterations in airway epithelial phenotype in COPD will provide a logical basis for novel therapeutic approaches.

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase.

PubMed

Nelson, Jennifer; Francom, Lyndee L; Anderson, Lynn; Damm, Kelly; Baker, Ryan; Chen, Joseph; Franklin, Sarah; Hamaker, Amy; Izidoro, Izadora; Moss, Eric; Orton, Mikayla; Stevens, Evan; Yeung, Celestine; Judd, Allan M; Bell, John D

2012-05-01

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. Copyright © 2012 Elsevier B.V. All rights reserved.

COBRA-LIKE2, a Member of the Glycosylphosphatidylinositol-Anchored COBRA-LIKE Family, Plays a Role in Cellulose Deposition in Arabidopsis Seed Coat Mucilage Secretory Cells1,2[OPEN

PubMed Central

Ben-Tov, Daniela; Abraham, Yael; Stav, Shira; Thompson, Kevin; Loraine, Ann; Elbaum, Rivka; de Souza, Amancio; Pauly, Markus; Kieber, Joseph J.; Harpaz-Saad, Smadar

2015-01-01

Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE2 (COBL2), a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE gene family in Arabidopsis (Arabidopsis thaliana), as coexpressed with other genes involved in cellulose deposition in mucilage secretory cells. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised, as demonstrated by scanning electron microscopy and in situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole-seed crystalline cellulose content compared with the wild type. These data establish that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation. PMID:25583925

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.

Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

PubMed Central

Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

2011-01-01

Background In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. PMID:21383842

A histidine-rich linker region in peptidylglycine α-amidating monooxygenase has the properties of a pH sensor.

PubMed

Vishwanatha, Kurutihalli; Bäck, Nils; Mains, Richard E; Eipper, Betty A

2014-05-02

Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.

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

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils.

PubMed

Carmo, Lívia A S; Dias, Felipe F; Malta, Kássia K; Amaral, Kátia B; Shamri, Revital; Weller, Peter F; Melo, Rossana C N

2015-10-01

SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of ER proteins involved in the functional organisation of the early secretory pathway in Drosophila cells by a targeted RNAi screen.

PubMed

Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

2011-02-23

In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for "more and smaller Golgi") upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation.

Morphological and functional studies on the epidermal cells of amphioxus ( Branchiostoma belcheri tsingtauense) at different developmental stages

NASA Astrophysics Data System (ADS)

Mao, Bing-Yu; Sun, Xiao-Yang; Zhang, Hong-Wei; Zhang, Shi-Cui; Wu, Xian-Han

1997-09-01

Epidermal cells of amphioxus at different developmental stages were investigated by electron microscopy and colloidal carbon tracing experiments. Amphioxus epidermal cells showed different ultrastructural characteristics at larval and adult stages. The epidermal cells at all larval stages studied (24 96 h) had numerous vesicles containing electron dense materials in their apical cytoplasm. In tracing experiments, carbon particles were found in apical vesicles and interoellular spaces. Under scanning electron microscope, many crater-like protrusions were observed on the surface of the cells. These results indicated that amphioxus larval epidermal cells may be capable of endocytosis. The epidermal cells of 3-month and adult amphioxus were obviously secretory ones characterized by well-developed peripheral filaments, a prominent Golgi apparatus and abundant apical secretory vesicles. This study also showed that adult amphioxus body surface mucus contained lectin that could agglutinate human red blood cells. The authors propose that the epidermal cells of amphioxus larva and adult may contribute to the immune defense of the amimal by different means.

Tergal glands in termite soldiers of the subfamily Syntermitinae (Isoptera: Termitidae).

PubMed

Costa-Leonardo, Ana Maria; Haifig, Ives; Laranjo, Lara Teixeira

2012-02-01

The subfamily Syntermitinae comprises 14 genera of termites that are exclusively neotropical. The present study reports morphological data about mandibulate nasute soldiers from termite species belonging to three different genera within this subfamily. We describe tergal glands that were present under all tergites of soldiers of the following species: Cornitermes cumulans, Procornitermes araujoi, Syntermes nanus, and Syntermes wheeleri. The tergal glands were composed of class 2 and class 3 cells. Class 2 cells never reached the cuticle and were located below a flat layer of epidermal cells. Class 3 cells, composed of secretory cells and canal cells, were sporadic, whereas class 2 secretory cells were abundant. Secretory cells of class 3 were narrow and their cytoplasms were filled with several clear, oval-shaped vesicles with limiting membranes. The ultrastructure of class 2 cells showed well-developed smooth endoplasmic reticulum, Golgi apparatus, elongated mitochondria, several electron-lucent vesicles, and electron-dense granules that contain paracrystalline structures in S. nanus. Scanning electron micrographs displayed pores, campaniform sensilla and hairs in the outer cuticle of the soldier tergites. We hypothesize that soldier tergal glands may be involved in the production of defensive compounds, which occur in similar glands of certain cockroaches, or of primer pheromones, that might act in the regulation of soldier differentiation in the termite colony. To date, tergal glands have only been described in termite imagoes, and their occurrence in these soldiers of basal Syntermitinae implies a specific role in this caste that is still speculative and needs to be clarified. Copyright © 2011 Elsevier Ltd. All rights reserved.

Morphology, ultrastructure, and chemical compounds of the osmeterium of Heraclides thoas (Lepidoptera: Papilionidae).

PubMed

Martínez, Luis Carlos; Plata-Rueda, Angelica; da Silva Neves, Guilherme; Cossolin, Jamile Fernanda; Dos Santos, Marcelo Henrique; Zanuncio, José Cola; Serrão, José Eduardo

2018-05-11

The osmeterium, found in papilionoid larvae, is an eversible organ with an exocrine gland that produces substances in response to the mechanical disturbances caused by natural enemies. The anatomy, histology and ultrastructure of the osmeterium, and the chemical composition of its secretion in Heraclides thoas (Lepidoptera: Papilionidae) were studied. Heraclides thoas larvae have a Y-shaped osmeterium in the thorax. The surface of the osmeterium has a rough cuticle lining cells with papillae and irregular folds, whereas the cells that limited the gland pores are irregular, folded, and devoid of papillae. Two types of cells are found: (i) cuticular epidermal cells on the surface of the tubular arms of the osmeterium and (ii) secretory cells of the ellipsoid gland within the region of the glandular pore. Cuticular epidermal cells show a thick cuticle, with several layers divided into epicuticle and lamellar endocuticle. Secretory cells are polygonal, with extensive folds in the basal plasma membrane that formed extracellular channels. The cytoplasm has mitochondria, ribosomes, and numerous vacuoles, whereas the nucleus is irregular in shape with decondensed chromatin. The chemical composition of the osmeterial secretion comprised (Z)-α-bisabolene (25.4%), α-bisabol (20.6%), β-bisabolene (13.1%), (E)-α-bisabolene 8%), β-pinene (9.91%), longipinene epoxide (8.92%), (Z)-β-farnesene (6.96%), β-caryophyllene (2.05%), farnesol (1.86%), linalyl propionate (1.86%), and 1-octyn-4-ol (1.07%). The morphological features suggest that the cuticular epidermal cells play a major role in the maintenance and protection of the osmeterium, whereas secretory cells are responsible for production of osmeterial secretions.

Secretome of Differentiated PC12 Cells Restores the Monocrotophos-Induced Damages in Human Mesenchymal Stem Cells and SHSY-5Y Cells: Role of Autophagy and Mitochondrial Dynamics.

PubMed

Srivastava, A; Singh, S; Rajpurohit, C S; Srivastava, P; Pandey, A; Kumar, D; Khanna, V K; Pant, A B

2018-06-01

A perturbed cellular homeostasis is a key factor associated with xenobiotic exposure resulting in various ailments. The local cellular microenvironment enriched with secretory components aids in cell-cell communication that restores this homeostasis. Deciphering the underlying mechanism behind this restorative potential of secretome could serve as a possible solution to many health hazards. We, therefore, explored the protective efficacy of the secretome of differentiated PC12 cells with emphasis on induction of autophagy and mitochondrial biogenesis. Monocrotophos (MCP), a widely used neurotoxic organophosphate, was used as the test compound at sublethal concentration. The conditioned medium (CM) of differentiated PC12 cells comprising of their secretome restored the cell viability, oxidative stress and apoptotic cell death in MCP-challenged human mesenchymal stem cells and SHSY-5Y, a human neuroblastoma cell line. Delving further to identify the underlying mechanism of this restorative effect we observed a marked increase in the expression of autophagy markers LC3, Beclin-1, Atg5 and Atg7. Exposure to autophagy inhibitor, 3-methyladenine, led to a reduced expression of these markers with a concomitant increase in the expression of pro-apoptotic caspase-3. Besides that, the increased mitochondrial fission in MCP-exposed cells was balanced with increased fusion in the presence of CM facilitated by AMPK/SIRT1/PGC-1α signaling cascade. Mitochondrial dysfunctions are strongly associated with autophagy activation and as per our findings, cellular secretome too induces autophagy. Therefore, connecting these three potential apices can be a major breakthrough in repair and rescue of xenobiotic-damaged tissues and cells.

The Harderian gland, its secretory duct and porphyrin content in the mongolian gerbil (Meriones unguiculatus).

PubMed Central

Johnston, H S; McGadey, J; Thompson, G G; Moore, M R; Payne, A P

1983-01-01

The Harderian gland, its secretory duct and porphyrin content were examined in the mongolian gerbil (Meriones unguiculatus). The gland consisted of tubules lined by a single layer of epithelial cells and a myoepithelial network. The tubule cells were often binucleate and possessed lipid vacuoles in the apical half of the cell, a corona of granular endoplasmic reticulum surrounding the nucleus, and cytoplasmic 'slashes'. The latter are probably derived from dense membranous couplets and may be precursors of the lipid vacuoles. Holocrine and merocrine secretion was observed. Interstitial cells included plasma cells, mast cells and (predominantly) melanocytes which render the gland black. The gland was surrounded by a collagen capsule and an outer layer of highly attenuated (possibly endothelioid) cells. Within the gland, the secretory duct was lined by a single layer of normal tubule cells. Outside the gland, the duct enlarged to form an ampulla, from which clefts led off to deep crypts. The ampulla and clefts were lined by cells with small dense apical granules and stubby microvilli; some possessed lipid vacuoles. The crypts were lined by serous cells with active Golgi regions. At the duct opening, ampullary cells became squamous and goblet cells occurred. Geometric crystalloid deposits (with a layered structure of 7.6 nm periodicity) occurred at cleft-crypt junctions. Islets of extra-glandular ductal tissue were occasionally found within the gland. Porphyrins were detectable both by chemical assay and fluorescence microscopy. There was a trend for female glands to have a higher content than males. Solid intraluminal accretions of porphyrin and/or lipid were present. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 12 Fig. 13 Fig. 14 Fig. 15 Fig. 16 PMID:6654750

Wide-range high-resolution transmission electron microscopy reveals morphological and distributional changes of endomembrane compartments during log to stationary transition of growth phase in tobacco BY-2 cells.

PubMed

Toyooka, Kiminori; Sato, Mayuko; Kutsuna, Natsumaro; Higaki, Takumi; Sawaki, Fumie; Wakazaki, Mayumi; Goto, Yumi; Hasezawa, Seiichiro; Nagata, Noriko; Matsuoka, Ken

2014-09-01

Rapid growth of plant cells by cell division and expansion requires an endomembrane trafficking system. The endomembrane compartments, such as the Golgi stacks, endosome and vesicles, are important in the synthesis and trafficking of cell wall materials during cell elongation. However, changes in the morphology, distribution and number of these compartments during the different stages of cell proliferation and differentiation have not yet been clarified. In this study, we examined these changes at the ultrastructural level in tobacco Bright yellow 2 (BY-2) cells during the log and stationary phases of growth. We analyzed images of the BY-2 cells prepared by the high-pressure freezing/freeze substitution technique with the aid of an auto-acquisition transmission electron microscope system. We quantified the distribution of secretory and endosomal compartments in longitudinal sections of whole cells by using wide-range gigapixel-class images obtained by merging thousands of transmission electron micrographs. During the log phase, all Golgi stacks were composed of several thick cisternae. Approximately 20 vesicle clusters (VCs), including the trans-Golgi network and secretory vesicle cluster, were observed throughout the cell. In the stationary-phase cells, Golgi stacks were thin with small cisternae, and only a few VCs were observed. Nearly the same number of multivesicular body and small high-density vesicles were observed in both the stationary and log phases. Results from electron microscopy and live fluorescence imaging indicate that the morphology and distribution of secretory-related compartments dramatically change when cells transition from log to stationary phases of growth. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

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 increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue.

Dermal glands in freshwater mites Limnesia undulata (O.F. Müller, 1776) and Limnesia fulgida (C.L. Koch, 1836) (Acariformes, Limnesiidae).

PubMed

Shatrov, Andrew B; Soldatenko, Elena V

2016-07-01

Dermal glands in the water mites Limnesia undulata (O.F. Müller, 1776) and Limnesia fulgida (C.L. Koch, 1836) and their secretion were studied by means of light microscopical, transmission electron microscopical (TEM) and scanning electron microscopical (SEM) methods. These mites possess two types of dermal glands - the 'common' dermal glands in a number of 14 pairs and one pair of the so-called 'idiosomal' dermal glands. The common dermal glands are bi-lobed organs and consist of high prismatic secretory cells directed to the gland mouth and mostly replacing the intra-alveolar lumen. The cells contain numerous cisterns of rough endoplasmic reticulum (RER) and specifically organized Golgi bodies (GB) producing electron-dense elongated secretory granules. These granules are released from the cells via apocrine secretion and come to the gland mouth, where they are sometimes accompanied by secretory cell cytoplasm. The final secretion may show a fibrous character. The idiosomal glands are sac-like organs stretched along the ventral body wall in posterior direction from the gland orifice corresponding to the epimeroglandularia 4. The secretory epithelium leaves a large intra-alveolar lumen filled with an electron-dense secretory material. Golgi bodies are organized identically with those in the common glands, which indicates the general homology of these two types of dermal glands. The glands' orifices are organized similarly in all glands and possess an internal funnel-shaped sclerite with muscle armament, an internal valve, medial epicuticular flaps and an external circular cuticular ring. All glandularia, except for E4 and V1, are accompanied with a long and thin sensitive seta. During fixation, secretion of the common dermal glands is extruded to the exterior in the form of large amounts of convoluted tube-like structures. In the living organisms, being secreted in mass from the glands, this secretion acquires the form of long rigid mostly hollow un-branched threads comparable with the similar silken threads of other water arthropods. The function of the idiosomal glands secretion still remains unknown. Copyright © 2016 Elsevier Ltd. All rights reserved.

The skeletal phenotype of achondrogenesis type 1A is caused exclusively by cartilage defects.

PubMed

Bird, Ian M; Kim, Susie H; Schweppe, Devin K; Caetano-Lopes, Joana; Robling, Alexander G; Charles, Julia F; Gygi, Steven P; Warman, Matthew L; Smits, Patrick J

2018-01-08

Inactivating mutations in the ubiquitously expressed membrane trafficking component GMAP-210 (encoded by Trip11 ) cause achondrogenesis type 1A (ACG1A). ACG1A is surprisingly tissue specific, mainly affecting cartilage development. Bone development is also abnormal, but as chondrogenesis and osteogenesis are closely coupled, this could be a secondary consequence of the cartilage defect. A possible explanation for the tissue specificity of ACG1A is that cartilage and bone are highly secretory tissues with a high use of the membrane trafficking machinery. The perinatal lethality of ACG1A prevents investigating this hypothesis. We therefore generated mice with conditional Trip11 knockout alleles and inactivated Trip11 in chondrocytes, osteoblasts, osteoclasts and pancreas acinar cells, all highly secretory cell types. We discovered that the ACG1A skeletal phenotype is solely due to absence of GMAP-210 in chondrocytes. Mice lacking GMAP-210 in osteoblasts, osteoclasts and acinar cells were normal. When we inactivated Trip11 in primary chondrocyte cultures, GMAP-210 deficiency affected trafficking of a subset of chondrocyte-expressed proteins rather than globally impairing membrane trafficking. Thus, GMAP-210 is essential for trafficking specific cargoes in chondrocytes but is dispensable in other highly secretory cells. © 2018. Published by The Company of Biologists Ltd.

Glucokinase is an integral component of the insulin granules in glucose-responsive insulin secretory cells and does not translocate during glucose stimulation.

PubMed

Arden, Catherine; Harbottle, Andrew; Baltrusch, Simone; Tiedge, Markus; Agius, Loranne

2004-09-01

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

Ubiquitin Fold Modifier 1 (UFM1) and Its Target UFBP1 Protect Pancreatic Beta Cells from ER Stress-Induced Apoptosis

PubMed Central

Granvik, Mikaela; Igoillo-Esteve, Mariana; Hohmeier, Hans E.; Hendrickx, Nico; Newgard, Christopher B.; Waelkens, Etienne; Cnop, Miriam; Schuit, Frans

2011-01-01

UFM1 is a member of the ubiquitin like protein family. While the enzymatic cascade of UFM1 conjugation has been elucidated in recent years, the biological function remains largely unknown. In this report we demonstrate that the recently identified C20orf116 [1], which we name UFM1-binding protein 1 containing a PCI domain (UFBP1), andCDK5RAP3 interact with UFM1. Components of the UFM1 conjugation pathway (UFM1, UFBP1, UFL1 and CDK5RAP3) are highly expressed in pancreatic islets of Langerhans and some other secretory tissues. Co-localization of UFM1 with UFBP1 in the endoplasmic reticulum (ER)depends on UFBP1. We demonstrate that ER stress, which is common in secretory cells, induces expression of Ufm1, Ufbp1 and Ufl1 in the beta-cell line INS-1E.siRNA-mediated Ufm1 or Ufbp1knockdown enhances apoptosis upon ER stress.Silencing the E3 enzyme UFL1, results in similar outcomes, suggesting that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis. Together, our data suggest that UFM1-UFBP1participate in preventing ER stress-induced apoptosis in protein secretory cells. PMID:21494687

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

Floral nectar production and carbohydrate composition and the structure of receptacular nectaries in the invasive plant Bunias orientalis L. (Brassicaceae).

PubMed

Denisow, Bożena; Masierowska, Marzena; Antoń, Sebastian

2016-11-01

The data relating to the nectaries and nectar secretion in invasive Brassicacean taxa are scarce. In the present paper, the nectar production and nectar carbohydrate composition as well as the morphology, anatomy and ultrastructure of the floral nectaries in Bunias orientalis were investigated. Nectary glands were examined using light, fluorescence, scanning electron and transmission electron microscopy. The quantities of nectar produced by flowers and total sugar mass in nectar were relatively low. Total nectar carbohydrate production per 10 flowers averaged 0.3 mg. Nectar contained exclusively glucose (G) and fructose (F) with overall G/F ratio greater than 1. The flowers of B. orientalis have four nectaries placed at the base of the ovary. The nectarium is intermediate between two nectary types: the lateral and median nectary type (lateral and median glands stay separated) and the annular nectary type (both nectaries are united into one). Both pairs of glands represent photosynthetic type and consist of epidermis and glandular tissue. However, they differ in their shape, size, secretory activity, dimensions of epidermal and parenchyma cells, thickness of secretory parenchyma, phloem supply, presence of modified stomata and cuticle ornamentation. The cells of nectaries contain dense cytoplasm, plastids with starch grains and numerous mitochondria. Companion cells of phloem lack cell wall ingrowths. The ultrastructure of secretory cells indicates an eccrine mechanism of secretion. Nectar is exuded throughout modified stomata.

The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

PubMed

Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

1995-07-15

The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process.

Excavating the surface-associated and secretory proteome of Mycobacterium leprae for identifying vaccines and diagnostic markers relevant immunodominant epitopes.

PubMed

Rana, Aarti; Thakur, Shweta; Bhardwaj, Nupur; Kumar, Devender; Akhter, Yusuf

2016-12-01

For centuries, Mycobacterium leprae, etiological agent of leprosy, has been afflicting mankind regardless of extensive use of live-attenuated vaccines and antibiotics. Surface-associated and secretory proteins (SASPs) are attractive targets against bacteria. We have integrated biological knowledge with computational approaches and present a proteome-wide identification of SASPs. We also performed computational assignment of immunodominant epitopes as coordinates of prospective antigenic candidates in most important class of SASPs, the outer membrane proteins (OMPs). Exploiting the known protein sequence and structural characteristics shared by the SASPs from bacteria, 17 lipoproteins, 11 secretory and 19 novel OMPs (including 4 essential proteins) were identified in M. leprae As OMPs represent the most exposed antigens on the cell surface, their immunoinformatics analysis showed that the identified 19 OMPs harbor T-cell MHC class I epitopes and class II epitopes against HLA-DR alleles (54), while 15 OMPs present potential T-cell class II epitopes against HLA-DQ alleles (6) and 7 OMPs possess T-cell class II epitopes against HLA-DP alleles (5) of humans. Additionally, 11 M. leprae OMPs were found to have B-cell epitopes and these may be considered as prime candidates for the development of new immunotherapeutics against M. leprae. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Intestinal immune system of young rats influenced by cocoa-enriched diet.

PubMed

Ramiro-Puig, Emma; Pérez-Cano, Francisco J; Ramos-Romero, Sara; Pérez-Berezo, Teresa; Castellote, Cristina; Permanyer, Joan; Franch, Angels; Izquierdo-Pulido, Maria; Castell, Margarida

2008-08-01

Gut-associated lymphoid tissue (GALT) maintains mucosal homeostasis by counteracting pathogens and inducing a state of nonresponsiveness when it receives signals from food antigens and commensal bacteria. We report for the first time the influence of continuous cocoa consumption on GALT function in rats postweaning. Weaned Wistar rats were fed cocoa-enriched diets (4% or 10% food intake) for 3 weeks. The function of the primary inductive sites of GALT, such as Peyer's patches (PP) and mesenteric lymph nodes (MLN), was evaluated through an analysis of IgA-secretory ability and lymphocyte composition (T, B and natural killer cells), activation (IL-2 secretion and IL-2 receptor alpha expression) and proliferation. T-helper effector cell balance was also established based on cytokine profile (interferon gamma, IL-4 and IL-10) after mitogen activation. A 10% cocoa intake induced significant changes in PP and MLN lymphocyte composition and function, whereas a 4% cocoa diet did not cause significant modifications in either tissues. Cocoa diet strongly reduced secretory IgA (S-IgA) in the intestinal lumen, although IgA's secretory ability was only slightly decreased in PP. In addition, the 10% cocoa diet increased T-cell-antigen receptor gammadelta cell proportion in both lymphoid tissues. Thus, cocoa intake modulates intestinal immune responses in young rats, influencing gammadelta T-cells and S-IgA production.

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

Shotgun Proteomics of Aspergillus niger Microsomes upon d-Xylose Induction▿ †

PubMed Central

de Oliveira, José Miguel P. Ferreira; van Passel, Mark W. J.; Schaap, Peter J.; de Graaff, Leo H.

2010-01-01

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which—many of them hypothetical proteins—were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles. PMID:20453123

Coordinated activation of the secretory pathway during notochord formation in the Xenopus embryo.

PubMed

Tanegashima, Kosuke; Zhao, Hui; Rebbert, Martha L; Dawid, Igor B

2009-11-01

We compared the transcriptome in the developing notochord of Xenopus laevis embryos with that of other embryonic regions. A coordinated and intense activation of a large set of secretory pathway genes was observed in the notochord, but not in notochord precursors in the axial mesoderm at early gastrula stage. The genes encoding Xbp1 and Creb3l2 were also activated in the notochord. These two transcription factors are implicated in the activation of secretory pathway genes during the unfolded protein response, where cells react to the stress of a build-up of unfolded proteins in their endoplasmic reticulum. Xbp1 and Creb3l2 are differentially expressed but not differentially activated in the notochord. Reduction of expression of Xbp1 or Creb3l2 by injection of antisense morpholinos led to strong deficits in notochord but not somitic muscle development. In addition, the expression of some, but not all, genes encoding secretory proteins was inhibited by injection of xbp1 morpholinos. Furthermore, expression of activated forms of Xbp1 or Creb3l2 in animal explants could activate a similar subset of secretory pathway genes. We conclude that coordinated activation of a battery of secretory pathway genes mediated by Xbp1 and Creb/ATF factors is a characteristic and necessary feature of notochord formation.

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.

[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.

Characterisation of secretory calcium-binding phosphoprotein-proline-glutamine-rich 1: a novel basal lamina component expressed at cell-tooth interfaces.

PubMed

Moffatt, Pierre; Wazen, Rima M; Dos Santos Neves, Juliana; Nanci, Antonio

2014-12-01

Functional genomic screening of the rat enamel organ (EO) has led to the identification of a number of secreted proteins expressed during the maturation stage of amelogenesis, including amelotin (AMTN) and odontogenic ameloblast-associated (ODAM). In this study, we characterise the gene, protein and pattern of expression of a related protein called secretory calcium-binding phosphoprotein-proline-glutamine-rich 1 (SCPPPQ1). The Scpppq1 gene resides within the secretory calcium-binding phosphoprotein (Scpp) cluster. SCPPPQ1 is a highly conserved, 75-residue, secreted protein rich in proline, leucine, glutamine and phenylalanine. In silico data mining has revealed no correlation to any known sequences. Northern blotting of various rat tissues suggests that the expression of Scpppq1 is restricted to tooth and associated tissues. Immunohistochemical analyses show that the protein is expressed during the late maturation stage of amelogenesis and in the junctional epithelium where it localises to an atypical basal lamina at the cell-tooth interface. This discrete localisation suggests that SCPPPQ1, together with AMTN and ODAM, participates in structuring the basal lamina and in mediating attachment of epithelia cells to mineralised tooth surfaces.

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.

Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.

PubMed

Li, Jingsong; Luo, Ruihua; Hooi, Shing Chuan; Ruga, Pilar; Zhang, Jiping; Meda, Paolo; Li, GuoDong

2005-03-22

Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.

Comparative histology of floral elaiophores in the orchids Rudolfiella picta (Schltr.) Hoehne (Maxillariinae sensu lato) and Oncidium ornithorhynchum H.B.K. (Oncidiinae sensu lato).

PubMed

Davies, Kevin L; Stpiczyńska, Malgorzata

2009-08-01

Floral elaiophores, although widespread amongst orchids, have not previously been described for Maxillariinae sensu lato. Here, two claims that epithelial, floral elaiophores occur in the genus Rudolfiella Hoehne (Bifrenaria clade) are investigated. Presumed elaiophores were compared with those of Oncidiinae Benth. and the floral, resin-secreting tissues of Rhetinantha M.A. Blanco and Heterotaxis Lindl., both genera formerly assigned to Maxillaria Ruiz & Pav. (Maxillariinae sensu stricto). Putative, floral elaiophore tissue of Rudolfiella picta (Schltr.) Hoehne and floral elaiophores of Oncidium ornithorhynchum H.B.K. were examined by means of light microscopy, histochemistry, scanning electron microscopy and transmission electron microscopy. Floral, epithelial elaiophores are present in Rudolfiella picta, indicating, for the first time, that oil secretion occurs amongst members of the Bifrenaria clade (Maxillariinae sensu lato). However, whereas the elaiophore of R. picta is borne upon the labellar callus, the elaiophores of O. ornithorhynchum occur on the lateral lobes of the labellum. In both species, the elaiophore comprises a single layer of palisade secretory cells and parenchymatous, subsecretory tissue. Cell wall cavities are absent from both and there is no evidence of cuticular distension in response to oil accumulation between the outer tangential wall and the overlying cuticle in R. picta. Distension of the cuticle, however, occurs in O. ornithorhynchum. Secretory cells of R. picta contain characteristic, spherical or oval plastids with abundant plastoglobuli and these more closely resemble plastids found in labellar, secretory cells of representatives of Rhetinantha (formerly Maxillaria acuminata Lindl. alliance) than elaiophore plastids of Oncidiinae. In Rhetinantha, such plastids are involved in the synthesis of resin-like material or wax. Despite these differences, the elaiophore anatomy of both R. picta (Bifrenaria clade) and O. ornithorhynchum (Oncidiinae) fundamentally resembles that of several representatives of Oncidiinae. These, in their possession of palisade secretory cells, in turn, resemble the floral elaiophores of certain members of Malpighiaceae, indicating that convergence has occurred here in response to similar pollination pressures.

β-Arrestin2 plays a key role in the modulation of the pancreatic beta cell mass in mice.

PubMed

Ravier, Magalie A; Leduc, Michele; Richard, Joy; Linck, Nathalie; Varrault, Annie; Pirot, Nelly; Roussel, Morgane M; Bockaert, Joël; Dalle, Stéphane; Bertrand, Gyslaine

2014-03-01

Beta cell failure due to progressive secretory dysfunction and limited expansion of beta cell mass is a key feature of type 2 diabetes. Beta cell function and mass are controlled by glucose and hormones/neurotransmitters that activate G protein-coupled receptors or receptor tyrosine kinases. We have investigated the role of β-arrestin (ARRB)2, a scaffold protein known to modulate such receptor signalling, in the modulation of beta cell function and mass, with a specific interest in glucagon-like peptide-1 (GLP-1), muscarinic and insulin receptors. β-arrestin2-knockout mice and their wild-type littermates were fed a normal or a high-fat diet (HFD). Glucose tolerance, insulin sensitivity and insulin secretion were assessed in vivo. Beta cell mass was evaluated in pancreatic sections. Free cytosolic [Ca(2+)] and insulin secretion were determined using perifused islets. The insulin signalling pathway was evaluated by western blotting. Arrb2-knockout mice exhibited impaired glucose tolerance and insulin secretion in vivo, but normal insulin sensitivity compared with wild type. Surprisingly, the absence of ARRB2 did not affect glucose-stimulated insulin secretion or GLP-1- and acetylcholine-mediated amplifications from perifused islets, but it decreased the islet insulin content and beta cell mass. Additionally, there was no compensatory beta cell mass expansion through proliferation in response to the HFD. Furthermore, Arrb2 deletion altered the islet insulin signalling pathway. ARRB2 is unlikely to be involved in the regulation of insulin secretion, but it is required for beta cell mass plasticity. Additionally, we provide new insights into the mechanisms involved in insulin signalling in beta cells.

COBRA-LIKE2, a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE family, plays a role in cellulose deposition in arabidopsis seed coat mucilage secretory cells.

PubMed

Ben-Tov, Daniela; Abraham, Yael; Stav, Shira; Thompson, Kevin; Loraine, Ann; Elbaum, Rivka; de Souza, Amancio; Pauly, Markus; Kieber, Joseph J; Harpaz-Saad, Smadar

2015-03-01

Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE2 (COBL2), a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE gene family in Arabidopsis (Arabidopsis thaliana), as coexpressed with other genes involved in cellulose deposition in mucilage secretory cells. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised, as demonstrated by scanning electron microscopy and in situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole-seed crystalline cellulose content compared with the wild type. These data establish that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation. © 2015 American Society of Plant Biologists. All Rights Reserved.

Adrenal Chromaffin Cells Exposed to 5-ns Pulses Require Higher Electric Fields to Porate Intracellular Membranes than the Plasma Membrane: An Experimental and Modeling Study.

PubMed

Zaklit, Josette; Craviso, Gale L; Leblanc, Normand; Yang, Lisha; Vernier, P Thomas; Chatterjee, Indira

2017-10-01

Nanosecond-duration electric pulses (NEPs) can permeabilize the endoplasmic reticulum (ER), causing release of Ca 2+ into the cytoplasm. This study used experimentation coupled with numerical modeling to understand the lack of Ca 2+ mobilization from Ca 2+ -storing organelles in catecholamine-secreting adrenal chromaffin cells exposed to 5-ns pulses. Fluorescence imaging determined a threshold electric (E) field of 8 MV/m for mobilizing intracellular Ca 2+ whereas whole-cell recordings of membrane conductance determined a threshold E-field of 3 MV/m for causing plasma membrane permeabilization. In contrast, a 2D numerical model of a chromaffin cell, which was constructed with internal structures representing a nucleus, mitochondrion, ER, and secretory granule, predicted that exposing the cell to the same 5-ns pulse electroporated the plasma and ER membranes at the same E-field amplitude, 3-4 MV/m. Agreement of the numerical simulations with the experimental results was obtained only when the ER interior conductivity was 30-fold lower than that of the cytoplasm and the ER membrane permittivity was twice that of the plasma membrane. A more realistic intracellular geometry for chromaffin cells in which structures representing multiple secretory granules and an ER showed slight differences in the thresholds necessary to porate the membranes of the secretory granules. We conclude that more sophisticated cell models together with knowledge of accurate dielectric properties are needed to understand the effects of NEPs on intracellular membranes in chromaffin cells, information that will be important for elucidating how NEPs porate organelle membranes in other cell types having a similarly complex cytoplasmic ultrastructure.

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila

PubMed Central

Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P.

2017-01-01

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1-knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. PMID:28381425

Role of Local Cytokines in Increased Gastric Expression of the Secretory Component in Helicobacter pylori Infection

PubMed Central

Ahlstedt, Ingela; Lindholm, Catharina; Lönroth, Hans; Hamlet, Annika; Svennerholm, Ann-Mari; Quiding-Järbrink, Marianne

1999-01-01

Using immunohistochemical staining, we examined the presence of secretory component (SC) on epithelial cells in gastric and duodenal biopsy specimens collected from Helicobacter pylori-infected individuals and healthy controls. Gastric epithelial cells from healthy volunteers expressed low, but detectable, levels of SC. In contrast, significantly higher level of expression of SC (P < 0.001) was observed on epithelial cells in the antra of H. pylori-infected individuals. The antral SC expression correlated with staining for gamma interferon of intraepithelial and lamina propria lymphocytes (rs = 0.76 and 0.69, respectively, P < 0.001) and correlated weakly with production of tumor necrosis factor alpha (rs = 0.43, P < 0.05), but it did not correlate at all with interleukin-4 production. PMID:10456951

E-cadherin interactions regulate beta-cell proliferation in islet-like structures.

PubMed

Carvell, Melanie J; Marsh, Phil J; Persaud, Shanta J; Jones, Peter M

2007-01-01

Islet function is dependent on cells within the islet interacting with each other. E-cadherin (ECAD) mediates Ca(2+)-dependent homophilic cell adhesion between b-cells within islets and has been identified as a tumour suppressor. We generated clones of the MIN6 beta-cell line that stably over- (S) and under-express (alphaS) ECAD. Modified expression of ECAD was confirmed by quantitative RT-PCR, immunoblotting and immunocytochemistry. Preproinsulin mRNA, insulin content and basal rates of insulin secretion were higher in S cells compared to aS and control (V) cells. However, stimulated insulin secretory responses were unaffected by ECAD expression levels. ECAD expression did affect proliferation, with enhanced ECAD expression being associated with reduced proliferation and vice versa. Formation of islet-like structures was associated with a significant reduction in proliferation of V and S cells but not alphaS cells. These data suggest that ECAD expression levels do not modulate insulin secretory function but are consistent with a role for ECAD in the regulation of beta-cell proliferation. Copyright (c) 2007 S. Karger AG, Basel.

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria.

PubMed

Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P; Yi, Ling; Kaler, Stephen G; Lutsenko, Svetlana; Ralle, Martina

2016-08-05

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

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

Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria,more » whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).« less

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria*

PubMed Central

Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P.; Yi, Ling; Kaler, Stephen G.; Lutsenko, Svetlana; Ralle, Martina

2016-01-01

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia). PMID:27226607

Bone Marrow Cells Expressing Clara Cell Secretory Protein Increase Epithelial Repair After Ablation of Pulmonary Clara Cells

PubMed Central

Bustos, Martha L; Mura, Marco; Marcus, Paula; Hwang, David; Ludkovski, Olga; Wong, Amy P; Waddell, Thomas K

2013-01-01

We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP+ cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP+ cells is beneficial after ablation of lung CCSP+ cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP+ or CCSP− BMC. Compared with mice administered CCSP− cells, mice treated with CCSP+ cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP+ cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP+ BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP+ BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised. PMID:23609017

The human uterotubal junction: a scanning electron microscope study during different phases of the menstrual cycle.

PubMed

Fadel, H E; Berns, D; Zaneveld, L J; Wilbanks, G D; Brueschke, E E

1976-10-01

Uterotubal junctions from surgically extirpated human uteri were examined. The specimens were obtained during different phases of the menstrual cycle. The interstitial portions of the tubes together with the cornual areas were dissected, excised, and their luminal surfaces exposed. The specimens were then processed for scanning electron microscopy. The surface epithelium of both the cornual endometrium and interstitial endosalpins. Ciliated cells were more numerous in the endosalpinx. Cyclic changes in ciliated cells were minimal, while cyclic secretory activity was demonstrated, especially in the endometrium. The transitional area between the endometrium and the endosalpinx was characterized by a marked increase in the number of ciliated cells, and a tendency of the secretory cells to assume a flattened, polygonal shape. These morphologic features suggest a possible role in the transport and/or maintenance of spermatozoa and/or ova.

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.

The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

PubMed Central

Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

1995-01-01

The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626033

Calcium-dependent transferrin receptor recycling in bovine chromaffin cells.

PubMed

Knight, Derek E

2002-04-01

The release of regulated secretory granules is known to be calcium dependent. To examine the Ca2+-dependence of other exocytic fusion events, transferrin recycling in bovine chromaffin cells was examined. Internalised 125I-transferrin was released constitutively from cells with a half-time of about 7 min. Secretagogues that triggered catecholamine secretion doubled the rate of 125I-transferrin release, the time courses of the two triggered secretory responses being similar. The triggered 125I-transferrin release came from recycling endosomes rather than from sorting endosomes or a triggered secretory vesicle pool. Triggered 125I-transferrin release, like catecholamine secretion from the same cells, was calcium dependent but the affinities for calcium were very different. The extracellular calcium concentrations that gave rise to half-maximal evoked secretion were 0.1 mm for 125I-transferrin and 1.0 mm for catecholamine, and the intracellular concentrations were 0.1 microm and 1 microm, respectively. There was significant 125I-transferrin recycling in the virtual absence of intracellular Ca2+, but the rate increased when Ca2+ was raised above 1 nm, and peaked at 1 microm when the rate had doubled. Botulinum toxin type D blocked both transferrin recycling and catecholamine secretion. These results indicate that a major component of the vesicular transport required for the constitutive recycling of transferrin in quiescent cells is calcium dependent and thus under physiological control, and also that some of the molecular machinery involved in transferrin recycling/fusion processes is shared with that for triggered neurosecretion.

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.

Chromogranin A promotes peptide hormone sorting to mobile granules in constitutively and regulated secreting cells: role of conserved N- and C-terminal peptides.

PubMed

Montero-Hadjadje, Maité; Elias, Salah; Chevalier, Laurence; Benard, Magalie; Tanguy, Yannick; Turquier, Valérie; Galas, Ludovic; Yon, Laurent; Malagon, Maria M; Driouich, Azeddine; Gasman, Stéphane; Anouar, Youssef

2009-05-01

Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N- and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N- and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.

The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis.

PubMed

Jing, Lan; Guo, Dandan; Hu, Wenjie; Niu, Xiaofan

2017-03-11

Many plant pathogen secretory proteins are known to be elicitors or pathogenic factors,which play an important role in the host-pathogen interaction process. Bioinformatics approaches make possible the large scale prediction and analysis of secretory proteins from the Puccinia helianthi transcriptome. The internet-based software SignalP v4.1, TargetP v1.01, Big-PI predictor, TMHMM v2.0 and ProtComp v9.0 were utilized to predict the signal peptides and the signal peptide-dependent secreted proteins among the 35,286 ORFs of the P. helianthi transcriptome. 908 ORFs (accounting for 2.6% of the total proteins) were identified as putative secretory proteins containing signal peptides. The length of the majority of proteins ranged from 51 to 300 amino acids (aa), while the signal peptides were from 18 to 20 aa long. Signal peptidase I (SpI) cleavage sites were found in 463 of these putative secretory signal peptides. 55 proteins contained the lipoprotein signal peptide recognition site of signal peptidase II (SpII). Out of 908 secretory proteins, 581 (63.8%) have functions related to signal recognition and transduction, metabolism, transport and catabolism. Additionally, 143 putative secretory proteins were categorized into 27 functional groups based on Gene Ontology terms, including 14 groups in biological process, seven in cellular component, and six in molecular function. Gene ontology analysis of the secretory proteins revealed an enrichment of hydrolase activity. Pathway associations were established for 82 (9.0%) secretory proteins. A number of cell wall degrading enzymes and three homologous proteins specific to Phytophthora sojae effectors were also identified, which may be involved in the pathogenicity of the sunflower rust pathogen. This investigation proposes a new approach for identifying elicitors and pathogenic factors. The eventual identification and characterization of 908 extracellularly secreted proteins will advance our understanding of the molecular mechanisms of interactions between sunflower and rust pathogen and will enhance our ability to intervene in disease states.

The cellular response to vascular endothelial growth factors requires co-ordinated signal transduction, trafficking and proteolysis

PubMed Central

Smith, Gina A.; Fearnley, Gareth W.; Tomlinson, Darren C.; Harrison, Michael A.; Ponnambalam, Sreenivasan

2015-01-01

VEGFs (vascular endothelial growth factors) are a family of conserved disulfide-linked soluble secretory glycoproteins found in higher eukaryotes. VEGFs mediate a wide range of responses in different tissues including metabolic homoeostasis, cell proliferation, migration and tubulogenesis. Such responses are initiated by VEGF binding to soluble and membrane-bound VEGFRs (VEGF receptor tyrosine kinases) and co-receptors. VEGF and receptor splice isoform diversity further enhances complexity of membrane protein assembly and function in signal transduction pathways that control multiple cellular responses. Different signal transduction pathways are simultaneously activated by VEGFR–VEGF complexes with membrane trafficking along the endosome–lysosome network further modulating signal output from multiple enzymatic events associated with such pathways. Balancing VEGFR–VEGF signal transduction with trafficking and proteolysis is essential in controlling the intensity and duration of different intracellular signalling events. Dysfunction in VEGF-regulated signal transduction is important in chronic disease states including cancer, atherosclerosis and blindness. This family of growth factors and receptors is an important model system for understanding human disease pathology and developing new therapeutics for treating such ailments. PMID:26285805

NFIB regulates embryonic development of submandibular glands.

PubMed

Mellas, R E; Kim, H; Osinski, J; Sadibasic, S; Gronostajski, R M; Cho, M; Baker, O J

2015-02-01

NFIB (nuclear factor I B) is a NFI transcription factor family member, which is essential for the development of a variety of organ systems. Salivary gland development occurs through several stages, including prebud, bud, pseudoglandular, canalicular, and terminal. Although many studies have been done to understand mouse submandibular gland (SMG) branching morphogenesis, little is known about SMG cell differentiation during the terminal stages. The goal of this study was to determine the role of NFIB during SMG development. We analyzed SMGs from wild-type and Nfib-deficient mice (Nfib (-/-)). At embryonic (E) day 18.5, SMGs from wild-type mice showed duct branching morphogenesis and differentiation of tubule ductal cells into tubule secretory cells. In contrast, SMGs from Nfib (-/-) mice at E18.5 failed to differentiate into tubule secretory cells while branching morphogenesis was unaffected. SMGs from wild-type mice at E16.5 displayed well-organized cuboidal inner terminal tubule cells. However, SMGs from Nfib (-/-) at E16.5 displayed disorganized inner terminal tubule cells. SMGs from wild-type mice at E18.5 became fully differentiated, as indicated by a high degree of apicobasal polarization (i.e., presence of apical ZO-1 and basolateral E-cadherin) and columnar shape. Furthermore, SMGs from wild-type mice at E18.5 expressed the protein SMGC, a marker for tubule secretory cells. However, SMGs from Nfib (-/-) mice at E18.5 showed apicobasal polarity, but they were disorganized and lost the ability to secrete SMGC. These findings indicate that the transcription factor NFIB is not required for branching morphogenesis but plays a key role in tubule cell differentiation during mouse SMG development. © International & American Associations for Dental Research 2014.

SOX10-positive salivary gland tumors: a growing list, including mammary analogue secretory carcinoma of the salivary gland, sialoblastoma, low-grade salivary duct carcinoma, basal cell adenoma/adenocarcinoma, and a subgroup of mucoepidermoid carcinoma.

PubMed

Hsieh, Min-Shu; Lee, Yi-Hsuan; Chang, Yih-Leong

2016-10-01

Transcription factor SRY-related HMG-box 10 (SOX10) is an important marker for melanocytic, schwannian, myoepithelial, and some salivary gland tumors. The aim of this study was to investigate SOX10 expression more thoroughly in the salivary gland neoplasms, including mammary analogue secretory carcinoma and hyalinizing clear cell carcinoma harboring specific genetic rearrangements. A new rabbit monoclonal anti-SOX10 antibody (clone EP268) was used to examine SOX10 expression in 14 different types of salivary gland tumors. We found that acinic cell carcinoma (AciCC), adenoid cystic carcinoma, mammary analogue secretory carcinoma (MASC), epithelial-myoepithelial carcinoma, low-grade salivary duct carcinoma, sialoblastoma, basal cell adenocarcinoma, basal cell adenoma, and pleomorphic adenoma were SOX10 positive. Salivary duct carcinoma, lymphoepithelial carcinoma, hyalinizing clear cell carcinoma, and oncocytoma were SOX10 negative. Earlier, mucoepidermoid carcinoma (MEC) was considered a SOX10-negative tumor. This study identified a subgroup of SOX10-positive MEC cases with characteristic polygonal epithelial cells, pale-to-eosinophilic cytoplasm, and colloid-like dense eosinophilic material. Our data show SOX10 expression can be observed in salivary gland tumors with either one of the 4 cell types: acinic cells, cuboidal ductal cells with low-grade cytologic features, basaloid cells, and myoepithelial cells. In this article we thoroughly evaluated SOX10 expression in salivary gland tumors. SOX10 is useful in the differential diagnosis between myoepithelial carcinoma with clear cell features and hyalinizing clear cell carcinoma. It can also be used to discriminate low-grade salivary duct carcinoma from high-grade ones. Pathologists should be cautious with the interpretation of SOX10 positivity in salivary gland tumors, and correlation with histologic feature is mandatory. Copyright © 2016 Elsevier Inc. All rights reserved.

ADIPOCYTOKINES AND OBESITY-LINKED DISORDERS

PubMed Central

OUCHI, NORIYUKI; OHASHI, KOJI; SHIBATA, REI; MUROHARA, TOYOAKI

2012-01-01

ABSTRACT Obesity is closely associated with an increased risk for metabolic and cardiovascular diseases. Adipose tissue produces a number of secretory bioactive substances, also known as adipocytokines or adipokines, which directly affect adjacent or distant organs. Most adipocytokines are pro-inflammatory, thereby promoting the obesity-linked disorders. In contrast, there are a small number of adipocytokines that exhibit anti-inflammatory properties. It is now recognized that dysregulated production or secretion of adipocytokines caused by adipocyte dysfunction leads to the development of obesity-linked complications. In this review, we focus on the functional role of several adipocytokines in metabolic and cardiovascular diseases. PMID:22515108

Identification of Catechin, Syringic Acid, and Procyanidin B2 in Wine as Stimulants of Gastric Acid Secretion.

PubMed

Liszt, Kathrin Ingrid; Eder, Reinhard; Wendelin, Sylvia; Somoza, Veronika

2015-09-09

Organic acids of wine, in addition to ethanol, have been identified as stimulants of gastric acid secretion. This study characterized the influence of other wine compounds, particularly phenolic compounds, on proton secretion. Forty wine parameters were determined in four red wines and six white wines, including the contents of organic acids and phenolic compounds. The secretory activity of the wines was determined in a gastric cell culture model (HGT-1 cells) by means of a pH-sensitive fluorescent dye. Red wines stimulated proton secretion more than white wines. Lactic acid and the phenolic compounds syringic acid, catechin, and procyanidin B2 stimulated proton secretion and correlated with the pro-secretory effect of the wines. Addition of the phenolic compounds to the least active white wine sample enhanced its proton secretory effect by 65 ± 21% (p < 0.05). These results indicate that not only malic and lactic acid but also bitter and astringent tasting phenolic compounds in wine contribute to its stimulatory effect on gastric acid secretion.

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

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens.

PubMed Central

Schweitzer-Stenner, R; Tamir, I; Pecht, I

1997-01-01

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response. PMID:9168023

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila.

PubMed

Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P

2017-06-01

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1 -knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. © 2017 Kaur et al. 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).

Ultrastructure of the salivary glands in Lithobius forficatus (Myriapoda, Chilopoda, Lithobiidae) according to seasonal and circadian rhythms.

PubMed

Kamińska, K; Włodarczyk, A; Sonakowska, L; Ostróżka, A; Marchewka, A; Rost-Roszkowska, M

2016-11-01

The salivary glands (mandibular epidermal glands) of adult males and females of Lithobius forficatus (Myriapoda, Chilopoda) were isolated during spring, summer and autumn. In addition, the organs were isolated at different times of the day - at about 12:00 (noon) and about 00:00 (midnight). The ultrastructure of these organs depending on seasonal and circadian rhythms was analyzed using transmission and scanning electron microscopy and histochemical methods. The paired salivary glands of L. forficatus are situated in the vicinity of the foregut and they are formed by numerous acini that are surrounded by the fat body, hemocytes and tracheolae. The salivary glands are composed of a terminal acinar component and a system of tubular ducts that are lined with a cuticle. The glandular part is composed of secretory epithelial cells that are at various stages of their secretory activity. The saliva that is produced by the secretory cells of the acini is secreted into the salivary ducts, which are lined with a simple epithelium that is based on the non-cellular basal lamina. The ultrastructural variations suggest that salivary glands function differently depending on seasonal rhythms and prepare the animal for overwintering. Therefore, the salivary glands of the centipedes that were analyzed participate in the accumulation of proteins, lipids and polysaccharides during the spring, summer and autumn. Subtle differences in the ultrastructure of the secretory cells of the salivary glands during the circadian cycle must be related to the physiological reactions of the organism. The salivary ducts showed no differences in the specimens that were analyzed during the day/night cycle or during the seasonal cycle. Copyright © 2016 Elsevier Ltd. All rights reserved.

Small-molecule MDM2 antagonists attenuate the senescence-associated secretory phenotype.

PubMed

Wiley, Christopher D; Schaum, Nicholas; Alimirah, Fatouma; Lopez-Dominguez, Jose Alberto; Orjalo, Arturo V; Scott, Gary; Desprez, Pierre-Yves; Benz, Christopher; Davalos, Albert R; Campisi, Judith

2018-02-05

Processes that have been linked to aging and cancer include an inflammatory milieu driven by senescent cells. Senescent cells lose the ability to divide, essentially irreversibly, and secrete numerous proteases, cytokines and growth factors, termed the senescence-associated secretory phenotype (SASP). Senescent cells that lack p53 tumor suppressor function show an exaggerated SASP, suggesting the SASP is negatively controlled by p53. Here, we show that increased p53 activity caused by small molecule inhibitors of MDM2, which promotes p53 degradation, reduces inflammatory cytokine production by senescent cells. Upon treatment with the MDM2 inhibitors nutlin-3a or MI-63, human cells acquired a senescence-like growth arrest, but the arrest was reversible. Importantly, the inhibitors reduced expression of the signature SASP factors IL-6 and IL-1α by cells made senescent by genotoxic stimuli, and suppressed the ability of senescent fibroblasts to stimulate breast cancer cell aggressiveness. Our findings suggest that MDM2 inhibitors could reduce cancer progression in part by reducing the pro-inflammatory environment created by senescent cells.

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.

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

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

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.

Protein disulfide isomerase externalization in endothelial cells follows classical and unconventional routes.

PubMed

Araujo, Thaís L S; Zeidler, Julianna D; Oliveira, Percíllia V S; Dias, Matheus H; Armelin, Hugo A; Laurindo, Francisco R M

2017-02-01

Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

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 basidiomycete-specific secretory protein family. We found that some of these proteins are cell type specific, thus contributing to the heterogeneity of a mating colony. Our study also demonstrates the importance of examining the protein expression pattern at the individual-cell level in addition to population gene expression profiling for the investigation of a heterogeneous community. Copyright © 2017 American Society for Microbiology.

PC5-A-mediated processing of pro-neurotensin in early compartments of the regulated secretory pathway of PC5-transfected PC12 cells.

PubMed

Barbero, P; Rovère, C; De Bie, I; Seidah, N; Beaudet, A; Kitabgi, P

1998-09-25

Among the members of the proprotein convertase (PC) family, PC1 and PC2 have well established roles as prohormone convertases. Another good candidate for this role is PC5-A that has been shown to be present in the regulated secretory pathway of certain neuroendocrine tissues, but evidence that it can process prohormones is lacking. To determine whether PC5-A could function as a prohormone convertase and to compare its cleavage specificity with that of PC1 and PC2, we stably transfected the rat pheochromocytoma PC12 cell line with PC5-A and analyzed the biosynthesis and subcellular localization of the enzyme, as well as its ability to process pro-neurotensin/neuromedin N (pro-NT/NN) into active peptides. Our data showed that in transfected PC12 cells, PC5-A was converted from its 126-kDa precursor form into a 117-kDa mature form and, to a lesser extent, into a C-terminally truncated 65-kDa form of the 117-kDa product. Metabolic and immunochemical studies showed that PC5-A was sorted to early compartments of the regulated secretory pathway where it colocalized with immunoreactive NT. Furthermore, pro-NT/NN was processed in these compartments according to a pattern that differed from that previously described in PC1- and PC2-transfected PC12 cells. This pattern resembled that previously reported for pro-NT/NN processing in the adrenal medulla, a tissue known to express high levels of PC5-A. Altogether, these data demonstrate for the first time the ability of PC5-A to function as a prohormone convertase in the regulated secretory pathway and suggest a role for this enzyme in the physiological processing of pro-NT/NN.

A histochemical study of rat salivary gland acid phosphatase.

PubMed

Isacsson, G

1986-01-01

Male Sprague-Dawley rats received 4 mg pilocarpine/100 g body wt intraperitoneally or physiological saline as control and were killed at various intervals. Acid phosphatase was reacted on frozen sections from soft palate, parotid and submandibular glands using sodium-alpha-naphthyl acid phosphate as substrate. Various inhibitors were added to the incubation medium. The strongest acid phosphatase activity was in the parotid gland acinar and proximal secretory duct cells; the mucous minor glands of the palate were completely negative. Activity was found in the acinar cells, proximal secretory duct cells, granular and striated duct and excretory duct cells. Pilocarpine injection slightly reduced the activity up to 6 h after injection. Cupric chloride added to the incubation medium lowered the overall activity. Fluoride and molybdate inhibited the acid phosphatase reaction in all structures. Tartrate inhibited the reaction in all structures except the submandibular striated duct cells. The tartrate-resistant activity may be a Na+K+-dependent ATPase involved in re-absorbing water and electrolytes from the primary saliva.

Multi‐omic profiling ­of EPO‐producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

PubMed Central

Ley, Daniel; Seresht, Ali Kazemi; Engmark, Mikael; Magdenoska, Olivera; Nielsen, Kristian Fog; Kildegaard, Helene Faustrup

2015-01-01

ABSTRACT Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi‐omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO‐K1 cells under growth‐limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO‐producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)+, adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT‐PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)+ and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post‐translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time‐course analysis of high‐ and low‐producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:25995028

Megamitochondria in the serous acinar cells of the submandibular gland of the neotropical fruit bat, Artibeus obscurus.

PubMed

Tandler, B; Nagato, T; Phillips, C J

1997-05-01

As part of a continuing investigation of the comparative ultrastructure of chiropteran salivary glands, we examined the submandibular glands of eight species of neotropical fruit bats in the genus Artibeus. We previously described secretory granules of unusual substructure in the seromucous demilunar cells of this organ in some species in this genus. In the present study, we turned our attention to the serous acinar cells in the same glands. Specimens of eight species of Artibeus were collected in neotropical localities. Salivary glands were extirpated in the field and thin slices were fixed by immersion in triple aldehyde-DMSO or in modified half-strength Karnovsky's fixative. Tissues were further processed for electron microscopy by conventional means. In contrast to seromucous cells, which exhibit species-specific diversification in bats of this genus, the secretory apparatus and secretory granules in the serous acinar cells are highly conserved across all seven species. The single exception involves the mitochondria in one species. In this instance, some of the serous cell mitochondria in Artibeus obscurus are modified into megamitochondria. Such organelles usually have short, peripheral cristae; a laminar inclusion is present in the matrix compartment of every outsized organelle. Inclusions of this nature never are present in normal-size mitochondria in the serous cells. None of the megamitochondria were observed in the process of degeneration. The giant mitochondria in A. obscurus have a matrical structure that is radically different from that of the only other megamitochondria reported to occur in bat salivary glands. The factors that lead to variation in megamitochondrial substructure in different species, as well as the functional capacities of such giant organelles, are unknown.

Electrical parameters and water permeability properties of monolayers formed by T84 cells cultured on permeable supports.

PubMed

Ozu, M; Toriano, R; Capurro, C; Parisi, M

2005-01-01

T84 is an established cell line expressing an enterocyte phenotype whose permeability properties have been widely explored. Osmotic permeability (POSM), hydraulic permeability (PHYDR) and transport-associated net water fluxes (JW-transp), as well as short-circuit current (ISC), transepithelial resistance (RT), and potential difference (deltaVT) were measured in T84 monolayers with the following results: POSM 1.3 +/- 0.1 cm.s-1 x 10-3; PHYDR 0.27 +/- 0.02 cm.s-1; RT 2426 +/- 109 omega.cm2, and deltaVT 1.31 +/- 0.38 mV. The effect of 50 microM 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a "net Cl- secretory agent", on T84 cells was also studied. We confirm the reported important increase in ISC induced by DCEBIO which was associated here with a modest secretory deltaJW-transp. The present results were compared with those reported using the same experimental approach applied to established cell lines originating from intestinal and renal epithelial cells (Caco-2, LLC-PK1 and RCCD-1). No clear association between PHYDR and RT could be demonstrated and high PHYDR values were observed in an electrically tight epithelium, supporting the view that a "water leaky" barrier is not necessarily an "electrically leaky" one. Furthermore, the modest secretory deltaJW-transp was not consistent with previous results obtained with RCCD-1 cells stimulated with vasopressin (absorptive fluxes) or with T84 cells secreting water under the action of Escherichia coli heat stable enterotoxin. We conclude that, while the presence of aquaporins is necessary to dissipate an external osmotic gradient, coupling between water and ion transport cannot be explained by a simple and common underlying mechanism.

Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1-7 cell line.

PubMed

Chappell, Patrick E; White, Rachel S; Mellon, Pamela L

2003-12-03

Although it has long been established that episodic secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for normal gonadotropin release, the molecular and cellular mechanisms underlying the synchronous release of GnRH are primarily unknown. We used the GT1-7 mouse hypothalamic cell line as a model for GnRH secretion, because these cells release GnRH in a pulsatile pattern similar to that observed in vivo. To explore possible molecular mechanisms governing secretory timing, we investigated the role of the molecular circadian clock in regulation of GnRH secretion. GT1-7 cells express many known core circadian clock genes, and we demonstrate that oscillations of these components can be induced by stimuli such as serum and the adenylyl cyclase activator forskolin, similar to effects observed in fibroblasts. Strikingly, perturbation of circadian clock function in GT1-7 cells by transient expression of the dominant-negative Clock-Delta19 gene disrupts normal ultradian patterns of GnRH secretion, significantly decreasing mean pulse frequency. Additionally, overexpression of the negative limb clock gene mCry1 in GT1-7 cells substantially increases GnRH pulse amplitude without a commensurate change in pulse frequency, demonstrating that an endogenous biological clock is coupled to the mechanism of neurosecretion in these cells and can regulate multiple secretory parameters. Finally, mice harboring a somatic mutation in the Clock gene are subfertile and exhibit a substantial increase in estrous cycle duration as revealed by examination of vaginal cytology. This effect persists in normal light/dark (LD) cycles, suggesting that a suprachiasmatic nucleus-independent endogenous clock in GnRH neurons is required for eliciting normal pulsatile patterns of GnRH secretion.

RFP tags for labeling secretory pathway proteins

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

Han, Liyang; Zhao, Yanhua; Zhang, Xi

2014-05-09

Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to anmore » environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.« less

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

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

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

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

Irradiation induces glioblastoma cell senescence and senescence-associated secretory phenotype.

PubMed

Jeon, Hee-Young; Kim, Jun-Kyum; Ham, Seok Won; Oh, Se-Yeong; Kim, Jaebong; Park, Jae-Bong; Lee, Jae-Yong; Kim, Sung-Chan; Kim, Hyunggee

2016-05-01

Glioblastoma multiforme (GBM) is one of the most aggressive and fatal primary brain tumors in humans. The standard therapy for the treatment of GBM is surgical resection, followed by radiotherapy and/or chemotherapy. However, the frequency of tumor recurrence in GBM patients is very high, and the survival rate remains poor. Delineating the mechanisms of GBM recurrence is essential for therapeutic advances. Here, we demonstrate that irradiation rendered 17-20 % of GBM cells dead, but resulted in 60-80 % of GBM cells growth-arrested with increases in senescence markers, such as senescence-associated beta-galactosidase-positive cells, H3K9me3-positive cells, and p53-p21(CIP1)-positive cells. Moreover, irradiation induced expression of senescence-associated secretory phenotype (SASP) mRNAs and NFκB transcriptional activity in GBM cells. Strikingly, compared to injection of non-irradiated GBM cells into immune-deficient mice, the co-injection of irradiated and non-irradiated GBM cells resulted in faster growth of tumors with the histological features of human GBM. Taken together, our findings suggest that the increases in senescent cells and SASP in GBM cells after irradiation is likely one of main reasons for tumor recurrence in post-radiotherapy GBM patients.

[Characterization of epithelial primary culture from human conjunctiva].

PubMed

Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

2014-01-01

To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells.

PubMed

Login, G R; Yang, J; Bryan, K P; Digenis, E C; McBride, J; Elovic, A; Quissell, D O; Dvorak, A M; Wong, D T

1997-03-01

Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.

Blood-cerebrospinal fluid barrier permeability in Alzheimer's disease.

PubMed

Chalbot, Sonia; Zetterberg, Henrik; Blennow, Kaj; Fladby, Tormod; Andreasen, Niels; Grundke-Iqbal, Inge; Iqbal, Khalid

2011-01-01

The role of blood-cerebrospinal fluid barrier (BCB) dysfunction in Alzheimer's disease (AD) has been addressed but not yet established. We evaluated the BCB integrity in 179 samples of cerebrospinal fluid (CSF) retrospectively collected from AD patients and control cases using both CSF/serum albumin ratio (QAlb) and CSF secretory Ca2+-dependent phospholipase A2 (sPLA2) activity. These analyses were supplemented with the measurement of total tau, amyloid-β1-42 (Aβ1-42), and ubiquitin CSF levels. We found that due to its higher sensitivity, CSF sPLA2 activity could 1) discriminate AD from healthy controls and 2) showed BCB impairment in neurological control cases while QAlb could not. Moreover, the CSF sPLA2 activity measurement showed that around half of the AD patients were characterized by a BCB impairment. The BCB dysfunction observed in AD was independent from Mini-Mental State Examination score as well as CSF levels of total tau, Aβ1-42, and ubiquitin. Finally, the BCB dysfunction was not limited to any of the CSF biomarkers-based previously identified subgroups of AD. These results suggest that the BCB damage occurs independent of and probably precedes both Aβ and tau pathologies in a restricted subgroup of AD patients.

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.

[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) and between SCE and HGOSC; 4) ALDH1 between the intact epithelium and SCE, between and SCE and STIC, and between STIC and HGOSC. SCE was shown to be an independent intraepithelial lesion. The incidence of this abnormality increased with age and significantly differed in the patients with fallopian tubal lesions in extraovarian diseases from that in those with malignant ovarian serous tumors (by 5.3 times), while these groups showed a three-fold difference in SCO. Thus, SCP may serve as a more sensitive marker for the early stages of the pathogenesis of ovarian serous carcinoma. The studied types of SCE demonstrated multiple molecular events (loss of PAX2 expression and increased Bcl-2, beta-catenin, and ALDH1 expressions), some of which underwent considerable changes, by increasing the severity of a pathological process (loss of ALDH1, and beta-catenin, and bcl-2 expressions). Thus, therapeutic exposure in the early stages of pathogenesis may have a few points of application and just several molecules can serve as independent markers for early pathological changes in the fallopian tubal epithelium.

Comparative histology of floral elaiophores in the orchids Rudolfiella picta (Schltr.) Hoehne (Maxillariinae sensu lato) and Oncidium ornithorhynchum H.B.K. (Oncidiinae sensu lato)

PubMed Central

Davies, Kevin L.; Stpiczyńska, Malgorzata

2009-01-01

Background and Aims Floral elaiophores, although widespread amongst orchids, have not previously been described for Maxillariinae sensu lato. Here, two claims that epithelial, floral elaiophores occur in the genus Rudolfiella Hoehne (Bifrenaria clade) are investigated. Presumed elaiophores were compared with those of Oncidiinae Benth. and the floral, resin-secreting tissues of Rhetinantha M.A. Blanco and Heterotaxis Lindl., both genera formerly assigned to Maxillaria Ruiz & Pav. (Maxillariinae sensu stricto). Methods Putative, floral elaiophore tissue of Rudolfiella picta (Schltr.) Hoehne and floral elaiophores of Oncidium ornithorhynchum H.B.K. were examined by means of light microscopy, histochemistry, scanning electron microscopy and transmission electron microscopy. Key Results and Conclusions Floral, epithelial elaiophores are present in Rudolfiella picta, indicating, for the first time, that oil secretion occurs amongst members of the Bifrenaria clade (Maxillariinae sensu lato). However, whereas the elaiophore of R. picta is borne upon the labellar callus, the elaiophores of O. ornithorhynchum occur on the lateral lobes of the labellum. In both species, the elaiophore comprises a single layer of palisade secretory cells and parenchymatous, subsecretory tissue. Cell wall cavities are absent from both and there is no evidence of cuticular distension in response to oil accumulation between the outer tangential wall and the overlying cuticle in R. picta. Distension of the cuticle, however, occurs in O. ornithorhynchum. Secretory cells of R. picta contain characteristic, spherical or oval plastids with abundant plastoglobuli and these more closely resemble plastids found in labellar, secretory cells of representatives of Rhetinantha (formerly Maxillaria acuminata Lindl. alliance) than elaiophore plastids of Oncidiinae. In Rhetinantha, such plastids are involved in the synthesis of resin-like material or wax. Despite these differences, the elaiophore anatomy of both R. picta (Bifrenaria clade) and O. ornithorhynchum (Oncidiinae) fundamentally resembles that of several representatives of Oncidiinae. These, in their possession of palisade secretory cells, in turn, resemble the floral elaiophores of certain members of Malpighiaceae, indicating that convergence has occurred here in response to similar pollination pressures. PMID:19447811

Study of the rat adrenal renin-angiotensin system at a cellular level.

PubMed Central

Chiou, C Y; Williams, G H; Kifor, I

1995-01-01

To address the question as to how zona glomerulosa (ZG) cell angiotensin II (Ang II) secretion is regulated, we developed an immuno-cell blot assay to measure its secretion from single cells. We compared these results with those obtained from population studies using a superfusion system. Modulation of Ang II secretion was investigated acutely (by administrating potassium [K+] or captopril) and chronically (by feeding the animals low or high sodium diets). The area of secretory cells, halo areas, and halo intensities varied widely but were highly significantly correlated (P < 0.001) with each other. A disproportionate amount of Ang II was secreted by a small number of large cells. When K+ concentration was increased from 3.6 to 0 mM, superfused ZG cells increased their Ang II secretion 2.32 +/- 0.59-fold. Administration of captopril reduced the K(+)-stimulated Ang II secretion 1.24 +/- 0.07 fold. These findings were reflected in the cell blot assay as a change in the frequency distribution of halo area by K+ and captopril in the same direction as in the population study. In both conditions, the percentage of secretory cells did not change significantly from control. Superfused ZG cells from rats on a low sodium diet secreted 1.85 +/- 0.58-fold more Ang II than cells from sodium-loaded rats (p < 0.05, n = 6). The cell blot assay confirmed these findings with sodium restriction significantly increasing (P < 0.001) both the halo area and its frequency distribution to a larger portion of high secreting cells. However, in contrast to acute treatment with K+ or captopril, the number of secretory cells also doubled. Thus, the individual ZG cell uses two mechanisms to modify Ang II production. In response to acute stimulation and suppression, the amount of Ang II secreted per cell is modified without changing the number of secretary cells. With chronic stimulation, both the amount of Ang II secreted per cell and the number of secretary cells increase. Images PMID:7657812

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

PubMed Central

MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

2015-01-01

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

Human Endometrial DNA Methylome Is Cycle-Dependent and Is Associated With Gene Expression Regulation

PubMed Central

Houshdaran, Sahar; Zelenko, Zara; Irwin, Juan C.

2014-01-01

Human endometrium undergoes major gene expression changes, resulting in altered cellular functions in response to cyclic variations in circulating estradiol and progesterone, largely mediated by transcription factors and nuclear receptors. In addition to classic modulators, epigenetic mechanisms regulate gene expression during development in response to environmental factors and in some diseases and have roles in steroid hormone action. Herein, we tested the hypothesis that DNA methylation plays a role in gene expression regulation in human endometrium in different hormonal milieux. High throughput, genome-wide DNA methylation profiling of endometrial samples in proliferative, early secretory, and midsecretory phases revealed dynamic DNA methylation patterns with segregation of proliferative from secretory phase samples by unsupervised cluster analysis of differentially methylated genes. Changes involved different frequencies of gain and loss of methylation within or outside CpG islands. Comparison of changes in transcriptomes and corresponding DNA methylomes from the same samples revealed association of DNA methylation and gene expression in a number of loci, some important in endometrial biology. Human endometrial stromal fibroblasts treated in vitro with estradiol and progesterone exhibited DNA methylation changes in several genes observed in proliferative and secretory phase tissues, respectively. Taken together, the data support the observation that epigenetic mechanisms are involved in gene expression regulation in human endometrium in different hormonal milieux, adding endometrium to a small number of normal adult tissues exhibiting dynamic DNA methylation. The data also raise the possibility that the interplay between steroid hormone and methylome dynamics regulates normal endometrial functions and, if abnormal, may result in endometrial dysfunction and associated disorders. PMID:24877562

Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

PubMed

Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

2016-08-01

The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. © 2016 International Federation for Cell Biology.

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

The acid test of fluoride: how pH modulates toxicity.

PubMed

Sharma, Ramaswamy; Tsuchiya, Masahiro; Skobe, Ziedonis; Tannous, Bakhos A; Bartlett, John D

2010-05-28

It is not known why the ameloblasts responsible for dental enamel formation are uniquely sensitive to fluoride (F(-)). Herein, we present a novel theory with supporting data to show that the low pH environment of maturating stage ameloblasts enhances their sensitivity to a given dose of F(-). Enamel formation is initiated in a neutral pH environment (secretory stage); however, the pH can fall to below 6.0 as most of the mineral precipitates (maturation stage). Low pH can facilitate entry of F(-) into cells. Here, we asked if F(-) was more toxic at low pH, as measured by increased cell stress and decreased cell function. Treatment of ameloblast-derived LS8 cells with F(-) at low pH reduced the threshold dose of F(-) required to phosphorylate stress-related proteins, PERK, eIF2alpha, JNK and c-jun. To assess protein secretion, LS8 cells were stably transduced with a secreted reporter, Gaussia luciferase, and secretion was quantified as a function of F(-) dose and pH. Luciferase secretion significantly decreased within 2 hr of F(-) treatment at low pH versus neutral pH, indicating increased functional toxicity. Rats given 100 ppm F(-) in their drinking water exhibited increased stress-mediated phosphorylation of eIF2alpha in maturation stage ameloblasts (pH<6.0) as compared to secretory stage ameloblasts (pH approximately 7.2). Intriguingly, F(-)-treated rats demonstrated a striking decrease in transcripts expressed during the maturation stage of enamel development (Klk4 and Amtn). In contrast, the expression of secretory stage genes, AmelX, Ambn, Enam and Mmp20, was unaffected. The low pH environment of maturation stage ameloblasts facilitates the uptake of F(-), causing increased cell stress that compromises ameloblast function, resulting in dental fluorosis.

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.

Stem Cell Therapy in Bladder Dysfunction: Where Are We? And Where Do We Have to Go?

PubMed Central

Lee, Sang-Rae; Song, Yun Seob; Lee, Hong Jun

2013-01-01

To date, stem cell therapy for the bladder has been conducted mainly on an experimental basis in the areas of bladder dysfunction. The therapeutic efficacy of stem cells was originally thought to be derived from their ability to differentiate into various cell types. Studies about stem cell therapy for bladder dysfunction have been limited to an experimental basis and have been less focused than bladder regeneration. Bladder dysfunction was listed in MESH as “urinary bladder neck obstruction”, “urinary bladder, overactive”, and “urinary bladder, neurogenic”. Using those keywords, several articles were searched and studied. The bladder dysfunction model includes bladder outlet obstruction, cryoinjured, diabetes, ischemia, and spinal cord injury. Adipose derived stem cells (ADSCs), bone marrow stem cells (BMSCs), and skeletal muscle derived stem cells (SkMSCs) are used for transplantation to treat bladder dysfunction. The main mechanisms of stem cells to reconstitute or restore bladder dysfunction are migration, differentiation, and paracrine effects. The aim of this study is to review the stem cell therapy for bladder dysfunction and to provide the status of stem cell therapy for bladder dysfunction. PMID:24151627

The bioactive effects of casein proteins on enteroendocrine cell health, proliferation and incretin hormone secretion.

PubMed

Gillespie, Anna L; Green, Brian D

2016-11-15

Previous studies suggest that casein exerts various anti-diabetic effects. However, it is not known which casein proteins are bioactive, nor their effects on enteroendocrine cells. This study evaluated the effects of intact whole casein, intact individual proteins (alpha, beta and kappa casein) and hydrolysates on an enteroendocrine cell line. High content analysis accurately monitored changes in cell health and intracellular glucagon-like peptide-1 (GLP-1) content. Cheese ripening duration and GLP-1 secretory responses were also considered. Beta casein significantly stimulated enteroendocrine cell proliferation and all caseins were potent GLP-1 secretagogues (except kappa casein). Interestingly the GLP-1 secretory activity was almost always lost or significantly reduced upon hydrolysis with proteolytic enzymes. Only pepsin-derived beta casein hydrolysates had significantly increased potency compared with the intact protein, but this was diminished with prolonged hydrolysis. In conclusion casein proteins are not detrimental to enteroendocrine cells, and alpha and beta casein are particularly beneficial stimulating proliferation and GLP-1 secretion. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs

PubMed Central

Terasaki, Mark; Shemesh, Tom; Kasthuri, Narayanan; Klemm, Robin W.; Schalek, Richard; Hayworth, Kenneth J.; Hand, Arthur R.; Yankova, Maya; Huber, Greg; Lichtman, Jeff W.; Rapoport, Tom A.; Kozlov, Michael M.

2013-01-01

The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used novel staining and automated ultra-thin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell. PMID:23870120

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.

Fine structure and development of the collar enamel in gars, Lepisosteus oculatus, Actinopterygii

NASA Astrophysics Data System (ADS)

Sasagawa, Ichiro; Ishiyama, Mikio; Yokosuka, Hiroyuki; Mikami, Masato

2008-06-01

The fine structure of collar enamel and the cells constituting the enamel organ during amelogenesis in Lepisosteus oculatus was observed by light, scanning electron and transmission electron microscopy. In the enamel, slender crystals were arranged perpendicular to the surface and the stripes that were parallel to the surface were observed, suggesting that the enamel in Lepisosteus shares common morphological features with that in sarcopterygian fish and amphibians. Ameloblasts containing developed Golgi apparatus, rough endoplasmic reticulum (rER) and secretory granules were found in the secretory stage. In the maturation stage, a ruffled border was not seen at the distal end of the ameloblasts, while many mitochondria and lysosome-like granules were obvious in the distal cytoplasm. The enamel organ consisted of the outer dental epithelial cells, stratum reticulum cells and ameloblasts, but there was no stratum intermedium. It is likely that the ameloblasts have less absorptive function in comparison with the inner dental epithelial cells facing cap enameloid.

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

Wnt signaling potentiates nevogenesis

PubMed Central

Pawlikowski, Jeff S.; McBryan, Tony; van Tuyn, John; Drotar, Mark E.; Hewitt, Rachael N.; Maier, Andrea B.; King, Ayala; Blyth, Karen; Wu, Hong; Adams, Peter D.

2013-01-01

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway (senescence-associated secretory phenotype). Cellular senescence is also a tumor suppressor mechanism, to which both proliferation arrest and senescence-associated secretory phenotype are thought to contribute. The melanocytes within benign human nevi are a paradigm for tumor-suppressive senescent cells in a premalignant neoplasm. Here a comparison of proliferating and senescent melanocytes and melanoma cell lines by RNA sequencing emphasizes the importance of senescence-associated proliferation arrest in suppression of transformation. Previous studies showed that activation of the Wnt signaling pathway can delay or bypass senescence. Consistent with this, we present evidence that repression of Wnt signaling contributes to melanocyte senescence in vitro. Surprisingly, Wnt signaling is active in many senescent human melanocytes in nevi, and this is linked to histological indicators of higher proliferative and malignant potential. In a mouse, activated Wnt signaling delays senescence-associated proliferation arrest to expand the population of senescent oncogene-expressing melanocytes. These results suggest that Wnt signaling can potentiate nevogenesis in vivo by delaying senescence. Further, we suggest that activated Wnt signaling in human nevi undermines senescence-mediated tumor suppression and enhances the probability of malignancy. PMID:24043806

Wnt signaling potentiates nevogenesis.

PubMed

Pawlikowski, Jeff S; McBryan, Tony; van Tuyn, John; Drotar, Mark E; Hewitt, Rachael N; Maier, Andrea B; King, Ayala; Blyth, Karen; Wu, Hong; Adams, Peter D

2013-10-01

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway (senescence-associated secretory phenotype). Cellular senescence is also a tumor suppressor mechanism, to which both proliferation arrest and senescence-associated secretory phenotype are thought to contribute. The melanocytes within benign human nevi are a paradigm for tumor-suppressive senescent cells in a premalignant neoplasm. Here a comparison of proliferating and senescent melanocytes and melanoma cell lines by RNA sequencing emphasizes the importance of senescence-associated proliferation arrest in suppression of transformation. Previous studies showed that activation of the Wnt signaling pathway can delay or bypass senescence. Consistent with this, we present evidence that repression of Wnt signaling contributes to melanocyte senescence in vitro. Surprisingly, Wnt signaling is active in many senescent human melanocytes in nevi, and this is linked to histological indicators of higher proliferative and malignant potential. In a mouse, activated Wnt signaling delays senescence-associated proliferation arrest to expand the population of senescent oncogene-expressing melanocytes. These results suggest that Wnt signaling can potentiate nevogenesis in vivo by delaying senescence. Further, we suggest that activated Wnt signaling in human nevi undermines senescence-mediated tumor suppression and enhances the probability of malignancy.

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.

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.

Colleters in Rubiaceae from forest and savanna: the link between secretion and environment

NASA Astrophysics Data System (ADS)

Tresmondi, Fernanda; Canaveze, Yve; Guimarães, Elza; Machado, Silvia Rodrigues

2017-04-01

This study aims to investigate colleters' secretory function, on cellular level, in Rubiaceae species from contrasting environments looking to explore the association between secretion and environment. We collected samples from eight species of Rubiaceae growing in forest and savanna having standard-type colleters with diverse histochemistry (hydrophilic, lipophilic and mixed secretions) and processed for both conventional and cytochemical study under transmission electron microscopy (TEM). The standard colleters, although similar in morphology and anatomy, exhibited marked differences on cellular level, especially in the abundance and topology of Golgi bodies, endoplasmic reticulum and plastids when comparing forest and savanna species. These differences were clearly aligned with the chemical nature of the secretions they produce, with predominance of hydrophilic secretions in forest species and lipophilic or mixed secretions in savanna species. The combination of methods in electron microscopy revealed the sites of synthesis and intracellular compartmentation of substances, the mechanisms of their secretion from the protoplast and confirmed the involvement of the outer walls of the epithelial cells in the elimination of exudates to the gland surface. Our study suggests a potential environment-associated plasticity of the secretory cells of standard-type colleters in modulating their secretory function performance.

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

Targeting inflammation in diabetes: Newer therapeutic options

PubMed Central

Agrawal, Neeraj Kumar; Kant, Saket

2014-01-01

Inflammation has been recognised to both decrease beta cell insulin secretion and increase insulin resistance. Circulating cytokines can affect beta cell function directly leading to secretory dysfunction and increased apoptosis. These cytokines can also indirectly affect beta cell function by increasing adipocyte inflammation.The resulting glucotoxicity and lipotoxicity further enhance the inflammatory process resulting in a vicious cycle. Weight reduction and drugs such as metformin have been shown to decrease the levels of C-Reactive Protein by 31% and 13%, respectively. Pioglitazone, insulin and statins have anti-inflammatory effects. Interleukin 1 and tumor necrosis factor-α antagonists are in trials and NSAIDs such as salsalate have shown an improvement in insulin sensitivity. Inhibition of 12-lipo-oxygenase, histone de-acetylases, and activation of sirtuin-1 are upcoming molecular targets to reduce inflammation. These therapies have also been shown to decrease the conversion of pre-diabetes state to diabetes. Drugs like glicazide, troglitazone, N-acetylcysteine and selective COX-2 inhibitors have shown benefit in diabetic neuropathy by decreasing inflammatory markers. Retinopathy drugs are used to target vascular endothelial growth factor, angiopoietin-2, various proteinases and chemokines. Drugs targeting the proteinases and various chemokines are pentoxifylline, inhibitors of nuclear factor-kappa B and mammalian target of rapamycin and are in clinical trials for diabetic nephropathy. Commonly used drugs such as insulin, metformin, peroxisome proliferator-activated receptors, glucagon like peptide-1 agonists and dipeptidyl peptidase-4 inhibitors also decrease inflammation. Anti-inflammatory therapies represent a potential approach for the therapy of diabetes and its complications. PMID:25317247

Tolerance and Exhaustion: Defining Mechanisms of T cell Dysfunction

PubMed Central

Schietinger, Andrea; Greenberg, Philip D.

2013-01-01

CD8 T cell activation and differentiation is tightly controlled, and dependent on the context in which naïve T cells encounter antigen, can either result in functional memory or T cell dysfunction, including exhaustion, tolerance, anergy, or senescence. With the identification of phenotypic and functional traits shared in different settings of T cell dysfunction, distinctions between such dysfunctional `states' have become blurred. Here, we discuss distinct states of CD8 T cell dysfunction, with emphasis on (i) T cell tolerance to self-antigens (self-tolerance), (ii) T cell exhaustion during chronic infections, and (iii) tumor-induced T cell dysfunction. We highlight recent findings on cellular and molecular characteristics defining these states, cell-intrinsic regulatory mechanisms that induce and maintain them, and strategies that can lead to their reversal. PMID:24210163

AN ELECTRON MICROSCOPE STUDY OF MATURE AND DIFFERENTIATING PANETH CELLS IN THE RAT, ESPECIALLY OF THEIR ENDOPLASMIC RETICULUM AND LYSOSOMES

PubMed Central

Behnke, O.; Moe, H.

1964-01-01

In an electron microsope study, the morphology of mature Paneth cells from the small intestine of adult rats is compared with that of differentiating Paneth cells from young rats 2 to 4 weeks old. All mature cells exhibit a marked polarity similar to that of other exocrine gland cells and contain a well developed endoplasmic reticulum, an elaborate Golgi complex, and numerous large secretory granules; they also possess an abundance of lysosomes. The most conspicuous occurrence in the process of differentiation is the development of the endoplasmic reticulum. The most immature Paneth cells possess an endoplasmic reticulum of the vesicular type, which, during maturation, is replaced by the characteristic lamellated ergastoplasm of the mature cell. At a certain stage of differentiation the cavities of the developing cisternae show numerous communications with the perinuclear space, suggesting an outgrowth of the ergastoplasm from the nuclear envelope. Furthermore, the cavities and the perinuclear space at this particular stage contain a material which shows a remarkable intrinsic periodicity. An identical periodicity was exhibited by material contained in Golgi cisternae and secretory granules. Lysosomes are also present in the differentiating cells. PMID:14206428

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.

Hyperforin accumulates in the translucent glands of Hypericum perforatum.

PubMed

Soelberg, Jens; Jørgensen, Lise Bolt; Jäger, Anna K

2007-06-01

Hypericum perforatum contains the therapeutically important compounds hypericin and hyperforin. Hypericin is known to accumulate in the dark glands. This investigation aimed to determine the accumulation site of hyperforin. Dark and translucent glands as well as non-secretory tissue in leaves were manually isolated under the microscope. Hyperforin content was quantified by UV HPLC. Secretory structures were surveyed anatomically. The hyperforin content of intact leaves was found to be about 3 mg g(-1) fresh tissue, whereas a content of about 7 mg g(-1) fresh material was found in isolated translucent glands. Hyperforin was found only to occur in minute amounts in dark glands (approx. 0.4 mg g(-1) fresh tissue). In non-secretory tissue no hyperforin was detected. The accumulation of hyperforin detected in the translucent glands supports the proposed hypothesis that hyperforin is synthesized by the same biosynthetic machinery as monoterpenes in the chloroplasts of cells delimiting the gland.

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

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.

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.

Distribution profile of inositol 1,4,5-trisphosphate receptor isoforms in adrenal chromaffin cells.

PubMed

Huh, Yang Hoon; Yoo, Jie Ae; Bahk, Sook Jin; Yoo, Seung Hyun

2005-05-09

Given the importance of inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels in the control of intracellular Ca(2+) concentrations, we determined the relative concentrations of the IP(3)R isoforms in subcellular organelles, based on serially sectioned electron micrographs. The endoplasmic reticulum (ER) was estimated to contain 15-20% of each of the three IP(3)R isoforms while secretory granules contained 58-69%. The nucleus contained approximately 15% each of IP(3)R-1 and -2, but 25% of IP(3)R-3, whereas the plasma membrane contained approximately 1% or less of each. These suggested that secretory granules, the nucleus and ER are at the center of IP(3)-dependent intracellular Ca(2+) control mechanisms in chromaffin cells.

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.

Cdk1-dependent control of membrane-trafficking dynamics

PubMed Central

McCusker, Derek; Royou, Anne; Velours, Christophe; Kellogg, Douglas

2012-01-01

Cyclin-dependent kinase 1 (Cdk1) is required for initiation and maintenance of polarized cell growth in budding yeast. Cdk1 activates Rho-family GTPases, which polarize the actin cytoskeleton for delivery of membrane to growth sites via the secretory pathway. Here we investigate whether Cdk1 plays additional roles in the initiation and maintenance of polarized cell growth. We find that inhibition of Cdk1 causes a cell surface growth defect that is as severe as that caused by actin depolymerization. However, unlike actin depolymerization, Cdk1 inhibition does not result in a massive accumulation of intracellular secretory vesicles or their cargoes. Analysis of post-Golgi vesicle dynamics after Cdk1 inhibition demonstrates that exocytic vesicles are rapidly mistargeted away from the growing bud, possibly to the endomembrane/vacuolar system. Inhibition of Cdk1 also causes defects in the organization of endocytic and exocytic zones at the site of growth. Cdk1 thus modulates membrane-trafficking dynamics, which is likely to play an important role in coordinating cell surface growth with cell cycle progression. PMID:22767578

Gross morphology and ultrastructure of salivary glands of the mute cicada Karenia caelatata Distant (Hemiptera: Cicadoidea).

PubMed

Zhong, Hai-ying; Wei, Cong; Zhang, Ya-lin

2013-02-01

Salivary glands of the cicada Karenia caelatata Distant were investigated using light microscopy and transmission electron microscopy. The salivary glands are paired structures and consist of principal glands and accessory glands. The principal gland is subdivided into anterior lobe and posterior lobe; the former contains about 34-39 long digitate lobules, while the latter contains approximately 30-33 long digitate lobules and 13-22 short digitate lobules. These short digitate lobules, about one fifth or sixth as long as the long digitate lobules, locate at the base of the long digitate lobules of posterior lobe. All of these digitate lobules vary in size, disposition, length and shape. The anterior lobe and the posterior lobe are connected by an anterior-posterior duct. Two efferent salivary ducts, which connect with the posterior lobe, fuse to form a common duct. The accessory gland is composed of three parts: a greatly tortuous and folded accessory salivary tube, a circlet of gular gland constituting of several acini of the same size, and a non-collapsible accessory salivary duct. The digitate lobules and gular glands possess secretory cells containing abundant secretory granules vary in size, shape, and electron density, as might indicate different materials are synthesized in different secretory regions. The anterior-posterior duct lines with a player of cuticular lining, and cells beneath the cuticular lining lack of basal infoldings, as suggests the duct serves just to transport secretions. The accessory salivary duct is lined with cuticular lining; cells of the duct have well developed basal infoldings associated with abundant mitochondria, as probably suggests the duct is a reabsorptive region of ions. The cells of the accessory salivary tube possess deep basal infoldings and well developed apical dense microvilli, indicating the cells of the tube are secretory in function. Concentric lamellar structures and a peculiar structure with abundant membrane-bound vesicles and secretory granules are observed for the first time, but their derivation and function remain unclear. The morphology and ultrastructure differences observed in the principal glands and accessory gland of the salivary glands of K. caelatata indicate that the sheath saliva was secreted by the principal glands, and the watery saliva was secreted by the accessory salivary glands. Rod-shaped microorganisms are found in the salivary glands (i.e., accessory salivary duct, gular gland, and long digitate lobule of salivary glands) for the first time, and their identity, function, and relationship to microorganisms residing in the salivary glands and/or other parts of alimentary canal of other cicadas need to be investigated further. Copyright © 2012 Elsevier Ltd. All rights reserved.

Extracellular small heat shock proteins: exosomal biogenesis and function.

PubMed

Reddy, V Sudhakar; Madala, Satish K; Trinath, Jamma; Reddy, G Bhanuprakash

2018-05-01

Small heat shock proteins (sHsps) belong to the family of heat shock proteins (Hsps): some are induced in response to multiple stressful events to protect the cells while others are constitutively expressed. Until now, it was believed that Hsps, including sHsps, are present inside the cells and perform intracellular functions. Interestingly, several groups recently reported the extracellular presence of Hsps, and sHsps have also been detected in sera/cerebrospinal fluids in various pathological conditions. Secretion into the extracellular milieu during many pathological conditions suggests additional or novel functions of sHsps in addition to their intracellular properties. Extracellular sHsps are implicated in cell-cell communication, activation of immune cells, and promoting anti-inflammatory and anti-platelet responses. Interestingly, exogenous administration of sHsps showed therapeutic effects in multiple disease models implying that extracellular sHsps are beneficial in pathological conditions. sHsps do not possess signal sequence and, hence, are not exported through the classical Endoplasmic reticulum-Golgi complex (ER-Golgi) secretory pathway. Further, export of sHsps is not inhibited by ER-Golgi secretory pathway inhibitors implying the involvement of a nonclassical secretory pathway in sHsp export. In lieu, lysoendosomal and exosomal pathways have been proposed for the export of sHsps. Heat shock protein 27 (Hsp27), αB-crystallin (αBC), and Hsp20 are shown to be exported by exosomes. Exosomes packaged with sHsps have beneficial effects in in vivo disease models. However, secretion mechanisms and therapeutic use of sHsps have not been elucidated in detail. Therefore, this review aimed at highlighting the current understanding of sHsps (Hsp27, αBC, and Hsp20) in the extracellular medium.

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.

Molecular Mechanism of Pancreatic and Salivary Glands Fluid and HCO3− Secretion

PubMed Central

Lee, Min Goo; Ohana, Ehud; Park, Hyun Woo; Yang, Dongki; Muallem, Shmuel

2013-01-01

Fluid and HCO3− secretion is a vital function of all epithelia and is required for the survival of the tissue. Aberrant fluid and HCO3− secretion is associated with many epithelial diseases, such as cystic fibrosis, pancreatitis, Sjögren’s syndrome and other epithelial inflammatory and autoimmune diseases. Significant progress has been made over the last 20 years in our understanding of epithelial fluid and HCO3− secretion, in particular by secretory glands. Fluid and HCO3− secretion by secretory glands is a two step process. Acinar cells secrete isotonic fluid in which the major salt is NaCl. Subsequently, the duct modifies the volume and electrolyte composition of the fluid to absorb the Cl− and secrete HCO3−. The relative volume secreted by acinar and duct cells and modification of electrolyte composition of the secreted fluids varies among secretory glands to meet their physiological functions. In the pancreas, acinar cells secrete small amount of NaCl-rich fluid, while the duct absorbs the Cl− and secretes HCO3− and the bulk of the fluid in the pancreatic juice. Fluid secretion appears to be driven by active HCO3− secretion. In the salivary glands, acinar cells secrete the bulk of the fluid in the saliva that contains high concentrations of Na+ and Cl− and fluid secretion is mediated by active Cl− secretion. The salivary glands duct absorbs both the Na+ and Cl− and secretes K+ and HCO3−. In this review, we focus on the molecular mechanism of fluid and HCO3− secretion by the pancreas and salivary glands, to highlight the similarities of the fundamental mechanisms of acinar and duct cell functions, and point the differences to meet glands specific secretions. PMID:22298651

[Histochemical characteristics of the secretory cells of gastric glands compared].

PubMed

Shubich, M G; Mogil'naia, G M; Dudetskiĭ, V I; Bogatyr', L Ia

1978-02-01

The work is dedicated to complex histological studies of the secreting cells in the gastric fundal glands, in their comparative aspect. In the representatives of Amphibia, Reptilians and birds, histochemical differentiation of oxyntopeptic cells was demonstrated to be independent on the peculiarities of the animal nutrition. In mammals, histochemical characteristic of the carbohydrate component in the glandular secreting cells depends on the type of nutrition.

Neuroendocrine, Metabolic and Cardiovascular Responses to Exercise Differ Among Healthy Men

DTIC Science & Technology

1995-12-20

Dose Response in AtT-20 Cells ...... 104 2. Acute and Long Term Effects of Lactate on AtT-20 Cells ...106 3. Secretory Effects of Lactate and Secretogogues, in Combination, on AtT-20 Cells . ......... ............. 106 a. CRH and Lactate...whereas, corticosterone serves the same function in rats. The actions of glucocorticoids are primarily permissive allowing 7 cells to achieve a higher

Targeted p120-catenin ablation disrupts dental enamel development.

PubMed

Bartlett, John D; Dobeck, Justine M; Tye, Coralee E; Perez-Moreno, Mirna; Stokes, Nicole; Reynolds, Albert B; Fuchs, Elaine; Skobe, Ziedonis

2010-09-16

Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows.

Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

PubMed

MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

2015-04-24

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The adverse effects of aldrin and dieldrin on both myometrial contractions and the secretory functions of bovine ovaries and uterus in vitro

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

Wrobel, Michał H., E-mail: m.wrobel@pan.olsztyn.pl; Grzeszczyk, Marlena; Mlynarczuk, Jaroslaw

Aldrin and dieldrin are chloroorganic insecticides which are recognised as endocrine disruptors. The aim of the study was to investigate their effect on the secretory functions of the uterus and ovary and on myometrial contractions. Myometrial strips and uterine and ovarian cells from nonpregnant cows were incubated with the xenobiotics (0.1, 1 or 10 ng/ml) for 24 or 72 h. Next, their effect on viability of myometrial, endometrial, granulosa and luteal cells, myometrial strip contractions, the synthesis and secretion of prostaglandins (PGs: PGF2α and PGE2) from uterine cells, the secretion of oestradiol (E2), testosterone (T) and oxytocin (OT) from granulosamore » cells and the secretion of progesterone (P4) and OT from luteal cells were determined. Neither of the xenobiotics (10 ng/ml) affected (P > 0.05) the viability of the ovarian and uterine cells, while both (0.1–10 ng/ml) decreased (P < 0.05) the basal and OT-stimulated myometrial contractions. In spite of these effects, neither of the insecticides affected (P > 0.05) the synthesis and the secretion of PGs from the myometrial cells. Although they also did not impair the secretion of the PGs from the endometrial cells, they abolished (P < 0.05) the stimulatory effect of OT (P < 0.05) on the secretion of the PGs and stimulated (P < 0.05) the secretion of OT from the granulosa and luteal cells. Moreover, aldrin and dieldrin stimulated secretion of E2 and T from the granulosa cells, while only dieldrin increased (P < 0.05) the secretion of P4 from luteal cells. The data show that aldrin and dieldrin stimulated the secretory function of the cultured granulosa and luteal cells and inhibited the myometrial contractions of cows in vitro, which may affect on natural parturition. - Highlights: • Aldrin and dieldrin inhibited bovine myometrial contractions. • The studied xenobiotics stimulated steroids and oxytocin secretion from ovaries. • Prostaglandins are not involved in adverse effect of the xenobiotics on uterine contractions. • On this basis, we believe that aldrin and dieldrin can hinder the course and onset of parturition.« less

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

Sensorimotor cortex ablation induces time-dependent response of ACTH cells in adult rats: behavioral, immunohistomorphometric and hormonal study.

PubMed

Lavrnja, Irena; Trifunovic, Svetlana; Ajdzanovic, Vladimir; Pekovic, Sanja; Bjelobaba, Ivana; Stojiljkovic, Mirjana; Milosevic, Verica

2014-02-10

Traumatic brain injury (TBI) represents a serious event with far reaching complications, including pituitary dysfunction. Pars distalis corticotropes (ACTH cells), that represent the active module of hypothalamo-pituitary-adrenocortical axis, seem to be affected as well. Since pituitary failure after TBI has been associated with neurobehavioral impairments the aim of this study was to evaluate the effects of TBI on recovery of motor functions, morphology and secretory activity of ACTH cells in the pituitary of adult rats. Wistar male rats, initially exposed to sensorimotor cortex ablation (SCA), were sacrificed at the 2nd, 7th, 14th and 30th days post-surgery (dps). A beam walking test was used to evaluate the recovery of motor functions. Pituitary glands and blood were collected for morphological and hormonal analyses. During the first two weeks post-injury increased recovery of locomotor function was detected, reaching almost the control value at day 30. SCA induces significant increase of pituitary weights compared to their time-matched controls. The volume of ACTH-immunopositive cells was reduced at the 7th dps, while at the 14th dps their volume was enlarged, in comparison to corresponding sham controls. Volume density of ACTH cells was increased only at 14th dps, while at day 30 this increase was insignificant. The plasma level of ACTH transiently increased after the injury. The most pronounced changes were observed at the 7th and 14th dps, and were followed by decrease toward control levels at the 30th dps. Thus, temporal changes in the hypothalamic-pituitary-adrenal axis after traumatic brain injury appear to correlate with the recovery process. Copyright © 2013 Elsevier Inc. All rights reserved.

New insights into the pathways initiating and driving pancreatitis

PubMed Central

Gukovskaya, Anna S.; Pandol, Stephen J.; Gukovsky, Ilya

2016-01-01

Purpose of review In this article, we discuss recent studies that advance our understanding of molecular and cellular factors initiating and driving pancreatitis, with the emphasis on the role of acinar cell organelle disorders. Recent findings The central physiologic function of the pancreatic acinar cell – to synthesize, store, and secrete digestive enzymes – critically relies on coordinated actions of the endoplasmic reticulum (ER), the endolysosomal system, mitochondria, and autophagy. Recent studies begin to unravel the roles of these organelles’ disordering in the mechanism of pancreatitis. Mice deficient in key autophagy mediators Atg5 or Atg7, or lysosome-associated membrane protein-2, exhibit dysregulation of multiple signaling and metabolic pathways in pancreatic acinar cells and develop spontaneous pancreatitis. Mitochondrial dysfunction caused by sustained opening of the permeability transition pore is shown to mediate pancreatitis in several clinically relevant experimental models, and its inhibition by pharmacologic or genetic means greatly reduces local and systemic pathologic responses. Experimental pancreatitis is also alleviated with inhibitors of ORAI1, a key component of the plasma membrane channel mediating pathologic rise in acinar cell cytosolic Ca2+. Pancreatitis-promoting mutations are increasingly associated with the ER stress. These findings suggest novel pathways and drug targets for pancreatitis treatment. In addition, the recent studies identify new mediators (e.g., neutrophil extracellular traps) of the inflammatory and other responses of pancreatitis. Summary The recent findings illuminate a critical role of organelles regulating the autophagic, endolysosomal, mitochondrial, and ER pathways in maintaining pancreatic acinar cell homeostasis and secretory function; provide compelling evidence that organelle disordering is a key pathogenic mechanism initiating and driving pancreatitis; and identify molecular and cellular factors that could be targeted to restore organellar functions and thus alleviate or treat pancreatitis. PMID:27428704

The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles.

PubMed

Hook, V Y; Sei, C; Yasothornsrikul, S; Toneff, T; Kang, Y H; Efthimiopoulos, S; Robakis, N K; Van Nostrand, W

1999-01-29

Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

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.

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

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils

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

Carmo, Lívia A.S.; Dias, Felipe F.; Malta, Kássia K.

Background: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Methods: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. Results: STX17more » was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. Conclusions: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. - Highlights: • First demonstration of the Qa-SNARE syntaxin-17 (STX17) in human eosinophils. • High resolution immunogold EM shows STX17 in granules and tubular vesicles. • Unstimulated, TNF-α or CCL11-stimulated eosinophils express STX17. • Our findings suggest a role for STX17 in the transport of granule-derived cargos.« less

Modelling the metabolism of protein secretion through the Tat route in Streptomyces lividans.

PubMed

Valverde, José R; Gullón, Sonia; Mellado, Rafael P

2018-06-14

Streptomyces lividans has demonstrated its value as an efficient host for protein production due to its ability to secrete functional proteins directly to the media. Secretory proteins that use the major Sec route need to be properly folded outside the cell, whereas secretory proteins using the Tat route appear outside the cell correctly folded. This feature makes the Tat system very attractive for the production of natural or engineered Tat secretory proteins. S. lividans cells are known to respond differently to overproduction and secretion of Tat versus Sec proteins. Increased understanding of the impact of protein secretion through the Tat route can be obtained by a deeper analysis of the metabolic impact associated with protein production, and its dependence on protein origin, composition, secretion mechanisms, growth phases and nutrients. Flux Balance Analysis of Genome-Scale Metabolic Network models provides a theoretical framework to investigate cell metabolism under different constraints. We have built new models for various S. lividans strains to better understand the mechanisms associated with overproduction of proteins secreted through the Tat route. We compare models of an S. lividans Tat-dependent agarase overproducing strain with those of the S. lividans wild-type, an S. lividans strain carrying the multi-copy plasmid vector and an α-amylase Sec-dependent overproducing strain. Using updated genomic, transcriptomic and experimental data we could extend existing S. lividans models and produce a new model which produces improved results largely extending the coverage of S. lividans strains, the number of genes and reactions being considered, the predictive behaviour and the dependence on specification of exchange constraints. Comparison of the optimized solutions obtained highlights numerous changes between Tat- and Sec-dependent protein secreting strains affecting the metabolism of carbon, amino acids, nucleotides, lipids and cofactors, and variability analysis predicts a large potential for protein overproduction. This work provides a detailed look to metabolic changes associated to Tat-dependent protein secretion reproducing experimental observations and identifying changes that are specific to each secretory route, presenting a novel, improved, more accurate and strain-independent model of S. lividans, thus opening the way for enhanced metabolic engineering of protein overproduction in S. lividans.

[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.

Autophagy wins the 2016 Nobel Prize in Physiology or Medicine: Breakthroughs in baker's yeast fuel advances in biomedical research

PubMed Central

Levine, Beth; Klionsky, Daniel J.

2017-01-01

Autophagy is an ancient pathway in which parts of eukaryotic cells are self-digested within the lysosome or vacuole. This process has been studied for the past seven decades; however, we are only beginning to gain a molecular understanding of the key steps required for autophagy. Originally characterized as a hormonal and starvation response, we now know that autophagy has a much broader role in biology, including organellar remodeling, protein and organelle quality control, prevention of genotoxic stress, tumor suppression, pathogen elimination, regulation of immunity and inflammation, maternal DNA inheritance, metabolism, and cellular survival. Although autophagy is usually a degradative pathway, it also participates in biosynthetic and secretory processes. Given that autophagy has a fundamental role in many essential cellular functions, it is not surprising that autophagic dysfunction is associated with a wide range of human diseases. Genetic studies in various fungi, particularly Saccharomyces cerevisiae, provided the key initial breakthrough that led to an explosion of research on the basic mechanisms and the physiological connections of autophagy to health and disease. The Nobel Committee has recognized this breakthrough by the awarding of the 2016 Nobel Prize in Physiology or Medicine for research in autophagy. PMID:28039434

Tumors of the endocrine/neuroendocrine system: an overview.

PubMed

Erlandson, R A; Nesland, J M

1994-01-01

For the sake of discussion, the markedly diversified tumors of the endocrine/neuroendocrine system are classified as those originating in classic epithelial endocrine organs (eg, adrenal cortical adenomas), from the diffuse endocrine cells (eg, jejunal carcinoid tumors), or from clusters of these cells (eg, islet cell tumors); and those arising from neurosecretory neurons (eg, neuroblastoma) or paraganglia (eg, carotid body tumor). Although traditional transmission electron microscopy is useful for identifying neurosecretory or endosecretory granules as such, with few exceptions (eg, insulin-containing granules with a complex paracrystalline core) it is not possible to ascribe a granule type (size, shape, or ultrastructure) to a distinct nosologic entity or secretory product because of their overlapping fine structures in different cell types. Immunoelectron microscopy methods utilizing colloidal gold-labeled secondary antibodies can be used to localize virtually any antigen (peptide or neuroamine) to a specific neurosecretory or endosecretory granule or other cell structure. General endocrine/neuroendocrine cell markers such as neuron-specific enolase, the chromogranins, and synaptophysin are useful in identifying neuroendocrine differentiation in a neoplasm using routine immunohistochemical procedures. The current relevance of the APUD concept of Pearse as well as the biologic importance of endocrine/neuroendocrine secretory products such as bombesin and insulinlike growth factors also are discussed.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells.

PubMed

Ehre, Camille; Rossi, Andrea H; Abdullah, Lubna H; De Pestel, Kathleen; Hill, Sandra; Olsen, John C; Davis, C William

2005-01-01

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G(q)-coupled P2Y(2) receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of beta- and gamma-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of beta- or gamma-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca(2+)-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

Human Rhinovirus 16 Causes Golgi Apparatus Fragmentation without Blocking Protein Secretion

PubMed Central

Mousnier, Aurelie; Swieboda, Dawid; Pinto, Anaïs; Guedán, Anabel; Rogers, Andrew V.; Walton, Ross; Johnston, Sebastian L.

2014-01-01

ABSTRACT The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins. PMID:25100828

Organization of the ER–Golgi interface for membrane traffic control

PubMed Central

Brandizzi, Federica; Barlowe, Charles

2014-01-01

Coat protein complex I (COPI) and COPII are required for bidirectional membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. While these core coat machineries and other transport factors are highly conserved across species, high-resolution imaging studies indicate that the organization of the ER–Golgi interface is varied in eukaryotic cells. Regulation of COPII assembly, in some cases to manage distinct cellular cargo, is emerging as one important component in determining this structure. Comparison of the ER–Golgi interface across different systems, particularly mammalian and plant cells, reveals fundamental elements and distinct organization of this interface. A better understanding of how these interfaces are regulated to meet varying cellular secretory demands should provide key insights into the mechanisms that control efficient trafficking of proteins and lipids through the secretory pathway. PMID:23698585

Comparison of the structure of floral nectaries in two Euonymus L. species (Celastraceae).

PubMed

Konarska, Agata

2015-05-01

The inconspicuous Euonymus L. flowers are equipped with open receptacular floral nectaries forming a quadrilateral green disc around the base of the superior ovary. The morphology and anatomy of the nectaries in Euonymus fortunei (Turcz.) Hand.-Mazz. and Euonymus europaeus L. flowers were analysed under a bright-field light microscope as well as stereoscopic and scanning electron microscopes. Photosynthetic nectaries devoid of the vascular tissue were found in both species. Nectar was exuded through typical nectarostomata (E. fortunei) or nectarostomata and secretory cell cuticle (E. europaeus). The nectaries of the examined species differed in their width and height, number of layers and thickness of secretory parenchyma, and the height of epidermal cells. Moreover, there were differences in the location and abundance of nectarostomata and the content of starch and phenolic compounds.

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.

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.

Neuropeptide S reduces duodenal bicarbonate secretion and ethanol-induced increases in duodenal motility in rats

PubMed Central

Wan Saudi, Wan Salman

2017-01-01

Alcohol disrupts the intestinal mucosal barrier by inducing metabolic and functional changes in epithelial cells. Recently, we showed that neuropeptide S (NPS) decreases duodenal motility and increases mucosal paracellular permeability, suggesting a role of NPS in the pathogenesis of disorders and dysfunctions in the small intestine. The aim of the present study was to investigate the effects of NPS on ethanol- and HCl-induced changes of duodenal mucosal barrier function and motility. Rats were anaesthetized with thiobarbiturate, and a 30-mm segment of the proximal duodenum with an intact blood supply was perfused in situ. The effects on duodenal bicarbonate secretion, the blood-to-lumen clearance of 51Cr-EDTA, motility and transepithelial net fluid flux were investigated. Intravenous (i.v.) administration of NPS significantly reduced duodenal mucosal bicarbonate secretion and stimulated mucosal transepithelial fluid absorption, mechanisms dependent on nitrergic signaling. NPS dose-dependently reduced ethanol-induced increases in duodenal motility. NPS (83 pmol·kg-1·min-1, i.v.) reduced the bicarbonate and fluid secretory response to luminal ethanol, whereas a 10-fold higher dose stimulated fluid secretion but did not influence bicarbonate secretion. In NPS-treated animals, duodenal perfusion of acid (pH 3) induced greater bicarbonate secretory rates than in controls. Pre-treating animals with Nω-nitro-L-arginine methyl ester (L-NAME) inhibited the effect of NPS on bicarbonate secretion. In response to luminal acid, NPS-treated animals had significantly higher paracellular permeability compared to controls, an effect that was abolished by L-NAME. Our findings demonstrate that NPS reduces basal and ethanol-induced increases in duodenal motility. In addition, NPS increases luminal alkalinization and mucosal permeability in response to luminal acid via mechanisms that are dependent on nitric oxide signaling. The data support a role for NPS in neurohumoral regulation of duodenal mucosal barrier function and motility. PMID:28384243

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.

X-ray microanalysis of exocrine glands in animal models for cystic fibrosis

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

Mueller, R.M.R.; Roomans, G.M.

1985-01-01

Elemental distribution and ultrastructure of the submandibular gland, the parotid gland and the pancreas were investigated in three suggested animal models of the disease cystic fibrosis: the chronically reserpinized rat, the chronically isoproterenol-treated rat, and the chronically pilocarpine-treated rat. To elucidate the cellular mechanism underlying the effects of these treatments, chronic effects of specific alpha - and beta -adrenergic agonists, as well as acute effects of reserpine and various agonists were also investigated. Reserpine, isoproterenol, and pilocarpine cause an increase in the calcium concentration in submandibular gland acinar cells, due to an increased calcium content of the intracellular mucus. Inmore » the parotid gland, reserpine and isoproterenol cause a decrease of the calcium concentration in acinar cells, due to a lower calcium content of the zymogen granules. In the submandibular gland, a decreased cellular Na concentration was noted after chronic treatment with isoproterenol or pilocarpine, and after a single dose of reserpine or isoproterenol. Ultrastructural changes in the exocrine glands investigated included excessive accumulation of intracellular secretory material and formation of abnormal uncondensed secretion granules. A common pattern in the animal models appears to be (1) inhibition of secretion resulting in intracellular accumulation of secretory material, (2) synthesis of secretory macromolecules with altered cation-binding properties.« less

Modification of hormonal secretion in clinically silent pituitary adenomas.

PubMed

Daems, Tania; Verhelst, Johan; Michotte, Alex; Abrams, Pascale; De Ridder, Dirk; Abs, Roger

2009-01-01

Silent pituitary adenomas are a subtype of adenomas characterized by positive immunoreactivity for one or more hormones classically secreted by normal pituitary cells but without clinical expression, although in some occasions enhanced or changed secretory activity can develop over time. Silent corticotroph adenomas are the classical example of this phenomenon. A series of about 500 pituitary adenomas seen over a period of 20 years were screened for modification in hormonal secretion. Biochemical and immunohistochemical data were reviewed. Two cases were retrieved, one silent somatotroph adenoma and one thyrotroph adenoma, both without specific clinical features or biochemical abnormalities, which presented 20 years after initial surgery with evidence of acromegaly and hyperthyroidism, respectively. While the acromegaly was controlled by a combination of somatostatin analogs and growth hormone (GH) receptor antagonist therapy, neurosurgery was necessary to manage the thyrotroph adenoma. Immunohistochemical examination demonstrated an increase in the number of thyroid stimulating hormone (TSH)-immunoreactive cells compared to the first tissue. Apparently, the mechanisms responsible for the secretory modifications are different, being a change in secretory capacity in the silent somatotroph adenoma and a quantitative change in the silent thyrotroph adenoma. These two cases, one somatotroph and one thyrotroph adenoma, are an illustration that clinically silent pituitary adenomas may in rare circumstances evolve over time and become active, as previously demonstrated in silent corticotroph adenomas.

Circadian Rhythms Regulate Amelogenesis

PubMed Central

Zheng, Li; Seon, Yoon Ji; Mourão, Marcio A.; Schnell, Santiago; Kim, Doohak; Harada, Hidemitsu; Papagerakis, Silvana; Papagerakis, Petros

2013-01-01

Ameloblasts, the cells responsible for making enamel, modify their morphological features in response to specialized functions necessary for synchronized ameloblast differentiation and enamel formation. Secretory and maturation ameloblasts are characterized by the expression of stage-specific genes which follows strictly controlled repetitive patterns. Circadian rhythms are recognized as key regulators of development and diseases of many tissues including bone. Our aim was to gain novel insights on the role of clock genes in enamel formation and to explore the potential links between circadian rhythms and amelogenesis. Our data shows definitive evidence that the main clock genes (Bmal1, Clock, Per1 and Per2) oscillate in ameloblasts at regular circadian (24h) intervals both at RNA and protein levels. This study also reveals that two markers of ameloblast differentiation i.e. amelogenin (Amelx; a marker of secretory ameloblasts) and kallikrein-related peptidase 4 (Klk4, a marker of maturation ameloblasts) are downstream targets of clock genes. Both, Amelx and Klk4 show 24h oscillatory expression patterns and their expression levels are up-regulated after Bmal1 over-expression in HAT-7 ameloblast cells. Taken together, these data suggest that both the secretory and the maturation stage of amelogenesis might be under circadian control. Changes in clock genes expression patterns might result in significant alterations of enamel apposition and mineralization. PMID:23486183

Auxin Deprivation Induces Synchronous Golgi Differentiation in Suspension-Cultured Tobacco BY-2 Cells1

PubMed Central

Winicur, Zev M.; Feng Zhang, Guo; Andrew Staehelin, L.

1998-01-01

To date, the lack of a method for inducing plant cells and their Golgi stacks to differentiate in a synchronous manner has made it difficult to characterize the nature and extent of Golgi retailoring in biochemical terms. Here we report that auxin deprivation can be used to induce a uniform population of suspension-cultured tobacco (Nicotiana tabacum cv BY-2) cells to differentiate synchronously during a 4-d period. Upon removal of auxin, the cells stop dividing, undergo elongation, and differentiate in a manner that mimics the formation of slime-secreting epidermal and peripheral root-cap cells. The morphological changes to the Golgi apparatus include a proportional increase in the number of trans-Golgi cisternae, a switch to larger-sized secretory vesicles that bud from the trans-Golgi cisternae, and an increase in osmium staining of the secretory products. Biochemical alterations include an increase in large, fucosylated, mucin-type glycoproteins, changes in the types of secreted arabinogalactan proteins, and an increase in the amounts and types of molecules containing the peripheral root-cap-cell-specific epitope JIM 13. Taken together, these findings support the hypothesis that auxin deprivation can be used to induce tobacco BY-2 cells to differentiate synchronously into mucilage-secreting cells. PMID:9625703

Matching native electrical stimulation by graded chemical stimulation in isolated mouse adrenal chromaffin cells.

PubMed

Fulop, Tiberiu; Smith, Corey

2007-11-30

Adrenal chromaffin cells release multiple transmitters in response to sympathetic stimulation. Modest cell firing, matching sympathetic tone, releases small freely soluble catecholamines. Elevated electrical firing rates matching input under sympathetic stress results in release of catecholamines as well as semi-soluble vaso- and neuro-active peptides packaged within the dense core of the secretory granule. This activity-dependent differential transmitter release has been shown to rely on a mechanistic shift in the mode of exocytosis through the regulated dilation of the secretory fusion pore between granule and cell surface membranes. However, biochemical description of the mechanism regulating fusion pore dilation remains elusive. In the experimental setting, electrical stimulation designed to mimic sympathetic input, is achieved through single-cell voltage-clamp. While precise, this approach is incompatible with biochemical and proteomic analysis, both of which require large sample sizes. We address this limitation in the current study. We describe a bulk chemical stimulation paradigm calibrated to match defined electrical activity. We utilize calcium and single-cell amperometric measurements to match extracellular potassium concentrations to physiological electrical stimulation under sympathetic tone as well as acute stress conditions. This approach provides larger samples of uniformly stimulated cells for determining molecular players in activity-dependent differential transmitter release from adrenal chromaffin cells.

Effect of zinc on nectar secretion of Hibiscus rosa -sinensis L.

PubMed

Sawidis, Thomas; Papadopoulou, Alexandra; Voulgaropoulou, Maria

2014-05-01

Zinc toxicity in secretory cells caused a range of effects, mainly depending on metal concentration. Low concentrations activated nectary function increasing nectar secretion but secretion was greatly inhibited or stopped entirely by ongoing concentration. Water loss rate of zinc treated flower parts was significantly reduced whereas green sepals were dehydrated more rapidly in comparison to colored petals. The content of zinc, calcium, magnesium and manganese increased mainly in sepals under excess of zinc, but in the secreted nectar this metal was not evident. Morphological changes were observed in mucilage cells concerning the mucilage structure and appearance. The parenchymatic, subglandular cells displayed an early vacuolarization and cytoplasm condensation. Secretory hairs appeared to be thinner, the apical cell folded inwards and plasmolytic shrinkage became severe in all cells. The waxy cuticula showed an increased electron density. A plasmalemma detachment from the external cell walls was observed creating a gap between cell wall and plasmalemma. ER cisterns of all treated nectary hairs dominated the cytoplasm and electron dense deposits were seen within its profiles. A great number of other organelles were also present, showing electron dense deposits in their membranes as well. The vacuome was drastically reduced in all cells, except in the subglandular ones and electron dense membrane remnants were observed.

Immunohistochemical localization of Clara cell secretory proteins (CC10-CC26) and Annexin-1 protein in rat major salivary glands

PubMed Central

Cecchini, Maria Paola; Merigo, Flavia; Cristofoletti, Mirko; Osculati, Francesco; Sbarbati, Andrea

2009-01-01

The oral cavity is continuously bathed by saliva secreted by the major and minor salivary glands. Saliva is the first biological medium to confront external materials that are taken into the body as part of food or drink or inhaled volatile substances, and it contributes to the first line of oral defence. In humans, it has been shown that sputum and a variety of biological fluids contain Clara cell secretory proteins (CC10–CC26). Various studies of the respiratory apparatus have suggested their protective effect against inflammatory response and oxidative stress. Recently, CC10 deficiency has been related to the protein Annexin-1 (ANXA1), which has immunomodulatory and anti-inflammatory properties. Considering the defensive role of both Clara cell secretory proteins and ANXA1 in the respiratory apparatus, and the importance of salivary gland secretion in the first line of oral defence, we decided to evaluate the expression of CC10, CC26 and ANXA1 proteins in rat major salivary glands using immunohistochemistry. CC10 expression was found only in the ductal component of the sublingual gland. Parotid and submandibular glands consistently lacked CC10 immunoreactivity. In the parotid gland, both acinar and ductal cells were always CC26-negative, whereas in the submandibular gland, immunostaining was localized in the ductal component and in the periodic acid Schiff (PAS)-positive area. In the sublingual gland, ductal cells were always positive. Acinar cells were not immunostained at all. ANXA1 was expressed in ductal cells in all three major glands. In parotid and sublingual glands, acinar cells were negative. In submandibular glands, immunostaining was present in the mucous PAS-positive portion, whereas serous acinar cells were consistently negative. The existence of some CC10-CC26–ANXA1-positive cells in rat salivary glandular tissue is an interesting preliminary finding which could support the hypothesis, suggested for airway tissue, that these proteins have a defensive and protective role. Protein expression heterogeneity in the different portions of the glands could be an important clue in further investigations of their role. PMID:19438769

Rab3a Is Critical for Trapping Alpha-MSH Granules in the High Ca2+-Affinity Pool by Preventing Constitutive Exocytosis

PubMed Central

Sedej, Simon; Klemen, Maša Skelin; Schlüter, Oliver M.; Rupnik, Marjan Slak

2013-01-01

Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca2+-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and β-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of α-MSH. PMID:24205339

Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs.

PubMed

Terasaki, Mark; Shemesh, Tom; Kasthuri, Narayanan; Klemm, Robin W; Schalek, Richard; Hayworth, Kenneth J; Hand, Arthur R; Yankova, Maya; Huber, Greg; Lichtman, Jeff W; Rapoport, Tom A; Kozlov, Michael M

2013-07-18

The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell. Copyright © 2013 Elsevier Inc. All rights reserved.

Specific and Nonspecific B-Cell Function in the Small Intestines of Patients with Whipple's Disease ▿

PubMed Central

Geelhaar, Anika; Moos, Verena; Schinnerling, Katina; Allers, Kristina; Loddenkemper, Christoph; Fenollar, Florence; LaScola, Bernard; Raoult, Didier; Schneider, Thomas

2010-01-01

Whipple's disease is a chronic multisystemic infection caused by Tropheryma whipplei that is characterized by arthritis, weight loss, and diarrhea. The immunological defects in the duodenal mucosa, the site of major replication of the agent underlying the pathogenesis of Whipple's disease, are poorly understood. Mucosal immunoglobulins are essential for the defense against intestinal pathogens; therefore, we analyzed the B-cell response in duodenal specimens and sera of Whipple's disease patients. Whereas systemic immunoglobulin production was affected only marginally, duodenal biopsy specimens of Whipple's disease patients contained reduced numbers of immunoglobulin-positive plasma cells and secreted less immunoglobulin compared to healthy controls but showed a weak secretory IgA response toward T. whipplei. This T. whipplei-specific intestinal immune response was not observed in controls. Thus, we were able to demonstrate that general mucosal immunoglobulin production in Whipple's disease patients is impaired. However, this deficiency does not completely abolish T. whipplei-specific secretory IgA production that nonetheless does not protect from chronic infection. PMID:20696822

Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation

USDA-ARS?s Scientific Manuscript database

Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our objective was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretor...

Does autophagy in the midgut epithelium of centipedes depend on the day/night cycle?

PubMed

Rost-Roszkowska, M M; Chajec, Ł; Vilimova, J; Tajovský, K; Kszuk-Jendrysik, M

2015-01-01

The midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata, is composed of digestive, secretory and regenerative cells. In L. forficatus, the autophagy occurred only in the cytoplasm of the digestive cells as a sporadic process, while in S. cingulata, it occurred intensively in the digestive, secretory and regenerative cells of the midgut epithelium. In both of the species that were analyzed, this process proceeded in a continuous manner and did not depend on the day/night cycle. Ultrastructural analysis showed that the autophagosomes and autolysosomes were located mainly in the apical and perinuclear cytoplasm of the digestive cells in L. forficatus. However, in S. cingulata, the entire cytoplasm was filled with autophagosomes and autolysosomes. Initially the membranes of phagophores surround organelles during autophagosome formation. Autolysosomes result from the fusion of autophagosomes and lysosomes. Residual bodies which are the last stage of autophagy were released into the midgut lumen due to necrosis. Autophagy in the midgut epithelia that were analyzed was confirmed using acid phosphatase and mono-dansyl-cadaverine stainings. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

The Acid Test of Fluoride: How pH Modulates Toxicity

PubMed Central

Sharma, Ramaswamy; Tsuchiya, Masahiro; Skobe, Ziedonis; Tannous, Bakhos A.; Bartlett, John D.

2010-01-01

Background It is not known why the ameloblasts responsible for dental enamel formation are uniquely sensitive to fluoride (F−). Herein, we present a novel theory with supporting data to show that the low pH environment of maturating stage ameloblasts enhances their sensitivity to a given dose of F−. Enamel formation is initiated in a neutral pH environment (secretory stage); however, the pH can fall to below 6.0 as most of the mineral precipitates (maturation stage). Low pH can facilitate entry of F− into cells. Here, we asked if F− was more toxic at low pH, as measured by increased cell stress and decreased cell function. Methodology/Principal Findings Treatment of ameloblast-derived LS8 cells with F− at low pH reduced the threshold dose of F− required to phosphorylate stress-related proteins, PERK, eIF2α, JNK and c-jun. To assess protein secretion, LS8 cells were stably transduced with a secreted reporter, Gaussia luciferase, and secretion was quantified as a function of F− dose and pH. Luciferase secretion significantly decreased within 2 hr of F− treatment at low pH versus neutral pH, indicating increased functional toxicity. Rats given 100 ppm F− in their drinking water exhibited increased stress-mediated phosphorylation of eIF2α in maturation stage ameloblasts (pH<6.0) as compared to secretory stage ameloblasts (pH∼7.2). Intriguingly, F−-treated rats demonstrated a striking decrease in transcripts expressed during the maturation stage of enamel development (Klk4 and Amtn). In contrast, the expression of secretory stage genes, AmelX, Ambn, Enam and Mmp20, was unaffected. Conclusions The low pH environment of maturation stage ameloblasts facilitates the uptake of F−, causing increased cell stress that compromises ameloblast function, resulting in dental fluorosis. PMID:20531944

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-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.

Pseudoislets as primary islet replacements for research: report on a symposium at King's College London, London UK.

PubMed

Persaud, Shanta J; Arden, Catherine; Bergsten, Peter; Bone, Adrian J; Brown, James; Dunmore, Simon; Harrison, Moira; Hauge-Evans, Astrid; Kelly, Catriona; King, Aileen; Maffucci, Tania; Marriott, Claire E; McClenaghan, Neville; Morgan, Noel G; Reers, Christina; Russell, Mark A; Turner, Mark D; Willoughby, Emma; Younis, Mustafa Y G; Zhi, Z L; Jones, Peter M

2010-01-01

Laboratory-based research aimed at understanding processes regulating insulin secretion and mechanisms underlying β-cell dysfunction and loss in diabetes often makes use of rodents, as these processes are in many respects similar between rats/mice and humans. Indeed, a rough calculation suggests that islets have been isolated from as many as 150,000 rodents to generate the data contained within papers published in 2009 and the first four months of 2010. Rodent use for islet isolation has been mitigated, to a certain extent, by the availability of a variety of insulin-secreting cell lines that are used by researchers world-wide. However, when maintained as monolayers the cell lines do not replicate the robust, sustained secretory responses of primary islets which limits their usefulness as islet surrogates. On the other hand, there have been several reports that configuration of MIN6 β-cells, derived from a mouse insulinoma, as three-dimensional cell clusters termed ‘pseudoislets’ largely recapitulates the function of primary islet β-cells. The Diabetes Research Group at King’s College London has been using the MIN6 pseudoislet model for over a decade and they hosted a symposium on “Pseudoislets as primary islet replacements for research”, which was funded by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London on 15th and 16th April 2010. This small, focused meeting was conceived as an opportunity to consolidate information on experiences of working with pseudoislets between different UK labs, and to introduce the theory and practice of pseudoislet culture to laboratories working with islets and/or β-cell lines but who do not currently use pseudoislets. This short review summarizes the background to the development of the cell line-derived pseudoislet model, the key messages arising from the symposium and emerging themes for future pseudoislet research.

Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells

PubMed Central

Robertson, Sarah Y. T.; Wen, Xin; Yin, Kaifeng; Chen, Junjun; Smith, Charles E.; Paine, Michael L.

2017-01-01

Calcium export is a key function for the enamel organ during all stages of amelogenesis. Expression of a number of ATPase calcium transporting, plasma membrane genes (ATP2B1-4/PMCA1-4), solute carrier SLC8A genes (sodium/calcium exchanger or NCX1-3), and SLC24A gene family members (sodium/potassium/calcium exchanger or NCKX1-6) have been investigated in the developing enamel organ in earlier studies. This paper reviews the calcium export pathways that have been described and adds novel insights to the spatiotemporal expression patterns of PMCA1, PMCA4, and NCKX3 during amelogenesis. New data are presented to show the mRNA expression profiles for the four Atp2b1-4 gene family members (PMCA1-4) in secretory-stage and maturation-stage rat enamel organs. These data are compared to expression profiles for all Slc8a and Slc24a gene family members. PMCA1, PMCA4, and NCKX3 immunolocalization data is also presented. Gene expression profiles quantitated by real time PCR show that: (1) PMCA1, 3, and 4, and NCKX3 are most highly expressed during secretory-stage amelogenesis; (2) NCX1 and 3, and NCKX6 are expressed during secretory and maturation stages; (3) NCKX4 is most highly expressed during maturation-stage amelogenesis; and (4) expression levels of PMCA2, NCX2, NCKX1, NCKX2, and NCKX5 are negligible throughout amelogenesis. In the enamel organ PMCA1 localizes to the basolateral membrane of both secretory and maturation ameloblasts; PMCA4 expression is seen in the basolateral membrane of secretory and maturation ameloblasts, and also cells of the stratum intermedium and papillary layer; while NCKX3 expression is limited to Tomes' processes, and the apical membrane of maturation-stage ameloblasts. These new findings are discussed in the perspective of data already present in the literature, and highlight the multiplicity of calcium export systems in the enamel organ needed to regulate biomineralization. PMID:28588505

Ascorbic acid increases SVCT2 localization at the plasma membrane by accelerating its trafficking from early secretory compartments and through the endocytic-recycling pathway.

PubMed

Covarrubias-Pinto, A; Acuña, A I; Boncompain, G; Papic, E; Burgos, P V; Perez, F; Castro, M A

2018-05-20

Ascorbic acid (Asc) is an antioxidant molecule essential for physiological functions. The concentration of extracellular Asc increases during synaptic transmission and renal reabsorption. These phenomena induce an increase of the Sodium-dependent-Vitamin-C-transporter 2 (SVCT2) at plasma membrane (PM) localization, as we previously demonstrated in neuronal and non-neuronal cells. Hence, the aim of this study was to evaluate intracellular SVCT2 trafficking kinetics in response to Asc. We observed two peaks of SVCT2 localization and function at the PM (at 5-10 min, "acute response", and 30-60 min, "post-acute response") when cells were incubated with Asc. We defined that the post-acute response was dependent on SVCT2 located in early secretory compartments, and its trafficking was abolished with Tunicamycin and Brefeldin A treatment. Moreover, using the RUSH system to retain and synchronize cargo secretion through the secretory pathway we demonstrated that the post-acute response increases SVCT2 trafficking kinetics from the ER to the PM suggesting the retention of SVCT2 at the early secretory pathway when Asc is absent. However, these observations do not explain the increased SVCT2 levels at the PM during the "acute" response, suggesting the involvement of a faster mechanism in close proximity with the PM. To investigate the possible role of endosomal compartments, we tested the effect of endocytosis inhibition. Expression of dominant-negative (DN) versions of the GTPase-dynamin II and clathrin-accessory protein AP180 showed a significant increase in SVCT2 levels at the PM. Moreover, expression of Rab11-DN, a GTPase implicated in cargo protein recycling from endosomes to the PM showed a similar outcome, strongly indicating that Asc impacts SVCT2 trafficking during the acute response. Therefore, our results revealed two mechanisms by which Asc modulates SVCT2 levels at the PM, one at the early secretory pathway and another at the endocytic compartments. We propose that these two mechanisms have key protective implications in the homeostasis of metabolically active and specialized tissues. Copyright © 2018 Elsevier Inc. All rights reserved.

A novel two-chain IGF-II-derived peptide from purified β-cell granules.

PubMed

Buchanan, Christina M; Phillips, Anthony R J; Cooper, Garth J S

2010-10-01

Insulin-like growth factor II (IGF-II) is a potent mitogen that regulates prenatal growth and development in both humans and rodents. Its role in post-natal life is less clear although immunohistochemical studies have observed IGF-II-like immunoreactivity (IGF-II-LI) associated with insulin-producing pancreatic β-cells. Here we isolated secretory granules from a β-cell line, βTC6-F7, and characterized the nature of the IGF-II-LI located therein. Secretory granules were isolated from cultured mouse βTC6-F7 cells by ultracentrifugation. Granule protein content was separated by reversed-phase HPLC, and assayed for IGF-II (radioimmunoassay) prior to identification by gas-phase NH(2)-terminal sequencing and MALDI-TOF MS. Effects of glucose incorporation into muscle glycogen were determined by incubating with isolated rat soleus muscle strips. βTC6-F7 cells contained 60 ± 8 pmol of IGF-II-LI per 10⁶ cells compared to 340 ± 44 pmol insulin-LI per 10⁶ cells. IGF-II immunoreactive fractions were found to contain an IGF-II-like molecule with a molecular mass of 6847.6 Da. The protein was found to be a two-chain insulin-like product of Igf2 that corresponds to mouse des(37-40)IGF-II, which we termed 'vesiculin'. This molecule was also detectable in βTC6-F7 cells by intact-cell mass spectrometry. Mouse vesiculin evoked concentration-dependent stimulation of muscle glycogen synthesis ex vivo with an EC(50) value of 131 nM ± 1.35. Vesiculin, des(37-40)IGF-II, is a novel two-chain insulin-like hormone and the major "IGF-II-like" peptide found in purified mouse βTC6-F7 secretory granules. It stimulated ex vivo muscle glycogen synthesis with an efficacy greater than or equal to the intrinsic potency of IGF-II when compared to insulin derived from the same species. Copyright © 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

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.

Global secretome characterization of A549 human alveolar epithelial carcinoma cells during Mycoplasma pneumoniae infection

PubMed Central

2014-01-01

Background Mycoplasma pneumoniae (M. pneumoniae) is one of the major etiological agents for community-acquired pneumonia (CAP) in all age groups. The early host response to M. pneumoniae infection relies on the concerted release of proteins with various biological activities. However, no comprehensive analysis of the secretory proteins has been conducted to date regarding the host response upon M. pneumoniae infection. Results We employed the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based label-free quantitative proteomic technology to identify and characterize the members of the human alveolar epithelial carcinoma A549 cell secretome during M. pneumoniae infection. A total of 256 proteins were identified, with 113 being differentially expressed (>1.5-fold change), among which 9 were only expressed in control cells, 10 only in M. pneumoniae-treated cells, while 55 were up-regulated and 39 down-regulated by M. pneumoniae. The changed expression of some of the identified proteins was validated by RT-PCR and immunoblot analysis. Cellular localization analysis of the secretome data revealed 59.38% of the proteins were considered as “putative secretory proteins”. Functional analysis revealed that the proteins affected upon M. pneumoniae infection were mainly related to metabolic process, stress response, and immune response. We further examined the level of one up-regulated protein, IL-33, in clinical samples. The result showed that IL-33 levels were significantly higher in the plasma and bronchoalveolar lavage fluid (BALF) of M. pneumoniae pneumonia (MPP) patients. Conclusions The present study provided systematic information about the changes in the expression of secretory proteins during M. pneumoniae infection, which is useful for the discovery of specific biomarkers and targets for pharmacological intervention. PMID:24507763

Photosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity.

PubMed

Laterre, Raphaëlle; Pottier, Mathieu; Remacle, Claire; Boutry, Marc

2017-04-01

Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO 2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundle-sheath (C4 plants), and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However, photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that tobacco ( Nicotiana tabacum ) trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M, which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas reinhardtii and purification of the full Rubisco complex showed that this isoform conferred higher V max and K m values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO 2 is released by the active specialized metabolism. © 2017 American Society of Plant Biologists. All Rights Reserved.

Targeted p120-Catenin Ablation Disrupts Dental Enamel Development

PubMed Central

Bartlett, John D.; Dobeck, Justine M.; Tye, Coralee E.; Perez-Moreno, Mirna; Stokes, Nicole; Reynolds, Albert B.; Fuchs, Elaine; Skobe, Ziedonis

2010-01-01

Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows. PMID:20862276

Hydrogen sulphide facilitates exocytosis by regulating the handling of intracellular calcium by chromaffin cells.

PubMed

de Pascual, Ricardo; Baraibar, Andrés M; Méndez-López, Iago; Pérez-Ciria, Martín; Polo-Vaquero, Ignacio; Gandía, Luis; Ohia, Sunny E; García, Antonio G; de Diego, Antonio M G

2018-05-02

Gasotransmitter hydrogen sulphide (H 2 S) has emerged as a regulator of multiple physiological and pathophysiological processes throughout. Here, we have investigated the effects of NaHS (fast donor of H 2 S) and GYY4137 (GYY, slow donor of H 2 S) on the exocytotic release of catecholamines from fast-perifused bovine adrenal chromaffin cells (BCCs) challenged with sequential intermittent pulses of a K + -depolarizing solution. Both donors caused a concentration-dependent facilitation of secretion. This was not due to an augmentation of Ca 2+ entry through voltage-activated Ca 2+ channels (VACCs) because, in fact, NaHS and GYY caused a mild inhibition of whole-cell Ca 2+ currents. Rather, the facilitation of exocytosis seemed to be associated to an augmented basal [Ca 2+ ] c and the K + -elicited [Ca 2+ ] c transients; such effects of H 2 S donors are aborted by cyclopiazonic acid (CPA), that causes endoplasmic reticulum (ER) Ca 2+ depletion through sarcoendoplasmic reticulum Ca2+ ATPase inhibition and by protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that impedes the ability of mitochondria to sequester cytosolic Ca 2+ during cell depolarization. Inasmuch as CPA and FCCP reversed the facilitation of secretion triggered by K + in the presence of NaHS and GYY, is seems that such facilitation is tightly coupled to Ca 2+ handling by the ER and mitochondria. On the basis of these results, we propose that H 2 S regulates catecholamine secretory responses triggered by K + in BCCs by (i) mobilisation of ER Ca 2+ and (ii) interference with mitochondrial Ca 2+ circulation. In so doing, the clearance of the [Ca 2+ ] c transient will be delayed and the Ca 2+ -dependent trafficking of secretory vesicles will be enhanced to overfill the secretory machinery with new vesicles to enhance exocytosis.

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

Molecular Determinants and Dynamics of Hepatitis C Virus Secretion

PubMed Central

Coller, Kelly E.; Heaton, Nicholas S.; Berger, Kristi L.; Cooper, Jacob D.; Saunders, Jessica L.; Randall, Glenn

2012-01-01

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells. PMID:22241992

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.

Localization of amylin-like immunoreactivity in melanocyte-stimulating hormone-containing cells of the pars intermedia but not those of the pars distalis in the axolotl (Ambystoma mexicanum) pituitary.

PubMed

Suzuki, Hirohumi; Yamamoto, Toshiharu

2016-04-01

Immunohistochemical techniques were employed to investigate the distribution of amylin-like immunoreactivity in the axolotl (Ambystoma mexicanum) pituitary. Amylin-immunoreactive cells were observed in the pars intermedia, and these cells were found to be immunoreactive for α-melanocyte-stimulating hormone (αMSH) as well. In contrast, αMSH-immunoreactive cells in the pars distalis were immuno-negaitive for amylin. These light microscopic findings were confirmed by immunoelectron microscopy. Amylin-immunoreactive signals were located on the haloes of presumable secretory granules in association with αMSH-immunoreactive signals in the amylin-positive cells. However, in the pars distalis, the αMSH-positive cells did not contain amylin-immunoreactive secretory granules. Western blot analysis of axolotl pituitary extracts revealed the labeling of a protein band at approximately 10.5-kDa by the anti-rat amylin serum, which was not labeled by the anti-αMSH antibody. These findings indicate that amylin secreted from MSH-producing cells in the pars intermedia may modulate MSH secretion in an autocrine fashion and may participate in MSH functions such as fatty homeostasis together with MSH. Copyright © 2016 Elsevier GmbH. All rights reserved.

Recombinant HE4 protein promotes proliferation of pancreatic and endometrial cancer cell lines.

PubMed

Lu, Qinsheng; Chen, Haibin; Senkowski, Christopher; Wang, Jianhao; Wang, Xue; Brower, Steven; Glasgow, Wayne; Byck, David; Jiang, Shi-Wen; Li, Jinping

2016-01-01

Pancreatic adenocarcinoma is one of the most deadly malignancies, and endometrial cancer represents the most common gynecologic cancer in the USA. Better understanding on the pathologic mechanisms and pathways is required for effective treatment of these malignancies. Recently, human epididymis protein 4 (HE4 or WFDC2), a secretory glycoprotein, was found to be overexpressed in pancreatic and endometrial cancers. In addition, studies have shown that HE4 overexpression in endometrial cancer cell lines led to faster cancer progression in a mouse subcutaneous model. These findings raise a question on the role(s) of secretory, extracellular HE4 in cancer development. In the present study, we found that treatment of pancreatic and endometrial cancer cell lines with purified, extracellular HE4 protein led to a significant increase in cell viability and proliferation. Moreover, extracellular HE4 protein was able to increase DNA synthesis, and modulate the mRNA and protein levels of cell cycle marker PCNA and cell cycle inhibitor p21. These effects appeared to be robust and sustainable and required a relatively low concentration of HE4 protein. The findings indicated the secreted, extracellular HE4 may carry some physiopathological functions. Via paracrine/endocrine actions, circulatory HE4 produced by malignant cells may contribute to pancreatic and endometrial cancer progression and/or metastasis.

Real-time detecting gelatinases activity in living cells by FRET imaging

NASA Astrophysics Data System (ADS)

Yang, Jie; Zhang, Zhihong; Liu, Bifeng; Luo, Qingming

2006-01-01

Degradation of the extracellular matrix by Matrix metalloproteinases (MMPs) not only enhances tumor invasion, but also affects tumor cell behaviour and leads to cancer progression. To monitor gelatinases (contain MMP2 and MMP9) activity in living cells, we constructed a vector that encoded a gelatinases recognition site (GRS) between citrine (mutation of EYFP Q69M) in N terminal and ECFP in C terminal. Because Gelatinases are secretory proteins and act outside of cell, an expressing vector displayed the fusion protein on cellular surface was used for this FRET gene probe. On expression of YFP-GRS-ECFP in MCF-7 cells that expressed no gelatinases, we were able to observe the efficient transfer of energy from excited ECFP to YFP within the YFP-GRS-ECFP molecule. However, the fusion protein YFP-GRS-ECFP was expressed in MDA-MB 453s cell line with high secretory gelatinases, so YFP-GRS-ECFP was cleaved by gelatinases, no such transfer of energy was detected and fluorescence signal disappeared in YFP channel since YFP protein was cut down. Moreover, Doxycycline, a MMP inhibitor, could make FRET signal increase and fluorescence signal appeared in YFP channel. Thus, the FRET probe YFP-GRS-ECFP can sensitively and reliably monitor gelatinases activation in living cells and can be used for screening MMP inhibitors.

O-linked N-acetylglucosamine transferase enhances secretory clusterin expression via liver X receptors and sterol response element binding protein regulation in cervical cancer.

PubMed

Kim, Min Jun; Choi, Mee Young; Lee, Dong Hoon; Roh, Gu Seob; Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Kim, Yoon Sook; Choi, Wan Sung

2018-01-12

O-linked N-acetylglucosamine transferase (OGT) expression is increased in various cancer types, indicating the potential importance of O-GlcNAcylation in tumorigenesis. Secretory clusterin (sCLU) is involved in cancer cell proliferation and drug resistance, and recently, liver X receptors (LXRs) and sterol response element binding protein-1 (SREBP-1) were reported to regulate sCLU transcription. Here, we found that sCLU is significantly increased in cervical cancer cell lines, which have higher expression levels of O-GlcNAc and OGT than keratinocytes. OGT knockdown decreased expression of LXRs, SREBP-1 and sCLU through hypo-O-GlcNAcylation of LXRs. Additionally, treatment with Thiamet G, O-GlcNAcase OGA inhibitor, increased expression of O-GlcNAcylation and sCLU, and high glucose increased levels of LXRs, SREBP-1 and sCLU in HeLa cells. Moreover, OGT knockdown induced G 0 /G 1 phase cell cycle arrest and late apoptosis in cisplatin-treated HeLa cells, and decreased viability compared to OGT intact HeLa cells. Taken together, these findings suggest that OGT, O-GlcNAcylated LXRs, and SREBP-1 increase sCLU expression in cervical cancer cells, which contributes to drug resistance.

Temporally and spatially restricted expression of a gland cell gene during regeneration and in vitro transdifferentiation in the hydrozoan Podocoryne carnea.

PubMed

Baader, C D; Schuchert, P; Schmid, V; Heiermann, Reinhard; Plickert, Günter

1995-01-01

An antiserum to transdifferentiated striated muscle cells from the medusa of Podocoryne carnea was prepared and used to screen a λ gt11-expression library prepared from gonozoids of P. carnea. We isolated a cDNA clone termed Pod-EPPT with at least 63 tandem repeats of the tetrapeptide-motive glu-pro-pro-thr, named Pod-EPPT. Using Pod-EPPT as a molecular marker for head quality the morphological relationship between the two metagenic life stages of this hydroid, the polyp and the medusa, was studied. In situ hybridization demonstrated that expression of the gene corresponding is restricted to secretory cells in the endoderm of the oral hypostome region of polyps and medusae and, presumably, to progenitor cells of this type. Cells expressing Pod-EPPT could not be observed in the larval stage. During head regeneration in polyps, Pod-EPPT expression is upregulated soon after head removal in previously non-expressing cells and in newly differentiating secretory cells. This activation of a head-specific gene precedes the morphologically obvious events of head regeneration. Pod-EPPT is one of the genes that are activated during manubrium (mouth) regeneration from experimentally combined subumbrellar plate endoderm and striated muscle of the medusa.

Ultrastructure of the Intramandibular Gland of Workers and Queens of the Stingless Bee, Melipona quadrifasciata

PubMed Central

Da Cruz-Landim, Carminda; Gracioli-Vitti, Luciana F.; Abdalla, Fábio C.

2011-01-01

The intramandibular glands of workers and queens of Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae), at different ages and from different functional groups, were studied using light and transmission electron microscopy. The results demonstrated that these glands are composed of two types of secretory structures: 1.A hypertrophied epidermis on the dorsal side of the mandible that is an epithelial gland. 2. Free secretory cells filling the inner spaces of the appendices that constitute a unicellular gland. The epithelial gland is larger in the young (1-2-day-old workers), and the gland becomes involuted during the nurse worker stage. The unicellular glands of the workers posses some secretion during all of the studied phases, but secretory activity is more intensive in the foraging workers. Vesicles of secretion are absent in the unicellular glands of queens. These results demonstrate that these glands show functional adaptations in different castes corresponding to the functions of each caste. PMID:22220493

Ultrastructure of the intramandibular gland of workers and queens of the stingless bee, Melipona quadrifasciata.

PubMed

Da Cruz-Landim, Carminda; Gracioli-Vitti, Luciana F; Abdalla, Fábio C

2011-01-01

The intramandibular glands of workers and queens of Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae), at different ages and from different functional groups, were studied using light and transmission electron microscopy. The results demonstrated that these glands are composed of two types of secretory structures: 1.A hypertrophied epidermis on the dorsal side of the mandible that is an epithelial gland. 2. Free secretory cells filling the inner spaces of the appendices that constitute a unicellular gland. The epithelial gland is larger in the young (1-2-day-old workers), and the gland becomes involuted during the nurse worker stage. The unicellular glands of the workers posses some secretion during all of the studied phases, but secretory activity is more intensive in the foraging workers. Vesicles of secretion are absent in the unicellular glands of queens. These results demonstrate that these glands show functional adaptations in different castes corresponding to the functions of each caste.

Hyperforin Accumulates in the Translucent Glands of Hypericum perforatum

PubMed Central

Soelberg, Jens; Jørgensen, Lise Bolt; Jäger, Anna K.

2007-01-01

Background and Aims Hypericum perforatum contains the therapeutically important compounds hypericin and hyperforin. Hypericin is known to accumulate in the dark glands. This investigation aimed to determine the accumulation site of hyperforin. Methods Dark and translucent glands as well as non-secretory tissue in leaves were manually isolated under the microscope. Hyperforin content was quantified by UV HPLC. Secretory structures were surveyed anatomically. Key Results The hyperforin content of intact leaves was found to be about 3 mg g−1 fresh tissue, whereas a content of about 7 mg g−1 fresh material was found in isolated translucent glands. Hyperforin was found only to occur in minute amounts in dark glands (approx. 0·4 mg g−1 fresh tissue). In non-secretory tissue no hyperforin was detected. Conclusions The accumulation of hyperforin detected in the translucent glands supports the proposed hypothesis that hyperforin is synthesized by the same biosynthetic machinery as monoterpenes in the chloroplasts of cells delimiting the gland. PMID:17468113

Electron-microscopic study of the secretion of the ependymal cells in the domestic cat (ependymin-beta cells).

PubMed

Gonzalez-Santander, R

1979-01-01

We have studied, by electron microscopy, the ultrastructural aspects of secretion (neurosecretion) of the ependyma of the third ventricle of the domestic cat. We have found cytoplasmic protrusions and isolated masses of cytoplasm, some with homogeneous cytoplasm and others with very dense granulation (protein-beta?). Axons, synaptic terminals and free secretory granules in the ventricular lumen were also seen. The existence of ependymin-beta cells (ependymocyte-beta) and axohormonal buttons is suggested. The ependymal cells are classified into seven types: (1) covering ependymocytes, (2) tanycyt ependymocytes, (3) secretory ependymocytes, (4) ependymocytes-beta, (5) neurosecretory ependymocytes, (6) neurosensorial ependymocytes (crown-like) and (7) supraependymal microgial ependymocytes. A neurohormonal hypothesis and the possible existence of one or more cerebral hormones (neurohormones) are suggested. These hormones would flow into the CSF through some of the ependymal cells (by microapocrine secretion, liberation of neurosecretion granules, or by axohormonal buttons): this could be the most important link in the endocrine system, assuring the functional unity throughout the ventricular system of the cerebrospinal axis which it winds through, although its basic influence is exercised) on the hypophysis level as a vertex of the classical endocrine system.

Myosin 5b loss of function leads to defects in polarized signaling: implication for microvillus inclusion disease pathogenesis and treatment

PubMed Central

Kravtsov, Dmitri; Mashukova, Anastasia; Forteza, Radia; Rodriguez, Maria M.; Ameen, Nadia A.

2014-01-01

Microvillus inclusion disease (MVID) is an autosomal recessive condition resulting in intractable secretory diarrhea in newborns due to loss-of-function mutations in myosin Vb (Myo5b). Previous work suggested that the apical recycling endosomal (ARE) compartment is the primary location for phosphoinositide-dependent protein kinase 1 (PDK1) signaling. Because the ARE is disrupted in MVID, we tested the hypothesis that polarized signaling is affected by Myo5b dysfunction. Subcellular distribution of PDK1 was analyzed in human enterocytes from MVID/control patients by immunocytochemistry. Using Myo5b knockdown (kd) in Caco-2BBe cells, we studied phosphorylated kinases downstream of PDK1, electrophysiological parameters, and net water flux. PDK1 was aberrantly localized in human MVID enterocytes and Myo5b-deficient Caco-2BBe cells. Two PDK1 target kinases were differentially affected: phosphorylated atypical protein kinase C (aPKC) increased fivefold and phosohoprotein kinase B slightly decreased compared with control. PDK1 redistributed to a soluble (cytosolic) fraction and copurified with basolateral endosomes in Myo5b kd. Myo5b kd cells showed a decrease in net water absorption that could be reverted with PDK1 inhibitors. We conclude that, in addition to altered apical expression of ion transporters, depolarization of PDK1 in MVID enterocytes may lead to aberrant activation of downstream kinases such as aPKC. The findings in this work suggest that PDK1-dependent signaling may provide a therapeutic target for treating MVID. PMID:25258405

Abdominal {gamma}-Radiation Induces an Accumulation of Function-Impaired Regulatory T Cells in the Small Intestine

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

Billiard, Fabienne; Buard, Valerie; Benderitter, Marc

Purpose: To assess the frequency and the functional characteristics of one major component of immune tolerance, the CD4{sup +}FoxP3{sup +} regulatory T cells (Tregs) in a mouse model of abdominal irradiation. Methods and Materials: Mice were exposed to a single abdominal dose of {gamma}-radiation (10 Gy). We evaluated small intestine Treg infiltration by Foxp3 immunostaining and the functional suppressive activity of Tregs isolated from mesenteric lymph nodes. Results: Foxp3 immunostaining showed that radiation induced a long-term infiltration of the intestine by Tregs (levels 5.5 times greater than in controls). Co-culture of Tregs from mesenteric lymph nodes with CD4{sup +} effectormore » cells showed that the Tregs had lost their suppressive function. This loss was associated with a significant decrease in the levels of Foxp3, TGF-{beta}, and CTLA-4 mRNA, all required for optimal Treg function. At Day 90 after irradiation, Tregs regained their suppressive activity as forkhead box P3 (Foxp3), transforming growth factor beta (TGF-{beta}), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression returned to normal. Analysis of the secretory function of mesenteric lymph node Tregs, activated in vitro with anti-CD3/anti-CD28 Abs, showed that this dysfunction was independent of a defect in interleukin-10 secretion. Conclusion: Radiation caused a long-term accumulation of function-impaired Foxp3{sup +}CD4{sup +} Tregs in the intestine. Our study provides new insights into how radiation affects the immune tolerance in peripheral tissues.« less

Dietary high-fat lard intake induces thyroid dysfunction and abnormal morphology in rats.

PubMed

Shao, Shan-shan; Zhao, Yuan-fei; Song, Yong-feng; Xu, Chao; Yang, Jian-mei; Xuan, Shi-meng; Yan, Hui-li; Yu, Chun-xiao; Zhao, Meng; Xu, Jin; Zhao, Jia-jun

2014-11-01

Excess dietary fat intake can induce lipotoxicity in non-adipose tissues. The aim of this study was to observe the effects of dietary high-fat lard intake on thyroid in rats. Male Sprague-Dawley rats were fed a high-fat lard diet for 24 weeks, and then the rats were fed a normal control diet (acute dietary modification) or the high-fat lard diet for another 6 weeks. The serum lipid profile, total thyroxine (TT4), free thyroxine (FT4) and thyrotropin (TSH) levels were determined at the 12, 18, 24 and 30 weeks. High-frequency ultrasound scanning of the thyroid glands was performed at the 24 or 30 weeks. After the rats were sacrificed, the thyroid glands were collected for histological and immunohistochemical analyses. The high-fat lard diet significantly increased triglyceride levels in both the serum and thyroid, and decreased serum TT4 and FT4 levels in parallel with elevated serum TSH levels. Ultrasonic imaging revealed enlarged thyroid glands with lowered echotexture and relatively heterogeneous features in the high-fat lard fed rats. The thyroid glands from the high-fat lard fed rats exhibited enlarged follicle cavities and flattened follicular epithelial cells under light microscopy, and dilated endoplasmic reticulum cisternae, twisted nuclei, fewer microvilli and secretory vesicles under transmission electron microscopy. Furthermore, the thyroid glands from the high-fat lard fed rats showed markedly low levels of thyroid hormone synthesis-related proteins TTF-1 and NIS. Acute dietary modification by withdrawal of the high-fat lard diet for 6 weeks failed to ameliorate the high-fat lard diet-induced thyroid changes. Dietary high-fat lard intake induces significant thyroid dysfunction and abnormal morphology in rats, which can not be corrected by short-term dietary modification.

Dietary glutamine and oral antibiotics each improve indexes of gut barrier function in rat short bowel syndrome.

PubMed

Tian, Junqiang; Hao, Li; Chandra, Prakash; Jones, Dean P; Willams, Ifor R; Gewirtz, Andrew T; Ziegler, Thomas R

2009-02-01

Short bowel syndrome (SBS) is associated with gut barrier dysfunction. We examined effects of dietary glutamine (GLN) or oral antibiotics (ABX) on indexes of gut barrier function in a rat model of SBS. Adult rats underwent a 60% distal small bowel + proximal colonic resection (RX) or bowel transection (TX; control). Rats were pair fed diets with or without l-GLN for 20 days after operation. Oral ABX (neomycin, metronidazole, and polymyxin B) were given in some RX rats fed control diet. Stool secretory immunoglobulin A (sIgA) was measured serially. On day 21, mesenteric lymph nodes (MLN) were cultured for gram-negative bacteria. IgA-positive plasma cells in jejunum, stool levels of flagellin- and lipopolysaccharide (LPS)-specific sIgA, and serum total, anti-flagellin- and anti-LPS IgG levels were determined. RX caused gram-negative bacterial translocation to MLN, increased serum total and anti-LPS IgG and increased stool total sIgA. After RX, dietary GLN tended to blunt bacterial translocation to MLN (-29%, P = NS) and significantly decreased anti-LPS IgG levels in serum, increased both stool and jejunal mucosal sIgA and increased stool anti-LPS-specific IgA. Oral ABX eliminated RX-induced bacterial translocation, significantly decreased total and anti-LPS IgG levels in serum, significantly decreased stool total IgA and increased stool LPS-specific IgA. Partial small bowel-colonic resection in rats is associated with gram-negative bacterial translocation from the gut and a concomitant adaptive immune response to LPS. These indexes of gut barrier dysfunction are ameliorated or blunted by administration of dietary GLN or oral ABX, respectively. Dietary GLN upregulates small bowel sIgA in this model.

Oxidative Stress Induced Inflammation Initiates Functional Decline of Tear Production

PubMed Central

Uchino, Yuichi; Kawakita, Tetsuya; Miyazawa, Masaki; Ishii, Takamasa; Onouchi, Hiromi; Yasuda, Kayo; Ogawa, Yoko; Shimmura, Shigeto; Ishii, Naoaki; Tsubota, Kazuo

2012-01-01

Oxidative damage and inflammation are proposed to be involved in an age-related functional decline of exocrine glands. However, the molecular mechanism of how oxidative stress affects the secretory function of exocrine glands is unclear. We developed a novel mev-1 conditional transgenic mouse model (Tet-mev-1) using a modified tetracycline system (Tet-On/Off system). This mouse model demonstrated decreased tear production with morphological changes including leukocytic infiltration and fibrosis. We found that the mev-1 gene encodes Cyt-1, which is the cytochrome b560 large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria (homologous to succinate dehydrogenase C subunit (SDHC) in humans). The mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in protein and aqueous secretory function. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease. Tear volume in Tet-mev-1 mice was lower than in wild type mice and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. These findings strongly suggest that oxidative stress can be a causative factor for the development of dry eye disease. PMID:23071526

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 some homologous proteins.

Indomethacin and flurbiprofen speed recovery of rat bronchial epithelium after exposure to cigarette smoke.

PubMed

Rogers, D F; Jeffery, P K

1986-01-01

The cigarette smoke-induced rat model of chronic bronchitis was used to study the time course of the return of cigarette smoke-induced secretory cell hyperplasia to the normal and the capacity of two non-steroidal anti-inflammatory drugs to speed this recovery. Cigarette smoke alone significantly increased (P less than 0.05) the number of secretory cells in all of the eight airway levels studied to between 52-225% above control values. After cessation of exposure, recovery was complete by 9 days in the trachea, between 10-21 days in 'proximal' intrapulmonary airways and 43-84 days in distal bronchioli. Indomethacin and flurbiprofen, given by intraperitoneal injection at 4 mg/kg body weight for 21 days of the recovery period, significantly reduced the time taken for recovery to between 4 and 9 days in intrapulmonary airways but had no effect in the trachea.

Immunohistochemical localization of fatty acid transporters and MCT1 in the sebaceous glands of mouse skin.

PubMed

Zheng, Miao; Lee, Shinhye; Tsuzuki, Satoshi; Inoue, Kazuo; Masuda, Daisaku; Yamashita, Shizuya; Iwanaga, Toshihiko

2016-01-01

The sebaceous glands secrete sebum to protect the epidermis and hairs by the oily products. The glands express several transporters and binding proteins for the production of fatty acids and uptake of their sources. The present immunohistochemical study examined the expression and localization of CD36, MCT1, FATP4, and E-FABP in the sebaceous glands, including the meibomian and preputial glands of mice. CD36 and MCT1 in sebaceous glands were largely co-localized along the plasma membrane of secretory cells, while they were separately expressed in the glandular portion of meibomian and preputial glands. Immunoreactivities for FATP4 and E-FABP appeared diffusely in the cytoplasm of secretory cells. Genetic deletion of CD36 did not affect the immunolocalization of the three other molecules. The sebaceous glands were judged to be useful for analyzing the functions and relation of fatty acid transporters and binding proteins.

Ultrastructural characterisation of the olfactory mucosa of the armadillo Dasypus hybridus (Dasypodidae, Xenarthra)

PubMed Central

FERRARI, C. C.; CARMANCHAHI, P. D.; ALDANA MARCOS, H. J.; AFFANNI, J. M.

2000-01-01

The ultrastructure of the olfactory mucosa of the armadillo Dasypus hybridus was studied. A comparison with the olfactory mucosa of another armadillo (Chaetophractus villosus) was made. The olfactory mucosa of D. hybridus shows many features which are similar to those of other mammals. Interestingly, it differs from the olfactory mucosa of the armadillo C. villosus. A suggestion is made that these differences may be due to differences in the digging habits of these species. In Dasypus, the supporting cells (SCs) showed dense vacuoles, multivesicular bodies and lysosome-like bodies probably related with the endocytotic system. The SCs show a dense network of SER presumably associated with xenobiotic mechanisms. The olfactory receptor neurons exhibit lysosome-like bodies and multivesicular bodies in their perikarya. These organelles suggest the presence of an endocytotic system. Duct cells of Bowman's glands exhibit secretory activities. Bowman's glands are compound-branched tubulo-acinar mixed glands with merocrine secretory mechanisms. PMID:10739023

Impaired local immune response in vitamin A-deficient rats.

PubMed Central

Sirisinha, S; Darip, M D; Moongkarndi, P; Ongsakul, M; Lamb, A J

1980-01-01

The functional integrity of the local immune system in vitamin A-deficient (A-) rats was investigated. Secretory IgA levels in the intestinal fluid of A- rats were significantly lower than in controls. This and the decrease in intensity of immunofluorescent staining for secretory component (SC) in the intestinal cells was related to the duration of vitamin A deprivation. IgG levels in the intestinal fluid, and serum IgA and IgG levels were unaffected in deficiency. Moreover, when the response of animals to DNP50-BGG was evaluated, the local anti-DNP response in the intestine was markedly depressed. These defects may result from impaired synthesis of SC by epithelial cells. On the other hand, the serum antibody response in deficient animals was not noticeably different from that of the controls; if any, htere was a slight reduction in the affinity of antibody. PMID:7389210

Sequestration of Mutated α1-Antitrypsin into Inclusion Bodies Is a Cell-protective Mechanism to Maintain Endoplasmic Reticulum Function

PubMed Central

Granell, Susana; Baldini, Giovanna; Mohammad, Sameer; Nicolin, Vanessa; Narducci, Paola; Storrie, Brian

2008-01-01

A variant α1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, α1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated α1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated α1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated α1-antitrypsin in the ER. In hepatoma cells, shift of mutated α1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated α1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway. PMID:18045994

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.

Kinetics of milk lipid droplet transport, growth, and secretion revealed by intravital imaging: lipid droplet release is intermittently stimulated by oxytocin | Center for Cancer Research

Cancer.gov

Description of the cover: The micrograph shows lipid droplets (red) accumulating at the apical surface of secretory cells (green) between oxytocin-induced contractions in a transgenic mouse line that expresses green fluorescent protein in the cytoplasm of most cells.

Influence of low-power laser radiation on mast cells function and biogenic amines content in stress

NASA Astrophysics Data System (ADS)

Brill, Gregory E.; Dobrovolsky, Gennady A.; Romanova, Tatyana P.; Brill, Alexander G.

1997-06-01

In experiments on white male rats stress was modeled by combined action of immobilization and sound stimulus during 2 h. It was established that stress induced storage of secretory material and accumulation of biogenic amines in mast cells. Preliminary transcutaneous He-Ne laser irradiation prevented stress-induced changes.

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 hydrophobic domain of the IBV E protein, a putative viroporin, causes disruption of the mammalian secretory pathway when exogenously expressed in cells. Understanding the mechanism of this disruption could lead to the identification of novel antiviral therapeutics. Here, we present biochemical evidence for two distinct oligomeric forms of IBV E, one essential for assembly and the other with a role in disruption of the secretory pathway. Discovery of two forms of CoV E protein will provide additional targets for antiviral therapeutics. PMID:26136577

Morphology and Histology of the Ductus Receptaculi and Accessory Glands in the Reproductive Tract of the Female Cricket, Teleogryllus commodus

PubMed Central

Sturm, Robert

2008-01-01

The morphology and histology of the ductus receptaculi and accessory glands in females of the black field cricket, Teleogryllus commodus Walker (Orthoptera: Gryllidae) are described. Both are reproductive organs situated in the 7th and 8th abdominal segment that join the posterior part of the genital chamber. The ductus receptaculi is a long (up to 25 mm) homogeneous tube, and the accessory glands (total length: 4 to 12 mm) are a complex system of tubes and end lobes with various numbers of ramifications. Based on their external shapes the accessory glands may be subdivided into three distinct regions, a distal region mainly producing the gland's secretion, a middle conducting region, and a basal region serving for the storage and release of the secretory substances into the genital chamber of the female. In histological respects, both organs have an outer muscle coat followed by a basal lamina, one or two cell layers, the cuticular intima, and the inner lumen. The ductus receptaculi is subdivided into three histologically different regions. The region located adjacent to the receptaculum and the region neighbouring the terminal papilla consist of a single, epithelial cell layer that is not secretory. The epithelium of the middle region contains two cell layers, glandular cells and cuticula-forming cells, which are responsible for the production of the cuticular intima. The secretion of the gland cells is released into an extracellular cavity, through which it reaches the lumen via a complex network of canals running through the intima. The histology of the accessory glands is rather homogeneous among the different regions, as one layer of epithelial cells produces both the secretion and the cuticular intima. Histological variations in the distal, middle, and basal gland sections mainly concern the height of the epithelium, the thickness of the basal lamina and the cuticular intima as well as the variable presence of the outer muscle coat. In contrast to the ductus receptaculi, secretory substances produced by the accessory gland cells accumulate in the lumen by a diffusive permeation of the intima. PMID:20298118

Post-embryonic larval development and metamorphosis of the hydroid Eudendrium racemosum (Cavolini) (Hydrozoa, Cnidaria)

NASA Astrophysics Data System (ADS)

Sommer, C.

1990-09-01

The morphology and histology of the planula larva of Eudendrium racemosum (Cavolini) and its metamorphosis into the primary polyp are described from light microscopic observations. The planula hatches as a differentiated gastrula. During the lecithotrophic larval period, large ectodermal mucous cells, embedded between epitheliomuscular cells, secrete a sticky slime. Two granulated cell types occur in the ectoderm that are interpreted as secretory and sensorynervous cells, but might also be representatives of only one cell type with a multiple function. The entoderm consists of yolk-storing gastrodermal cells, digestive gland cells, interstitial cells, cnidoblasts, and premature cnidocytes. The larva starts metamorphosis by affixing its blunt aboral pole to a substratum. While the planula flattens down, the mucous cells penetrate the mesolamella and migrate through the entoderm into the gastral cavity where they are lysed. Subsequently, interstitial cells, cnidoblasts, and premature cnidocytes migrate in the opposite direction, i.e. from entoderm to ectoderm. Then, the polypoid body organization, comprising head (hydranth), stem and foot, all covered by peridermal secretion, becomes recognisable. An oral constriction divides the hypostomal portion of the gastral cavity from the stomachic portion. Within the hypostomal entoderm, cells containing secretory granules differentiate. Following growth and the multiplication of tentacles, the head periderm disappears. A ring of gland cells differentiates at the hydranth's base. The positioning of cnidae in the tentacle ectoderm, penetration of the mouth opening and the multiplication of digestive gland cells enable the polyp to change from lecithotrophic to planktotrophic nutrition.

Relationship between infectious agents for vulvovaginitis and skin color.

PubMed

Nomelini, Rosekeila Simoes; Carrijo, Ana Paula Borges; Adad, Sheila Jorge; Nunes, Altacílio Aparecido; Murta, Eddie Fernando Candido

2010-12-01

Many factors influence occurrences of vulvovaginitis. The aims here were to assess skin color and age-related differences in the vaginal flora and occurrences of vulvovaginitis. Cross-sectional study; tertiary referral hospital (Universidade Federal do Triângulo Mineiro, Uberaba). Healthy women who underwent routine outpatient gynecological assessments were assessed for vulvovaginitis and vaginal flora and then divided into whites (n = 13,881) and nonwhites (n = 5,295). Statistical analysis was performed using the X² test, logistic regression and odds ratios. The vaginal microflora was skin-color dependent, with greater occurrence of clue cells, Trichomonas vaginalis and coccobacilli in nonwhite women (p < 0.0001). Döderlein bacilli and cytolytic flora were more prevalent in white women (p < 0.0001 and p < 0.05, respectively). The vaginal microflora was age-dependent within the skin color groups. Among the nonwhite women, clue cells were more prevalent in women aged 21 to 50 years; Trichomonas in women up to 40 years and coccobacili in women between 21 and 40 years (P < 0.05). During the proliferative and secretory phases, the nonwhite women were more likely to present clue cells, Trichomonas, Candida and coccobacilli (OR, proliferative phase: 1.31, 1.79, 1.6 and 1.25 respectively; secretory phase: 1.31, 2.88, 1.74 and 1.21 respectively), while less likely to present Döderlein flora (OR, proliferative phase: 0.76; secretory phase: 0.66), compared with white women, irrespective of age. There are differences in vulvovaginitis occurrence relating to skin color, which may be associated with variations in vaginal flora.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

PubMed Central

Saitoh, Sei; Ohno, Nobuhiko; Saitoh, Yurika; Terada, Nobuo; Shimo, Satoshi; Aida, Kaoru; Fujii, Hideki; Kobayashi, Tetsuro; Ohno, Shinichi

2018-01-01

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets. PMID:29622846

Apigenin suppresses the senescence-associated secretory phenotype and paracrine effects on breast cancer cells.

PubMed

Perrott, Kevin M; Wiley, Christopher D; Desprez, Pierre-Yves; Campisi, Judith

2017-04-01

Apigenin (4',5,7,-trihydroxyflavone) is a flavonoid found in certain herbs, fruits, and vegetables. Apigenin can attenuate inflammation, which is associated with many chronic diseases of aging. Senescent cells-stressed cells that accumulate with age in mammals-display a pro-inflammatory senescence-associated secretory phenotype (SASP) that can drive or exacerbate several age-related pathologies, including cancer. Flavonoids, including apigenin, were recently shown to reduce the SASP of a human fibroblast strain induced to senesce by bleomycin. Here, we confirm that apigenin suppresses the SASP in three human fibroblast strains induced to senesce by ionizing radiation, constitutive MAPK (mitogen-activated protein kinase) signaling, oncogenic RAS, or replicative exhaustion. Apigenin suppressed the SASP in part by suppressing IL-1α signaling through IRAK1 and IRAK4, p38-MAPK, and NF-κB. Apigenin was particularly potent at suppressing the expression and secretion of CXCL10 (IP10), a newly identified SASP factor. Further, apigenin-mediated suppression of the SASP substantially reduced the aggressive phenotype of human breast cancer cells, as determined by cell proliferation, extracellular matrix invasion, and epithelial-mesenchymal transition. Our results support the idea that apigenin is a promising natural product for reducing the impact of senescent cells on age-related diseases such as cancer.

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.

Origin of clear cell carcinoma: nature or nurture?

PubMed

Kolin, David L; Dinulescu, Daniela M; Crum, Christopher P

2018-02-01

A rare but serious complication of endometriosis is the development of carcinoma, and clear cell and endometrioid carcinomas of the ovary are the two most common malignancies which arise from endometriosis. They are distinct diseases, characterized by unique morphologies, immunohistochemical profiles, and responses to treatment. However, both arise in endometriosis and can share common mutations. The overlapping mutational profiles of clear cell and endometrioid carcinomas suggest that their varied histologies may be due to a different cell of origin which gives rise to each type of cancer. Cochrane and colleagues address this question in a recent article in this journal. They show that a marker of ovarian clear cell carcinoma, cystathionine gamma lyase, is expressed in ciliated cells. Similarly, they show that markers of secretory cells (estrogen receptor and methylenetetrahydrofolate dehydrogenase 1) are expressed in ovarian endometrioid carcinoma. Taken together, they suggest that endometrioid and clear cell carcinomas arise from cells related to secretory and ciliated cells, respectively. We discuss Cochrane et al's work in the context of other efforts to determine the cell of origin of gynecological malignancies, with an emphasis on recent developments and challenges unique to the area. These limitations complicate our interpretation of tumor differentiation; does it reflect nature imposed by a specific cell of origin or nurture, by either mutation(s) or environment? Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Regenerative and reparative effects of human chorion-derived stem cell conditioned medium on photo-aged epidermal cells

PubMed Central

Li, Qiankun; Chen, Yan; Ma, Kui; Zhao, Along; Zhang, Cuiping; Fu, Xiaobing

2016-01-01

ABSTRACT Epidermal cells are an important regenerative source for skin wound healing. Aged epidermal cells have a low ability to renew themselves and repair skin injury. Ultraviolet (UV) radiation, particularly UVB, can cause photo-aging of the skin by suppressing the viability of human epidermal cells. A chorion-derived stem cell conditioned medium (CDSC-CNM) is thought to have regenerative properties. This study aimed to determine the regenerative effects of CDSC-CNM on UVB-induced photo-aged epidermal cells. Epidermal cells were passaged four times and irradiated with quantitative UVB, and non-irradiated cells served as a control group. Cells were then treated with different concentrations of CDSC-CNM. Compared to the non-irradiated group, the proliferation rates and migration rates of UVB-induced photo-aged epidermal cells significantly decreased (p < 0.05) with increasing intracellular radical oxygen species (ROS) generation and DNA damage. After treatment with CDSC-CNM, photo-aged epidermal cells significantly improved their viability, and their ROS generation and DNA damage decreased. The secretory factors in CDSC-CNM, including epidermal growth factor (EGF), transforming growth factor-β (TGF-β), interleukin (IL)-6, and IL-8 and the related signaling pathway protein levels, increased compared to the control medium (CM). The potential regenerative and reparative effects of CDSC-CNM indicate that it may be a candidate material for the treatment of prematurely aged skin. The functions of the secretory factors and the mechanisms of CDSC-CNM therapy deserve further attention. PMID:27097375

Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

PubMed

Green, Alastair D; Vasu, Srividya; Moffett, R Charlotte; Flatt, Peter R

2016-06-01

We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

The Malarial Host-Targeting Signal Is Conserved in the Irish Potato Famine Pathogen

PubMed Central

Liolios, Konstantinos; Win, Joe; Kanneganti, Thirumala-Devi; Young, Carolyn; Kamoun, Sophien; Haldar, Kasturi

2006-01-01

Animal and plant eukaryotic pathogens, such as the human malaria parasite Plasmodium falciparum and the potato late blight agent Phytophthora infestans, are widely divergent eukaryotic microbes. Yet they both produce secretory virulence and pathogenic proteins that alter host cell functions. In P. falciparum, export of parasite proteins to the host erythrocyte is mediated by leader sequences shown to contain a host-targeting (HT) motif centered on an RxLx (E, D, or Q) core: this motif appears to signify a major pathogenic export pathway with hundreds of putative effectors. Here we show that a secretory protein of P. infestans, which is perceived by plant disease resistance proteins and induces hypersensitive plant cell death, contains a leader sequence that is equivalent to the Plasmodium HT-leader in its ability to export fusion of green fluorescent protein (GFP) from the P. falciparum parasite to the host erythrocyte. This export is dependent on an RxLR sequence conserved in P. infestans leaders, as well as in leaders of all ten secretory oomycete proteins shown to function inside plant cells. The RxLR motif is also detected in hundreds of secretory proteins of P. infestans, Phytophthora sojae, and Phytophthora ramorum and has high value in predicting host-targeted leaders. A consensus motif further reveals E/D residues enriched within ~25 amino acids downstream of the RxLR, which are also needed for export. Together the data suggest that in these plant pathogenic oomycetes, a consensus HT motif may reside in an extended sequence of ~25–30 amino acids, rather than in a short linear sequence. Evidence is presented that although the consensus is much shorter in P. falciparum, information sufficient for vacuolar export is contained in a region of ~30 amino acids, which includes sequences flanking the HT core. Finally, positional conservation between Phytophthora RxLR and P. falciparum RxLx (E, D, Q) is consistent with the idea that the context of their presentation is constrained. These studies provide the first evidence to our knowledge that eukaryotic microbes share equivalent pathogenic HT signals and thus conserved mechanisms to access host cells across plant and animal kingdoms that may present unique targets for prophylaxis across divergent pathogens. PMID:16733545

Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism.

PubMed

Lee, Young Ah; Nam, Young Hee; Min, Arim; Kim, Kyeong Ah; Nozaki, Tomoyoshi; Saito-Nakano, Yumiko; Mirelman, David; Shin, Myeong Heon

2014-01-01

Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis. © Y.A. Lee et al., published by EDP Sciences, 2014.

The integrity of the plant Golgi apparatus depends on cell growth-controlled activity of GNL1.

PubMed

Du, Wenyan; Tamura, Kentaro; Stefano, Giovanni; Brandizzi, Federica

2013-05-01

Membrane traffic and organelle integrity in the plant secretory pathway depend on ARF-GTPases, which are activated by guanine-nucleotide exchange factors (ARF-GEFs). While maintenance of conserved roles, evolution of unique functions as well as tissue-specific roles have been shown for a handful of plant ARF-GEFs, a fundamental yet unanswered question concerns the extent to which their function overlaps during cell growth. To address this, we have characterized pao, a novel allele of GNOM-like 1 (GNL1), a brefeldin A (BFA)-insensitive ARF-GEF, isolated through a confocal microscopy-based forward genetics screen of the Golgi in Arabidopsis thaliana. Specifically, we have analyzed the dependence of the integrity of trafficking routes and secretory organelles on GNL1 availability during expansion stages of cotyledon epidermal cells, an exquisite model system for vegetative cell growth analyses in intact tissues. We show that Golgi traffic is influenced largely by GNL1 availability at early stages of cotyledon cell expansion but by BFA-sensitive GEFs when cell growth terminates. These data reveal an unanticipated level of complexity in the biology of GNL1 by showing that its cellular roles are correlated with cell growth. These results also indicate that the cell growth stage is an important element weighting into functional analyses of the cellular roles of ARF-GEFs.

Circadian rhythms regulate amelogenesis.

PubMed

Zheng, Li; Seon, Yoon Ji; Mourão, Marcio A; Schnell, Santiago; Kim, Doohak; Harada, Hidemitsu; Papagerakis, Silvana; Papagerakis, Petros

2013-07-01

Ameloblasts, the cells responsible for making enamel, modify their morphological features in response to specialized functions necessary for synchronized ameloblast differentiation and enamel formation. Secretory and maturation ameloblasts are characterized by the expression of stage-specific genes which follows strictly controlled repetitive patterns. Circadian rhythms are recognized as key regulators of the development and diseases of many tissues including bone. Our aim was to gain novel insights on the role of clock genes in enamel formation and to explore the potential links between circadian rhythms and amelogenesis. Our data shows definitive evidence that the main clock genes (Bmal1, Clock, Per1 and Per2) oscillate in ameloblasts at regular circadian (24 h) intervals both at RNA and protein levels. This study also reveals that the two markers of ameloblast differentiation i.e. amelogenin (Amelx; a marker of secretory stage ameloblasts) and kallikrein-related peptidase 4 (Klk4, a marker of maturation stage ameloblasts) are downstream targets of clock genes. Both, Amelx and Klk4 show 24h oscillatory expression patterns and their expression levels are up-regulated after Bmal1 over-expression in HAT-7 ameloblast cells. Taken together, these data suggest that both the secretory and the maturation stages of amelogenesis might be under circadian control. Changes in clock gene expression patterns might result in significant alterations of enamel apposition and mineralization. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Gene expression and localization of two types of AQP5 in Xenopus tropicalis under hydration and dehydration.

PubMed

Shibata, Yuki; Sano, Takahiro; Tsuchiya, Nobuhito; Okada, Reiko; Mochida, Hiroshi; Tanaka, Shigeyasu; Suzuki, Masakazu

2014-07-01

Two types of aquaporin 5 (AQP5) genes (aqp-xt5a and aqp-xt5b) were identified in the genome of Xenopus tropicalis by synteny comparison and molecular phylogenetic analysis. When the frogs were in water, AQP-xt5a mRNA was expressed in the skin and urinary bladder. The expression of AQP-xt5a mRNA was significantly increased in dehydrated frogs. AQP-xt5b mRNA was also detected in the skin and increased in response to dehydration. Additionally, AQP-xt5b mRNA began to be slightly expressed in the lung and stomach after dehydration. For the pelvic skin of hydrated frogs, immunofluorescence staining localized AQP-xt5a and AQP-xt5b to the cytoplasm of secretory cells of the granular glands and the apical plasma membrane of secretory cells of the small granular glands, respectively. After dehydration, the locations of both AQPs in their respective glands did not change, but AQP-xt5a was visualized in the cytoplasm of secretory cells of the small granular glands. For the urinary bladder, AQP-xt5a was observed in the apical plasma membrane and cytoplasm of a number of granular cells under normal hydration. After dehydration, AQP-xt5a was found in the apical membrane and cytoplasm of most granular cells. Injection of vasotocin into hydrated frogs did not induce these changes in the localization of AQP-xt5a in the small granular glands and urinary bladder, however. The results suggest that AQP-xt5a might be involved in water reabsorption from the urinary bladder during dehydration, whereas AQP-xt5b might play a role in water secretion from the small granular gland. Copyright © 2014 the American Physiological Society.

A Single Polar Residue and Distinct Membrane Topologies Impact the Function of the Infectious Bronchitis Coronavirus E Protein

PubMed Central

Ruch, Travis R.; Machamer, Carolyn E.

2012-01-01

The coronavirus E protein is a small membrane protein with a single predicted hydrophobic domain (HD), and has a poorly defined role in infection. The E protein is thought to promote virion assembly, which occurs in the Golgi region of infected cells. It has also been implicated in the release of infectious particles after budding. The E protein has ion channel activity in vitro, although a role for channel activity in infection has not been established. Furthermore, the membrane topology of the E protein is of considerable debate, and the protein may adopt more than one topology during infection. We previously showed that the HD of the infectious bronchitis virus (IBV) E protein is required for the efficient release of infectious virus, an activity that correlated with disruption of the secretory pathway. Here we report that a single residue within the hydrophobic domain, Thr16, is required for secretory pathway disruption. Substitutions of other residues for Thr16 were not tolerated. Mutations of Thr16 did not impact virus assembly as judged by virus-like particle production, suggesting that alteration of secretory pathway and assembly are independent activities. We also examined how the membrane topology of IBV E affected its function by generating mutant versions that adopted either a transmembrane or membrane hairpin topology. We found that a transmembrane topology was required for disrupting the secretory pathway, but was less efficient for virus-like particle production. The hairpin version of E was unable to disrupt the secretory pathway or produce particles. The findings reported here identify properties of the E protein that are important for its function, and provide insight into how the E protein may perform multiple roles during infection. PMID:22570613

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

The Dual Role of Cellular Senescence in Developing Tumors and Their Response to Cancer Therapy

PubMed Central

Schosserer, Markus; Grillari, Johannes; Breitenbach, Michael

2017-01-01

Cellular senescence describes an irreversible growth arrest characterized by distinct morphology, gene expression pattern, and secretory phenotype. The final or intermediate stages of senescence can be reached by different genetic mechanisms and in answer to different external and internal stresses. It has been maintained in the literature but never proven by clearcut experiments that the induction of senescence serves the evolutionary purpose of protecting the individual from development and growth of cancers. This hypothesis was recently scrutinized by new experiments and found to be partly true, but part of the gene activities now known to happen in senescence are also needed for cancer growth, leading to the view that senescence is a double-edged sword in cancer development. In current cancer therapy, cellular senescence is, on the one hand, intended to occur in tumor cells, as thereby the therapeutic outcome is improved, but might, on the other hand, also be induced unintentionally in non-tumor cells, causing inflammation, secondary tumors, and cancer relapse. Importantly, organismic aging leads to accumulation of senescent cells in tissues and organs of aged individuals. Senescent cells can occur transiently, e.g., during embryogenesis or during wound healing, with beneficial effects on tissue homeostasis and regeneration or accumulate chronically in tissues, which detrimentally affects the microenvironment by de- or transdifferentiation of senescent cells and their neighboring stromal cells, loss of tissue specific functionality, and induction of the senescence-associated secretory phenotype, an increased secretory profile consisting of pro-inflammatory and tissue remodeling factors. These factors shape their surroundings toward a pro-carcinogenic microenvironment, which fuels the development of aging-associated cancers together with the accumulation of mutations over time. We are presenting an overview of well-documented stress situations and signals, which induce senescence. Among them, oncogene-induced senescence and stress-induced premature senescence are prominent. New findings about the role of senescence in tumor biology are critically reviewed with respect to new suggestions for cancer therapy leveraging genetic and pharmacological methods to prevent senescence or to selectively kill senescent cells in tumors. PMID:29218300

[Cylindroma of the breast. A histochemical and histogenetic study (author's transl)].

PubMed

van Bogaert, L J; Maldague, P; Pham-Maldague, H; Staquet, J P

1975-10-20

The histochemical study of mucopolysaccharide components of the ground substance of the cylindroma lends force to their epithelial origine. The myoepithelial cell, which is ectodermal in origin, plays an essential role in their secretion. Silver impregnation of normal and dysplastic breast illustrates the secretory function of myoepithelial cells and their possible implication in the histogenesis of cylindroma.

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.

Unconventional Transport Routes of Soluble and Membrane Proteins and Their Role in Developmental Biology

PubMed Central

Pompa, Andrea; De Marchis, Francesca; Pallotta, Maria Teresa; Benitez-Alfonso, Yoselin; Jones, Alexandra; Schipper, Kerstin; Moreau, Kevin; Žárský, Viktor; Di Sansebastiano, Gian Pietro; Bellucci, Michele

2017-01-01

Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on “Unconventional Protein and Membrane Traffic” (UPMT) during 4–7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes. PMID:28346345

Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

PubMed Central

Strasser, Jane E.; Arribas, Monica; Blagoveshchenskaya, Anastasia D.; Cutler, Daniel F.

1999-01-01

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase. PMID:10436017

Postnatal telomere dysfunction induces cardiomyocyte cell-cycle arrest through p21 activation

PubMed Central

Aix, Esther; Gutiérrez-Gutiérrez, Óscar; Sánchez-Ferrer, Carlota; Aguado, Tania

2016-01-01

The molecular mechanisms that drive mammalian cardiomyocytes out of the cell cycle soon after birth remain largely unknown. Here, we identify telomere dysfunction as a critical physiological signal for cardiomyocyte cell-cycle arrest. We show that telomerase activity and cardiomyocyte telomere length decrease sharply in wild-type mouse hearts after birth, resulting in cardiomyocytes with dysfunctional telomeres and anaphase bridges and positive for the cell-cycle arrest protein p21. We further show that premature telomere dysfunction pushes cardiomyocytes out of the cell cycle. Cardiomyocytes from telomerase-deficient mice with dysfunctional telomeres (G3 Terc−/−) show precocious development of anaphase-bridge formation, p21 up-regulation, and binucleation. In line with these findings, the cardiomyocyte proliferative response after cardiac injury was lost in G3 Terc−/− newborns but rescued in G3 Terc−/−/p21−/− mice. These results reveal telomere dysfunction as a crucial signal for cardiomyocyte cell-cycle arrest after birth and suggest interventions to augment the regeneration capacity of mammalian hearts. PMID:27241915

Integration of Golgi trafficking and growth factor signaling by the lipid phosphatase SAC1

PubMed Central

Blagoveshchenskaya, Anastasia; Cheong, Fei Ying; Rohde, Holger M.; Glover, Greta; Knödler, Andreas; Nicolson, Teresa; Boehmelt, Guido; Mayinger, Peter

2008-01-01

When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I–mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor–induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery. PMID:18299350

Mechanisms of stress-induced cellular HSP72 release: implications for exercise-induced increases in extracellular HSP72.

PubMed

Lancaster, Graeme I; Febbraio, Mark A

2005-01-01

The heat shock proteins are a family of highly conserved proteins with critical roles in maintaining cellular homeostasis and in protecting the cell from stressful conditions. While the critical intracellular roles of heat shock proteins are undisputed, evidence suggests that the cell possess the necessary machinery to actively secrete specific heat shock proteins in response to cellular stress. In this review, we firstly discuss the evidence that physical exercise induces the release of heat shock protein 72 from specific tissues in humans. Importantly, it appears as though this release is the result of an active secretory process, as opposed to non-specific processes such as cell lysis. Next we discuss recent in vitro evidence that has identified a mechanistic basis for the observation that cellular stress induces the release of a specific subset of heat shock proteins. Importantly, while the classical protein secretory pathway does not seem to be involved in the stress-induced release of HSP72, we discuss the evidence that lipid-rafts and exosomes are important mediators of the stress-induced release of HSP72.

The clypeal gland: a new exocrine gland in termite imagoes (Isoptera: Serritermitidae, Rhinotermitidae, Termitidae).

PubMed

Křížková, Barbora; Bourguignon, Thomas; Vytisková, Blahoslava; Sobotník, Jan

2014-11-01

Social insects possess a rich set of exocrine organs producing diverse pheromones and defensive compounds. This is especially true for termite imagoes, which are equipped with several glands producing, among others, sex pheromones and defensive compounds protecting imagoes during the dispersal flight and colony foundation. Here, we describe the clypeal gland, a new termite exocrine organ occurring in the labro-clypeal region of imagoes of most Rhinotermitidae, Serritermitidae and Termitidae species. The clypeal gland of Coptotermes testaceus consists of class 1 (modified epidermal cell) and class 3 (bicellular gland unit) secretory cells. Ultrastructural features suggest that the gland secretes volatile compounds and proteins, probably after starting the reproduction. One peculiar feature of the gland is the presence of multiple secretory canals in a single canal cell, a feature never observed before in other insect glands. Although the function of the gland remains unknown, we hypothesize that it could produce secretion signalling the presence of functional reproductives or their need to be fed. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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

The neuronal porosome complex in health and disease

PubMed Central

Naik, Akshata R; Lewis, Kenneth T

2015-01-01

Cup-shaped secretory portals at the cell plasma membrane called porosomes mediate the precision release of intravesicular material from cells. Membrane-bound secretory vesicles transiently dock and fuse at the base of porosomes facing the cytosol to expel pressurized intravesicular contents from the cell during secretion. The structure, isolation, composition, and functional reconstitution of the neuronal porosome complex have greatly progressed, providing a molecular understanding of its function in health and disease. Neuronal porosomes are 15 nm cup-shaped lipoprotein structures composed of nearly 40 proteins, compared to the 120 nm nuclear pore complex composed of >500 protein molecules. Membrane proteins compose the porosome complex, making it practically impossible to solve its atomic structure. However, atomic force microscopy and small-angle X-ray solution scattering studies have provided three-dimensional structural details of the native neuronal porosome at sub-nanometer resolution, providing insights into the molecular mechanism of its function. The participation of several porosome proteins previously implicated in neurotransmission and neurological disorders, further attest to the crosstalk between porosome proteins and their coordinated involvement in release of neurotransmitter at the synapse. PMID:26264442

Cryopreservation and hypothermic storage of lacrimal gland: towards enabling delivery of regenerative medicine therapies for treatment of dry eye syndrome.

PubMed

Massie, I; Spaniol, K; Geerling, G; Schrader, S

2017-12-01

Severe dry eye syndrome (DES) can cause painful loss of vision and may result from lacrimal gland dysfunction. Current treatments are palliative, so a causative therapy is desirable. The ability to (cryo)preserve lacrimal gland tissue or epithelial cells would simplify this. Here, lacrimal gland tissue was cryopreserved in 10% dimethylsulphoxide in liquid nitrogen, or stored at 4 °C in culture medium for up to 7 days, and compared with fresh tissue using immunohistochemistry. Cultures were initiated from fresh and stored tissue, and cells characterised in P1 for proliferation (WST-1), colony-forming efficiency (CFE) and secretory capacity (immunocytochemistry and β-hexosaminidase activity assay). Tissue stored for > 3 days at 4 °C displayed grossly altered tissue architecture when compared with fresh tissue, decreased acinus density and increased caspase-3 activity. Cryopreserved tissue showed less obvious signs of damage without caspase-3 activation. Storage at 4 °C and cryopreservation delayed epithelial outgrowth compared with that from fresh tissue initially (p  <  0.05) but, by day 9, all explants showed comparable outgrowth (~90%), except tissue stored at 4 °C for 3 or 7 days (p  <  0.05 compared with fresh tissue). Epithelial cell yields per explant were similar from fresh and stored tissue, apart from tissue stored at 4 °C for 7 days (p  <  0.01). In P1, epithelial cells from fresh and stored tissue were largely equivalent in terms of: proliferation; CFE (~21%); Rab3D, HexA and lysozyme expression; mucin production; and β-hexosaminidase activity. These data demonstrate that cryo(preservation) of lacrimal gland tissue and cells is possible, which may enable use of autologous cells in regenerative medicine approaches to treating DES. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Walter Reed Army Institute of Research Annual Progress Report, Fiscal Year 1981.

DTIC Science & Technology

1981-10-01

induce mononuclear cell-mediated antibacterial activity against X-16 shigella has been further in- vestigated. Monocytes, but not K or T lymphocytes are...M.J. Bertovich, and S.B. Formal. Antibody-dependent cell-mediated antibacterial activity : K lymphocytes, monocytes and granulo- cytes are effective...Wilson, S.R., Formal, S.B., Keren, D.F. and Lowell, G.H. Secretory IgA- dependent mononuclear cell-mediated antibacterial activity . Fed. Proc. 4787

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse.

PubMed

Schulte, B A; Spicer, S S

1983-12-01

Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal alpha-N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate beta-galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20-50% of these cells in all glands contained terminal N-acetylglucosamine residues. In contrast, terminal alpha-N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.

Old and sticky—adhesive mechanisms in the living fossil Nautilus pompilius (Mollusca, Cephalopoda)

PubMed Central

von Byern, Janek; Wani, Ryoji; Schwaha, Thomas; Grunwald, Ingo; Cyran, Norbert

2012-01-01

Nautiloidea is the oldest group within the cephalopoda, and modern Nautilus differs much in its outer morphology from all other recent species; its external shell and pinhole camera eye are the most prominent distinguishing characters. A further unique feature of Nautilus within the cephalopods is the lack of suckers or hooks on the tentacles. Instead, the animals use adhesive structures present on the digital tentacles. Earlier studies focused on the general tentacle morphology and put little attention on the adhesive gland system. Our results show that the epithelial parts on the oral adhesive ridge contain three secretory cell types (columnar, goblet, and cell type 1) that differ in shape and granule size. In the non-adhesive aboral epithelium, two glandular cell types (cell types 2 and 3) are present; these were not mentioned in any earlier study and differ from the cells in the adhesive area. The secretory material of all glandular cell types consists mainly of neutral mucopolysaccharide units, whereas one cell type in the non-adhesive epithelium also reacts positive for acidic mucopolysaccharides. The present data indicate that the glue in Nautilus consists mainly of neutral mucopolysaccharides. The glue seems to be a viscous carbohydrate gel, as known from another cephalopod species. De-attachment is apparently effectuated mechanically, i.e., by muscle contraction of the adhesive ridges and tentacle retraction. PMID:22221553

Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts.

PubMed

Motté, Evi; Szepessy, Edit; Suenens, Krista; Stangé, Geert; Bomans, Myriam; Jacobs-Tulleneers-Thevissen, Daniel; Ling, Zhidong; Kroon, Evert; Pipeleers, Daniel

2014-11-01

β-Cells generated from large-scale sources can overcome current shortages in clinical islet cell grafts provided that they adequately respond to metabolic variations. Pancreatic (non)endocrine cells can develop from human embryonic stem (huES) cells following in vitro derivation to pancreatic endoderm (PE) that is subsequently implanted in immune-incompetent mice for further differentiation. Encapsulation of PE increases the proportion of endocrine cells in subcutaneous implants, with enrichment in β-cells when they are placed in TheraCyte-macrodevices and predominantly α-cells when they are alginate-microencapsulated. At posttransplant (PT) weeks 20-30, macroencapsulated huES implants presented higher glucose-responsive plasma C-peptide levels and a lower proinsulin-over-C-peptide ratio than human islet cell implants under the kidney capsule. Their ex vivo analysis showed the presence of single-hormone-positive α- and β-cells that exhibited rapid secretory responses to increasing and decreasing glucose concentrations, similar to isolated human islet cells. However, their insulin secretory amplitude was lower, which was attributed in part to a lower cellular hormone content; it was associated with a lower glucose-induced insulin biosynthesis, but not with lower glucagon-induced stimulation, which together is compatible with an immature functional state of the huES-derived β-cells at PT weeks 20-30. These data support the therapeutic potential of macroencapsulated huES implants but indicate the need for further functional analysis. Their comparison with clinical-grade human islet cell grafts sets references for future development and clinical translation. Copyright © 2014 the American Physiological Society.

IL-6, IL-8, MMP-2, MMP-9 are overexpressed in Fanconi anemia cells through a NF-κB/TNF-α dependent mechanism.

PubMed

Epanchintsev, Alexey; Shyamsunder, Pavithra; Verma, Rama S; Lyakhovich, Alex

2015-12-01

Fanconi anemia (FA) is a rare autosomal recessive genetic disorder associated with a bone-marrow failure, genome instability, hypersensitivity to DNA crosslinking agents and a predisposition to cancer. Mutations have been documented in 16 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its symptoms. FA cells have been characterized by an overproduction of cytokines, MAPKs, and Interleukins. Through this study we have identified the overexpression of additional secretory factors such as IL-6, IL-8, MMP-2, and MMP-9 in FA cells and in cells depleted of FANCA or FANCC and proved that their expression is under the control of NF-κB/TNF-α signaling pathways. We also demonstrated that these overexpressed secretory factors were effective in promoting the proliferation, migration, and invasion of surrounding tumor cells a fundamental event in the process of epithelial mesenchymal transition (EMT) and that they also modulated the expression of EMT markers such as E-cadherin and SNAIL. Overall our data suggest that the upregulation of EMT promoting factors in FA may contribute to predisposing FA patients to cancer, thereby providing new insights into possible therapeutic interventions. © 2014 Wiley Periodicals, Inc.

Ultrastructure of the labrum and foregut of Derocheilocaris remanei (Crustacea, Mystacocarida).

PubMed

Herrera-Alvarez, Letizia; Fernández, Isabel; Benito, Jesús; Pardos, Fernando

1996-11-01

The cuticle-lined foregut of Derocheilocaris remanei consists of the mouth with its associated labrum, and an undifferentiated esophagus. It is separated from the midgut by an esophageal valve. The labrum is a conspicuous structure moved by five pairs of muscles (four dorsoventral and one longitudinal). Four pairs of subcuticular glands open to its inner face forming two longitudinal, lateral rows of cuticular pores. Each secretory unit is composed of a glandular component (with one or two secretory cells), a neck cell, and a duct cell. In addition, a single gland cell opens mesially into the buccal cavity. The ventrally located mouth is a complex structure characterized by a filter-like system, a sensory organ, and epithelial cells with highly developed microvilli. The esophagus is a simple tube with a characteristic curvature following the mouth. It has a rounded cross section and a triradiate lumen. A layer of circular musculature surrounds this region. The end of the esophagus protrudes into the midgut lumen forming the so-called esophageal valve. The ultrastructural features of the foregut, with the presence of a mucus-trapping mechanism, a relatively well-developed filter system and associated structures and an esophagus lacking glands confirm the microphagic feeding habits of mystacocarids. © 1996 Wiley-Liss, Inc. Copyright © 1996 Wiley-Liss, Inc.

Exocyst sec5 regulates exocytosis of newcomer insulin granules underlying biphasic insulin secretion.

PubMed

Xie, Li; Zhu, Dan; Kang, Youhou; Liang, Tao; He, Yu; Gaisano, Herbert Y

2013-01-01

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca(2+) channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.