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Sample records for human cysteine-rich secretory

  1. Cysteine-rich mini-proteins in human biology.

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    Lavergne, Vincent; Taft, Ryan J; Alewood, Paul F

    2012-01-01

    Understanding the relationship between structure and function underpins both biochemistry and chemical biology, and has enabled the discovery of numerous agricultural and therapeutic agents. Small cysteine-rich proteins, which form a unique set of protein frameworks and folds, are found in all living organisms and often play crucial roles as hormones, growth factors, ion channel modulators and enzyme inhibitors in various biological pathways. Here we review secreted human cysteine-rich mini-proteins, classify them into broad families and briefly describe their structure and function. To systematically investigate this protein sub-class we designed a step-wise high throughput algorithm that is able to isolate the mature and active forms of human secreted cysteine-rich proteins (up to 200 amino acids in length) and extract their cysteine scaffolds. We limited our search to frameworks that contain an even number of cysteine residues (5% cysteine residues led to the identification of 22 cysteine-rich frameworks representing 21 protein families. Analysis of their molecular targets showed that these mini-proteins are frequently ligands for G protein- and enzyme-coupled receptors, transporters, extracellular enzyme inhibitors, and antimicrobial peptides. It is clear that these human secreted mini-proteins possess a wide diversity of frameworks and folds, some of which are conserved across the phylogenetic spectrum. Further study of these proteins will undoubtedly lead to insights into unresolved questions of basic biology, and the development of system-specific human therapeutics.

  2. Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy

    DEFF Research Database (Denmark)

    Horne, A W; Duncan, W C; King, A E

    2009-01-01

    gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT-PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human...... chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P ... to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression...

  3. Cobra venom contains a pool of cysteine-rich secretory proteins.

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    Osipov, Alexey V; Levashov, Mikhail Yu; Tsetlin, Victor I; Utkin, Yuri N

    2005-03-04

    A large family of cysteine-rich secretory proteins (CRISPs) includes proteins of different origin, the function of the majority of CRISPs being unknown. For CRISPs isolated from snake venom, two types of activities were found: two proteins blocked cyclic nucleotide-gated ion channels, several others blocked potassium-stimulated smooth muscle contraction. Thus, snake CRISPs represent potentially valuable tools for studies of ion channels, which makes promising a search for new CRISPs. Here we report on the isolation of several novel CRISPs from the venoms of Asian cobra Naja kaouthia and African cobra Naja haje using a combination of different types of liquid chromatography. Four CRISP variants were identified in N. kaouthia venom and three proteins, one of them acidic, were found in N. haje venom. Acidic CRISP was found in a reptilian venom for the first time. Our data suggest that each cobra venom contains a pool of different CRISPs.

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

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    Sergey Shabelnikov

    Full Text Available 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.

  5. Cysteine-Rich Secretory Proteins (CRISP and their role in mammalian fertilization

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    Débora J Cohen

    2011-01-01

    Full Text Available Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2. In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.

  6. Crovirin, a snake venom cysteine-rich secretory protein (CRISP with promising activity against Trypanosomes and Leishmania.

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    Camila M Adade

    2014-10-01

    Full Text Available The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP from Cvv venom with promising activity against trypanosomes and Leishmania.Crude venom extract was loaded onto a reverse phase analytical (C8 column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml. A considerably higher concentration (20 µg/ml of crovirin was required to elicit only limited toxicity on mammalian cells.This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

  7. Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

    Science.gov (United States)

    Adade, Camila M; Carvalho, Ana Lúcia O; Tomaz, Marcelo A; Costa, Tatiana F R; Godinho, Joseane L; Melo, Paulo A; Lima, Ana Paula C A; Rodrigues, Juliany C F; Zingali, Russolina B; Souto-Padrón, Thaïs

    2014-10-01

    The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

  8. Expression and localization of cysteine-rich secretory protein-3 (CRISP-3) in the prepubertal and postpubertal male horse.

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    Fedorka, C E; Scoggin, K E; Squires, E L; Ball, B A; Troedsson, M H T

    2017-01-01

    The seminal plasma protein, cysteine-rich secretory protein-3 (CRISP-3), has been correlated with increased fertility and first-cycle conception rates, and has been suggested to be involved in the modulation of polymorphonuclear neutrophil and phagocytosis of spermatozoa during the inflammatory response to breeding in the horse. Previous research demonstrated that equine CRISP-3 is located in both the ampulla of the vas deferens and the seminal vesicles. However, this was done with nonquantitative laboratory techniques. In humans and rodents, CRISP-3 has been described as an androgen-dependent protein, but the effect of androgens on the expression of CRISP-3 has not been investigated in the horse. The objectives of this study were to (a) confirm and quantify the expression of CRISP-3 in the male equine reproductive tract, (b) describe the localization of CRISP-3 within the specific tissues which express it, and (c) determine if expression of CRISP-3 increases after puberty. We hypothesized that expression of CRISP-3 would be expressed in both the ampulla of the vas deferens and the seminal vesicles, and expression would increase after puberty. Tissues were collected postmortem from three prepubertal colts (3 years). Tissue samples were collected from the ampulla of vas deferens, seminal vesicles, bulbourethral gland, prostate gland, testis, as well as the cauda, corpus, and caput aspects of the epididymis. Quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) were performed using an equine-specific CRISP-3 designed primer and monocolonal antibody. A mixed linear additive model was used to compare mRNA expression between age groups, and significance was set to P CRISP-3 mRNA was found primarily in the ampulla of vas deferens with lesser expression in the seminal vesicles. Expression of CRISP-3 was higher in the postpubertal stallion when compared with the prepubertal colt for the ampulla (P CRISP-3 is primarily located in the glandular

  9. Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

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    Suzuki, Nobuhiro [Department of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan); Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Yamazaki, Yasuo [Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Brown, R. Lane [Neurological Science Institute, Oregon Health and Science University, Beaverton, Oregon 97006 (United States); Fujimoto, Zui [Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Morita, Takashi, E-mail: tmorita@my-pharm.ac.jp [Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Mizuno, Hiroshi, E-mail: tmorita@my-pharm.ac.jp [Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); VALWAY Technology Center, NEC Soft Ltd, Koto-ku, Tokyo 136-8627 (Japan); Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, Tsukuba, Ibaraki 305-8566 (Japan); Department of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan)

    2008-10-01

    The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn{sup 2+}-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn{sup 2+} ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn{sup 2+} binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.

  10. Investigating possible biological targets of Bj-CRP, the first cysteine-rich secretory protein (CRISP) isolated from Bothrops jararaca snake venom.

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    Lodovicho, Marina E; Costa, Tássia R; Bernardes, Carolina P; Menaldo, Danilo L; Zoccal, Karina F; Carone, Sante E; Rosa, José C; Pucca, Manuela B; Cerni, Felipe A; Arantes, Eliane C; Tytgat, Jan; Faccioli, Lúcia H; Pereira-Crott, Luciana S; Sampaio, Suely V

    2017-01-04

    Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Cysteine-rich domain of human ADAM 12 (meltrin alpha) supports tumor cell adhesion

    DEFF Research Database (Denmark)

    Iba, K; Albrechtsen, R; Gilpin, B J

    1999-01-01

    The ADAMs (A disintegrin and metalloprotease) comprise a family of membrane-anchored cell surface proteins with a putative role in cell-cell and/or cell-matrix interactions. By immunostaining, ADAM 12 (meltrin alpha) was up-regulated in several human carcinomas and could be detected along the tumor...... cell membranes. Because of this intriguing staining pattern, we investigated whether human ADAM 12 supports tumor cell adhesion. Using an in vitro assay using recombinant polypeptides expressed in Escherichia coli, we examined the ability of individual domains of human ADAM 12 and ADAM 15 to support...... tumor cell adhesion. We found that the disintegrin-like domain of human ADAM 15 supported adhesion of alphavbeta3-expressing A375 melanoma cells. In the case of human ADAM 12, however, recombinant polypeptides of the cysteine-rich domain but not the disintegrin-like domain supported cell adhesion...

  12. Blocking effect and crystal structure of natrin toxin, a cysteine-rich secretory protein from Naja atra venom that targets the BKCa channel.

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    Wang, Jing; Shen, Bing; Guo, Min; Lou, Xiaohua; Duan, Yuanyuan; Cheng, Xin Ping; Teng, Maikun; Niu, Liwen; Liu, Qun; Huang, Qingqiu; Hao, Quan

    2005-08-02

    Cysteine-rich secretory proteins (CRISPs) are widespread in snake venoms. Some members of these CRISPs recently have been found to block L-type Ca(2+) channels or cyclic nucleotide-gated ion (CNG) channels. Here, natrin purified from Naja atra venom, a member of the CRISP family, can induce a further contractile response in the endothelium-denuded thoracic aorta of mouse which has been contracted by a high-K(+) solution. Further experiments show it can block the high-conductance calcium-activated potassium (BK(Ca)) channel in a concentration-dependent manner with an IC(50) of 34.4 nM and a Hill coefficient of 1.02, which suggests that only a single natrin molecule is required to bind an ion channel to block BK(Ca) current. The crystal structure of natrin displaying two domains in tandem shows its cysteine-rich domain (CRD) has relatively independent flexibility, especially for the C-terminal long loop (loop I) of CRD to participate in the interface of two domains. On the basis of previous studies of CNG channel and L-Ca(2+) channel blockers, and the sequence and structural comparison of natrin and stecrisp, the deviation of the vital loop I of CRD is suggested to contribute to different effects of some CRISPs in protein-protein interaction.

  13. Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins*

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    Mehus, Aaron A.; Muhonen, Wallace W.; Garrett, Scott H.; Somji, Seema; Sens, Donald A.; Shabb, John B.

    2014-01-01

    Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14N- or 15N-iodoacetamide. Absolute quantitation was achieved using 15N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These

  14. Quantitation of human metallothionein isoforms: a family of small, highly conserved, cysteine-rich proteins.

    Science.gov (United States)

    Mehus, Aaron A; Muhonen, Wallace W; Garrett, Scott H; Somji, Seema; Sens, Donald A; Shabb, John B

    2014-04-01

    Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with ¹⁴N- or ¹⁵N-iodoacetamide. Absolute quantitation was achieved using ¹⁵N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells

  15. Molecular mechanisms involved in gamete interaction: evidence for the participation of cysteine-rich secretory proteins (CRISP) in sperm-egg fusion.

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    Da Ros, V; Busso, D; Cohen, D J; Maldera, J; Goldweic, N; Cuasnicu, P S

    2007-01-01

    Epididymal protein DE and testicular protein Tpx-1 are two cysteine-rich secretory proteins also known as CRISP-1 and CRISP-2, respectively. DE/ CRISP-1 is localised on the equatorial segment of acrosome-reacted sperm and participates in rat gamete fusion through its binding to egg-complementary sites. Recent results using bacterially-expressed recombinant fragments of DE as well as synthetic peptides revealed that the ability of DE to bind to the egg surface and inhibit gamete fusion resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family (Signature 2). Given the high degree of homology between DE/CRISP-1 and Tpx-1/CRISP-2, we also explored the potential participation of the testicular intra-acrosomal protein in gamete fusion. Results showing the ability of recombinant Tpx-1 to bind to the surface of rat eggs (evaluated by indirect immunofluorescence) and to significantly inhibit zona-free egg penetration, support the participation of this protein in gamete fusion through its interaction with egg-binding sites. Interestingly, rat Tpx-1 exhibits only two substitutions in Signature 2 when compared to this region in DE. Together, these results provide evidence for the involvement of both epididymal DE/CRISP-1 and testicular Tpx-1/CRISP-2 in gamete fusion suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilisation.

  16. Association of the cysteine-rich secretory protein-3 (CRISP-3) and some of its polymorphisms with the quality of cryopreserved stallion semen.

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    Usuga, Alexandra; Rojano, Benjamín A; Restrepo, Giovanni

    2017-08-31

    Contribution of seminal plasma proteins to semen freezability has been reported in several species, suggesting these proteins as genetic markers. The aim of this study was to evaluate the relationship between cysteine-rich secretory protein-3 (CRISP-3) and some of its single-nucleotide polymorphisms (SNPs) with post-thawing semen quality in stallions. DNA was obtained from 100 stallions, regions of interest were amplified by polymerase chain reaction and sequenced. Evaluated SNPs within the equine CRISP-3 gene were CRISP3c.+199A>G (SNP1), CRISP3c.+566C>A (SNP2), CRISP3c.+622G>A (SNP3) and CRISP3c.+716A>G (SNP4). CRISP-3 protein content in seminal plasma was determined by enzyme-linked immunosorbent assay. Semen from 30 stallions was cryopreserved and post-thaw motility, kinetics, abnormal morphology (AM), sperm vitality (SV) and membrane integrity (MI) were evaluated. Generalized linear models were fitted and means were compared using Tukey's test. Correlation and regression analyses were performed. For SNP1 and SNP3, the AA genotype had the highest results for motility and MI; for SNP2, the best results for motility and AM were obtained with the CC genotype. For SNP4, the GG genotype had the lowest results, except for MI. A high level of CRISP-3 protein in seminal plasma had the best results for motility, kinetics, SV and AM. In conclusion, there was a relationship between CRISP-3 genotype and seminal plasma protein and post-thawing semen quality in stallions.

  17. Characterization of a novel human scavenger receptor cysteine-rich molecule SCART1 expressed by lymphocytes

    DEFF Research Database (Denmark)

    Holm, D.; Fink, D. R.; Steffensen, M. A.

    2013-01-01

    of hSCART1 in the small intestine and colon. An antibody raised against an N-terminal hSCART1 peptide stains a subset of cells in the small intestine, stomach, and gall bladder, and it also stains placental villi. In conclusion, the characterization of hSCART1 at the mRNA and protein level suggests...... that displays synteny to the position of mSCART1 in the murine genome. The primary structure of hSCART1 was established by molecular cloning. The longest cDNA sequence of hSCART1 that was found is 2200bp and encodes a protein composed of a signal peptide, 5 SRCR domains, and an in-frame potential cytoplasmic...... domain. Shorter splice forms have also been isolated. Quantitative real-time PCR analysis on human blood-fractions has shown that hSCART1 is expressed primarily by CD4(+) and CD8(+) T lymphocytes with either αβ or γδ T cell receptors, and real-time PCR on 22 different human tissues showed high expression...

  18. Three-dimensional solution structure and conformational plasticity of the N-terminal scavenger receptor cysteine-rich domain of human CD5.

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    Garza-Garcia, Acely; Esposito, Diego; Rieping, Wolfgang; Harris, Richard; Briggs, Cherry; Brown, Marion H; Driscoll, Paul C

    2008-04-18

    The lymphocyte receptor CD5 influences cell activation by modifying the strength of the intracellular response initiated by antigen engagement. Regulation through CD5 involves the interaction of one or more of its three scavenger receptor cysteine-rich domains present in the extracellular region. Here, we present the 3D solution structure of a non-glycosylated double mutant of the N-terminal domain of human CD5 expressed in Escherichia coli (eCD5d1m), which has enhanced solubility compared to the non-glycosylated wild-type (eCD5d1). In common with a glycosylated form expressed in Pichia pastoris, the [(15)N,(1)H]-correlation spectra of both eCD5d1 and eCD5d1m exhibit non-uniform temperature-dependent signal intensities, indicating extensive conformational fluctuations on the micro-millisecond timescale. Although approximately one half of the signals expected for the domain are absent at 298 K, essentially complete resonance assignments and a solution structure could be obtained at 318 K. Because of the sparse nature of the experimental restraint data and the potentially important contribution of conformational exchange to the nuclear Overhauser effect peak intensity, we applied inferential structure determination to calculate the eCD5d1m structure. The inferential structure determination ensemble has similar features to that obtained by traditional simulated annealing methods, but displays superior definition and structural quality. The eCD5d1m structure is similar to other members of the scavenger receptor cysteine-rich superfamily, but the position of the lone alpha helix differs due to interactions with the unique N-terminal region of the domain. The availability of an experimentally tractable form of CD5d1, together with its 3D structure, provides new tools for further investigation of its function within intact CD5.

  19. Combined treatment with troglitazone and lovastatin inhibited epidermal growth factor-induced migration through the downregulation of cysteine-rich protein 61 in human anaplastic thyroid cancer cells.

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    Li-Han Chin

    Full Text Available Our previous studies have demonstrated that epidermal growth factor (EGF can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61 in human anaplastic thyroid cancer (ATC cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB and extracellular signal-regulated kinase (ERK, were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC.

  20. Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma

    DEFF Research Database (Denmark)

    Udby, Lene; Sørensen, Ole E; Pass, Jesper

    2004-01-01

    (28 kDa), we hypothesized that CRISP-3 in plasma was bound to another component. This was supported by size-exclusion chromatography and immunoprecipitation of plasma proteins. The binding partner was identified by mass spectrometry as alpha(1)B-glycoprotein (A1BG), which is a known plasma protein...... and is held together by strong electrostatic forces. Similar complexes have been described between toxins from snake venom and A1BG-like plasma proteins from opossum species. In these cases, complex formation inhibits the toxic effect of snake venom metalloproteinases or myotoxins and protects the animal from...... of unknown function and a member of the immunoglobulin superfamily. We demonstrate that CRISP-3 is a specific and high-affinity ligand of A1BG with a dissociation constant in the nanomolar range as evidenced by surface plasmon resonance. The A1BG-CRISP-3 complex is noncovalent with a 1:1 stoichiometry...

  1. Cloning and characterization of SCART1, a novel scavenger receptor cysteine-rich type I transmembrane molecule

    DEFF Research Database (Denmark)

    Holm, Dorte; Fink, Dorte Rosenbek; Grønlund, Jørn

    2009-01-01

    We have cloned and characterized a novel murine transmembrane molecule, mSCART1 belonging to the scavenger receptor cysteine-rich superfamily. The cDNA encodes a polypeptide chain of 989 amino acids, organized as a type I transmembrane protein that contains eight extracellular SRCR domains followed...... family of the SRCR superfamily. Finally, a novel human scavenger receptor cysteine-rich molecule with high homology to mSCART1 was identified by searching in the human genomic databases using the mSCART1 cDNA sequence....

  2. Chemical detection of cysteine-rich circular petides in selected ...

    African Journals Online (AJOL)

    Cysteine-rich circular peptides (CRCs) comprise a large family of gene encoded and low molecular weight polypeptides that has recently engaged the attention of scientists. This class of peptides exhibit a continuous circular configuration and a cystine knot backbone, which defines their resilient nature-directed structural ...

  3. Cysteine-rich venom proteins from the snakes of Viperinae subfamily - molecular cloning and phylogenetic relationship.

    Science.gov (United States)

    Ramazanova, Anna S; Starkov, Vladislav G; Osipov, Alexey V; Ziganshin, Rustam H; Filkin, Sergey Yu; Tsetlin, Victor I; Utkin, Yuri N

    2009-01-01

    Cysteine-rich proteins found in animal venoms (CRISP-Vs) are members of a large family of cysteine-rich secretory proteins (CRISPs). CRISP-Vs acting on different ion channels were found in venoms or mRNA (cDNA) encoding CRISP-Vs were cloned from snakes of three main families (Elapidae, Colubridae and Viperidae). About thirty snake CRISP-Vs were sequenced so far, however no complete sequence for CRISP-V from Viperinae subfamily was reported. We have cloned and sequenced for the first time cDNAs encoding CRISP-Vs from Vipera nikolskii and Vipera berus vipers (Viperinae). The deduced mature CRISP-V amino acid sequences consist of 220 amino acid residues. Phylogenetic analysis showed that viper proteins are closely related to those of Crotalinae snakes. The presence of CRISP-V in the V. berus venom was revealed using a combination of gel-filtration chromatography, electrophoresis and MALDI mass spectrometry. The finding of the putative channel blocker in viper venom may indicate its action on prey nervous system.

  4. Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms.

    Science.gov (United States)

    Jin, Yang; Lu, Qiumin; Zhou, Xingding; Zhu, Shaowen; Li, Rui; Wang, Wanyu; Xiong, Yuliang

    2003-10-01

    Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes.

  5. A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica.

    Science.gov (United States)

    Zhong, Jian; Wang, Wenhong; Yang, Xiaomei; Yan, Xiuwen; Liu, Rui

    2013-01-01

    Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  6. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

    Directory of Open Access Journals (Sweden)

    Berlin Londono-Renteria

    2015-10-01

    Full Text Available Dengue virus (DENV is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379, whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

  7. Antibody mapping and tissue localization of globular and cysteine-rich regions of perlecan domain III

    DEFF Research Database (Denmark)

    Couchman, J R; Ljubimov, A V; Sthanam, M

    1995-01-01

    Perlecan is the best-characterized basement membrane heparan sulfate proteoglycan. It has a large (approximately 400 KD) core protein consisting of five distinct domains. Domain III, a centrally located domain, contains three globular domains separated by cysteine-rich epidermal growth factor (EG...

  8. Accurate disulfide-bonding network predictions improve ab initio structure prediction of cysteine-rich proteins.

    Science.gov (United States)

    Yang, Jing; He, Bao-Ji; Jang, Richard; Zhang, Yang; Shen, Hong-Bin

    2015-12-01

    Cysteine-rich proteins cover many important families in nature but there are currently no methods specifically designed for modeling the structure of these proteins. The accuracy of disulfide connectivity pattern prediction, particularly for the proteins of higher-order connections, e.g., >3 bonds, is too low to effectively assist structure assembly simulations. We propose a new hierarchical order reduction protocol called Cyscon for disulfide-bonding prediction. The most confident disulfide bonds are first identified and bonding prediction is then focused on the remaining cysteine residues based on SVR training. Compared with purely machine learning-based approaches, Cyscon improved the average accuracy of connectivity pattern prediction by 21.9%. For proteins with more than 5 disulfide bonds, Cyscon improved the accuracy by 585% on the benchmark set of PDBCYS. When applied to 158 non-redundant cysteine-rich proteins, Cyscon predictions helped increase (or decrease) the TM-score (or RMSD) of the ab initio QUARK modeling by 12.1% (or 14.4%). This result demonstrates a new avenue to improve the ab initio structure modeling for cysteine-rich proteins. http://www.csbio.sjtu.edu.cn/bioinf/Cyscon/ zhng@umich.edu or hbshen@sjtu.edu.cn. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  9. Molecular variability in the cysteine rich protein of potato virus M

    OpenAIRE

    Tabasinejad, Fatemeh; Jafarpour, Behrooz; Zakiaghl, Mohammad; Siampour, Majid; Rowhani, Hamid; Mehrvar, Mohsen

    2015-01-01

    The potato virus M (PVM), belonging to the genus Carlavirus, is a worldwide endemic pathogen in potato fields. p11 is an 11–16 kDa protein encoded by the last open reading frame of PVM which contains cysteine rich proteins (CRPs) motif. CRPs have been identified as suppressors of gene silencing. In this study the p11 gene from 28 PVM isolates, including 16 new isolates from Iran, were used to determine the global genetic structure of PVM populations. Pairwise nucleotide sequence identity scor...

  10. Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx{sub a}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Chul Won; Kim, Jim Il [Chonnam National Univ., Gwangju (Korea, Republic of); Sato, Kazuki [Fukuoka Women' s Univ., Fukuoka (Japan)

    2013-06-15

    Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTx{sub a}), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca{sup 2+} release from sarcoplasmic reticulum (SR). IpTx{sub a} increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states.

  11. Molecular variability in the cysteine rich protein of potato virus M.

    Science.gov (United States)

    Tabasinejad, Fatemeh; Jafarpour, Behrooz; Zakiaghl, Mohammad; Siampour, Majid; Rowhani, Hamid; Mehrvar, Mohsen

    2015-09-01

    The potato virus M (PVM), belonging to the genus Carlavirus, is a worldwide endemic pathogen in potato fields. p11 is an 11-16 kDa protein encoded by the last open reading frame of PVM which contains cysteine rich proteins (CRPs) motif. CRPs have been identified as suppressors of gene silencing. In this study the p11 gene from 28 PVM isolates, including 16 new isolates from Iran, were used to determine the global genetic structure of PVM populations. Pairwise nucleotide sequence identity scores showed that global PVM CRP sequence similarity was between 69.3 and 100 %. This genetic diversity divided the 28 isolates into two main divergent phylogenetic clades. The rate of genetic diversity and non-synonymous to synonymous mutations (dN/dS) were significantly different between these two clades. Analysis showed that PVM CP is under significant negative selection pressure with the global ω value of 0.260.

  12. Establishing human lacrimal gland cultures with secretory function.

    Directory of Open Access Journals (Sweden)

    Shubha Tiwari

    Full Text Available PURPOSE: Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in-vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures. METHODS: Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM, mesenchymal (Vimentin, CD90 and myofibroblastic (α-SMA, S-100 origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme post carbachol (100 µM stimulation by ELISA. RESULTS: Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%, high ALDH1 (3.8±1.26% and c-kit (6.7±2.0%. Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15-20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed 'spherules' with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml, lysozyme (24.36 to 144.74 ng/ml and lactoferrin (32.45 to 40.31 ng/ml in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons. CONCLUSION: The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also

  13. Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

    Energy Technology Data Exchange (ETDEWEB)

    Nakashima, Yukiko; Morimoto, Mayuka [Department of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women' s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179 (Japan); Toda, Ken-ichi [Department of Dermatology, Kitano Hospital, The Tazuke Kofukai Nedical Institute, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8480 (Japan); Shinya, Tomohiro; Sato, Keizo [Department of Clinical Biochemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Nobeoka, Miyazaki 882-8508 (Japan); Takahashi, Satoru, E-mail: imwalrus@mukogawa-u.ac.jp [Department of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women' s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179 (Japan); Institute for Biosciences, Mukogawa Women' s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179 (Japan)

    2015-07-03

    Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed, because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells.

  14. Arabidopsis cysteine-rich receptor-like kinase 45 positively regulates disease resistance to Pseudomonas syringae.

    Science.gov (United States)

    Zhang, Xiujuan; Han, Xiaomin; Shi, Rui; Yang, Guanyu; Qi, Liwang; Wang, Ruigang; Li, Guojing

    2013-12-01

    Arabidopsis cysteine-rich receptor-like protein kinase 45 (CRK45) was found to be involved in ABA signaling in Arabidopsis thaliana previously. Here, we reported that it also positively regulates disease resistance. The CRK45 overexpression plants increased expression of the defense genes, and enhanced resistance to Pseudomonas syringae whereas the crk45 mutant were more sensitive to P. syringae and weakened expression of the defense genes, compared to the wild type. We also found that treatment with P. syringae leads to a declined expression of CRK45 in the npr1 mutant and the NahG transgenic plants. At the same time, significantly decreased expression of CRK45 transcript in the wrky70 mutant than that in the wild type was also detected. Our results suggested that CRK45 acted as a positive regulator in Arabidopsis disease resistance, and was regulated downstream of NPR1 and WRKY70 at the transcriptional level. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  15. Cysteine-rich protein 1 (CRP1 regulates actin filament bundling

    Directory of Open Access Journals (Sweden)

    Fraley Tamara S

    2005-12-01

    Full Text Available Abstract Background Cysteine-rich protein 1 (CRP1 is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified. Results CRP1 expression increased actin bundling in rat embryonic fibroblasts. Although CRP1 did not affect the bundling activity of α-actinin, CRP1 was found to stabilize the interaction of α-actinin with actin bundles and to directly bundle actin microfilaments. Using confocal and photobleaching fluorescence resonance energy transfer (FRET microscopy, we demonstrate that there are two populations of CRP1 localized along actin stress fibers, one associated through interaction with α-actinin and one that appears to bind the actin filaments directly. Consistent with a role in regulating actin filament cross-linking, CRP1 also localized to the membrane ruffles of spreading and PDGF treated fibroblasts. Conclusion CRP1 regulates actin filament bundling by directly cross-linking actin filaments and stabilizing the interaction of α-actinin with actin filament bundles.

  16. Sortase A-aided Escherichia coli expression system for functional osteoprotegerin cysteine-rich domain.

    Science.gov (United States)

    Jin, Mengmeng; Chen, Yuan; Zhao, Yunfeng; Che, Luyang; Ma, Yanyan; Li, Jingzhe; Wang, Yi; Tao, Hua; Ma, Juan; Pan, Bing; Liu, Changzhen; Huang, Peng

    2017-06-01

    As a natural inhibitor of the receptor activator of nuclear factor-кB ligand (RANKL), osteprotegerin (OPG) is considered a promising treatment for metabolic bone diseases. Typical approaches for preparing recombinant OPG or its derivatives employ eukaryotic expression systems. Due to the advantages of a prokaryotic expression system, which include its convenience, low cost, and abundant production, in this study, we establish a strategy for preparing functional OPG using the Escherichia coli expression system. After initial failures in preparation of OPG and its truncation, OPG cysteine-rich domain (OPG-CRD/OPGT) by using pET and pGEX vectors, we constructed a sortase A (SrtA)-aided E. coli expression system, in which the expressed protein was a self-cleaving SrtA fusion protein. Using this system, we successfully prepared the recombinant OPGT protein. The BIAcore analyses indicated that the prepared OPGT had high affinities in binding with RANKL and TRAIL. Cell experiments confirmed the inhibitory effects of the prepared OPGT on RANKL-induced osteoclast differentiation and TRAIL-induced tumor cell apoptosis. The sortase A-aided E. coli expression system for OPGT established in this study may contribute to further studies and commercial applications of OPG.

  17. Bleogens: Cactus-Derived Anti-Candida Cysteine-Rich Peptides with Three Different Precursor Arrangements

    Directory of Open Access Journals (Sweden)

    Shining Loo

    2017-12-01

    Full Text Available Cysteine-rich peptides (CRPs play important host-defense roles in plants. However, information concerning CRPs in the Cactaceae (cactus family is limited, with only a single cactus-derived CRP described to date. Here, we report the identification of 15 novel CRPs with three different precursor architectures, bleogens pB1-15 from Pereskia bleo of the Cactaceae family. By combining proteomic and transcriptomic methods, we showed that the prototype, bleogen pB1, contained 36 amino acid residues, a six-cysteine motif typical of the six-cysteine-hevein-like peptide (6C-HLP family, and a type I two-domain precursor consisting of an endoplasmic reticulum (ER and a mature domain. In contrast, the precursors of the other 14 bleogens contained a type II three-domain architecture with a propeptide domain inserted between the ER and the mature bleogen domain. Four of these 14 bleogens display a third type of architecture with a tandemly repeating bleogen domain. A search of the Onekp database revealed that <1% plant species possess three different precursor architectures for the biosynthesis of 6C-HLPs, including Lophophora williamsii, Pereskia aculeate, Portulaca cryptopetala, Portulaca oleracea, Portulaca suffruticosa, and Talinum sp. NMR analysis confirmed that bleogen pB1 has cystine-knot disulfide connectivity as well as a two-beta-sheet and a four-loop structural fold that is similar to other 6C-HLPs. Sequence analysis, structural studies, and in silico modeling revealed that bleogen pB1 has a cation-polar-cation motif, a signature heparin-binding motif that was confirmed by heparin affinity chromatography. Cell-based assays showed that bleogen pB1 is non-toxic to mammalian cells but functions as an anti-Candida peptide. Taken together, our findings provide insight into the occurrence, functions and precursor architectures of CRPs in the cactus family.

  18. Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways.

    NARCIS (Netherlands)

    Aarbiou, J.; Schadewijk, A. van; Stolk, J.; Sont, J.K.; Boer, W.I.; Rabe, K.F.; Krieken, J.H.J.M. van; Mauad, T.; Hiemstra, P.S.

    2004-01-01

    OBJECTIVE: The aim of this study was to analyze a possible contribution of human neutrophil defensins and secretory leukocyte proteinase inhibitor (SLPI) to the induction of airway epithelial changes such as squamous cell metaplasia. MATERIALS AND METHODS: The presence of these molecules and the

  19. Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    You Wanhui

    2012-04-01

    Full Text Available Abstract Background In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. Results We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs. In leaves, the CRP genes are silent and their coding regions contain dense, transposon-like methylation in CG, CHG and CHH contexts, which depends partly on the Pol IV/Pol V pathway and small RNAs. Methylation in the coding region is reduced, however, in the synergid cells of the female gametophyte, where the CRP genes are specifically expressed. Further demonstrating that expressed CRP genes lack gene body methylation, a CRP4-GFP fusion gene under the control of the constitutive 35 S promoter remains unmethylated in leaves and is transcribed to produce a translatable mRNA. By contrast, a CRP4-GFP fusion gene under the control of a CRP4 promoter fragment acquires CG and non-CG methylation in the CRP coding region in leaves similar to the silent endogenous CRP4 gene. Conclusions Unlike CG methylation in gene bodies, which does not dramatically affect Pol II transcription, combined CG and non-CG methylation in CRP coding regions is likely to

  20. The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors

    Directory of Open Access Journals (Sweden)

    Nicholas eYap

    2015-07-01

    Full Text Available The class A Scavenger Receptor (cA-SR family is a group of five evolutionarily related innate immune receptors. The cA-SRs are known for their promiscuous ligand binding; as they have been shown to bind bacteria such as Streptococcus pneumoniae, and Escherichia coli, as well as different modified forms of low-density lipoprotein. Three of the five family members possess a Scavenger Receptor Cysteine Rich (SRCR domain while the remaining two receptors lack the domain. Previous work has suggested that the Macrophage Associated Receptor with COllagenous structure (MARCO shares a recent common ancestor with the non-SRCR-containing receptors; however the origin of the SRCR domain within the cA-SRs remains unknown. We hypothesize that the SRCR domains of the cA-SRs have a common origin that predates teleost fish. Using the newly available sequence data from sea lamprey and ghost shark genome projects, we have shown that MARCO shares a common ancestor with the SRCR-containing proteins. In addition, we explored the evolutionary relationships within the SRCR domain by reconstructing the ancestral SRCR domains of the cA-SRs. We identified a motif that is highly conserved between the cA-SR SRCR domains and the ancestral SRCR domain that consist of WGTVCDD. We also show that the GRAEVYY motif, a functionally important motif within MARCO, is poorly conserved in the other cA-SRs and in the reconstructed ancestral domain. Further, we identified three sites within MARCO’s SRCR domain which are under positive selection. Two of these sites lie adjacent to the conserved WGTVCDD motif, and may indicate a potential biological function for these sites. Together these findings indicate a common origin of the SRCR domain within the cA-SRs; however different selective pressures between the proteins may have caused MARCOs SRCR domain to evolve to contain different functional motifs when compared to the other SRCR-containing cA-SRs.

  1. NFAP2, a novel cysteine-rich anti-yeast protein from Neosartorya fischeri NRRL 181: isolation and characterization

    OpenAIRE

    T?th, Lili?na; Kele, Zolt?n; Borics, Attila; Nagy, L?szl? G.; V?radi, Gy?rgyi; Vir?gh, M?t?; Tak?, Mikl?s; V?gv?lgyi, Csaba; Galg?czy, L?szl?

    2016-01-01

    The increasing incidence of fungal infections and damages due to drug-resistant fungi urges the development of new antifungal strategies. The cysteine-rich antifungal proteins from filamentous ascomycetes provide a feasible base for protection against molds due to their potent antifungal activity on them. In contrast to this, they show no or weak activity on yeasts, hence their applicability against this group of fungi is questionable. In the present study a 5.6?kDa anti-yeast protein (NFAP2)...

  2. Cellular source of human platelet secretory phospholipase A2.

    Science.gov (United States)

    Emadi, S; Mirshahi, M; Elalamy, I; Nicolas, C; Vargaftig, B B; Hatmi, M

    1998-02-01

    Platelets are one source of the group II extracellular form of phospholipase A2 (sPLA2) which is involved in the amplification of local and systemic inflammation. Although sPLA2 protein has been described in human platelets, its presence in human megakaryocytes has not been yet established. We demonstrated in this study that the human erythroleukaemia (HEL) cell line, which has megakaryoblastic features, constitutively expresses sPLA2. Using an anti-rhsPLA2 monoclonal antibody (mAb BA11) and dot-blot detection, we showed that HEL cells and platelets release sPLA2 into incubation medium upon stimulation by thrombin. Similar results were obtained for sPLA2 activity detected by a spectrofluorescence assay. Enzymatic activity was abolished by mAb BA11 and by protamine. In both cell types, although released, the major part of sPLA2 remained in the cell pellet, and was probably adsorbed at non-specific membrane sites. Double labelling experiments using mAb BA11 and an anti-GPIIb antiserum revealed the presence of sPLA2 in human bone-marrow megakaryocytes. The use of reverse transcription-polymerase chain reaction conjugated with hybridization analysis demonstrated the presence of mRNA encoding for sPLA2 in platelets and HEL cells. Expression of sPLA2 in platelets and megakaryocytes at both transcriptional and post-translational levels strongly argues in favour of a megakaryocytic origin of platelet sPLA2 and rules out a role for endocytosis of the enzyme from plasma by circulating platelets.

  3. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation

    DEFF Research Database (Denmark)

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G

    2016-01-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages...... and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD m......RNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA...

  4. Dual effect of neutrophils on secretory component production by human bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    C. Pilette

    2006-12-01

    Full Text Available A decreased bronchial expression of secretory component (SC was demonstrated in severe COPD, and correlated with neutrophils. Mechanisms of epithelial cell/neutrophils interactions remain however poorly understood. Calu-3 (human bronchial epithelial cells were incubated after confluence (in triplicate conditions with various ratios of activated neutrophils (0.5:1 to 15:1, neutrophils: Calu-3 cells. After 48hrs of co-culture supernatants were assayed for SC by ELISA. SC production by Calu-3 cells increased at intermediate neutrophil numbers (316±32 versus 193±19ng·ml–1, ratio of 5:1 versus control, mean±SEM of 3 experiments, p = 0.05. In contrast, a trend for decrease in SC was observed with high neutrophil numbers (111±19 versus 193±19ng·ml–1, ratio of 15:1 versus control, p = 0.06. The addition of secretory leukocyte protease inhibitor further increased SC upregulation at intermediate ratios, and inhibited the SC decrease at high neutrophil numbers. The mechanism of SC up-regulation by neutrophils did not implicate TNF-alpha or IL-1beta. This study provides direct evidence of a dual effect of neutrophils on epithelial SC. Our data suggest that neutrophils could differently affect epithelial immune secretory function according to the extent of neutrophil influx and/or to the reactivity of airway epithelial cells.

  5. Comparison in gene expression of secretory human endometrium using laser microdissection

    Directory of Open Access Journals (Sweden)

    Aida Tadateru

    2004-09-01

    Full Text Available Abstract Background The endometrium prepares for implantation under the control of steroid hormones. It has been suggested that there are complicated interactions between the epithelium and stroma in the endometrium during menstrual cycle. In this study, we demonstrate a difference in gene expression between the epithelial and stromal areas of the secretory human endometrium using microdissection and macroarray technique. Methods The epithelial and stromal areas were microdissected from the human endometrium during the secretory phase. RNA was extracted and amplified by PCR. Macroarray analysis of nearly 1000 human genes was carried out in this study. Some genes identified by macroarray analysis were verified using real-time PCR. Results In this study, changes in expression 5-fold were WAP four-disulfide core domain 2 (44.1 fold, matrix metalloproteinase 7 (40.1 fold, homeo box B5 (19.8 fold, msh homeo box homolog (18.8 fold, homeo box B7 (12.7 fold and protein kinase C, theta (6.4 fold. On the other hand, decorin (55.6 fold, discoidin domain receptor member 2 (17.3 fold, tissue inhibitor of metalloproteinase 1 (9 fold, ribosomal protein S3A (6.3 fold, and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (5.2 fold were strongly expressed in the stromal areas. WAP four-disulfide core domain 2 (19.4 fold, matrix metalloproteinase 7 (9.7-fold, decorin (16.3-fold and tissue inhibitor of metalloproteinase 1 (7.2-fold were verified by real-time PCR. Conclusions Some of the genes we identified with differential expression are related to the immune system. These results are telling us the new information for understanding the secretory human endometrium.

  6. Soilborne wheat mosaic virus (SBWMV 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing

    Directory of Open Access Journals (Sweden)

    Howard Amanda

    2005-03-01

    Full Text Available Abstract Amino acid sequence analyses indicate that the Soilborne wheat mosaic virus (SBWMV 19K protein is a cysteine-rich protein (CRP and shares sequence homology with CRPs derived from furo-, hordei-, peclu- and tobraviruses. Since the hordei- and pecluvirus CRPs were shown to be pathogenesis factors and/or suppressors of RNA silencing, experiments were conducted to determine if the SBWMV 19K CRP has similar activities. The SBWMV 19K CRP was introduced into the Potato virus X (PVX viral vector and inoculated to tobacco plants. The SBWMV 19K CRP aggravated PVX-induced symptoms and restored green fluorescent protein (GFP expression to GFP silenced tissues. These observations indicate that the SBWMV 19K CRP is a pathogenicity determinant and a suppressor of RNA silencing.

  7. Identification and Gene Expression Analysis of a Taxonomically Restricted Cysteine-Rich Protein Family in Reef-Building Corals

    Science.gov (United States)

    Sunagawa, Shinichi; DeSalvo, Michael K.; Voolstra, Christian R.; Reyes-Bermudez, Alejandro; Medina, Mónica

    2009-01-01

    The amount of genomic sequence information continues to grow at an exponential rate, while the identification and characterization of genes without known homologs remains a major challenge. For non-model organisms with limited resources for manipulative studies, high-throughput transcriptomic data combined with bioinformatics methods provide a powerful approach to obtain initial insights into the function of unknown genes. In this study, we report the identification and characterization of a novel family of putatively secreted, small, cysteine-rich proteins herein named Small Cysteine-Rich Proteins (SCRiPs). Their discovery in expressed sequence tag (EST) libraries from the coral Montastraea faveolata required the performance of an iterative search strategy based on BLAST and Hidden-Markov-Model algorithms. While a discernible homolog could neither be identified in the genome of the sea anemone Nematostella vectensis, nor in a large EST dataset from the symbiotic sea anemone Aiptasia pallida, we identified SCRiP sequences in multiple scleractinian coral species. Therefore, we postulate that this gene family is an example of lineage-specific gene expansion in reef-building corals. Previously published gene expression microarray data suggest that a sub-group of SCRiPs is highly responsive to thermal stress. Furthermore, data from microarray experiments investigating developmental gene expression in the coral Acropora millepora suggest that different SCRiPs may play distinct roles in the development of corals. The function of these proteins remains to be elucidated, but our results from in silico, transcriptomic, and phylogenetic analyses provide initial insights into the evolution of SCRiPs, a novel, taxonomically restricted gene family that may be responsible for a lineage-specific trait in scleractinian corals. PMID:19283069

  8. Identification and gene expression analysis of a taxonomically restricted cysteine-rich protein family in reef-building corals.

    Directory of Open Access Journals (Sweden)

    Shinichi Sunagawa

    Full Text Available The amount of genomic sequence information continues to grow at an exponential rate, while the identification and characterization of genes without known homologs remains a major challenge. For non-model organisms with limited resources for manipulative studies, high-throughput transcriptomic data combined with bioinformatics methods provide a powerful approach to obtain initial insights into the function of unknown genes. In this study, we report the identification and characterization of a novel family of putatively secreted, small, cysteine-rich proteins herein named Small Cysteine-Rich Proteins (SCRiPs. Their discovery in expressed sequence tag (EST libraries from the coral Montastraea faveolata required the performance of an iterative search strategy based on BLAST and Hidden-Markov-Model algorithms. While a discernible homolog could neither be identified in the genome of the sea anemone Nematostella vectensis, nor in a large EST dataset from the symbiotic sea anemone Aiptasia pallida, we identified SCRiP sequences in multiple scleractinian coral species. Therefore, we postulate that this gene family is an example of lineage-specific gene expansion in reef-building corals. Previously published gene expression microarray data suggest that a sub-group of SCRiPs is highly responsive to thermal stress. Furthermore, data from microarray experiments investigating developmental gene expression in the coral Acropora millepora suggest that different SCRiPs may play distinct roles in the development of corals. The function of these proteins remains to be elucidated, but our results from in silico, transcriptomic, and phylogenetic analyses provide initial insights into the evolution of SCRiPs, a novel, taxonomically restricted gene family that may be responsible for a lineage-specific trait in scleractinian corals.

  9. Ciliary and secretory differentiation of normal human middle ear epithelial cells.

    Science.gov (United States)

    Choi, Jae Young; Kim, Chang-Hoon; Lee, Won-Sang; Kim, Hee-Nam; Song, Kyoung-Seob; Yoon, Joo-Heon

    2002-04-01

    Recent technical advances now permit the serial culture of normal human middle ear epithelial (NHMEE) cells. However, the ciliary differentiation of these cells has not been achieved. The purpose of this study was to establish a culture system in order to differentiate serially cultured NHMEE cells into ciliated cells. If ciliated cells developed, the percentages of ciliated cells and secretory cells were measured throughout the duration of culture. We also examined the levels of mucin and lysozyme secretion and their mRNAs in a time-dependent manner. Human middle ear mucosa with a normal appearance was harvested and serially cultured after enzymatic disaggregation. These cells were cultured in an air-liquid interface (ALI) culture system for 2, 7, 14, 21 and 28 days after confluence. Ciliogenesis usually began 16-18 days after confluence. The percentage of ciliated cells detected by means of immunohistochemical staining increased over time up to a maximum of 10.6% but the percentage of secretory cells remained stable at approximately 40% throughout the duration of culture. By Day 14 after confluence, the amounts of mucin and lysozyme secretion, as measured by dot-blotting analysis, had increased significantly and then remained stable. The expression levels of mucin gene 5B (MUC5B), MUC8 and lysozyme increased with the duration of culture. MUC8 in particular showed a dramatic increase on Day 28 after confluence. In contrast, the level of MUC5AC mRNA peaked on Day 14 after confluence, and then decreased. In conclusion, ciliary differentiation of NHMEE cells can be induced using an ALI culture system. Our study also suggests that secretory function develops earlier than ciliogenesis, and that the expressions of MUC5B and MUC8 mRNAs increase as a function of differentiation.

  10. Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains

    DEFF Research Database (Denmark)

    Bikker, Floris J; Ligtenberg, Antoon J M; End, Caroline

    2004-01-01

    The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight fo...

  11. Structural studies of human glioma pathogenesis-related protein 1

    Energy Technology Data Exchange (ETDEWEB)

    Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu [College of Medicine, Nebraska Medical Center, Omaha, NE 68198-6495 (United States); Koski, Raymond A.; Bonafé, Nathalie [L2 Diagnostics LLC, 300 George Street, New Haven, CT 06511 (United States); College of Medicine, Nebraska Medical Center, Omaha, NE 68198-6495 (United States)

    2011-10-01

    Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

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

    Directory of Open Access Journals (Sweden)

    Stephen eSalton

    2013-08-01

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

  13. Salivary agglutinin and lung scavenger receptor cysteine-rich glycoprotein 340 have broad anti-influenza activities and interactions with surfactant protein D that vary according to donor source and sialylation

    DEFF Research Database (Denmark)

    Hartshorn, Kevan L.; Ligtenberg, Antoon; White, Mitchell R.

    2006-01-01

    has co-operative interactions with SP-D in viral neutralization and aggregation assays. We now report that salivary gp-340 can, in some cases, strongly antagonize certain antiviral activities of SP-D. This effect was associated with greater binding of salivary gp-340 to the carbohydrate recognition......We previously found that scavenger receptor cysteine-rich gp-340 (glycoprotein-340), isolated from lung or saliva, directly inhibits human IAVs (influenza A viruses). We now show that salivary gp-340 has broad antiviral activity against human, equine and porcine IAV strains. Although lung...... and salivary gp-340 are identical in protein sequence, salivary gp-340 from one donor had significantly greater antiviral activity against avian-like IAV strains which preferentially bind sialic acids in alpha(2,3) linkage. A greater density of alpha(2,3)-linked sialic acids was present on the salivary gp-340...

  14. Patterns of divergence of a large family of nodule cysteine-rich peptides in accessions of Medicago truncatula.

    Science.gov (United States)

    Nallu, Sumitha; Silverstein, Kevin A T; Zhou, Peng; Young, Nevin D; Vandenbosch, Kathryn A

    2014-05-01

    The nodule cysteine-rich (NCR) groups of defensin-like (DEFL) genes are one of the largest gene families expressed in the nodules of some legume plants. They have only been observed in the inverted repeat loss clade (IRLC) of legumes, which includes the model legume Medicago truncatula. NCRs are reported to play an important role in plant-microbe interactions. To understand their diversity we analyzed their expression and sequence polymorphisms among four accessions of M. truncatula. A significant expression and nucleotide variation was observed among the genes. We then used 26 accessions to estimate the selection pressures shaping evolution among the accessions by calculating the nucleotide diversity at non-synonymous and synonymous sites in the coding region. The mature peptides of the orthologous NCRs had signatures of both purifying and diversifying selection pressures, unlike the seed DEFLs, which predominantly exhibited purifying selection. The expression, sequence variation and apparent diversifying selection in NCRs within the Medicago species indicates rapid and recent evolution, and suggests that this family of genes is actively evolving to adapt to different environments and is acquiring new functions. © 2014 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  15. Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

    Energy Technology Data Exchange (ETDEWEB)

    Stiegler, A.; Burden, S; Hubbard, S

    2009-01-01

    Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 {angstrom} resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.

  16. Production of Secretory Leucocyte Protease Inhibitor (SLPI in Human Pancreatic β-Cells

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    Max Nyström

    1999-01-01

    Full Text Available Secretory leucocyte protease inhibitor (SLPI is a potent inhibitor of granulocyte elastase and cathepsin G, and also an inhibitor of pancreatic enzymes like trypsin, chymotrypsin and pancreatic elastase. SLPI has also been shown to inhibit HIV-1 infections by blocking viral DNA synthesis. Since SLPI is an inhibitor of pancreatic proteases we wished to investigate whether SLPI was also actually produced in the pancreas. M-RNA from human pancreatic tissue showed evidence of SLPI production using the reverse transcriptase polymer chain reaction technique (RTPCR. Using immunohistochemical methods SLPI was demonstrated in the β-ce1ls of the islets of Langerhans. The function could be local protease/antiprotease regulation or antiviral/antibacterial defence in the close vicinity of the cell surface, or even inside the β-cell itself.

  17. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

    Science.gov (United States)

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Praetorius, Jeppe; Borregaard, Niels; Petersen, Steen V

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  19. Arabidopsis cysteine-rich receptor-like kinase 45 functions in the responses to abscisic acid and abiotic stresses

    KAUST Repository

    Zhang, Xiujuan

    2013-06-01

    The phytohormone abscisic acid (ABA) regulates seed germination, plant growth and development, and response to abiotic stresses such as drought and salt stresses. Receptor-like kinases are well known signaling components that mediate plant responses to developmental and environmental stimuli. Here, we characterized the biological function of an ABA and stress-inducible cysteine-rich receptor-like protein kinase, CRK45, in ABA signaling in Arabidopsis thaliana. The crk45 mutant was less sensitive to ABA than the wild type during seed germination and early seedling development, whereas CRK45 overexpression plants were more sensitive to ABA compared to the wild type. Furthermore, overexpression of CRK45 led to hypersensitivity to salt and glucose inhibition of seed germination, whereas the crk45 mutant showed the opposite phenotypes. In addition, CRK45 overexpression plants had enhanced tolerance to drought. Gene expression analyses revealed that the expression of representative stress-responsive genes was significantly enhanced in CRK45 overexpression plants in response to salt stress. ABA biosynthetic genes such as NCED3,. 22NCED3, 9-Cis-Epoxycarotenoid Dioxygenase 3.NCED5,. 33NCED5, 9-Cis-Epoxycarotenoid Dioxygenase 5.ABA2,. 44ABA2, Abscisic Acid Deficient 2. and AAO355AAO3, Abscisic Aldehyde Oxidase 3. were also constitutively elevated in the CRK45 overexpression plants. We concluded that CRK45 plays an important role in ABA signaling that regulates Arabidopsis seeds germination, early seedling development and abiotic stresses response, by positively regulating ABA responses in these processes. © 2013 Elsevier Masson SAS.

  20. Two Microplitis demolitor polydnavirus mRNAs expressed in hemocytes of Pseudoplusia includens contain a common cysteine-rich domain.

    Science.gov (United States)

    Strand, M R; Witherell, R A; Trudeau, D

    1997-03-01

    Microplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. A previous study indicated that M. demolitor polydnavirus (MdPDV) infects primarily hemocytes in parasitized hosts. Thereafter, several alterations that compromise the immune response of P. includens toward the developing parasitoid occur in hemocytes. In this study, we identified two MdPDV mRNAs (1.0 and 1.5 kb) expressed in P. includens hemocytes that have homology to the viral genomic clone pMd-2. Corresponding 1.0- and 1.5-kb cDNA clones (MdPi455 and MdPi59) were isolated from an MdPDV-infected hemocyte cDNA library. Nucleotide sequence analysis of the cDNA clones confirmed that the 1.5- and 1.0-kb mRNAs have significant regions of homology. Sequence alignment revealed that the gene, OMd1.0, encoding the 1.0-kb mRNA is present in pMd-2. This gene contains two introns and three exons that agree with the sequence for MdPi455. In contrast, the 1.5-kb mRNA is likely encoded by a related gene located on the same MdPDV genomic DNA as is OMd1.0. The predicted peptide sequences for the 1.0- and 1.5-kb transcripts contain a cysteine-rich region at their 5' ends that have some similarity with epidermal growth factor-like motifs. Hybridization studies revealed that both mRNAs are expressed in granular cells and plasmatocytes, the primary classes of hemocytes involved in defense against M. demolitor and other parasites.

  1. Lamprey buccal gland secretory protein-2 (BGSP-2 inhibits human T lymphocyte proliferation

    Directory of Open Access Journals (Sweden)

    Jing SUN, Shuiyan YU, Zhuang XUE, Cenjie LIU, Yu WU, Xin LIU, Qingwei LI

    2010-04-01

    Full Text Available Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2 (BGSP-2 from a buccal gland cDNA library of Lampetra japonica. The full-length BGSP-2 gene was cloned and the recombinant BGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects on human T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducing G1/S cell cycle arrest. Furthermore, it inhibited the proliferation of human T lymphocytes stimulated by phytohemagglutinin (PHA at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of human T lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes [Current Zoology 56 (2: 252–258, 2010].

  2. High-affinity aptamers selectively inhibit human nonpancreatic secretory phospholipase A2 (hnps-PLA2).

    Science.gov (United States)

    Bridonneau, P; Chang, Y F; O'Connell, D; Gill, S C; Snyder, D W; Johnson, L; Goodson, T; Herron, D K; Parma, D H

    1998-03-12

    A family of sequence-related 2'-aminopyrimidine, 2'-hydroxylpurine aptamers, developed by oligonucleotide-based combinatorial chemistry, SELEX (systematic evolution of ligand by exponential enrichment) technology, binds human nonpancreatic secretory phospholipase A2 (hnps-PLA2) with nanomolar affinities and inhibits enzymatic activity. Aptamer 15, derived from the family, binds hnps-PLA2 with a Kd equal to 1.7 +/- 0.2 nM and, in a standard chromogenic assay of enzymatic activity, inhibits hnps-PLA2 with an IC50 of 4 nM, at a mole fraction of substrate concentration of 4 x 10(-6) and a calculated Ki of 0.14 nM. Aptamer 15 is selective for hnps-PLA2, having a 25- and 2500-fold lower affinity, respectively, for the unrelated proteins human neutrophil elastase and human IgG. Contractions of guinea pig lung pleural strips induced by hnps-PLA2 are abolished by 0.3 microM aptamer 15, whereas contractions induced by arachidonic acid are not altered. The structure that is essential for binding and inhibition appears to be a 40-base hairpin/loop motif with an asymmetrical internal loop. The affinity and activity of the aptamers demonstrate the ability of the SELEX process to isolate antagonists of nonnucleic-acid-binding proteins from vast oligonucleotide combinatorial libraries.

  3. Cysteine-rich secretory protein-3 (CRISP3 is strongly up-regulated in prostate carcinomas with the TMPRSS2-ERG fusion gene.

    Directory of Open Access Journals (Sweden)

    Franclim R Ribeiro

    Full Text Available A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (n = 16 and without (n = 8 TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (r(s = 0.65, p<0.001 and both were associated with pT3 disease staging. Furthermore, immunohistochemistry results showed CRISP3 protein overexpression in 63% of the carcinomas and chromatin immunoprecipitation with an anti-ERG antibody showed that CRISP3 is a direct target of the transcription factor ERG. We conclude that ERG rearrangement is associated with significant expression alterations in genes involved in critical cellular pathways that define a subset of locally advanced PCa. In particular, we show that CRISP3 is a direct target of ERG that is strongly overexpressed in PCa with the TMPRSS2-ERG fusion gene.

  4. In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts

    DEFF Research Database (Denmark)

    Le Bihan, Marie-Catherine; Bigot, Anne; Jensen, Søren Skov

    2012-01-01

    Efficient muscle regeneration requires cross talk between multiple cell types via secreted signaling molecules. However, as yet there has been no comprehensive analysis of this secreted signaling network in order to understand how it regulates myogenesis in humans. Using integrated proteomic...... and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling...

  5. Secretory leukocyte protease inhibitor in punch biopsies from human colonic mucosa

    Directory of Open Access Journals (Sweden)

    Max Nyström

    2001-01-01

    Full Text Available Secretory leukocyte protease inhibitor (SLPI is a wellknown protease inhibitor. Its function is thought to be protease/protease-inhibitor balance. Free proteolytic activity, mainly pancreatic elastase, anionic trypsin and granulocytic elastase, has been demonstrated in faecal extracts from patients with ulcerative colitis. We wanted to verify that SLPI is actually secreted from normal human colonic mucosa. Also, we wanted to ascertain whether studies of SLPI secretion based on punch biopsies were dependent on biopsy area or on biopsy circumference. Normal colonic mucosa was sampled during surgery for colonic cancer. A total of 36 samples from four patients were used. Mucosa preparation was carried out using a punch biopsy technique, and samples of 3, 4 and 6 mm diameter were used. All media contained SLPI at varying concentrations. When expressed in terms of the sample area, the secretion per millimetre-squared seemed to decrease with increasing area. When calculated as secretion per circumference, secretion seemed to be constant. In conclusion, SLPI was secreted from normal human colonic mucosa. The SLPI secretion seemed dependent on the circumference of the biopsy rather than on the area of the biopsy.

  6. Expression of ODC Antizyme Inhibitor 2 (AZIN2 in Human Secretory Cells and Tissues.

    Directory of Open Access Journals (Sweden)

    Tiina Rasila

    Full Text Available Ornithine decarboxylase (ODC antizyme inhibitor 2 (AZIN2, originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3 to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.

  7. Secretory products of breast cancer cells specifically affect human osteoblastic cells: partial characterization of active factors.

    Science.gov (United States)

    Siwek, B; Lacroix, M; De Pollak, C; Marie, P; Body, J J

    1997-04-01

    The pathogenesis of tumor-induced osteolysis (TIO) following breast cancer metastases in bone remains unclear. We postulated that osteoblasts could be target cells for the secretory products of breast cancer cells. We previously showed that serum-free conditioned medium (CM) of the breast cancer cell line MCF-7 inhibits DNA synthesis by 75% of control values in osteoblast-like cells SaOS-2 and that this effect is only in a minor part due to transforming growth factor beta secretion. To establish the specificity of our observations and to look for other biologically active factors, we have tested the effects of medium conditioned by several cancer and noncancer cell lines (breast, colon, placenta, or fibrosarcoma) on the proliferation of osteoblast-like cells (SaOS-2, MG-63), normal human osteoblasts, human fibrosarcoma cells, and normal human fibroblasts. Culture medium (1:2) of the breast cancer cell lines MCF-7, T-47D, MDA-MB-231, and SK-BR-3 inhibited by 25-50% the proliferation of osteoblast-like cells SaOS-2, MG-63, and normal osteoblasts as evaluated by the MTT survival test or [3H]thymidine incorporation. MCF-7 cells completely inhibited the proliferation of normal human osteoblasts in coculture. This inhibitory effect was reversible and not due to cytotoxicity. Moreover, the cyclic adenosine monophosphate (cAMP) response to parathyroid hormone (PTH) of osteoblast-like cells SaOS-2 was also increased by 100-240% by the same CM. Such activities were, however, not detected in medium from the breast noncancer cell line HBL-100 or in the medium conditioned by non-breast cancer cell lines (COLO 320DM, HT-29, JAR, or HT-1080). Medium from the breast cancer cells had no effect on normal human fibroblasts or fibrosarcoma cells (HT-1080), suggesting the specificity of their action on human osteoblasts. After partial purification by ultrafiltration and size-exclusion chromatography, we found that medium of T-47D cells contained at least three nonprostanoid factors of

  8. The challenge of improved secretory production of active pharmaceutical ingredients in Saccharomyces cerevisiae: a case study on human insulin analogs.

    Science.gov (United States)

    Kazemi Seresht, Ali; Palmqvist, Eva A; Schluckebier, Gerd; Pettersson, Ingrid; Olsson, Lisbeth

    2013-10-01

    The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. Great attention has been put on improved secretory production of active pharmaceutical ingredients, and the secretory pathway of this eukaryotic host has been the playground of diverse strain engineering studies, aiming at enhanced cellular capacities for folding and trafficking of the target proteins. However, the cellular quality assessment for secretory proteins remains mostly unpredictable, and different target proteins often do not picture similar secretion yields, underlining the dependency of efficient secretion on the physicochemical properties of the protein of interest. In this study, two human insulin analog precursors (IAPs) with minor differences in their amino acid sequences were used as model secretory proteins. No differences between cells expressing these two proteins were found in the IAP transcript levels, gene copy numbers, or intra-cellularly accumulated proteins, yet a more than sevenfold difference in their secretion yields was found. Physiological characterization of cells expressing these proteins in batch processes revealed no significant difference in their specific growth rate, but an altered overflow metabolism. Global transcriptome analysis carried out in chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated and indicated an increased degradation of the IAP with the low secretion yield. In silico protein structure modeling of the IAPs suggested a difference in conformational stability, induced by the amino acid substitution, which most likely resulted in disparity in trafficking through the secretory pathway and thus a large difference in secretion yields. Copyright © 2013 Wiley Periodicals, Inc.

  9. CD63 is tightly associated with intracellular, secretory events chaperoning piecemeal degranulation and compound exocytosis in human eosinophils.

    Science.gov (United States)

    Carmo, Lívia A S; Bonjour, Kennedy; Ueki, Shigeharu; Neves, Josiane S; Liu, Linying; Spencer, Lisa A; Dvorak, Ann M; Weller, Peter F; Melo, Rossana C N

    2016-08-01

    Eosinophil activation leads to secretion of presynthesized, granule-stored mediators that determine the course of allergic, inflammatory, and immunoregulatory responses. CD63, a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) and present on the limiting membranes of eosinophil-specific (secretory) granules, is considered a potential surface marker for eosinophil degranulation. However, the intracellular secretory trafficking of CD63 in eosinophils and other leukocytes is not understood. Here, we provide a comprehensive investigation of CD63 trafficking at high resolution within human eosinophils stimulated with inflammatory stimuli, CCL11 and tumor necrosis factor α, which induce distinctly differing secretory processes in eosinophils: piecemeal degranulation and compound exocytosis, respectively. By using different transmission electron microscopy approaches, including an immunonanogold technique, for enhanced detection of CD63 at subcellular compartments, we identified a major intracellular pool of CD63 that is directly linked to eosinophil degranulation events. Transmission electron microscopy quantitative analyses demonstrated that, in response to stimulation, CD63 is concentrated within granules undergoing secretion by piecemeal degranulation or compound exocytosis and that CD63 tracks with the movements of vesicles and granules in the cytoplasm. Although CD63 was observed at the cell surface after stimulation, immunonanogold electron microscopy revealed that a strong CD63 pool remains in the cytoplasm. It is remarkable that CCL11 and tumor necrosis factor α triggered increased formation of CD63(+) large vesiculotubular carriers (eosinophil sombrero vesicles), which fused with granules in the process of secretion, likely acting in the intracellular translocation of CD63. Altogether, we identified active, intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways. This is important for understanding the

  10. High specificity of human secretory class II phospholipase A2 for phosphatidic acid.

    Science.gov (United States)

    Snitko, Y; Yoon, E T; Cho, W

    1997-02-01

    Lysophosphatidic acid (LPA) is a potent lipid second messenger which stimulates platelet aggregation, cell proliferation and smooth-muscle contraction. The phospholipase A2 (PLA2)-catalysed hydrolysis of phosphatidic acid (PA) is thought to be a primary synthetic route for LPA. Of the multiple forms of PLA2 present in human tissues, human secretory class-II PLA2 (hs-PLA2) has been implicated in the production of LPA from platelets and whole blood cells challenged with inflammatory stimuli. To explore further the possibility that hs-PLA2 is involved in the production of LPA, we rigorously measured the phospholipid head group specificity of hs-PLA2 by a novel PLA2 kinetic system using polymerized mixed liposomes. Kinetic analysis of recombinant hs-PLA2 demonstrates that hs-PLA2 strongly prefers PA as substrate over other phospholipids found in the mammalian plasma membrane including phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The order of preference is PA > PE approximately PS > PC. To identify amino acid residues of hs-PLA2 that are involved in its unique substrate specificity, we mutated two residues, Glu-56 and Lys-69, which were shown to interact with the phospholipid head group in the X-ray-crystallographic structure of the hs-PLA2-transition-state-analogue complex. The K69Y mutant showed selective inactivation toward PA whereas the E56K mutant displayed a most pronounced inactivation to PE. Thus it appears that Lys-69 is at least partially involved in the PA specificity of hs-PLA2 and Glu-56 in the distinction between PE and PC. In conjunction with a recent cell study [Fourcade, Simon, Viode, Rugani, Leballe, Ragab, Fournie, Sarda and Chap (1995) Cell 80, 919-927], these studies suggest that hs-PLA2 can rapidly hydrolyse PA molecules exposed to the outer layer of cell-derived microvesicles and thereby produce LPA.

  11. Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region

    DEFF Research Database (Denmark)

    Madsen, Mette; Møller, Holger J; Nielsen, Marianne Jensby

    2004-01-01

    CD163 is the macrophage receptor for endocytosis of haptoglobin.hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteine rich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163...... to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant...

  12. Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

    Directory of Open Access Journals (Sweden)

    Campbell Kevin P

    2007-08-01

    Full Text Available Abstract Background Polymorphonuclear neutrophils (PMN constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.

  13. Secretory products from thrombin-stimulated human platelets exert an inhibitory effect on NK-cytotoxic activity

    DEFF Research Database (Denmark)

    Skov Madsen, P; Hokland, P; Hokland, M

    1987-01-01

    We have investigated the interaction between human platelets and the NK-system, with special emphasis on the action of secretory products from platelets in an NK assay with 51Cr-labelled K562 as target cells. Supernatants from thrombin-stimulated platelets added to the NK assay consistently...... with thrombin, are capable of secreting different, yet undefined factors, which significantly inhibit NK activity in vitro. The results also suggest that the role of products from contaminating in vitro activated platelets should be borne in mind when performing conventional NK assays. Udgivelsesdato: 1986-Oct...

  14. Rotavirus specific plasma secretory immunoglobulin in children with acute gastroenteritis and children vaccinated with an attenuated human rotavirus vaccine

    OpenAIRE

    Herrera Daniel; Vásquez Camilo; Corthésy Blaise E.; Franco M. A.; Angel Juana

    2013-01-01

    Rotavirus (RV)-specific secretory immunoglobulin (RV-sIg) has been previously detected in serum of naturally RV infected children and shown to reflect the intestinal Ig immune response. Total plasma sIga and plasma RV-sIg were evaluated by eLIsa in children with gastroenteritis due or not due to RV infection and in 50 children vaccinated with the attenuated RIX4414 human RV vaccine and 62 placebo recipients. RV-sIg was only detected in children with evidence of previous RV infection or with a...

  15. Impact of simulated microgravity on the secretory and adhesive activity of cultured human vascular endothelial cells.

    Science.gov (United States)

    Rudimov, Evgeny; Buravkova, Ludmila; Pogodina, Margarita; Andrianova, Irina

    The layer of vascular endothelial cells (ECs) is a dynamic,disseminated organ that perform the function of an interface between the blood and vascular wall. The endothelial monolayer is able to quickly respond to changes in the microenvironment due to its synthesis of vasoactive substances, chemokines, adhesion molecules expression, etc. ECs are highly sensitive to gravitational changes and capable of short-term and long-term responses (Sangha et al., 2001; Buravkova et al., 2005; Infanger et al., 2006, 2007. However, the question remains how to reflect the impact of microgravity on endothelium under the inflammatory process. Therefore, the aim of this study was to investigate secretory and adhesive activity of human umbilical vein endothelial cells (HUVECs) during simulated microgravity and TNF-a activation. HUVECs were isolated according to Gimbrone et al. (1978) in modification A. Antonov (1981) and used for experiments at 2-4 passages. HUVECs were activated by low level of TNF-a (2 ng/ml). Microgravity was generated by Random Positioning Machine (RPM, Dutch Space, Leiden) placed into the thermostat at 37°C. After 24 hours of clinorotation we measured adhesion molecules expression on the cell surface (ICAM-1, VCAM-1, PECAM-1, E-selectin, CD144, endoglin (CD105)) and cell viability using a flow cytometry. To evaluate the level of target gene expression was used the real time RT-PCR. IL-6 and IL-8 concentration was measured in the conditioned medium of HUVECs by using the ELISA test. We found that simulated microgravity within 24 hours caused a decrease of ICAM-1, CD144, and E-selectin expression, at the same time not affect the cell viability, endoglin and PECAM-1 expression on the surface HUVEC. Furthermore, there were no changes of the level of IL-6 and IL-8 gene expression and their products in the culture medium. TNF-activated HUVECs showed an increase in gene expression of interleukins and molecules involved in the adhesion process, which also was confirmed

  16. Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily

    DEFF Research Database (Denmark)

    Bikker, Floris J; Ligtenberg, Antoon J M; Nazmi, Kamran

    2002-01-01

    Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300-400 kDa glycoprotein is composed of conserved peptide motifs: 14...... SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment....... Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate...

  17. Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production.

    Science.gov (United States)

    Chaiyadet, Sujittra; Smout, Michael; Johnson, Michael; Whitchurch, Cynthia; Turnbull, Lynne; Kaewkes, Sasithorn; Sotillo, Javier; Loukas, Alex; Sripa, Banchob

    2015-10-01

    Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south

  18. Trichinella britovi human infection in Spain : antibody response to surface, excretory/secretory and somatic antigens

    Directory of Open Access Journals (Sweden)

    Rodríguez-Osorio M.

    2003-06-01

    Full Text Available A third outbreak of Trichinella britovi with 140 people involved, occurred in Granada Spain (December 1998. The source of infection was sausage made from uninspected wild boar meat. Fifty-two patients agreed to participated in this study. An elevated eosinophil level (> 5 % was detected in 59.6 % of patients, and persisted in most of these cases for two months. A moderate IgG response was observed. At the onset of symptoms, Western blot (WB test detected more positive cases than Enzyme linked immunosorbent assay (ELISA and indirect immunofluorescence (IIF. Six months from infection, ELISA revealed fewer positive cases than the other two tests. It would appear that the response to somatic antigens starts earlier than those to cuticular and excretory/secretory (ES antigens and that the response to ES antigens is the first to decrease.

  19. A new family of giardial cysteine-rich non-VSP protein genes and a novel cyst protein.

    Directory of Open Access Journals (Sweden)

    Barbara J Davids

    2006-12-01

    Full Text Available Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, "HCNCp" (High Cysteine Non-variant Cyst protein, that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many "CxxC" tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple "CxC" motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp C-terminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp containing > or = 20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents

  20. Rotavirus specific plasma secretory immunoglobulin in children with acute gastroenteritis and children vaccinated with an attenuated human rotavirus vaccine.

    Science.gov (United States)

    Herrera, Daniel; Vásquez, Camilo; Corthésy, Blaise; Franco, Manuel A; Angel, Juana

    2013-11-01

    Rotavirus (RV)-specific secretory immunoglobulin (RV-SIg) has been previously detected in serum of naturally RV infected children and shown to reflect the intestinal Ig immune response. Total plasma SIgA and plasma RV-SIg were evaluated by ELISA in children with gastroenteritis due or not due to RV infection and in 50 children vaccinated with the attenuated RIX4414 human RV vaccine and 62 placebo recipients. RV-SIg was only detected in children with evidence of previous RV infection or with acute RV gastroenteritis. Vaccinees had higher RV-SIg titers than placebo recipients and RV-SIg titers increased after the second vaccine dose. RV-SIg measured after the second dose correlated with protection when vaccinees and placebo recipients were analyzed jointly. RV-SIg may serve as a valuable correlate of protection for RV vaccines.

  1. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, R.L.; Austen, K.F. (Brigham and Women' s Hospital, Boston, MA (USA)); Fox, C.C.; Lichtenstein, L.M. (Johns Hopkins School of Medicine, Baltimore, MD (USA))

    1988-04-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of {sup 35}S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although ({sup 35}S)heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate ({sup 35}S)sulfate into separate heparin and chondroitin sulfate proteoglycans in an {approx}2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin ({sup 35}S)sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin ({sup 35}S)sulfate E proteoglycans and the ({sup 35}S)heparin proteoglycans were exocytosed from the ({sup 35}S)sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of {sup 35}S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

  2. Weight Loss Improves the Adipogenic Capacity of Human Preadipocytes and Modulates Their Secretory Profile

    Science.gov (United States)

    Rossmeislová, Lenka; Mališová, Lucia; Kračmerová, Jana; Tencerová, Michaela; Kováčová, Zuzana; Koc, Michal; Šiklová-Vítková, Michaela; Viquerie, Nathalie; Langin, Dominique; Štich, Vladimír

    2013-01-01

    Calorie restriction–induced weight loss is accompanied by profound changes in adipose tissue characteristics. To determine the effect of weight loss on differentiation of preadipocytes and secretory capacity of in vitro differentiated adipocytes, we established cultures of these cells from paired subcutaneous adipose tissue biopsies obtained before and at the end of weight-reducing dietary intervention (DI) in 23 obese women. Based on lipid accumulation and the expression of differentiation markers, in vitro adipogenesis increased after weight loss and it was accompanied by enhanced expression of genes involved in de novo lipogenesis. This effect of weight loss was not driven by changes of peroxisome proliferator–activated receptor γ sensitivity to rosiglitazone. Weight loss also enhanced the expression of adiponectin and leptin while reducing that of monocyte chemoattractant protein 1 and interleukin-8 by cultured adipocytes. Thus, the weight-reducing (DI) increased adipogenic capacity of preadipocytes and shifted their secretion toward lower inflammatory profile. Reprogramming of preadipocytes could represent an adaptation to weight loss leading to partial restoration of preobese adipose tissue traits and thus contribute to the improvement of metabolic status. However, enhanced adipogenesis could also contribute to the unwanted weight regain after initial weight loss. PMID:23378611

  3. Characterization of the excretory/secretory products of Dermatobia hominis larvae, the human bot fly.

    Science.gov (United States)

    Brant, M P R; Guimarães, S; Souza-Neto, J A; Ribolla, P E M; Oliveira-Sequeira, T C G

    2010-03-25

    Proteolytic activity in excretory/secretory products (ESP) of first- (L1), second- (L2) and third-instar (L3) larvae of Dermatobia hominis was analyzed through gelatin-gel and colorimetric enzyme assays with the chromogenic substrates azocasein and BApNA. The functional characterization of proteases was based on inhibition assays including synthetic inhibitors. ESP were obtained from new-hatched larvae reared in the laboratory and from second- and third-instar larvae removed from naturally infested cattle. Gelatin-gel analysis evidenced few bands of proteolysis, predominantly of high apparent molecular masses, in ESP of L1, whereas in the gel of L2 and L3 ESP there was a wide range of proteolytic activity most of them not resolved in a single species. Azocasein assays revealed a progressive increase of protease activity from first- to third-instar larvae. Protease inhibitor assays revealed a predominance of metalloproteases in L1 ESP that could be related to a skin penetration process and to a diversion of host immune response. The predominance of serine proteases in L2 and L3 and the great tryptic activity presented by L3 ESP were attributed to an increasing trophic activity by the growing larvae, since the viability of adult flies strictly depends on larval abilities to assimilate nutrients from the host. Taking together, these results suggest that Dematobia larvae secrete/excrete different proteases that may be related to diverse functions during host penetration and infestation, which reinforces the relevance of the study of such proteolytic enzymes.

  4. Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant.

    Science.gov (United States)

    Horváth, Beatrix; Domonkos, Ágota; Kereszt, Attila; Szűcs, Attila; Ábrahám, Edit; Ayaydin, Ferhan; Bóka, Károly; Chen, Yuhui; Chen, Rujin; Murray, Jeremy D; Udvardi, Michael K; Kondorosi, Éva; Kaló, Péter

    2015-12-08

    Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

  5. The Arabidopsis thaliana cysteine-rich receptor-like kinase CRK20 modulates host responses to Pseudomonas syringae pv. tomato DC3000 infection

    KAUST Repository

    Ederli, Luisa

    2011-10-01

    In plants, the cysteine-rich repeat kinases (CRKs) are a sub-family of receptor-like protein kinases that contain the DUF26 motif in their extracellular domains. It has been shown that in Arabidopsis thaliana, CRK20 is transcriptionally induced by pathogens, salicylic acid and ozone (O3). However, its role in responses to biotic and abiotic stress remains to be elucidated. To determine the function of CRK20 in such responses, two CRK20 loss-of-function mutants, crk20-1 and crk20-2, were isolated from public collections of Arabidopsis T-DNA tagged lines and examined for responses to O3 and Pseudomonas syringae pv. tomato (Pst) DC3000. crk20-1 and crk20-2 showed similar O3 sensitivities and no differences in the expression of defense genes when compared with the wild-type. However, pathogen growth was significantly reduced, while there were no differences in the induction of salicylic acid related defense genes or salicylic acid accumulation. Furthermore, correlation analysis of CRK20 gene expression suggests that it has a role in the control of H2O and/or nutrient transport. We therefore propose that CRK20 promotes conditions that are favorable for Pst DC3000 growth in Arabidopsis, possibly through the regulation of apoplastic homeostasis, and consequently, of the environment of this biotrophic pathogen. © 2011 Elsevier GmbH.

  6. DMSA-Coated Iron Oxide Nanoparticles Greatly Affect the Expression of Genes Coding Cysteine-Rich Proteins by Their DMSA Coating.

    Science.gov (United States)

    Zhang, Ling; Wang, Xin; Zou, Jinglu; Liu, Yingxun; Wang, Jinke

    2015-10-19

    The dimercaptosuccinic acid (DMSA) was widely used to coat iron oxide nanoparticles (FeNPs); however, its intracellular cytotoxicity remains to be adequately elucidated. This study analyzed the differentially expressed genes (DEGs) in four mammalian cells treated by a DMSA-coated magnetite FeNP at various doses at different times. The results revealed that about one-fourth of DEGs coded cysteine-rich proteins (CRPs) in all cells under each treatment, indicating that the nanoparticles greatly affected the expressions of CRP-coding genes. Additionally, about 26% of CRP-coding DEGs were enzyme genes in all cells, indicating that the nanoparticles greatly affected the expression of enzyme genes. Further experiments with the nanoparticles and a polyethylenimine (PEI)-coated magnetite FeNP revealed that the effect mainly resulted from DMSA carried into cells by the nanoparticles. This study thus first reported the cytotoxicity of DMSA at the gene transcription level as coating molecules of FeNPs. This study provides new insight into the molecular mechanism by which the DMSA-coated nanoparticles resulted in the transcriptional changes of many CRP-coding genes in cells. This study draws attention toward the intracellular cytotoxicity of DMSA as a coating molecule of nanoparticles, which has very low toxicity as an orally administered antidote due to its extracellular distribution.

  7. Transcriptome Analysis Revealed Highly Expressed Genes Encoding Secondary Metabolite Pathways and Small Cysteine-Rich Proteins in the Sclerotium of Lignosus rhinocerotis.

    Directory of Open Access Journals (Sweden)

    Hui-Yeng Y Yap

    Full Text Available Lignosus rhinocerotis (Cooke Ryvarden (tiger milk mushroom has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications.

  8. A Genetic Screen Identifies a Requirement for Cysteine-Rich-Receptor-Like Kinases in Rice NH1 (OsNPR1-Mediated Immunity.

    Directory of Open Access Journals (Sweden)

    Mawsheng Chern

    2016-05-01

    Full Text Available Systemic acquired resistance, mediated by the Arabidopsis NPR1 gene and the rice NH1 gene, confers broad-spectrum immunity to diverse pathogens. NPR1 and NH1 interact with TGA transcription factors to activate downstream defense genes. Despite the importance of this defense response, the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined. Here we report the identification of a rice mutant, snim1, which suppresses NH1-mediated immunity and demonstrate that two genes encoding previously uncharacterized cysteine-rich-receptor-like kinases (CRK6 and CRK10, complement the snim1 mutant phenotype. Silencing of CRK6 and CRK10 genes individually in the parental genetic background recreates the snim1 phenotype. We identified a rice mutant in the Kitaake genetic background with a frameshift mutation in crk10; this mutant also displays a compromised immune response highlighting the important role of crk10. We also show that elevated levels of NH1 expression lead to enhanced CRK10 expression and that the rice TGA2.1 protein binds to the CRK10 promoter. These experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression.

  9. A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus).

    Science.gov (United States)

    Verma, Shiv S; Yajima, William R; Rahman, Muhammad H; Shah, Saleh; Liu, Jun-Jun; Ekramoddoullah, Abul K M; Kav, Nat N V

    2012-05-01

    Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it's in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.

  10. Variant size- and glycoforms of the scavenger receptor cysteine-rich protein gp-340 with differential bacterial aggregation

    DEFF Research Database (Denmark)

    Eriksson, Christer; Frängsmyr, Lars; Danielsson Niemi, Liza

    2007-01-01

    glycoforms I and II/III correlate with Se(-) and Se(+) phenotypes, respectively (p Streptococcus suis) or sialic acid- (Streptococcus mutans) binding bacteria......Glycoprotein gp-340 aggregates bacteria in saliva as part of innate defence at mucosal surfaces. We have detected size- and glycoforms of gp-340 between human saliva samples (n = 7) and lung gp-340 from a proteinosis patient using antibodies and lectins in Western blots and ELISA measurements...... bands. Purified I to IV proteins all revealed a N-terminal sequence TGGWIP upon Edman degradation. Moreover, purified gp-340 from the seven donors and lung gp-340 shared N-glycans, sialylated Galbeta1-3GalNAc and (poly)lactosamine structures. However, the larger size gp-340 grouping II/III (n = 4...

  11. Oxidation of the cysteine-rich regions of parkin perturbs its E3 ligase activity and contributes to protein aggregation

    Directory of Open Access Journals (Sweden)

    Ma Yuliang

    2011-05-01

    Full Text Available Abstract Background Accumulation of aberrant proteins to form Lewy bodies (LBs is a hallmark of Parkinson's disease (PD. Ubiquitination-mediated degradation of aberrant, misfolded proteins is critical for maintaining normal cell function. Emerging evidence suggests that oxidative/nitrosative stress compromises the precisely-regulated network of ubiquitination in PD, particularly affecting parkin E3 ligase activity, and contributes to the accumulation of toxic proteins and neuronal cell death. Results To gain insight into the mechanism whereby cell stress alters parkin-mediated ubiquitination and LB formation, we investigated the effect of oxidative stress. We found significant increases in oxidation (sulfonation and subsequent aggregation of parkin in SH-SY5Y cells exposed to the mitochondrial complex I inhibitor 1-methyl-4-phenlypyridinium (MPP+, representing an in vitro cell-based PD model. Exposure of these cells to direct oxidation via pathological doses of H2O2 induced a vicious cycle of increased followed by decreased parkin E3 ligase activity, similar to that previously reported following S-nitrosylation of parkin. Pre-incubation with catalase attenuated H2O2 accumulation, parkin sulfonation, and parkin aggregation. Mass spectrometry (MS analysis revealed that H2O2 reacted with specific cysteine residues of parkin, resulting in sulfination/sulfonation in regions of the protein similar to those affected by parkin mutations in hereditary forms of PD. Immunohistochemistry or gel electrophoresis revealed an increase in aggregated parkin in rats and primates exposed to mitochondrial complex I inhibitors, as well as in postmortem human brain from patients with PD with LBs. Conclusion These findings show that oxidative stress alters parkin E3 ligase activity, leading to dysfunction of the ubiquitin-proteasome system and potentially contributing to LB formation.

  12. Human milk secretory immunoglobulin a and lactoferrin N-glycans are altered in women with gestational diabetes mellitus.

    Science.gov (United States)

    Smilowitz, Jennifer T; Totten, Sarah M; Huang, Jincui; Grapov, Dmitry; Durham, Holiday A; Lammi-Keefe, Carol J; Lebrilla, Carlito; German, J Bruce

    2013-12-01

    Very little is known about the effects of gestational diabetes mellitus (GDM) on lactation and milk components. Recent reports suggested that hyperglycemia during pregnancy was associated with altered breast milk immune factors. Human milk oligosaccharides (HMOs) and N-glycans of milk immune-modulatory proteins are implicated in modulation of infant immunity. The objective of the current study was to evaluate the effect of GDM on HMO and protein-conjugated glycan profiles in breast milk. Milk was collected at 2 wk postpartum from women diagnosed with (n = 8) or without (n = 16) GDM at week 24-28 in pregnancy. Milk was analyzed for HMO abundances, protein concentrations, and N-glycan abundances of lactoferrin and secretory immunoglobulin A (sIgA). HMOs and N-glycans were analyzed by mass spectrometry and milk lactoferrin and sIgA concentrations were analyzed by the Bradford assay. The data were analyzed using multivariate modeling confirmed with univariate statistics to determine differences between milk of women with compared with women without GDM. There were no differences in HMOs between milk from women with vs. without GDM. Milk from women with GDM compared with those without GDM was 63.6% lower in sIgA protein (P milk free oligosaccharide abundances but decreased total protein and glycosylation of sIgA and increased glycosylation of lactoferrin in transitional milk. The results suggest that maternal glucose dysregulation during pregnancy has lasting consequences that may influence the innate immune protective functions of breast milk.

  13. The role of human epididymis secretory protein E4 in patients with endometrial cancer and premalignant endometrial lesions.

    Science.gov (United States)

    Yılmaz, Setenay Arzu; Altınkaya, Sündüz Özlem; Kerimoglu, Özlem Seçilmiş; Tazegül Pekin, Aybike; Akyürek, Fikret; Ilhan, Tolgay Tuyan; Benzer, Nilgün; Unlu, Ali; Yuksel, Hasan; Celik, Cetin

    2017-01-01

    We evaluated the concentrations of human epididymis secretory protein E4 (HE4) and Ca-125 in relation to clinicopathologic features in patients with endometrial cancer and premalignant endometrial lesions. Women with abnormal uterine bleeding (n = 167) who underwent endometrial sampling were divided into four groups. Group 1: endometrial cancer (n = 68), group 2: atypical endometrial hyperplasia (n = 12), group 3: endometrial hyperplasia without atypia (n = 39) and group 4: controls (n = 48). Women with endometrial cancer exhibited higher concentrations of HE4 levels than controls (91.4 pmol/L vs. 46.2 pmol/L, p endometrial cancer were 72.7%, 84.4%, 80% and 78.4%, respectively. Preoperative HE4 levels are more elevated in women with endometrial cancer than those with benign endometrium as well as in women with prognostic high-risk factors with endometrial cancer. HE4 may be used as an additional marker in combination with other clinicopathologic features for planning the treatment.

  14. Human Milk Secretory Immunoglobulin A and Lactoferrin N-Glycans Are Altered in Women with Gestational Diabetes Mellitus123

    Science.gov (United States)

    Smilowitz, Jennifer T.; Totten, Sarah M.; Huang, Jincui; Grapov, Dmitry; Durham, Holiday A.; Lammi-Keefe, Carol J.; Lebrilla, Carlito; German, J. Bruce

    2013-01-01

    Very little is known about the effects of gestational diabetes mellitus (GDM) on lactation and milk components. Recent reports suggested that hyperglycemia during pregnancy was associated with altered breast milk immune factors. Human milk oligosaccharides (HMOs) and N-glycans of milk immune-modulatory proteins are implicated in modulation of infant immunity. The objective of the current study was to evaluate the effect of GDM on HMO and protein-conjugated glycan profiles in breast milk. Milk was collected at 2 wk postpartum from women diagnosed with (n = 8) or without (n = 16) GDM at week 24–28 in pregnancy. Milk was analyzed for HMO abundances, protein concentrations, and N-glycan abundances of lactoferrin and secretory immunoglobulin A (sIgA). HMOs and N-glycans were analyzed by mass spectrometry and milk lactoferrin and sIgA concentrations were analyzed by the Bradford assay. The data were analyzed using multivariate modeling confirmed with univariate statistics to determine differences between milk of women with compared with women without GDM. There were no differences in HMOs between milk from women with vs. without GDM. Milk from women with GDM compared with those without GDM was 63.6% lower in sIgA protein (P lactoferrin total N-glycans (P lactoferrin fucose and sialic acid N-glycans (P lactoferrin in transitional milk. The results suggest that maternal glucose dysregulation during pregnancy has lasting consequences that may influence the innate immune protective functions of breast milk. PMID:24047700

  15. D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function.

    Directory of Open Access Journals (Sweden)

    Christoph Sonderegger

    Full Text Available The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19. We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF.

  16. Multi-environment QTL studies suggest a role for cysteine-rich protein kinase genes in quantitative resistance to blackleg disease in Brassica napus.

    Science.gov (United States)

    Larkan, Nicholas J; Raman, Harsh; Lydiate, Derek J; Robinson, Stephen J; Yu, Fengqun; Barbulescu, Denise M; Raman, Rosy; Luckett, David J; Burton, Wayne; Wratten, Neil; Salisbury, Philip A; Rimmer, S Roger; Borhan, M Hossein

    2016-08-24

    Resistance to the blackleg disease of Brassica napus (canola/oilseed rape), caused by the hemibiotrophic fungal pathogen Leptosphaeria maculans, is determined by both race-specific resistance (R) genes and quantitative resistance loci (QTL), or adult-plant resistance (APR). While the introgression of R genes into breeding material is relatively simple, QTL are often detected sporadically, making them harder to capture in breeding programs. For the effective deployment of APR in crop varieties, resistance QTL need to have a reliable influence on phenotype in multiple environments and be well defined genetically to enable marker-assisted selection (MAS). Doubled-haploid populations produced from the susceptible B. napus variety Topas and APR varieties AG-Castle and AV-Sapphire were analysed for resistance to blackleg in two locations over 3 and 4 years, respectively. Three stable QTL were detected in each population, with two loci appearing to be common to both APR varieties. Physical delineation of three QTL regions was sufficient to identify candidate defense-related genes, including a cluster of cysteine-rich receptor-like kinases contained within a 49 gene QTL interval on chromosome A01. Individual L. maculans isolates were used to define the physical intervals for the race-specific R genes Rlm3 and Rlm4 and to identify QTL common to both field studies and the cotyledon resistance response. Through multi-environment QTL analysis we have identified and delineated four significant and stable QTL suitable for MAS of quantitative blackleg resistance in B. napus, and identified candidate genes which potentially play a role in quantitative defense responses to L. maculans.

  17. D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function.

    Science.gov (United States)

    Sonderegger, Christoph; Fizil, Ádám; Burtscher, Laura; Hajdu, Dorottya; Muñoz, Alberto; Gáspári, Zoltán; Read, Nick D; Batta, Gyula; Marx, Florentine

    2017-01-01

    The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF.

  18. Substitution of a conserved cysteine-996 in a cysteine-rich motif of the laminin {alpha}2-chain in congenital muscular dystrophy with partial deficiency of the protein

    Energy Technology Data Exchange (ETDEWEB)

    Nissinen, M.; Xu Zhang; Tryggvason, K. [Univ. of Oulu (Finland)

    1996-06-01

    Congenital muscular dystrophies (CMDs) are autosomal recessive muscle disorders of early onset. Approximately half of CMD patients present laminin {alpha}2-chain (merosin) deficiency in muscle biopsies, and the disease locus has been mapped to the region of the LAMA2 gene (6q22-23) in several families. Recently, two nonsense mutations in the laminin {alpha}2-chain gene were identified in CMD patients exhibiting complete deficiency of the laminin {alpha}2-chain in muscle biopsies. However, a subset of CMD patients with linkage to LAMA2 show only partial absence of the laminin {alpha}2-chain around muscle fibers, by immunocytochemical analysis. In the present study we have identified a homozygous missense mutation in the {alpha}2-chain gene of a consanguineous Turkish family with partial laminin {alpha}2-chain deficiency. The T{r_arrow}C transition at position 3035 in the cDNA sequence results in a Cys996{r_arrow}Arg substitution. The mutation that affects one of the conserved cysteine-rich repeats in the short arm of the laminin {alpha}2-chain should result in normal synthesis of the chain and in formation and secretion of a heterotrimeric laminin molecule. Muscular dysfunction is possibly caused either by abnormal disulfide cross-links and folding of the laminin repeat, leading to the disturbance of an as yet unknown binding function of the laminin {alpha}2-chain and to shorter half-life of the muscle-specific laminin-2 and laminin-4 isoforms, or by increased proteolytic sensitivity, leading to truncation of the short arm. 42 refs., 7 figs.

  19. Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse

    Science.gov (United States)

    Volpert, Marianna; Mangum, Jonathan E.; Jamsai, Duangporn; D'Sylva, Rebecca; O'Bryan, Moira K.; McIntyre, Peter

    2014-02-01

    While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

  20. The Pseudomonas secretory product pyocyanin inhibits catalase activity in human lung epithelial cells.

    Science.gov (United States)

    O'Malley, Yunxia Q; Reszka, Krzysztof J; Rasmussen, George T; Abdalla, Maher Y; Denning, Gerene M; Britigan, Bradley E

    2003-11-01

    Pyocyanin, produced by Pseudomonas aeruginosa, has many deleterious effects on human cells that relate to its ability to generate reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. Human cells possess several mechanisms to protect themselves from ROS, including manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), and catalase. Given the link between pyocyanin-mediated epithelial cell injury and oxidative stress, we assessed pyocyanin's effect on MnSOD, CuZnSOD, and catalase levels in the A549 human alveolar epithelial cell line and in normal human bronchial epithelial cells. In both cell types, CuZnSOD and MnSOD were unaltered, but over 24 h pyocyanin significantly decreased cellular catalase activity and protein content. Pyocyanin also decreased catalase mRNA. Overexpression of MnSOD in A549 cells prevented pyocyanin-mediated loss of catalase protein, but catalase activity still declined. Furthermore, pyocyanin decreased catalase activity, but not protein, in A549 cells overexpressing human catalase. These data suggest a direct effect of pyocyanin on catalase activity. Addition of pyocyanin to catalase in a cell-free system also decreased catalase activity. Mammalian catalase binds four NADPH molecules, helping maintain enzyme activity. Spin-trapping data suggest that pyocyanin directly oxidizes this NADPH, producing superoxide. We conclude that pyocyanin may decrease cellular catalase activity via both transcriptional regulation and direct inactivation of the enzyme. Decreased cellular catalase activity and failure to augment MnSOD could contribute to pyocyanin-dependent cytotoxicity.

  1. NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands

    DEFF Research Database (Denmark)

    Nejsum, Lene Niemann; Prætorius, Jeppe; Nielsen, Søren

    2005-01-01

    1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na(+)-coupled acid-base transporters...... and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 m...... palmar skin by immunoblotting, whereas NKCC2, NHE2, and NHE3 proteins were not detected. Immunohistochemistry was performed using sections from rat, mouse, and human palmar tissue. Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat...

  2. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane

    DEFF Research Database (Denmark)

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels Henrik Helweg

    2013-01-01

    granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in...... subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes....

  3. The Effect of Secretory Factors of Adipose-Derived Stem Cells on Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Soo-Wan Nam

    2012-01-01

    Full Text Available The beneficial effects of adipose-derived stem cell conditioned medium (ADSC-CM on skin regeneration have been reported. Although the mechanism of how ADSC-CM promotes skin regeneration is unclear, ADSC-CM contained various growth factors and it is an excellent raw material for skin treatment. ADSC-CM produced in a hypoxia condition of ADSC—in other words, Advanced Adipose-Derived Stem cell Protein Extract (AAPE—has great merits for skin regeneration. In this study, human primary keratinocytes (HKs, which play fundamental roles in skin tissue, was used to examine how AAPE affects HK. HK proliferation was significantly higher in the experimental group (1.22 μg/mL than in the control group. DNA gene chip demonstrated that AAPE in keratinocytes (p < 0.05 notably affected expression of 290 identified transcripts, which were associated with cell proliferation, cycle and migration. More keratinocyte wound healing and migration was shown in the experimental group (1.22 μg/mL. AAPE treatment significantly stimulated stress fiber formation, which was linked to the RhoA-ROCK pathway. We identified 48 protein spots in 2-D gel analysis and selected proteins were divided into 64% collagen components and 30% non-collagen components as shown by the MALDI-TOF analysis. Antibody array results contained growth factor/cytokine such as HGF, FGF-1, G-CSF, GM-CSF, IL-6, VEGF, and TGF-β3 differing from that shown by 2-D analysis. Conclusion: AAPE activates HK proliferation and migration. These results highlight the potential of the topical application of AAPE in the treatment of skin regeneration.

  4. Human protein secretory pathway genes are expressed in a tissue-specific pattern to match processing demands of the secretome

    DEFF Research Database (Denmark)

    Feizi, Amir; Gatto, Francesco; Uhlén, Mathias

    2017-01-01

    -translational modifications in each tissue's secretome. Our findings conciliate both the housekeeping and tissue-specific nature of the protein secretory pathway, which we attribute to a fine-tuned regulation of defined gene families to support the diversity of secreted proteins and their modifications....

  5. Passive administration of purified secretory IgA from human colostrum induces protection against Mycobacterium tuberculosis in a murine model of progressive pulmonary infection

    Directory of Open Access Journals (Sweden)

    Alvarez Nadine

    2013-02-01

    Full Text Available Abstract Background Immunoglobulin A is the most abundant isotype in secretions from mucosal surfaces of the gastrointestinal, respiratory and genitourinary tracts and in external secretions such as colostrum, breast milk, tears and saliva. The high concentration of human secretory IgA (hsIgA in human colostrum strongly suggests that it should play an important role in the passive immune protection against gastrointestinal and respiratory infections. Materials and methods Human secretory IgA was purified from colostrum. The reactivity of hsIgA against mycobacterial antigens and its protective capacity against mycobacterial infection was evaluated. Results The passive administration of hsIgA reduces the pneumonic area before challenge with M. tuberculosis. The intratracheal administration of M. tuberculosis preincubated with hsIgA to mice greatly reduced the bacterial load in the lungs and diminished lung tissue injury. Conclusions HsIgA purified from colostrum protects against M. tuberculosis infection in an experimental mouse model.

  6. A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania.

    Science.gov (United States)

    Lyda, Todd A; Joshi, Manju B; Andersen, John F; Kelada, Andrew Y; Owings, Joshua P; Bates, Paul A; Dwyer, Dennis M

    2015-06-01

    Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts.

  7. The regulated secretory pathway in CD4(+ T cells contributes to human immunodeficiency virus type-1 cell-to-cell spread at the virological synapse.

    Directory of Open Access Journals (Sweden)

    Clare Jolly

    2011-09-01

    Full Text Available Direct cell-cell spread of Human Immunodeficiency Virus type-1 (HIV-1 at the virological synapse (VS is an efficient mode of dissemination between CD4(+ T cells but the mechanisms by which HIV-1 proteins are directed towards intercellular contacts is unclear. We have used confocal microscopy and electron tomography coupled with functional virology and cell biology of primary CD4(+ T cells from normal individuals and patients with Chediak-Higashi Syndrome and report that the HIV-1 VS displays a regulated secretion phenotype that shares features with polarized secretion at the T cell immunological synapse (IS. Cell-cell contact at the VS re-orientates the microtubule organizing center (MTOC and organelles within the HIV-1-infected T cell towards the engaged target T cell, concomitant with polarization of viral proteins. Directed secretion of proteins at the T cell IS requires specialized organelles termed secretory lysosomes (SL and we show that the HIV-1 envelope glycoprotein (Env localizes with CTLA-4 and FasL in SL-related compartments and at the VS. Finally, CD4(+ T cells that are disabled for regulated secretion are less able to support productive cell-to-cell HIV-1 spread. We propose that HIV-1 hijacks the regulated secretory pathway of CD4(+ T cells to enhance its dissemination.

  8. The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading

    DEFF Research Database (Denmark)

    Iba, K; Albrechtsen, R; Gilpin, B

    2000-01-01

    -dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading...

  9. Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

    Directory of Open Access Journals (Sweden)

    Chugh Dipti

    2010-05-01

    Full Text Available Abstract Background The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries. Results A synthetic insulin precursor (IP-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae α-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant. Using immobilized metal ion affinity chromatography (IMAC as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth. Conclusions A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.

  10. Salivary Secretory Immunoglobulin a secretion increases after 4-weeks ingestion of chlorella-derived multicomponent supplement in humans: a randomized cross over study

    Science.gov (United States)

    2011-01-01

    Background Chlorella, a unicellular green alga that grows in fresh water, contains high levels of proteins, vitamins, minerals, and dietary fibers. Some studies have reported favorable immune function-related effects on biological secretions such as blood and breast milk in humans who have ingested a chlorella-derived multicomponent supplement. However, the effects of chlorella-derived supplement on mucosal immune functions remain unclear. The purpose of this study was to investigate whether chlorella ingestion increases the salivary secretory immunoglobulin A (SIgA) secretion in humans using a blind, randomized, crossover study design. Methods Fifteen men took 30 placebo and 30 chlorella tablets per day for 4 weeks separated by a 12-week washout period. Before and after each trial, saliva samples were collected from a sterile cotton ball that was chewed after overnight fasting. Salivary SIgA concentrations were measured using ELISA. Results Compliance rates for placebo and chlorella ingestions were 97.0 ± 1.0% and 95.3 ± 1.6%, respectively. No difference was observed in salivary SIgA concentrations before and after placebo ingestion (P = 0.38). However, salivary SIgA concentrations were significantly elevated after chlorella ingestion compared to baseline (P chlorella ingestion than before intake (P chlorella-derived multicomponent supplement increases salivary SIgA secretion and possibly improves mucosal immune function in humans. PMID:21906314

  11. Effect of yoghurt containing Bifidobacterium lactis Bb12® on faecal excretion of secretory immunoglobulin A and human beta-defensin 2 in healthy adult volunteers

    Directory of Open Access Journals (Sweden)

    Kabeerdoss Jayakanthan

    2011-12-01

    Full Text Available Abstract Background Probiotics are used to provide health benefits. The present study tested the effect of a probiotic yoghurt on faecal output of beta-defensin and immunoglobulin A in a group of young healthy women eating a defined diet. Findings 26 women aged 18-21 (median 19 years residing in a hostel were given 200 ml normal yoghurt every day for a week, followed by probiotic yoghurt containing Bifidobacterium lactis Bb12® (109 in 200 ml for three weeks, followed again by normal yoghurt for four weeks. Stool samples were collected at 0, 4 and 8 weeks and assayed for immunoglobulin A and human beta-defensin-2 by ELISA. All participants tolerated both normal and probiotic yoghurt well. Human beta-defensin-2 levels in faeces were not altered during the course of the study. On the other hand, compared to the basal sample, faecal IgA increased during probiotic feeding (P = 0.0184 and returned to normal after cessation of probiotic yoghurt intake. Conclusions Bifidobacterium lactis Bb12® increased secretory IgA output in faeces. This property may explain the ability of probiotics to prevent gastrointestinal and lower respiratory tract infections.

  12. Effect of yoghurt containing Bifidobacterium lactis Bb12® on faecal excretion of secretory immunoglobulin A and human beta-defensin 2 in healthy adult volunteers.

    Science.gov (United States)

    Kabeerdoss, Jayakanthan; Devi, R Shobana; Mary, R Regina; Prabhavathi, D; Vidya, R; Mechenro, John; Mahendri, N V; Pugazhendhi, Srinivasan; Ramakrishna, Balakrishnan S

    2011-12-23

    Probiotics are used to provide health benefits. The present study tested the effect of a probiotic yoghurt on faecal output of beta-defensin and immunoglobulin A in a group of young healthy women eating a defined diet. 26 women aged 18-21 (median 19) years residing in a hostel were given 200 ml normal yoghurt every day for a week, followed by probiotic yoghurt containing Bifidobacterium lactis Bb12® (10⁹ in 200 ml) for three weeks, followed again by normal yoghurt for four weeks. Stool samples were collected at 0, 4 and 8 weeks and assayed for immunoglobulin A and human beta-defensin-2 by ELISA. All participants tolerated both normal and probiotic yoghurt well. Human beta-defensin-2 levels in faeces were not altered during the course of the study. On the other hand, compared to the basal sample, faecal IgA increased during probiotic feeding (P = 0.0184) and returned to normal after cessation of probiotic yoghurt intake. Bifidobacterium lactis Bb12® increased secretory IgA output in faeces. This property may explain the ability of probiotics to prevent gastrointestinal and lower respiratory tract infections.

  13. Evaluation of erythrocyte band 3 phosphotyrosine level, glutathione content, CA-125, and human epididymal secretory protein E4 as combined parameters in endometriosis.

    Science.gov (United States)

    Bordin, Luciana; Fiore, Cristina; Donà, Gabriella; Andrisani, Alessandra; Ambrosini, Guido; Faggian, Diego; Plebani, Mario; Clari, Giulio; Armanini, Decio

    2010-10-01

    To investigate the biochemical parameters of the erythrocyte response to diamide-induced oxidative stress, alone or as adjuncts to serum values of CA-125 and human epididymal secretory protein E4 (HE4), in the diagnosis and study of endometriosis. University of Padova. Prospective study. Forty-five patients of reproductive age undergoing laparoscopy. All women were studied for endometriotic foci during laparoscopic surgery. Forty-one had laparoscopically and histologically confirmed endometriosis, and four did not. Twenty women with confirmed endometriosis were reassessed 1-4 months later. CA-125 and HE4 and two new parameters evaluated in erythrocytes after diamide-induced stress, that is, band 3 tyrosine phosphorylation (Tyr-P) level and decrease in total glutathione content (ΔGSH), were assessed in all patients. In association with serum CA-125 levels but not with HE4, diamide-related erythrocyte band 3 Tyr-P and ΔGSH were significantly higher in patients with endometriosis and were able to discriminate with high sensitivity and specificity between patients before and after surgery. Endometriosis is associated with an increase in systemic oxidative stress, affecting the antioxidative defenses of circulating erythrocytes. All related implications, including evaluation of other oxidative stress-related changes, warrant further study. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  14. Partial sleep deprivation activates the DNA damage response (DDR) and the senescence-associated secretory phenotype (SASP) in aged adult humans.

    Science.gov (United States)

    Carroll, Judith E; Cole, Steven W; Seeman, Teresa E; Breen, Elizabeth C; Witarama, Tuff; Arevalo, Jesusa M G; Ma, Jeffrey; Irwin, Michael R

    2016-01-01

    Age-related disease risk has been linked to short sleep duration and sleep disturbances; however, the specific molecular pathways linking sleep loss with diseases of aging are poorly defined. Key cellular events seen with aging, which are thought to contribute to disease, may be particularly sensitive to sleep loss. We tested whether one night of partial sleep deprivation (PSD) would increase leukocyte gene expression indicative of DNA damage responses (DDR), the senescence-associated secretory phenotype (SASP), and senescence indicator p16(INK4a) in older adult humans, who are at increased risk for cellular senescence. Community-dwelling older adults aged 61-86years (n=29; 48% male) underwent an experimental partial sleep deprivation (PSD) protocol over 4 nights, including adaptation, an uninterrupted night of sleep, partial sleep deprivation (sleep restricted 3-7AM), and a subsequent full night of sleep. Blood samples were obtained each morning to assess peripheral blood mononuclear cell (PBMC) gene expression using Illumina HT-12 arrays. Analyses of microarray results revealed that SASP (psenescence marker p16(INK4a) (CDKN2A) was increased 1day after PSD compared to baseline (psenescence. These findings causally link sleep deprivation to the molecular processes associated with biological aging. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Validation of an excretory/secretory antigen based-ELISA for the diagnosis of Opisthorchis felineus infection in humans from low trematode endemic areas.

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    Maria Angeles Gómez-Morales

    Full Text Available Since opisthorchiasis does not show pathognomonic signs or symptoms, physicians can have serious problems to make a differential diagnosis of this infection in non endemic areas, in particular when there is a simultaneous occurrence with other seasonal infections. Moreover, symptomatic infections due to O. felineus can last a few weeks and then the signs and symptoms disappear, but the worms survive in the bile ducts for years causing hepatobiliary diseases including hepatomegaly, cholangitis, fibrosis of the periportal system, cholecystitis, and gallstones. Consequently, an early diagnosis prevents chronicity and loss of working days. The detection of specific antibodies has been considered as a complementary tool to the fecal examination to establish the definitive diagnosis of this infection and for the follow up. Therefore the aim of this work was the development and validation of an enzyme-linked immunosorbent assay (ELISA using excretory/secretory antigens (ESA from O. felineus adult worms to detect anti-Opisthorchis IgG in human sera. A total of 370 human sera were tested: 144 sera from persons with a confirmed diagnosis of opisthorchiasis, 110 sera from healthy Italian people, and 116 sera from people with other parasitic or non-parasitic infections. Results were analyzed by receiver-operator characteristic (ROC curve analysis. The accuracy of the test, calculated by the area under curve (AUC, yielded a 0.999 value, indicating the high performance of the test. The sensitivity was 100% (95% CI: 97.40% to 100% and no false-negative sera were detected; the specificity was 99.09% (95% CI: 95.02% to 99.83%. The validated ELISA shows a good performance in terms of sensitivity, repeatability and reproducibility, and it is suitable to detect anti-Opisthorchis IgG in human sera for diagnostic purposes and for the follow up to assess the efficacy of drug treatment.

  16. Distal M domain of cobra ADAM-like metalloproteinase mediates the binding of positively charged cysteine-rich domain to αvβ3 integrin in the suppression of cell migration.

    Science.gov (United States)

    Wu, Po-Long; Lin, Chien-Chu; Lin, Ting-Hui; Lee, Min-Shi; Wu, Wen-Guey

    2016-08-01

    We have previously identified two new P-III type ADAM-like snake venom metalloproteinases (SVMPs), i.e., atragin and kaouthiagin-like, from Taiwan cobra venom and determined their 3D structures with a distinct C- and I-shaped metalloproteinase/disintegrin-like/cysteine-rich (MDC) modular architecture. Herein, we investigated their functional targets to elucidate the role of cobra SVMPs in perturbing wound healing in snakebite victims. We showed that the non-RGD (Arg-Gly-Asp) C-shaped SVMP atragin binds about ten-fold stronger than the RGD-containing I-shaped SVMP kaouthiagin-like to αvβ3 integrin in the surface-immobilized form. Atragin binds to αvβ3 integrin through a novel interaction mode involving distal M and C domains via the RRN sequence motif in the hyper variable loop. In a cell adhesion assay, the adhesion of fibroblasts to atragin was mediated by αvβ3 integrin. Furthermore, atragin inhibited wound healing and suppressed cell migration in a αvβ3 integrin-dependent manner. These results, together with our previous demonstration of non-cytotoxic cobra CTX A5 in targeting αvβ3 integrin, suggest that cobra venom consists of several non-RGD toxins with integrin-binding specificity that could perturb wound healing in snakebite victims. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases

    Energy Technology Data Exchange (ETDEWEB)

    Miyakawa, Takuya; Sawano, Yoriko; Miyazono, Ken-ichi [Department of Applied Biochemical Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan); Hatano, Ken-ichi [Department of Chemistry and Chemical Biology, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biochemical Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2007-09-01

    Purification and crystallization of ginkbilobin-2 and its selenomethionine derivative allowed the collection of complete data to 2.38 Å resolution and multiwavelength anomalous diffraction data sets, respectively. The antifungal protein ginkbilobin-2 (Gnk2) from Ginkgo biloba seeds does not show homology to other pathogenesis-related proteins, but does show homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Native Gnk2 purified from ginkgo nuts and the selenomethionine derivative of recombinant Gnk2 (SeMet-rGnk2) were crystallized by the sitting-drop vapour-diffusion method using different precipitants. X-ray diffraction data were collected from Gnk2 at 2.38 Å resolution and from SeMet-rGnk2 at 2.79 Å resolution using a synchrotron-radiation source. The crystals of both proteins belonged to the primitive cubic space group P2{sub 1}3, with unit-cell parameters a = b = c = 143.2 Å.

  18. Age-Dependent Decline in the Coordinated [Ca2+] and Insulin Secretory Dynamics in Human Pancreatic Islets.

    Science.gov (United States)

    Westacott, Matthew J; Farnsworth, Nikki L; St Clair, Joshua R; Poffenberger, Greg; Heintz, Audrey; Ludin, Nurin W; Hart, Nathaniel J; Powers, Alvin C; Benninger, Richard K P

    2017-09-01

    Aging is associated with increased risk for type 2 diabetes, resulting from reduced insulin sensitivity and secretion. Reduced insulin secretion can result from reduced proliferative capacity and reduced islet function. Mechanisms underlying altered β-cell function in aging are poorly understood in mouse and human islets, and the impact of aging on intraislet communication has not been characterized. Here, we examine how β-cell [Ca2+] and electrical communication are impacted during aging in mouse and human islets. Islets from human donors and from mice were studied using [Ca2+] imaging, static and perifusion insulin secretion assays, and gap junction permeability measurements. In human islets, [Ca2+] dynamics were coordinated within distinct subregions of the islet, invariant with islet size. There was a marked decline in the coordination of [Ca2+] dynamics, gap junction coupling, and insulin secretion dynamics with age. These age-dependent declines were reversed by pharmacological gap junction activation. These results show that human islet function declines with aging, which can reduce insulin action and may contribute to increased risk of type 2 diabetes. © 2017 by the American Diabetes Association.

  19. Mammary Analogue Secretory Carcinoma.

    Science.gov (United States)

    Stevens, Todd M; Parekh, Vishwas

    2016-09-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor that shares the same histologic appearance and ETV6 gene (12p13) rearrangement as secretory carcinoma of the breast. Prior to its recognition, MASC cases were commonly labeled acinic cell carcinoma and adenocarcinoma, not otherwise specified. Despite distinctive histologic features, MASC may be difficult to distinguish from other salivary gland tumors, in particular zymogen-poor acinic cell carcinoma and low-grade salivary duct carcinoma. Although characteristic morphologic and immunohistochemical features form the basis of a diagnosis of MASC, the presence of an ETV6-NTRK3 gene fusion is confirmatory. Given its recent recognition the true prognostic import of MASC is not yet clearly defined.

  20. Human IgA as a heterovalent ligand: switching from the asialoglycoprotein receptor to secretory component during transport across the rat hepatocyte.

    Science.gov (United States)

    Schiff, J M; Fisher, M M; Jones, A L; Underdown, B J

    1986-03-01

    Asialoglycoproteins are taken up by the rat liver for degradation; rat polymeric IgA is taken up via a separate receptor, secretory component (SC), for quantitative delivery to bile. There is negligible uptake of these ligands by the converse receptor, and only a low level of missorting of ligands to opposite destinations. The two pathways are not cross-inhibitable and operate independently (Schiff, J.M., M. M. Fisher, and B. J. Underdown, 1984, J. Cell Biol., 98:79-89). We report here that when human IgA is presented as a ligand in the rat, it is processed using elements of both pathways. To study this in detail, different IgA fractions were prepared using two radiolabeling methods that provide separate probes for degradation or re-secretion. Behavior of intravenously injected human polymeric IgA in the rat depended on its binding properties. If deprived of SC binding activity by affinity adsorption or by reduction and alkylation, greater than 80% of human IgA was degraded in hepatic lysosomes; radioactive catabolites were released into bile by a leupeptin-inhibitable process. If prevented from binding to the asialoglycoprotein receptor by competition or by treatment with galactose oxidase, human IgA was cleared and transported to bile directly via SC, but its uptake was about fivefold slower than rat IgA. Untreated human IgA was taken up rapidly by the asialoglycoprotein receptor, but depended on SC binding to get to bile: the proportion secreted correlated 1:1 with SC binding activity determined in vitro, and the IgA was released into bile with SC still attached. These results demonstrate that human IgA is normally heterovalent: it is first captured from blood by the asialoglycoprotein receptor, but escapes the usual fate of asialoglycoproteins by switching to SC during transport. Since the biliary transit times of native human and rat IgA are the same, it is probable that the receptor switching event occurs en route. This implies that the two receptors briefly

  1. General method for selective labelling of double-chain cysteine-rich peptides with a lanthanide chelate via solid-phase synthesis.

    Science.gov (United States)

    Shabanpoor, Fazel; Separovic, Frances; Wade, John D

    2011-03-01

    The use of lanthanides in preference to radioisotopes as probes for various biological assays has gained enormous popularity. The introduction of lanthanide chelates to peptides/proteins can be carried out either in solution using a commercially available labelling kit or by solid-phase peptide synthesis using an appropriate lanthanide chelate. Herein, a detailed protocol for the latter is provided for the labelling of peptides or small proteins with diethylenetriamine-N, N, N″, N″-tetra-tert-butyl acetate-N'-acetic acid (DTPA) chelate or other similar chelates on a solid support using a chimeric insulin-like peptide composed of human insulin-like peptide 5 (INSL5) A-chain and relaxin-3 B-chain as a model peptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

  2. Directional secretory response of double stranded RNA-induced thymic stromal lymphopoetin (TSLP) and CCL11/eotaxin-1 in human asthmatic airways.

    Science.gov (United States)

    Nino, Gustavo; Huseni, Shehlanoor; Perez, Geovanny F; Pancham, Krishna; Mubeen, Humaira; Abbasi, Aleeza; Wang, Justin; Eng, Stephen; Colberg-Poley, Anamaris M; Pillai, Dinesh K; Rose, Mary C

    2014-01-01

    Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral) and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. Primary human bronchial epithelial cells (HBEC) from control (n = 3) and asthmatic (n = 3) donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI) conditions and treated apically with dsRNA (viral surrogate) or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC) from normal (n = 3) and asthmatic (n = 3) donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20) vs. non-asthmatic uninfected controls (n = 20). Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay. Our data demonstrate that: 1) Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2) TSLP exposure induces unidirectional (apical) secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3) Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1. There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations.

  3. Directional secretory response of double stranded RNA-induced thymic stromal lymphopoetin (TSLP and CCL11/eotaxin-1 in human asthmatic airways.

    Directory of Open Access Journals (Sweden)

    Gustavo Nino

    Full Text Available Thymic stromal lymphoproetin (TSLP is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state.Primary human bronchial epithelial cells (HBEC from control (n = 3 and asthmatic (n = 3 donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI conditions and treated apically with dsRNA (viral surrogate or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC from normal (n = 3 and asthmatic (n = 3 donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20 vs. non-asthmatic uninfected controls (n = 20. Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay.Our data demonstrate that: 1 Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2 TSLP exposure induces unidirectional (apical secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3 Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1.There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations.

  4. Partial Sleep Deprivation Activates the DNA Damage Response (DDR) and the Senescence-Associated Secretory Phenotype (SASP) in Aged Adult Humans

    Science.gov (United States)

    Carroll, Judith E.; Cole, Steven W.; Seeman, Teresa E.; Breen, Elizabeth C.; Witarama, Tuff; Arevalo, Jesusa M.G.; Ma, Jeffrey; Irwin, Michael R.

    2015-01-01

    Age-related disease risk has been linked to short sleep duration and sleep disturbances; however, the specific molecular pathways linking sleep loss with diseases of aging are poorly defined. Key cellular events seen with aging, which are thought to contribute to disease, may be particularly sensitive to sleep loss. We tested whether one night of partial sleep deprivation (PSD) would increase leukocyte gene expression indicative of DNA damage responses (DDR), the senescence-associated secretory phenotype (SASP), and senescence indicator p16INK4a in older adult humans, who are at increased risk for cellular senescence. Community-dwelling older adults aged 61-86 years (n=29; 48% male) underwent an experimental partial sleep deprivation (PSD) protocol over 4 nights, including adaptation, an uninterrupted night of sleep, partial sleep deprivation (sleep restricted 3 AM to 7 AM), and a subsequent full night of sleep. Blood samples were obtained each morning to assess peripheral blood mononuclear cell (PBMC) gene expression using Illumina HT-12 arrays. Analyses of microarray results revealed that SASP (p < .05) and DDR (p = .08) gene expression were elevated from baseline to PSD nights. Gene expression changes were also observed from baseline to PSD in NFKB2, NBS1 and CHK2 (all p's < .05). The senescence marker p16INK4a (CDKN2A) was increased one day after PSD compared to baseline (p < .01), however confirmatory RT-PCR did not replicate this finding. One night of partial sleep deprivation activates PBMC gene expression patterns consistent with biological aging in this older adult sample. PSD enhanced the SASP and increased the accumulation of damage that initiates cell cycle arrest and promotes cellular senescence. These findings causally link sleep deprivation to the molecular processes associated with biological aging. PMID:26336034

  5. Human peripheral late/exhausted memory B cells express a senescent-associated secretory phenotype and preferentially utilize metabolic signaling pathways.

    Science.gov (United States)

    Frasca, Daniela; Diaz, Alain; Romero, Maria; Blomberg, Bonnie B

    2017-01-01

    The percentage of late/exhausted memory (LM) B cells increases with age and we show here that this is associated with a lower influenza vaccine response. To identify novel contributors to the phenotypic and functional changes observed in aged B cells, we sorted the major peripheral B cell subsets [naïve, IgM memory, switched memory (swIg) and late/exhausted memory (LM)] and determined their percentages in the peripheral blood as well as their level of immune activation by measuring basal levels of expression of multiple senescence-associated secretory phenotype (SASP) markers, such as pro-inflammatory cytokines (TNF-α/IL-6/IL-8), inflammatory micro-RNAs (miRs, miR-155/16/93), cell cycle regulators (p16(INK4)). We found that only memory B cells express SASP markers, and especially the LM B cell subset, which is also showing spontaneous activation of AMP-activated protein kinase (AMPK), the energy sensing enzyme which is ubiquitously expressed in mammalian cells. LM B cells, but not IgM memory B cells, activate a p38MAPK signaling pathway, downstream of AMPK, leading to the expression of SASP mediators, while class switch recombination is downregulated. These data show that some B cell subsets are more inflammatory than others, that they are pre-activated and that this signaling through metabolic pathways is associated with a senescence phenotype, demonstrating for the first time in human B lymphocytes the link between aging, cellular senescence, SASP and metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Proliferation and Cytokine Production of Human Mesangial Cells Stimulated by Secretory IgA Isolated from Patients with IgA Nephropathy

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    Yan Liang

    2015-07-01

    Full Text Available Background/Aims: IgA nephropathy (IgAN is the most common form of primary glomerulonephritis, and often aggravates by mucosal infection. Secretory IgA (SIgA is the dominant immunoglobulin in mucosal immunity, and is deposited in the mesangium in IgAN. The biological effects of SIgA on mesangial cells are poorly understood. Methods: Deposition of SIgA in frozen renal sections from IgAN patients was detected and the association between deposition of SIgA and patients characteristics was analyzed. The biological effects of SIgA and polymeric IgA (pIgA on human renal mesangial cells were compared. We also studied the molecular mechanism of microRNA regulating the inflammatory effects of SIgA on mesangial cells. Results: Fifty-five of 176 patients had SIgA deposition with higher incidence of infection history and hematuria, lower serum cystatin C, β2 microglobulin, blood urea nitrogen and T-grade in the Oxford classification, compared with patients without SIgA deposition. SIgA stimulated mesangial cells at a higher ratio of proliferation and higher production of interleukin (IL-6, IL-8, monocyte chemotactic protein 1, transforming growth factor-β1 and fibronectin, compared with SIgA from healthy volunteers. The proliferation and cytokines production in mesangial cells stimulated by SIgA were significantly lower than that stimulated by pIgA. miR-16 targeted the 3′-untranslated region of IL-6 and suppressed its translation in mesangial cells induced by SIgA. Conclusions: The biological effects of SIgA on mesangial cells differ from those of pIgA. SIgA stimulates mesangial cell proliferation and production of proinflammatory cytokines. IL-6 production is regulated by miR-16 in mesangial cells.

  7. Value of detection of serum human epididymis secretory protein 4 and carbohydrate antigen 125 in diagnosis of early endometrial cancer of different pathological subtypes.

    Science.gov (United States)

    Liu, Xingui; Zhao, Fengting; Hu, Linli; Sun, Yingpu

    2015-01-01

    This study explored the value of detection of human epididymis secretory protein 4 (HE4) and carbohydrate antigen 125 (CA125) from serum in diagnosis of early endometrial cancer of different pathological subtypes and discussed the mechanism of HE4 and CA125 in diagnosis. In this study, enzyme-linked immunosorbent assay and chemiluminescent immunoassay were used to detect HE4 and CA125 from serum in endometrial cancer and control groups. Besides, the concentration of HE4 and CA125 was compared in these two groups, and the expression of CA125 and HE4 and clinicopathological characteristics in patients with endometrial cancer were also analyzed. Compared with the control group, the expression of HE4 was much higher in serum of patients with endometrial cancer, while there was no obvious change in the expression of CA125. The threshold detection value was acquired by receiver operating characteristic analysis method, that is, 141.5 pmol/L and 54.5 U/L, respectively. When comparing the concentration of HE4 in patients with endometrial cancer at the early stage (stage I) with healthy people, the difference therein had statistical significance, but there was no obvious difference in CA125. HE4 and CA125 in diagnosis of endometrial cancer in the stages I and II were found with no statistically significant difference. The difference of HE4 in the stages II and III had statistical significance while the difference of CA125 had no statistical significance. The specificity of both HE4 and CA125 was 95%, and the sensitivity of HE4 to uterine papillary serous carcinomas was higher than that to endometrioid adenocarcinoma. Thus, the serum HE4 is much better than CA125 in detecting the endometrial cancer at an early stage.

  8. Endoplasmic reticulum (ER stress inducible factor cysteine-rich with EGF-like domains 2 (Creld2 is an important mediator of BMP9-regulated osteogenic differentiation of mesenchymal stem cells.

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    Jiye Zhang

    Full Text Available Mesenchymal stem cells (MSCs are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2 in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

  9. Silencing of reversion-inducing cysteine-rich protein with Kazal motifs stimulates hyperplastic phenotypes through activation of epidermal growth factor receptor and hypoxia-inducible factor-2α.

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    You Mie Lee

    Full Text Available Reversion-inducing cysteine-rich protein with Kazal motifs (RECK, a tumor suppressor is down-regulated by the oncogenic signals and hypoxia, but the biological function of RECK in early tumorigenic hyperplastic phenotypes is largely unknown. Knockdown of RECK by small interfering RNA (siRECK or hypoxia significantly promoted cell proliferation in various normal epithelial cells. Hypoxia as well as knockdown of RECK by siRNA increased the cell cycle progression, the levels of cyclin D1 and c-Myc, and the phosphorylation of Rb protein (p-pRb, but decreased the expression of p21(cip1, p27(kip1, and p16(ink4A. HIF-2α was upregulated by knockdown of RECK, indicating HIF-2α is a downstream target of RECK. As knockdown of RECK induced the activation of epidermal growth factor receptor (EGFR and treatment of an EGFR kinase inhibitor, gefitinib, suppressed HIF-2α expression induced by the silencing of RECK, we can suggest that the RECK silenicng-EGFR-HIF-2α axis might be a key molecular mechanism to induce hyperplastic phenotype of epithelial cells. It was also found that shRNA of RECK induced larger and more numerous colonies than control cells in an anchorage-independent colony formation assay. Using a xenograft assay, epithelial cells with stably transfected with shRNA of RECK formed a solid mass earlier and larger than those with control cells in nude mice. In conclusion, the suppression of RECK may promote the development of early tumorigenic hyperplastic characteristics in hypoxic stress.

  10. Identification of a novel small cysteine-rich protein in the fraction from the biocontrol Fusarium oxysporum strain CS-20 that mitigates Fusarium wilt symptoms and triggers defense responses in tomato

    Directory of Open Access Journals (Sweden)

    Larisa A. Shcherbakova

    2016-01-01

    Full Text Available The biocontrol effect of the nonpathogenic F. oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP, which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed.

  11. Endometrial CRISP3 is regulated throughout the mouse estrous and human menstrual cycle and facilitates adhesion and proliferation of endometrial epithelial cells.

    Science.gov (United States)

    Evans, Jemma; D'Sylva, Rebecca; Volpert, Marianna; Jamsai, Duangporn; Merriner, Donna Jo; Nie, Guiying; Salamonsen, Lois A; O'Bryan, Moira K

    2015-04-01

    The endometrium (the mucosal lining of the uterus) is a dynamic tissue that undergoes extensive remodeling, secretory transformation in preparation for implantation of an embryo, inflammatory and proteolytic activity during menstruation, and rapid postmenstrual repair. A plethora of local factors influence these processes. Recently, a cysteine-rich protein, CRISP3, a clade of the CRISP, antigen 5, pathogenesis-related (CAP) protein superfamily, has been implicated in uterine function. The localization, regulation, and potential function of CRISP3 in both the human and mouse endometrium is described. CRISP3 localizes to the luminal and glandular epithelium of the endometrium within both species, with increased immunoreactivity during the proliferative phase of the human cycle. CRISP3 also localizes to neutrophils, particularly within the premenstrual human endometrium and during the postbreakdown repair phase of a mouse model of endometrial breakdown and repair. Endometrial CRISP3 is produced by primary human endometrial epithelial cells and secreted in vivo to accumulate in the uterine cavity. Secreted CRISP3 is more abundant in uterine lavage fluid during the proliferative phase of the menstrual cycle. Human endometrial epithelial CRISP3 is present in both a glycosylated and a nonglycosylated form in vitro and in vivo. Treatment of endometrial epithelial cells in vitro with recombinant CRISP3 enhances both adhesion and proliferation. These data suggest roles for epithelial and neutrophil-derived CRISP3 in postmenstrual endometrial repair and regeneration. © 2015 by the Society for the Study of Reproduction, Inc.

  12. Is there any association between secretory IgA and lactoferrin concentration in mature human milk and food allergy in breastfed children.

    Science.gov (United States)

    Hogendorf, Anna; Stańczyk-Przyłuska, Anna; Sieniwicz-Luzeńczyk, Katarzyna; Wiszniewska, Magdalena; Arendarczyk, Jerzy; Banasik, Małgorzata; Fendler, Wojciech; Kowalski, Marek; Zeman, Krzysztof

    2013-01-01

    Breastfeeding is recommended as a protective method against the development of allergy. However, some studies have reported an increased risk of allergies development in breastfed infants of atopic mothers, which implies that atopic mothers may have an altered composition of breast milk. The aim of the study was to determine the concentration of secretory immunoglobulin A (S-IgA) and lactoferrin in human mature milk and to evaluate the association between the levels of these proteins in breast milk with food allergy in children, depending on the allergy status of the breastfeeding mother. Medical data was collected from birth to 24 months of age from 84 mother-child pairs participating in an EU-funded project "EuroPrevall - The prevalence, cost and basis of food allergy across Europe". The diagnosis of food allergy in children was based on the positive result of a double-blind placebo-controlled food challenge (DBPCFC). S-IgA and lactoferrin levels were measured in the whey of mature breast milk with commercial enzyme-linked immunosorbent assay (ELISA) kits. Statistical analysis (the U Mann-Whitney and Kruskal-Wallis tests as well as the Spearman's rank correlation coefficient) was performed using STATISTICA 8.0 PL (Statsoft, Tulsa, USA). Ten out of eighty four participating children had positive skin prick tests (SPT) and/or sIgE to food antigens and in 7 (8.4%) DBPCFC confirmed food allergy. the median concentration of S-IgA was 476,83 μg/ml (range 6.51-1359.61 μg/ml). the median concentration of Lf was 15.68 μg/ml (range 11.68-36.43 μg/ml). The concentrations of S-IgA and Lf showed a moderate, negative, correlation R=-0.28; p=0.05. Mature breast milk of mothers of children with food allergy and of healthy children showed similar concentrations of both proteins. The level of S-IgA in the mature milk of mothers with atopic allergy was significantly lower, compared to non-atopic mothers. More studies are needed to reveal the mystery of the lack of protective

  13. Hydrolysis of Phosphatidylcholine-Isoprostanes (PtdCho-IP) by Peripheral Human Group IIA, V and X Secretory Phospholipases A2 (sPLA2).

    Science.gov (United States)

    Kuksis, Arnis; Pruzanski, Waldemar

    2017-06-01

    Biologically active F- and E/D-type-prostane ring isomers (F2-IP and E2/D2-IP, respectively) are produced in situ by non-enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho-IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet-activating factor acetylhydrolases (PAF-AH) are the main endogenous PLA2 involved in degradation of PtdCho-IP. The present study shows that the PtdCho-IP are also subject to hydrolysis by group IIA, V and X secretory PLA2, which also have a wide peripheral tissue distribution. For this demonstration, we compared the LC/MS profiles of PtdCho-IP of auto-oxidized plasma lipoproteins after incubation for 1-4 h (37 °C) in the absence or presence of recombinant human sPLA2 (1-2.5 µg/ml). In the absence of exogenously added sPLA2 the total PtdCho-IP level after 4 h incubation reached 15.9, 21.6 and 8.7 nmol/mg protein of LDL, HDL and HDL3, respectively. In the presence of group V or group X sPLA2 (2.5 µg/ml), the PtdCho-IP was completely hydrolyzed in 1 h, while in the presence of group IIA sPLA2 (2.5 µg/ml) the hydrolysis was less than 25% in 4 h, although it was complete after 8-24 h incubation. This report provides the first demonstration that PtdCho-IP are readily hydrolyzed by group IIA, V and X sPLA2. A co-location of sPLA2 and the substrates in various tissues has been recorded. Thus, the initiation of interaction and production of isoprostanes in situ are highly probable.

  14. A model of the complex between human beta-microseminoprotein and CRISP-3 based on NMR data

    DEFF Research Database (Denmark)

    Ghasriani, Houman; Fernlund, Per; Udby, Lene

    2008-01-01

    beta-Microseminoprotein (MSP), a 10kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human...... MSP and CRISP-3 with multidimensional NMR. (15)N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show...... that only one side of hMSP, comprising beta-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that beta-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model...

  15. A model of the complex between human beta-microseminoprotein and CRISP-3 based on NMR data.

    Science.gov (United States)

    Ghasriani, Houman; Fernlund, Per; Udby, Lene; Drakenberg, Torbjörn

    2009-01-09

    beta-Microseminoprotein (MSP), a 10kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human MSP and CRISP-3 with multidimensional NMR. (15)N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show that only one side of hMSP, comprising beta-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that beta-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model of CRISP-3.

  16. Purification and characterization of CRISP-3 from human seminal plasma and its real-time binding kinetics with PSP94.

    Science.gov (United States)

    Anklesaria, Jenifer H; Pandya, Rushika R; Pathak, Bhakti R; Mahale, Smita D

    2016-12-15

    Cysteine-rich secretory proteins (CRISPs) have been postulated to have a role in male reproduction and prostate pathophysiology. Of the mammalian CRISPs, CRISP-3 levels in particular have been shown to be upregulated in prostate cancer. Efforts have been made to obtain highly pure CRISP-3 for gaining structure-function information of this protein. However, well characterized and highly pure protein is not available yet. CRISPs from snake venom have been purified using prostate secretory protein of 94 amino acids (PSP94) has been reported earlier. In the present study, CRISP-3 was purified to homogeneity from human seminal plasma using human PSP94-immnobilized affinity column. The molecular mass of the purified protein was determined by SDS-PAGE followed by immunoblotting and found to be ∼26kDa and ∼28kDa. The purity was further verified using MALDI-TOF MS analysis, where two peaks at m/z 25509 and 27715 were obtained. The lower molecular weight peak corresponds to the calculated molecular mass of CRISP-3 (∼26kDa); whereas the higher molecular weight peak was confirmed to be the glycosylated form (∼28kDa) from the deglycosylation experiment. Binding of PSP94 in increasing concentrations to purified CRISP-3 immobilized chip was further validated using surface plasmon resonance. The kinetics data suggested that purified CRISP-3 binds specifically and with high affinity to PSP94. In conclusion, a homogeneous preparation of highly pure CRISP-3 protein is obtained from human seminal plasma. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Excretory/secretory products of anisakid nematodes

    DEFF Research Database (Denmark)

    Mehrdana, Foojan; Buchmann, Kurt

    2017-01-01

    as intermediate or transport hosts and mammals or birds as final hosts. Human consumption of raw or underprocessed seafood containing third stage larvae of anisakid parasites may elicit a gastrointestinal disease (anisakidosis) and allergic responses. Excretory and secretory (ES) compounds produced...... a therapeutic potential in immune-related diseases....... by the parasites are assumed to be key players in clinical manifestation of the disease in humans, but the molecules are likely to play a general biological role in invertebrates and lower vertebrates as well. ES products have several functions during infection, e.g. penetration of host tissues and evasion of host...

  18. Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins.

    Science.gov (United States)

    Laustsen, Andreas H; Gutiérrez, José María; Lohse, Brian; Rasmussen, Arne R; Fernández, Julián; Milbo, Christina; Lomonte, Bruno

    2015-06-01

    The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. High ω-3:ω-6 fatty acids ratio increases fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in human ectopic endometrial cells

    Science.gov (United States)

    Khanaki, Korosh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mehdi; Darabi, Masoud; Mehdizadeh, Amir; Shabani, Mahdi; Rahimipour, Ali; Nouri, Mohammad

    2014-01-01

    Background: Endometriosis, a common chronic inflammatory disorder, is defined by the atypical growth of endometrium- like tissue outside of the uterus. Secretory phospholipase A2 group IIa (sPLA2-IIa) and fatty acid binding protein4 (FABP4) play several important roles in the inflammatory diseases. Objective: Due to reported potential anti-inflammatory effects of ω-3 and ω-6 fatty acids, the purpose of the present study was to investigate the effects of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) on fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in cultured endometrial cells. Materials and Methods: Ectopic and eutopic endometrial tissues obtained from 15 women were snap frozen. After thawing and tissue digestion, primary mixed stromal and endometrial epithelial cell culture was performed for 8 days in culture mediums supplemented with normal and high ratios of ω-3 and ω-6 PUFA. sPLA2-IIa in the culture medium and FABP4 level was determined using enzyme immuno assay (EIA) technique. Results: Within ectopic endometrial cells group, the level of cellular FABP4 and extracellular sPLA2-IIa were remarkably increased under high ω-3 PUFA exposure compared with control condition (p=0.014 and p=0.04 respectively). Conclusion: ω-3 PUFAs may increase the level of cellular FABP4 and extracellular sPLA2-IIa in ectopic endometrial cells, since sPLAIIa and FABP4 may affect endometriosis via several mechanisms, more relevant studies are encouraged to know the potential effect of increased cellular FABP4 and extracellular sPLA2-IIa on endometriosis. PMID:25709631

  20. [The microbiology of secretory otitis].

    Science.gov (United States)

    Lacosta, J L; Infante, J C; Undabeitia, E; Gastañares, M J

    1995-01-01

    The AA. realize a comparative study on the differences between the nasopharyngeal microbial flora of 50 children suffering a secretory otitis and other 40 children without middle ear disease. In nasopharyngeal cultures the pathogenic flora (Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Streptococcus beta hemoliticus group A, Staphilococcus aureus) amounted for 96 percent in children with secretory otitis, which figure was reduced to 80 percent in healthy infants (p < 0.05). Haemophilus influenzae was the most identified microorganism in a both nasopharyngeal and otic flora. We have found a significative association (p < 0.001) among nasopharyngeal and otic flora of each individual.

  1. Overexpression of the secretory small GTPase Rab27B in human breast cancer correlates closely with lymph node metastasis and predicts poor prognosis

    Directory of Open Access Journals (Sweden)

    Zhang Jia-Xing

    2012-12-01

    Full Text Available Abstract Background The secretory small GTPase Rab27b was recently identified as an oncogene in breast cancer (BC in vivo and in vitro studies. This research was designed to further explore the clinical and prognostic significance of Rab27B in BC patients. Methods The mRNA/protein expression level of Rab27B was examined by performing Real-time PCR, western blot, and immunohistochemistry (IHC assays in 12 paired BC tissues and matched adjacent noncancerous tissues (NAT. Then we carried out IHC assay in a large cohort of 221 invasive BC tissues, 22 normal breast tissues, 40 fibroadenoma (FA, 30 ductual carcinoma in situ (DCIS and 40 metastatic lymph nodes (LNs. The receiver operating characteristic curve method was applied to obtain the optimal cutoff value for high Rab27B expression. Epithelial-mesenchymal transition (EMT marker expression levels were detected in relation to Rab27B expression. Results We observed that the increased expression of Rab27B was dependent upon the magnitude of cancer progression (P P  Conclusion Our findings suggest that overexpression of Rab27B in BC coincides with lymph node metastasis and acquisition of a poor prognostic phenotype.

  2. Cytokine, chemokine and secretory IgA levels in human milk in relation to atopic disease and IgA production in infants.

    Science.gov (United States)

    Böttcher, Malin F; Jenmalm, Maria C; Björkstén, Bengt

    2003-02-01

    The relationship between breast-feeding, IgA production and development of atopic disease in children is a matter of controversy. Some of this controversy might be due to individual differences in the composition of breast milk. The aim of this study was to relate the levels of cytokines, chemokines and secretory (S)-IgA antibodies in breast milk to the development of atopic manifestation and salivary IgA production in infants. Cytokine, chemokine and SIgA levels, as measured with enzyme-linked immunosorbent assay (ELISA), in colostrum and mature milk were analyzed in relation to the development of positive skin-prick tests (SPT), allergic symptoms and salivary IgA antibody production during the first 2 years of life in 53 infants. There was no association between levels of IL-4, -5, -6, -8, -10, -13, -16, IFN-gamma, TGF-beta1, -beta2, RANTES, eotaxin or SIgA levels in the breast milk with either SPT-positivity, development of allergic symptoms or salivary IgA levels during the first 2 years of life in the infants. Thus, differences in the composition of cytokines, chemokines and SIgA in breast milk did not, to any major degree, affect the development of a positive SPT, atopic symptoms, nor salivary IgA antibody production during the first 2 years of life.

  3. LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts.

    Science.gov (United States)

    Zhang, Hui; Sweezey, Neil B; Kaplan, Feige

    2015-02-15

    Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development. Copyright © 2015 the American Physiological

  4. Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

    Energy Technology Data Exchange (ETDEWEB)

    Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu [Pathology and Microbiology Department, 986495 Nebraska Medical Center, Omaha, NE 68198-6495 (United States)

    2011-05-01

    The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.

  5. Directional Secretory Response of Double Stranded RNA-Induced Thymic Stromal Lymphopoetin (TSLP) and CCL11/Eotaxin-1 in Human Asthmatic Airways

    OpenAIRE

    Gustavo Nino; Shehlanoor Huseni; Perez, Geovanny F.; Krishna Pancham; Humaira Mubeen; Aleeza Abbasi; Justin Wang; Stephen Eng; Colberg-Poley, Anamaris M.; Pillai, Dinesh K; Mary C. Rose

    2014-01-01

    BACKGROUND: Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral) and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. METHODS: Primary human bronchial epithelial cells (HBEC) from c...

  6. Secretory Products of the Human GI Tract Microbiome and Their Potential Impact on Alzheimer's Disease (AD: Detection of Lipopolysaccharide (LPS in AD Hippocampus

    Directory of Open Access Journals (Sweden)

    Yuhai Zhao

    2017-07-01

    Full Text Available Although the potential contribution of the human gastrointestinal (GI tract microbiome to human health, aging, and disease is becoming increasingly acknowledged, the molecular mechanics and signaling pathways of just how this is accomplished is not well-understood. Major bacterial species of the GI tract, such as the abundant Gram-negative bacilli Bacteroides fragilis (B. fragilis and Escherichia coli (E. coli, secrete a remarkably complex array of pro-inflammatory neurotoxins which, when released from the confines of the healthy GI tract, are pathogenic and highly detrimental to the homeostatic function of neurons in the central nervous system (CNS. For the first time here we report the presence of bacterial lipopolysaccharide (LPS in brain lysates from the hippocampus and superior temporal lobe neocortex of Alzheimer's disease (AD brains. Mean LPS levels varied from two-fold increases in the neocortex to three-fold increases in the hippocampus, AD over age-matched controls, however some samples from advanced AD hippocampal cases exhibited up to a 26-fold increase in LPS over age-matched controls. This “Perspectives” paper will further highlight some very recent research on GI tract microbiome signaling to the human CNS, and will update current findings that implicate GI tract microbiome-derived LPS as an important internal contributor to inflammatory degeneration in the CNS.

  7. Muscle as a secretory organ

    DEFF Research Database (Denmark)

    Pedersen, Bente K

    2013-01-01

    Skeletal muscle is the largest organ in the body. Skeletal muscles are primarily characterized by their mechanical activity required for posture, movement, and breathing, which depends on muscle fiber contractions. However, skeletal muscle is not just a component in our locomotor system. Recent...... evidence has identified skeletal muscle as a secretory organ. We have suggested that cytokines and other peptides that are produced, expressed, and released by muscle fibers and exert either autocrine, paracrine, or endocrine effects should be classified as "myokines." The muscle secretome consists...... of several hundred secreted peptides. This finding provides a conceptual basis and a whole new paradigm for understanding how muscles communicate with other organs such as adipose tissue, liver, pancreas, bones, and brain. In addition, several myokines exert their effects within the muscle itself. Many...

  8. Identification of the human Lewis(a) carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium myrtillus L.).

    Science.gov (United States)

    Melo, N S; Nimtz, M; Conradt, H S; Fevereiro, P S; Costa, J

    1997-09-29

    This paper reports for the first time the presence of the human Lewis(a) type determinant in glycoproteins secreted by plant cells. A single glycopeptide was identified in the tryptic hydrolysis of the peroxidase VMPxC1 from Vaccinium myrtillus L. by HPLC/ESI-MS. The oligosaccharide structures were elucidated by ESI-MS-MS and by methylation analysis before and after removal of fucose by mild acid hydrolysis. The major structure determined is of the biantennary plant complex type containing the outer chain motif Lewis(a) [structure in text]. A corresponding fucosyltransferase activity catalyzing the formation of Lewis(a) type structures in vitro was identified in cellular extracts of the suspension cultures.

  9. Humoral immunity to commensal oral bacteria in human infants: salivary secretory immunoglobulin A antibodies reactive with Streptococcus mitis biovar 1, Streptococcus oralis, Streptococcus mutans, and Enterococcus faecalis during the first two years of life.

    Science.gov (United States)

    Cole, M F; Bryan, S; Evans, M K; Pearce, C L; Sheridan, M J; Sura, P A; Wientzen, R L; Bowden, G H

    1999-04-01

    Secretory immunoglobulin A (SIgA) antibodies reactive with the pioneer oral streptococci Streptococcus mitis biovar 1 and Streptococcus oralis, the late oral colonizer Streptococcus mutans, and the pioneer enteric bacterium Enterococcus faecalis in saliva samples from 10 human infants from birth to age 2 years were analyzed. Low levels of salivary SIgA1 and SIgA2 antibodies reactive with whole cells of all four species were detected within the first month after birth, even though S. mutans and E. faecalis were not recovered from the mouths of the infants during the study period. Although there was a fivefold increase in the concentration of SIgA between birth and age 2 years, there were no differences between the concentrations of SIgA1 and SIgA2 antibodies reactive with the four species over this time period. When the concentrations of SIgA1 and SIgA2 antibodies reactive with all four species were normalized to the concentrations of SIgA1 and SIgA2 in saliva, SIgA1 and SIgA2 antibodies reactive with these bacteria showed a significant decrease from birth to 2 years of age. Adsorption of each infant's saliva with cells of one species produced a dramatic reduction of antibodies recognizing the other three species. Sequential adsorption of saliva samples removed all SIgA antibody to the bacteria, indicating that the SIgA antibodies were directed to antigens shared by all four species. The induction by the host of a limited immune response to common antigens that are likely not involved in adherence may be among the mechanisms that commensal streptococci employ to persist in the oral cavity.

  10. Effects of smoking, mother's age, body mass index, and parity number on lipid, protein, and secretory immunoglobulin A concentrations of human milk.

    Science.gov (United States)

    Bachour, Pamela; Yafawi, Rula; Jaber, Farouk; Choueiri, Elias; Abdel-Razzak, Ziad

    2012-06-01

    This study investigated the effect of smoking, mother's age, body mass index (BMI), and parity number on density, lipids, proteins, and secreted immunoglobulin A (SIgA) of human milk. Transitional and mature milk samples were collected from 23 nursing smoker mothers and 43 nursing nonsmoker mothers. Proteins, lipids, and SIgA concentrations were determined as well as the milk density and the general protein profile. Our investigation showed that the milk of smokers contained less lipids and proteins (statistically significant 26% and 12% decrease, respectively), whereas milk density was unchanged. SIgA concentration was 27% lower in milk from smokers, but the decrease was not statistically significant. The general protein profile showed no significant smoking-associated changes in the four identified proteins (β-casein, immunoglobulin A heavy chain, serum albumin, and lactoferrin). Mothers' age and residential area showed noticeable but statistically nonsignificant differences in some of the measured parameters. However, parity number, lactation stage, and BMI were associated with a significant modification of milk composition. Mature milk contained more lipids and less protein, whereas the increase of parity number was associated with an increase in lipid concentration. The group of overweight mothers showed lower milk protein concentration in comparison with the normal group. Multivariate analysis showed a statistically significant interaction effect of the variables (smoking, parity number, lactation stage, age, and BMI) on lipids and between some of them on proteins and SIgA. Our study showed that smoking was associated with lower milk lipid and protein concentrations and that the parity number and BMI were associated with a change in milk lipids and proteins content, respectively.

  11. Immunoglobulins in nasal secretions of healthy humans: structural integrity of secretory immunoglobulin A1 (IgA1) and occurrence of neutralizing antibodies to IgA1 proteases of nasal bacteria

    DEFF Research Database (Denmark)

    Kirkeby, L; Rasmussen, TT; Reinholdt, Jesper

    2000-01-01

    Certain bacteria, including overt pathogens as well as commensals, produce immunoglobulin A1 (IgA1) proteases. By cleaving IgA1, including secretory IgA1, in the hinge region, these enzymes may interfere with the barrier functions of mucosal IgA antibodies, as indicated by experiments in vitro. P...

  12. Ophiophagus hannah Venom: Proteome, Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv

    Directory of Open Access Journals (Sweden)

    Witchuda Danpaiboon

    2014-05-01

    Full Text Available Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase. N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

  13. Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

    Science.gov (United States)

    Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

    2014-05-13

    Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

  14. Secretory products of helminth parasites as immunomodulators.

    Science.gov (United States)

    Harnett, William

    2014-07-01

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

  15. Porosome: The Universal Secretory Portal in Cells

    Science.gov (United States)

    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

  16. Characterization and treatment of persistent hepatocellular secretory failure.

    Science.gov (United States)

    van Dijk, Remco; Kremer, Andreas E; Smit, Wouter; van den Elzen, Bram; van Gulik, Thomas; Gouma, Dirk; Lameris, Johan S; Bikker, Hennie; Enemuo, Valentine; Stokkers, Pieter C F; Feist, Mark; Bosma, Piter; Jansen, Peter L M; Beuers, Ulrich

    2015-04-01

    Hepatocellular secretory failure induced by drugs, toxins or transient biliary obstruction may sometimes persist for months after removal of the initiating factor and may then be fatal without liver transplantation. We characterized patients with severe persistent hepatocellular secretory failure (PHSF) and treated them with the pregnane X receptor (PXR) agonist, rifampicin. We also studied the effect of rifampicin on PXR-dependent expression of genes involved in biotransformation and secretion in vitro. Thirteen patients (age 18-81 years, 6 male) with hepatocellular secretory failure that persisted after removal of the inducing factor (drugs/toxin: 9) or biliary obstruction (4) were identified over 6 years. Six of these patients were screened for ATP8B1 or ABCB11 mutations. All were treated with rifampicin (300 mg daily) for 1-10 weeks. Expression of genes involved in biotransformation and secretion was determined by rtPCR in human hepatocytes and intestinal cells incubated with rifampicin (10 μmol/L). Serum bilirubin of patients with PHSF ranged from 264 to 755 μmol/L. Normal γGT was found in 10/13 patients of whom 3/6 tested positive for ATP8B1/ABCB11 mutations. Serum bilirubin declined to hepatocellular secretory failure may develop in carriers of transporter gene mutations. In severe cases, rifampicin may represent an effective therapeutic option of PHSF. PXR-dependent induction of CYP3A4, UGT1A1, MRP2 and OSTβ could contribute to the anticholestatic effect of rifampicin in PHSF. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Characterization and treatment of persistent hepatocellular secretory failure

    NARCIS (Netherlands)

    van Dijk, Remco; Kremer, Andreas E.; Smit, Wouter; van den Elzen, Bram; van Gulik, Thomas; Gouma, Dirk; Lameris, Johan S.; Bikker, Hennie; Enemuo, Valentine; Stokkers, Pieter C. F.; Feist, Mark; Bosma, Piter; Jansen, Peter L. M.; Beuers, Ulrich

    2015-01-01

    Hepatocellular secretory failure induced by drugs, toxins or transient biliary obstruction may sometimes persist for months after removal of the initiating factor and may then be fatal without liver transplantation. We characterized patients with severe persistent hepatocellular secretory failure

  18. Data for the inhibition effects of recombinant lamprey CRBGP on the tube formation of HUVECs and new blood vessel generation in CAM models

    Directory of Open Access Journals (Sweden)

    Qi Jiang

    2016-03-01

    Full Text Available In the present data article, lamprey cysteine-rich buccal gland protein (CRBGP which belongs to cysteine-rich secretory proteins (CRISPs family was recombinant and expressed in Rosetta blue cells. After identification, the recombinant protein was purified through affinity chromatograph. The inhibition effects of recombinant lamprey CRBGP (rL-CRBGP on tube formation of human umbilical vein endothelial cells (HUVECs and new blood vessel generation in chick chorioallantoic membrane (CAM models were analyzed. This paper contains data related to research concurrently published in “Anti-angiogenic activities of CRBGP from buccal glands of lampreys (Lampetra japonica” [1].

  19. Synaptic Control of Secretory Trafficking in Dendrites

    Directory of Open Access Journals (Sweden)

    Cyril Hanus

    2014-06-01

    Full Text Available Localized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. Here, we monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor KIF17 by Ca2+/calmodulin-dependent protein kinases (CaMK. Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport.

  20. Psychological distress and salivary secretory immunity

    NARCIS (Netherlands)

    Engeland, C.G.; Hugo, F.N.; Hilgert, J.B.; Nascimento, G.G.; Junges, R.; Lim, H.-J.; Marucha, P.T.; Bosch, J.A.

    Stress-induced impairments of mucosal immunity may increase susceptibility to infectious diseases. The present study investigated the association of perceived stress, depressive symptoms, and loneliness with salivary levels of secretory immunoglobulin A (S-IgA), the subclasses S-IgA1, S-IgA2, and

  1. Predicting Secretory Proteins with SignalP

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2017-01-01

    SignalP is the currently most widely used program for prediction of signal peptides from amino acid sequences. Proteins with signal peptides are targeted to the secretory pathway, but are not necessarily secreted. After a brief introduction to the biology of signal peptides and the history...

  2. Secretory immunity with special reference to the oral cavity

    Science.gov (United States)

    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

  3. Secretory immunity with special reference to the oral cavity

    Directory of Open Access Journals (Sweden)

    Per Brandtzaeg

    2013-03-01

    Full Text Available 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.

  4. Production of secretory IgA antibodies in plants.

    Science.gov (United States)

    Larrick, J W; Yu, L; Naftzger, C; Jaiswal, S; Wycoff, K

    2001-10-15

    Functional antibodies produced in tobacco plants were first reported over a decade ago (1989). The basic protocol used to generate these 'plantibodies' involved the independent cloning of H and L chain antibody genes in Agrobacterium tumefaciens vectors, the transformation of plant tissue in vitro with the recombinant bacterium, the reconstitution of whole plants expressing individual chains, and their sexual cross. In a 'Mendelian' fashion, a fully assembled and functional antibody was recovered from plant tissue in some double-transgenic plants. In mammalian cells, the antibody H and L chains are produced as precursor proteins that are translocated into the endoplasmic reticulum (ER), under the guidance of signal sequences. Within the ER, the signal peptides are proteolytically cleaved, and several stress proteins act as chaperonins to bind the unassembled antibody chains, and direct subsequent folding and tetramer formation. A similar process occurs in plant cells, and expression can be directed via signal sequences (even of foreign origin) into the aqueous environment of the apoplasm, or to be accumulated in other specific plant tissues, including tubers, fruit, or seed. Plants can facilely assemble secretory IgA, which is comprised of four chains, H and L chains, J chain and secretory component. Plant 'bioreactors' are expected to yield over 10 kg of therapeutic antibody/acre in tobacco, maize, soybean, and alfalfa [(Ann. NY Acad. Sci.)721(1994)235; (Biotechnol. Bioeng.)20(1999)135]. Compared with conventional steel tank bioreactors using mammalian cells, or microorganisms, the costs of GMP plantibodies are expected to perhaps one tenth. The differences in glycosylation patterns of plant and mammalian cell produced antibodies apparently have no effect on antigen-binding or specificity, but there is some concern about potential immunogenicity in humans. N-linked glycans of plants differ from human by having fucose-linked alpha 1,3 and the sugar xylose. No

  5. Docking of secretory vesicles is syntaxin dependent.

    Directory of Open Access Journals (Sweden)

    Heidi de Wit

    Full Text Available Secretory vesicles dock at the plasma membrane before they undergo fusion. Molecular docking mechanisms are poorly defined but believed to be independent of SNARE proteins. Here, we challenged this hypothesis by acute deletion of the target SNARE, syntaxin, in vertebrate neurons and neuroendocrine cells. Deletion resulted in fusion arrest in both systems. No docking defects were observed in synapses, in line with previous observations. However, a drastic reduction in morphologically docked secretory vesicles was observed in chromaffin cells. Syntaxin-deficient chromaffin cells showed a small reduction in total and plasma membrane staining for the docking factor Munc18-1, which appears insufficient to explain the drastic reduction in docking. The sub-membrane cortical actin network was unaffected by syntaxin deletion. These observations expose a docking role for syntaxin in the neuroendocrine system. Additional layers of regulation may have evolved to make syntaxin redundant for docking in highly specialized systems like synaptic active zones.

  6. Mammary Analogue Secretory Carcinoma Mimicking Salivary Adenoma

    OpenAIRE

    Williams, Lindsay; Chiosea, Simion I.

    2013-01-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor characterized by ETV6 translocation. It appears that prior studies have identified MASC by reviewing salivary gland carcinomas, such as acinic cell carcinoma and adenocarcinoma, not otherwise specified. To address the possibility of MASC mimicking benign salivary neoplasms we reviewed 12 salivary gland (cyst)adenomas diagnosed prior to the discovery of MASC. One encapsulated (cyst)adenoma of the parotid g...

  7. RFP tags for labeling secretory pathway proteins

    Energy Technology Data Exchange (ETDEWEB)

    Han, Liyang; Zhao, Yanhua [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Zhang, Xi; Peng, Jianxin [College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Xu, Pingyong, E-mail: pyxu@ibp.ac.cn [Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Huan, Shuangyan, E-mail: shuangyanhuan@163.com [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Zhang, Mingshu, E-mail: mingshu1984@gmail.com [Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    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 an 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.

  8. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Steffen Nyegaard

    Full Text Available Secretory phospholipase A2 (sPLA2 is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.

  9. Novel distribution of the secretory leucocyte proteinase inhibitor in kidney

    Directory of Open Access Journals (Sweden)

    Sophie Ohlsson

    2001-01-01

    Full Text Available The secretory leucocyte proteinase inhibitor (SLPI is a low molecular weight, tissue-specific inhibitor of, for example, elastase and cathepsin G, which also have antimicrobial capacity. SLPI has been localised to the respiratory, gastrointestinal and genital tracts, but so far not to the kidney. The presence of SLPI in renal tubuli cells was demonstrated using immunohistochemistry and, by means of in situ hybridisation on human renal biopsies, we were able to demonstrate SLPI production. In various inflammatory conditions in the kidneys, the protease-antiprotease balance is disturbed. For this reason, as well as the possible role in the defence against ascending urinary tract infections, it is interesting to establish a source of SLPI in renal tubuli cells.

  10. Rac1 Regulates Endometrial Secretory Function to Control Placental Development.

    Directory of Open Access Journals (Sweden)

    Juanmahel Davila

    2015-08-01

    Full Text Available 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

  11. Rac1 Regulates Endometrial Secretory Function to Control Placental Development

    Science.gov (United States)

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

    2015-01-01

    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

  12. Immunoassays of human trefoil factors 1 and 2

    DEFF Research Database (Denmark)

    Vestergaard, E M; Brynskov, J; Ejskjaer, K

    2004-01-01

    The trefoil factors (TFF1-3) are cysteine-rich peptides expressed in the gastrointestinal tract where they play a critical role in mucosal protection and repair. The expression is up-regulated at sites of ulceration in various chronic inflammatory diseases. Recently, we presented an ELISA method...... for measurement of TFF3. The aims of the present study were to develop and evaluate ELISAs for the other two known human trefoil peptides, TFF1 and TFF2, and to carry out a cross-sectional study on serum TFF levels in patients with inflammatory bowel disease (IBD)....

  13. Targeting ADAM12 in human disease: head, body or tail?

    DEFF Research Database (Denmark)

    Jacobsen, J; Wewer, U M

    2009-01-01

    properties. This functional trinity is reflected in the structure of ADAM12, which can be divided into head, body, and tail. The head of the protein (consisting of the pro and catalytic domains) mediates processing of growth factors and cytokines and has been implicated in epidermal growth factor (EGF......) and insulin-like growth factor receptor signaling. The body of the protein (consisting of the disintegrin, cysteine-rich, and EGF-like domains) is involved in contacts with the extracellular matrix and other cells through interactions with integrins and syndecans. Finally, the tail of the protein (consisting...... the possible approaches to targeting ADAM12 in human disease....

  14. ATP: The crucial component of secretory vesicles.

    Science.gov (United States)

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  15. Secretory proteins in the saliva of children.

    Science.gov (United States)

    Sivakumar, Thiruvanamalai; Hand, Arthur R; Mednieks, Maija

    2009-12-01

    The protein composition of oral fluid is modulated by environmental factors and physiological states, i.e. chemical, mechanical and pharmacologic stimuli, pathologic conditions, and psychological stress. Secretory protein concentrations in samples of whole saliva (WS) from children were measured and the results were subjected to statistical analysis. Protein expression was determined using electrophoresis and Western blotting. Protein profiles of children were significantly different from those of adults (n = 50, P saliva from children contained a group of high-molecular-weight (>90 kDa) proteins, whereas fewer than 5% of samples from adults had comparable bands. The ratio of the regulatory subunits (RII) of type II protein kinase A (an enzyme that regulates secretion) to total protein was stable in children's saliva, but variable in saliva from adults. Alpha amylase (alpha-amylase), an enzyme that digests carbohydrates, was less degraded in WS of children than in that of adults. Gingival crevicular fluid of both children and adults did not contain alpha-amylase or RII. No significant gender-based differences were found, but Caucasian children had higher salivary protein levels than children with an African background. Saliva collection is rapid, painless, non-invasive, economical, and yields findings that are reproducible. Objective, biochemical monitoring of secretory proteins in oral fluid of children may reveal responses to stressful stimuli.

  16. Influence of continuous light and darkness on the secretory ...

    Indian Academy of Sciences (India)

    Unknown

    nocturnal Indian catfish, ultrastructural indications for the presence of secretory activity in the pinealocytes of the pineal parenchyma were reported and two morpho- logical cell types: light and dark cells representing diffe- rent phases of the secretory process were demonstrated. (Srivastava 1999). The aim of the present ...

  17. Ameloblast transcriptome changes from secretory to maturation stages.

    Science.gov (United States)

    Simmer, James P; Richardson, Amelia S; Wang, Shih-Kai; Reid, Bryan M; Bai, Yongsheng; Hu, Yuanyuan; Hu, Jan C-C

    2014-08-01

    The purpose of this study was to identify the major molecular components in the secretory and maturation stages of amelogenesis through transcriptome analyses. Ameloblasts (40 sections per age group) were laser micro-dissected from Day 5 (secretory stage) and Days 11-12 (maturation stage) first molars. PolyA+ RNA was isolated from the lysed cells, converted to cDNA, and amplified to generate a cDNA library. DNA sequences were obtained using next generation sequencing and analyzed to identify genes whose expression had increased or decreased at least 1.5-fold in maturation stage relative to secretory stage ameloblasts. Among the 9198 genes that surpassed the quality threshold, 373 showed higher expression in secretory stage, while 614 genes increased in maturation stage ameloblasts. The results were cross-checked against a previously published transcriptome generated from tissues overlying secretory and maturation stage mouse incisor enamel and 34 increasing and 26 decreasing expressers common to the two studies were identified. Expression of F2r, which encodes protease activated receptor 1 (PAR1) that showed 10-fold higher expression during the secretory stage in our transcriptome analysis, was characterized in mouse incisors by immunohistochemistry. PAR1 was detected in secretory, but not maturation stage ameloblasts. We conclude that transcriptome analyses are a good starting point for identifying genes/proteins that are critical for proper dental enamel formation and that PAR1 is specifically expressed by secretory stage ameloblasts.

  18. Influence of continuous light and darkness on the secretory ...

    Indian Academy of Sciences (India)

    ... but not secretory active and dark cells showed a necrotic condition. Phagocytotic activity of the dark cells was also seen. Intense neural activity was also observed during exposure to both the artificial photoperiods. The results highlight the role of light on the secretory activities of the pinealocytes of the catfish pineal organ.

  19. Secretory Phospholipase A2-IIA and Cardiovascular Disease

    DEFF Research Database (Denmark)

    Holmes, Michael V; Simon, Tabassome; Exeter, Holly J

    2013-01-01

    This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease.......This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease....

  20. Secretory Structure, Histochemistry and Phytochemistry Analyses of Stimulant Plant

    Science.gov (United States)

    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

  1. Electroimmunodiffusion Studies of alpha Chain, Secretory Piece and Secretory IgA.

    Science.gov (United States)

    1977-03-15

    V of proteins in biological flui ds collected in wt amounts~ ‘thus eliminating the need for stimulation of secretions and sample concentration...useful for determining levels of iminunoglobulins in unconcentrated saliva . The main immuno- glc ulin in saliva and other external secretions is...throughout this study (Behring Diagnostics , Somerville, N.J.). According to the manufacturer antiserum to free secretory piece was produced by immunization

  2. Airway Secretory microRNAome Changes during Rhinovirus Infection in Early Childhood.

    Directory of Open Access Journals (Sweden)

    Maria J Gutierrez

    Full Text Available Innate immune responses are fine-tuned by small noncoding RNA molecules termed microRNAs (miRs that modify gene expression in response to the environment. During acute infections, miRs can be secreted in extracellular vesicles (EV to facilitate cell-to-cell genetic communication. The purpose of this study was to characterize the baseline population of miRs secreted in EVs in the airways of young children (airway secretory microRNAome and examine the changes during rhinovirus (RV infection, the most common cause of asthma exacerbations and the most important early risk factor for the development of asthma beyond childhood.Nasal airway secretions were obtained from children (≤3 yrs. old during PCR-confirmed RV infections (n = 10 and age-matched controls (n = 10. Nasal EVs were isolated with polymer-based precipitation and global miR profiles generated using NanoString microarrays. We validated our in vivo airway secretory miR data in an in vitro airway epithelium model using apical secretions from primary human bronchial epithelial cells (HBEC differentiated at air-liquid interface (ALI. Bioinformatics tools were used to determine the unified (nasal and bronchial signature airway secretory miRNAome and changes during RV infection in children.Multiscale analysis identified four signature miRs comprising the baseline airway secretory miRNAome: hsa-miR-630, hsa-miR-302d-3p, hsa- miR-320e, hsa-miR-612. We identified hsa-miR-155 as the main change in the baseline miRNAome during RV infection in young children. We investigated the potential biological relevance of the airway secretion of hsa-mir-155 using in silico models derived from gene datasets of experimental in vivo human RV infection. These analyses confirmed that hsa-miR-155 targetome is an overrepresented pathway in the upper airways of individuals infected with RV.Comparative analysis of the airway secretory microRNAome in children indicates that RV infection is associated with airway

  3. The secretory synapse: the secrets of a serial killer.

    Science.gov (United States)

    Bossi, Giovanna; Trambas, Christina; Booth, Sarah; Clark, Richard; Stinchcombe, Jane; Griffiths, Gillian M

    2002-11-01

    Cytotoxic T lymphocytes (CTLs) destroy their targets by a process involving secretion of specialized granules. The interactions between CTLs and target can be very brief; nevertheless, adhesion and signaling proteins segregate into an immunological synapse. Secretion occurs in a specialized secretory domain. Use of live and fixed cell microscopy allows this secretory synapse to be visualized both temporally and spatially. The combined use of confocal and electron microscopy has produced some surprising findings, which suggest that the secretory synapse may be important both in delivering the lethal hit and in facilitating membrane transfer from target to CTL. Studies on the secretory synapse in wild-type and mutant CTLs have been used to identify proteins involved in secretion. Further clues as to the signals required for secretion are emerging from comparisons of inhibitory and activating synapses formed by natural killer cells.

  4. Mammary analogue secretory carcinoma mimicking salivary adenoma.

    Science.gov (United States)

    Williams, Lindsay; Chiosea, Simion I

    2013-12-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor characterized by ETV6 translocation. It appears that prior studies have identified MASC by reviewing salivary gland carcinomas, such as acinic cell carcinoma and adenocarcinoma, not otherwise specified. To address the possibility of MASC mimicking benign salivary neoplasms we reviewed 12 salivary gland (cyst)adenomas diagnosed prior to the discovery of MASC. One encapsulated (cyst)adenoma of the parotid gland demonstrated features of MASC. The diagnosis was confirmed by fluorescence in situ hybridization with an ETV6 break-apart probe. An unusual complex pattern of ETV6 rearrangement with duplication of the telomeric/distal ETV6 probe was identified. This case illustrates that MASC may mimic salivary (cyst)adenomas. To more accurately assess true clinical and morphologic spectrum of MASC, future studies may have to include review of salivary (cyst)adenomas. The differential diagnosis of MASC may have to be expanded to include cases resembling salivary (cyst)adenomas.

  5. Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses

    Directory of Open Access Journals (Sweden)

    Sharp Tyler M

    2012-09-01

    Full Text Available Abstract Background Our previous report that the Norwalk virus nonstructural protein p22 is an antagonist of the cellular secretory pathway suggests a new aspect of norovirus/host interaction. To explore conservation of function of this highly divergent calicivirus protein, we examined the effects of p22 homologues from four human and two murine noroviruses, and feline calicivirus on the secretory pathway. Findings All human noroviruses examined induced Golgi disruption and inhibited protein secretion, with the genogroup II.4 Houston virus being the most potent antagonist. Genogroup II.6 viruses have a conserved mutation in the mimic of an Endoplasmic Reticulum export signal (MERES motif that is highly conserved in human norovirus homologues of p22 and is critical for secretory pathway antagonism, and these viruses had reduced levels of Golgi disruption and inhibition of protein secretion. p22 homologues from both persistent and nonpersistent strains of murine norovirus induced Golgi disruption, but only mildly inhibited cellular protein secretion. Feline calicivirus p30 did not induce Golgi disruption or inhibit cellular protein secretion. Conclusions These differences confirm a norovirus-specific effect on host cell secretory pathway antagonism by homologues of p22, which may affect viral replication and/or cellular pathogenesis.

  6. NON-SECRETORY MYELOMA: READY FOR A NEW DEFINITION?

    Directory of Open Access Journals (Sweden)

    Alessandro Corso

    2017-08-01

    Full Text Available Non-secretory myeloma is a myeloma subtype whose diagnosis, until a few years ago, was established by demonstration of monoclonal plasma cells in the bone marrow and by negative results on serum and urine electrophoresis and immunofixation studies. However, this type of myeloma could be misdiagnosed if the workup does not include an accurate study of serum free light chain test since some of the patients diagnosed as non-secretory could be light chain only with small amounts monoclonal proteinuria. Due to this limit in classification, all the information available today, generally coming from retrospective studies including patients studied in a complete and incomplete fashion, could be misleading. A new definition is thus needed to distinguish between the true non-secretory, with a possible better prognosis, and the other forms of oligosecretory myeloma with a prognosis more similar to the secretory form of myeloma. With all the data of the literature, the availability of laboratory and radiological tools, times are mature to depict a new definition of non-secretory myeloma which deserves, a peculiar work up and different response evaluation and, maybe, a different therapeutic approach.   Keywords: Non-secretory myeloma; Free light chain; Myeloma subtype.

  7. Platelet secretory products inhibit lipoprotein metabolism in macrophages

    Science.gov (United States)

    Phillips, David R.; Arnold, Kay; Innerarity, Thomas L.

    1985-08-01

    Macrophages possess a receptor that binds low-density lipoproteins (LDL) containing lysine residues modified by acetylation (Ac LDL), acetoacetylation (AcAc LDL) or malondialdehyde treatment1-3. This receptor (referred to as the Ac LDL receptor or scavenger receptor) internalizes the bound lipoprotein. As a consequence, massive amounts of cholesteryl esters accumulate so that macrophages in culture resemble foam cells found in atherosclerotic lesions4,5. In an effort to identify an unmodified mammalian macromolecule that binds to the Ac LDL receptor, we investigated whether platelet secretory products affect the receptor-mediated endocytosis of chemically modified lipoproteins. Platelets are a potential source of such activity because they exist in close association with foam cells in developing atherosclerotic lesions6,7. Our study demonstrates that human blood platelets secrete a product that inhibits the binding and uptake of AcAc LDL by mouse peritoneal macrophages and the subsequent accumulation of cholesteryl esters. This is the first indication that an endogenous macromolecule interacts with Ac LDL receptor on macrophages.

  8. Cytopathologic features of mammary analogue secretory carcinoma.

    Science.gov (United States)

    Bishop, Justin A; Yonescu, Raluca; Batista, Denise A S; Westra, William H; Ali, Syed Z

    2013-05-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland neoplasm that is defined by ETV6-NTRK3 gene fusion. To the best of the authors' knowledge, only rare case reports of the cytopathologic features of MASC have been published to date. A wide variety of archival salivary gland tumors were tested for ETV6 translocation by break-apart fluorescent in situ hybridization. Positive cases with preoperative fine-needle aspiration (FNA) specimens or intraoperative touch preparations were retrieved from the archives of The Johns Hopkins Hospital. All smears were reviewed and the cytologic characteristics were described. Five cases of MASC with cytopathologic material (4 FNA specimens and 1 touch preparation) were identified. The cases occurred in 3 men and 2 women ranging in age from 21 years to 78 years (mean, 52 years). On the cytologic smears, the MASCs were variably cellular and exhibited 2 different architectural patterns: 1) intact tissue fragments with isomorphic cells arranged in a sheet-like or papillary configuration; and 2) dispersed and dissociated cells with a mostly "histiocyte-like" appearance with large cells containing abundant vacuolated cytoplasm. No matrix tissue or stromal spindled cells were present. The cells did not display acinic differentiation in the form of cytoplasmic zymogen granules. In each case, the preoperative FNA correctly identified a neoplasm, and the most frequent diagnostic considerations were acinic cell carcinoma, mucoepidermoid carcinoma, and pleomorphic adenoma. MASC is a newly described salivary gland tumor that should be considered in the differential diagnosis of low-grade salivary gland neoplasms. Its cytologic features overlap considerably with those of other tumors, especially acinic cell carcinoma and mucoepidermoid carcinoma. Copyright © 2013 American Cancer Society.

  9. Secretory structure and histochemistry test of some Zingiberaceae plants

    Science.gov (United States)

    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

  10. Cysteine-rich 61-connective tissue growth factor-nephroblastoma-overexpressed 5 (CCN5)/Wnt-1-induced signaling protein-2 (WISP-2) regulates microRNA-10b via hypoxia-inducible factor-1α-TWIST signaling networks in human breast cancer cells.

    Science.gov (United States)

    Haque, Inamul; Banerjee, Snigdha; Mehta, Smita; De, Archana; Majumder, Monami; Mayo, Matthew S; Kambhampati, Suman; Campbell, Donald R; Banerjee, Sushanta K

    2011-12-16

    MicroRNAs (miRNAs) are naturally occurring single-stranded RNA molecules that post-transcriptionally regulate the expression of target mRNA transcripts. Many of these target mRNA transcripts are involved in regulating processes commonly altered during tumorigenesis and metastatic growth. These include cell proliferation, differentiation, apoptosis, migration, and invasion. Among the several miRNAs, miRNA-10b (miR-10b) expression is increased in metastatic breast cancer cells and positively regulates cell migration and invasion through the suppression of the homeobox D10 (HOXD10) tumor suppressor signaling pathway. In breast metastatic cells, miR-10b expression is enhanced by a transcription factor TWIST1. We find that miR-10b expression in breast cancer cells can be suppressed by CCN5, and this CCN5 effect is mediated through the inhibition of TWIST1 expression. Moreover, CCN5-induced inhibition of TWIST1 expression is mediated through the translational inhibition/modification of hypoxia-inducible factor-1α via impeding JNK signaling pathway. Collectively, these studies suggest a novel regulatory pathway exists through which CCN5 exerts its anti-invasive function. On the basis of these findings, it is plausible that reactivation of CCN5 in miR-10b-positive invasive/metastatic breast cancers alone or in combination with current therapeutic regimens could provide a unique, alternative strategy to existing breast cancer therapy.

  11. Stress modulates intestinal secretory immunoglobulin A

    Directory of Open Access Journals (Sweden)

    Rafael eCampos-Rodríguez

    2013-12-01

    Full Text Available Stress is a response of the central nervous system to environmental stimuli perceived as a threat to homeostasis. The stress response triggers the generation of neurotransmitters and hormones from the hypothalamus pituitary adrenal axis, sympathetic axis and brain gut axis, and in this way modulates the intestinal immune system. The effects of psychological stress on intestinal immunity have been investigated mostly with the restraint/immobilization rodent model, resulting in an up or down modulation of SIgA levels depending on the intensity and time of exposure to stress. SIgA is a protein complex formed by dimeric (dIgA or polymeric IgA (pIgA and the secretory component (SC, a peptide derived from the polymeric immunoglobulin receptor (pIgR. The latter receptor is a transmembrane protein expressed on the basolateral side of gut epithelial cells, where it uptakes dIgA or pIgA released by plasma cells in the lamina propria. As a result, the IgA-pIgR complex is formed and transported by vesicles to the apical side of epithelial cells. pIgR is then cleaved to release SIgA into the luminal secretions of gut. Down modulation of SIgA associated with stress can have negative repercussions on intestinal function and integrity. This can take the form of increased adhesion of pathogenic agents to the intestinal epithelium and/or an altered balance of inflammation leading to greater intestinal permeability. Most studies on the molecular and biochemical mechanisms involved in the stress response have focused on systemic immunity. The present review analyzes the impact of stress (mostly by restraint/immobilization, but also with mention of other models on the generation of SIgA, pIgR and other humoral and cellular components involved in the intestinal immune response. Insights into these mechanisms could lead to better therapies for protecting against pathogenic agents and avoiding epithelial tissue damage by modulating intestinal inflammation.

  12. Role of acidic stores in secretory epithelia.

    Science.gov (United States)

    Gerasimenko, Julia; Peng, Shuang; Gerasimenko, Oleg

    2014-06-01

    There is growing evidence that intracellular calcium plays a primary role in the pathophysiology of the pancreas in addition to its crucial importance in major physiological functions. Pancreatic acinar cells have a remarkably large amount of Ca(2+) stored in both the endoplasmic reticulum (ER) and the acidic stores. The vast majority of the classical ER Ca(2+) store is located in the basal part of the acinar cells with extensions protruding into the apical area, however, the acidic stores are exclusively located in the secretory granular area of the cells. Both types of Ca(2+) store respond to all three intracellular Ca(2+) messengers - inositol trisphosphate (InsP3), cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). The two stores interact with each other via calcium-induced calcium release; however, they can be separated using pharmacological tools. The ER relies on sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) that can be blocked by the specific inhibitor thapsigargin. The acidic store requires a low pH that can be modified by blocking vacuolar H(+)-ATPase. The acidic store is particularly important for pathological processes in the pancreas. Acute pancreatitis is initiated as a result of calcium overload in the apical pole, which leads to trypsinogen activation; two major causes are gall bladder stones and excessive alcohol consumption. Excessive Ca(2+) release from the acidic stores plays a major role in both scenarios; however NAADP-induced calcium release from acidic stores is particularly important for bile-induced pancreatitis. Cell-permeable calmodulin (CaM) activators such as CALP3 boost the natural protective effect of CaM by inhibiting excessive calcium release from the internal stores through inositol trisphosphate (InsP3R) and ryanodine receptors (RyR). Alternatively calcium overload can be dramatically reduced by inhibiting Ca(2+)-release-activated Ca(2+) (CRAC) currents that are required to reload the

  13. The Essential Oil Secretory Structures of Prostanthera ovalifolia (Lamiaceae)

    Science.gov (United States)

    GERSBACH, P. V.

    2002-01-01

    The structure of the essential oil secretory tissues of Prostanthera ovalifolia R.Br was investigated using bright‐ and dark‐field optical microscopy, and scanning and transmission electron microscopy. The leaves of P. ovalifolia have glandular trichomes of the peltate type common to many Lamiaceae species. The trichomes consist of a basal cell embedded in the epidermis, a stalk cell with heavily cutinized walls and a 16‐celled secretory head, but they differ from those of many previously reported Lamiaceae species in their morphological form defined by the elevated cuticle. The sub‐cuticular space contains a mixture of lipid and aqueous phases. Secretory cells have dense cytoplasm with many leucoplasts present. Volatile terpenoids are eliminated from the cytoplasm into the sub‐cuticular space, the site of essential oil accumulation, via granulocrine secretion. PMID:12096737

  14. Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

    Science.gov (United States)

    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.

  15. Secretory phospholipase A2-IIa upregulates HER/HER2-elicited signaling in lung cancer cells

    OpenAIRE

    DONG, ZHONGYUN; MELLER, JAROSLAW; SUCCOP, PAUL; WANG, JIANG; WIKENHEISER-BROKAMP, KATHRYN; STARNES, SANDRA; LU, SHAN

    2014-01-01

    Lung cancer is the leading cause of cancer death worldwide. There is an urgent need for early diagnostic tools and novel therapies in order to increase lung cancer survival. Secretory phospholipase A2 group IIa (sPLA2-IIa) is involved in inflammation, tumorigenesis and metastasis. We were the first to uncover that cancer cells secrete sPLA2-IIa. sPLA2-IIa is overexpressed in almost all specimens of human lung cancers examined and is significantly elevated in the plasma of lung cancer patients...

  16. Mammary analogue secretory carcinoma: A rare salivary gland tumour

    Directory of Open Access Journals (Sweden)

    B S Jackson

    2017-04-01

    Full Text Available Mammary analogue secretory carcinoma (MASC is a rare and recently described tumour of the salivary glands. MASC has similar histomorphological and immunohistochemical features of secretory carcinoma of the breast. MASC can be mistaken for other salivary gland tumours, especially acinic cell carcinoma. A 28-year-old man was diagnosed with a rare salivary gland tumour in Pretoria, South Africa (SA. To our knowledge, a report of MASC in SA has not previously been published. The surgeons dealing with salivary gland tumours should be aware of the clinical presentation. Current treatment is similar to that of other salivary gland malignancies.

  17. Mammary analogue secretory carcinoma: A rare salivary gland tumour.

    Science.gov (United States)

    Jackson, B S; Pratt, T L; Van Rooyen, A

    2017-03-29

    Mammary analogue secretory carcinoma (MASC) is a rare and recently described tumour of the salivary glands. MASC has similar histomorphological and immunohistochemical features of secretory carcinoma of the breast. MASC can be mistaken for other salivary gland tumours, especially acinic cell carcinoma. A 28-year-old man was diagnosed with a rare salivary gland tumour in Pretoria, South Africa (SA). To our knowledge, a report of MASC in SA has not previously been published. The surgeons dealing with salivary gland tumours should be aware of the clinical presentation. Current treatment is similar to that of other salivary gland malignancies.

  18. Identification and characterization of secretory proteins during ionizing radiation-induced premature senescence

    Energy Technology Data Exchange (ETDEWEB)

    Han, Na Kyung; Hong, Mi Na; Jung, Seung Hee; Kang, Kyoung Ah; Lee, Jae Seon [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Chi, Seong Gil [Korea University, Seoul (Korea, Republic of)

    2011-05-15

    Cellular senescence was first described by Hayflick and Moorhead in 1961 who observed that cultures of normal human fibroblasts had a limited replicative potential and eventually became irreversibly arrest. The majority of senescent cells assume a characteristic flattened and enlarged morphological change, senescence associated {beta} alactosidase positivity. Recently a large number of molecular phenotypes such as changes in gene expression, protein processing and chromatin organization have been also described. In contrast to apoptosis, senescence does not destroy the cells but leaves them metabolically and synthetically active and therefore able to affect their microenvironment. In particular, senescent fibroblasts and some cancer cells were found to secrete proteins with known or putative tumor-promoting functions such as growth factors or proteolytic enzymes. However, the knowledge about secreted proteins from senescent tumor cells and their functions to surrounding cells is still lacking. In this study, changes of senescence associated secretory protein expression profile were observed in MCF7 human breast cancer cells exposed to gamma-ray radiation using two dimensional electrophoresis. Also, we identified up-regulated secretory proteins during ionizing radiation-induced cellular senescence. Here, we show that senescent human breast cancer MCF7 cells promote the proliferation, invasion and migration of neighboring cells

  19. Matrix-dependent local retention of secretory vesicle cargo in cortical neurons

    NARCIS (Netherlands)

    de Wit, J.; Toonen, R.F.G.; Verhage, M.

    2009-01-01

    Neurons secrete many diffusible signals from synaptic and other secretory vesicles. We characterized secretion of guidance cues, neuropeptides, neurotrophins, and proteases from single secretory vesicles using pHluorin-tagged cargo in cortical neurons. Stimulation triggered transient and persistent

  20. SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kandaswamy, Krishna Kumar [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany); Graduate School for Computing in Medicine and Life Sciences, University of Luebeck, 23538 Luebeck (Germany); Pugalenthi, Ganesan [School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Hartmann, Enno; Kalies, Kai-Uwe [Centre for Structural and Cell Biology in Medicine, Institute of Biology, University of Luebeck, 23538 Luebeck (Germany); Moeller, Steffen [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany); Suganthan, P.N. [School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Martinetz, Thomas, E-mail: martinetz@inb.uni-luebeck.de [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany)

    2010-01-15

    Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at (http://www.inb.uni-luebeck.de/tools-demos/spred/spred).

  1. Generic sorting of raft lipids into secretory vesicles in yeast

    DEFF Research Database (Denmark)

    Surma, Michal A; Klose, Christian; Klemm, Robin W

    2011-01-01

    a complete lipid overview of the yeast late secretory pathway. We could show that vesicles captured with different baits carry the same cargo and have almost identical lipid compositions; being highly enriched in ergosterol and sphingolipids. This finding indicates that lipid raft sorting is a generic...... feature of vesicles carrying PM cargo and suggests a common lipid-based mechanism for their formation....

  2. Proteolytic processing in the secretory pathway of Aspergillus niger

    NARCIS (Netherlands)

    Jalving, R.

    2005-01-01

    A number of filamentous fungi are saprophytes and they secrete a wide spectrum of enzymes to degrade their complex substrates. Many secreted proteins enter the secretory pathway as proproteins and need some form of proteolytic processing before they obtain their mature active state. As described in

  3. Secretory phospholipase A(2) in newborn infants with sepsis

    NARCIS (Netherlands)

    Schrama, A. J. J.; De Beaufort, A. J.; Poorthuis, B. J. H. M.; Berger, H. M.; Walther, F. J.

    2008-01-01

    Objective: To investigate secretory phospholipase A(2) ( sPLA(2)) activity in neonatal sepsis. Study Design: Plasma sPLA(2) activity, C- reactive protein ( CRP) concentration, leukocyte count and immature/ total neutrophil ( I/ T) ratio were assessed in a group of 156 infants admitted for neonatal

  4. Mammary analogue secretory carcinoma: A rare salivary gland tumour

    African Journals Online (AJOL)

    Salivary gland malignancy is rare, with a global annual incidence of. 3 per 100 000 people.[1,2] A rare salivary gland tumour, mammary analogue secretory carcinoma (MASC), has only recently been described.[3] The few reports and studies concerning MASC have been published in several pathology journals. We report ...

  5. Secretory ribonucleases in the primitive ruminant chevrotain (Tragulus javanicus)

    NARCIS (Netherlands)

    Breukelman, HJ; Jekel, PA; Dubois, JYF; Mulder, PPMFA; Warmels, HW; Beintema, JJ

    Phylogenetic analyses of secretory ribonucleases or RNases 1 have shown that gene duplication events, giving rise to three paralogous genes (pancreatic, seminal and brain RNase), occurred during the evolution of ancestral ruminants. A higher number of paralogous sequences are present in chevrotain

  6. Secretory Phospholipase A(2)-IIA and Cardiovascular Disease

    NARCIS (Netherlands)

    Holmes, Michael V.; Simon, Tabassome; Exeter, Holly J.; Folkersen, Lasse; Asselbergs, Folkert W.; Guardiola, Montse; Cooper, Jackie A.; Palmen, Jutta; Hubacek, Jaroslav A.; Carruthers, Kathryn F.; Horne, Benjamin D.; Brunisholz, Kimberly D.; Mega, Jessica L.; Van Iperen, Erik P. A.; Li, Mingyao; Leusink, Maarten; Trompet, Stella; Verschuren, Jeffrey J. W.; Hovingh, G. Kees; Dehghan, Abbas; Nelson, Christopher P.; Kotti, Salma; Danchin, Nicolas; Scholz, Markus; Haase, Christiane L.; Rothenbacher, Dietrich; Swerdlow, Daniel I.; Kuchenbaecker, Karoline B.; Staines-Urias, Eleonora; Goel, Anuj; van 't Hooft, Ferdinand; Gertow, Karl; de Faire, Ulf; Panayiotou, Andrie G.; Tremoli, Elena; Baldassarre, Damiano; Veglia, Fabrizio; Holdt, Lesca M.; Beutner, Frank; Gansevoort, Ron T.; Navis, Gerjan J.; Mateo Leach, Irene; Breitling, Lutz P.; Brenner, Hermann; Thiery, Joachim; Dallmeier, Dhayana; Franco-Cereceda, Anders; Boer, Jolanda M. A.; Stephens, Jeffrey W.; Hofker, Marten H.; Tedgui, Alain; Hofman, Albert; Uitterlinden, Andre G.; Adamkova, Vera; Pitha, Jan; Onland-Moret, N. Charlotte; Cramer, Maarten J.; Nathoe, Hendrik M.; Spiering, Wilko; Klungel, Olaf H.; Kumari, Meena; Whincup, Peter H.; Morrow, David A.; Braund, Peter S.; Hall, Alistair S.; Olsson, Anders G.; Doevendans, Pieter A.; Trip, Mieke D.; Tobin, Martin D.; Hamsten, Anders; Watkins, Hugh; Koenig, Wolfgang; Nicolaides, Andrew N.; Teupser, Daniel; Day, Ian N. M.; Carlquist, John F.; Gaunt, Tom R.; Ford, Ian; Sattar, Naveed; Tsimikas, Sotirios; Schwartz, Gregory G.; Lawlor, Debbie A.; Morris, Richard W.; Sandhu, Manjinder S.; Poledne, Rudolf; Maitland-van der Zee, Anke H.; Khaw, Kay-Tee; Keating, Brendan J.; van der Harst, Pim; Price, Jackie F.; Mehta, Shamir R.; Yusuf, Salim; Witteman, Jaqueline C. M.; Franco, Oscar H.; Jukema, J. Wouter; de Knijff, Peter; Tybjaerg-Hansen, Anne; Rader, Daniel J.; Farrall, Martin; Samani, Nilesh J.; Kivimaki, Mika; Fox, Keith A. A.; Humphries, Steve E.; Anderson, Jeffrey L.; Boekholdt, S. Matthijs; Palmer, Tom M.; Eriksson, Per; Pare, Guillaume; Hingorani, Aroon D.; Sabatine, Marc S.; Mallat, Ziad; Casas, Juan P.; Talmud, Philippa J.

    2013-01-01

    Objectives This study sought to investigate the role of secretory phospholipase A(2) (sPLA(2))-IIA in cardiovascular disease. Background Higher circulating levels of sPLA(2)-IIA mass or sPLA(2) enzyme activity have been associated with increased risk of cardiovascular events. However, it is not

  7. Aliskiren accumulates in renin secretory granules and binds plasma prorenin

    NARCIS (Netherlands)

    M. Krop (Manne); I.M. Garrelds (Ingrid); R.J.A. de Bruin (René); J.M. van Gool (Jeanette); N.D.L. Fisher (Naomi); N.K. Hollenberg (Norman); A.H.J. Danser (Jan)

    2008-01-01

    textabstractThe vascular effects of aliskiren last longer than expected based on its half life, and this renin inhibitor has been reported to cause a greater renin rise than other renin-angiotensin system blockers. To investigate whether aliskiren accumulation in secretory granules contributes to

  8. Male accessory gland secretory protein polymorphism in natural ...

    Indian Academy of Sciences (India)

    Male accessory gland secretory protein polymorphism was analysed in natural populations of Drosophila nasuta nasuta and. D. sulfurigaster neonasuta for the first time, using SDS-PAGE to score polymorphism of these proteins in 2788 individuals of D. n. nasuta and 2232 individuals of D. s. neonasuta from 12 different ...

  9. Primary temporal bone secretory meningioma presenting as chronic otitis media.

    NARCIS (Netherlands)

    Marcelissen, T.A.; Bondt, R.B.J de; Lammens, M.M.Y.; Manni, J.J.

    2008-01-01

    We report an extremely rare case of a secretory meningioma primarily involving the temporal bone. A 56-year old female patient presented to us with a history of a chronic otitis media and unilateral hearing loss. Diagnostic investigations revealed a tumor arising from the temporal bone without signs

  10. Plant expression of chicken secretory antibodies derived from combinatorial libraries

    NARCIS (Netherlands)

    Wieland, W.H.; Lammers, A.; Schots, A.; Orzaéz, D.V.

    2006-01-01

    Delivery of secretory IgA antibodies (sIgA) to mucosal surfaces is a promising strategy to passively prevent infectious diseases. Plants have been proposed as biofactories for such complex immunoglobulin molecules. Recently, the molecular characterization of all four monomers of chicken sIgA (IgA

  11. Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Muhammad Umair Hanif

    2017-01-01

    Full Text Available Recombinant human Bone Morphogenetic Protein 2 (rhBMP2 has important applications in the spine fusion and ortho/maxillofacial surgeries. Here we first report the secretory expression of biological active dimerized rhBMP2 from Bacillus subtilis system. The mature domain of BMP2 gene was amplified from pTz57R/BMP2 plasmid. By using pHT43 expression vector two constructs, pHT43-BMP2-M (single BMP2 gene and pHT43-BMP2-D (two BMP2 genes coupled with a linker to produce a dimer, were designed. After primary cloning (DH5α strain and sequence analysis, constructs were transformed into Bacillus subtilis for secretory expression. Expression conditions like media (2xYT and temperature (30°C were optimized. Maximum 35% and 25% secretory expression of monomer (~13 kDa and dimer (~25 kDa, respectively, were observed on SDS-PAGE in SCK6 strain. The expression and dimeric nature of rhBMP2 were confirmed by western blot and native PAGE analysis. For rhBMP2 purification, 200 ml culture supernatant was freeze dried to 10 ml and dialyzed (Tris-Cl, pH 8.5 and Fast Protein Liquid Chromatography (6 ml, Resource Q column was performed. The rhBMP2 monomer and dimer were eluted at 0.9 M and 0.6 M NaCl, respectively. The alkaline phosphatase assay of rhBMP2 (0, 50, 100, 200, and 400 ng/ml was analyzed on C2C12 cells and maximum 200 ng/ml activity was observed in dose dependent manner.

  12. Angular distribution of cross-sectioned cell boundaries at the distal terminal web in differentiating preameloblasts, inner enamel secretory ameloblasts and outer enamel secretory ameloblasts.

    Science.gov (United States)

    Yuan, Xiaohong; Nishikawa, Sumio

    2014-02-01

    The cross-sectioned profiles of differentiating preameloblasts, inner enamel secretory ameloblasts and outer enamel secretory ameloblasts at the distal terminal web were quantitatively compared. First, the angles of each line constituting the sectioned cell polygons were measured, and the patterns of angular distribution histograms were compared. Second, all groups of line angles from one differentiating preameloblast population, two inner enamel secretory ameloblast and one outer enamel secretory ameloblast populations at the distal terminal web were compared statistically by the χ(2)-test using the multiple comparison method. The results showed that cell shapes between differentiating preameloblasts and inner enamel secretory ameloblasts were similar, but that those between differentiating preameloblasts and outer enamel secretory ameloblasts and between inner enamel secretory ameloblasts and outer enamel secretory ameloblasts were significantly different. Third, F-actin fluorescence microscopy in the distal terminal web was performed and was consistent with the angular distribution. These results suggest that cell shapes of inner enamel secretory ameloblasts and differentiating preameloblasts at the distal terminal web are specialized for sideways cell movement during decussating tooth enamel formation.

  13. Deficiency of ATP2C1, a golgi ion pump, induces secretory pathway defects in endoplasmic reticulum ( ER)-associated degradation and sensitivity to ER stress

    NARCIS (Netherlands)

    Ramos-Castaneda, J; Park, YN; Liu, M; Hauser, K; Rudolph, H; Shull, GE; Jonkman, MF; Mori, K; Ikeda, S; Ogawa, H; Arvan, P

    2005-01-01

    Relatively few clues have been uncovered to elucidate the cell biological role(s) of mammalian ATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitously expressed. Deficiency of ATP2C1 results in a human disease ( Hailey-Hailey), which primarily affects

  14. E-Cadherin Can Replace N-Cadherin during Secretory-Stage Enamel Development

    Science.gov (United States)

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

    2014-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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

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

    Directory of Open Access Journals (Sweden)

    Xiaomu Guan

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

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

    Directory of Open Access Journals (Sweden)

    Okulicz William C

    2004-07-01

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

  17. Genome-scale modeling of the protein secretory machinery in yeast

    DEFF Research Database (Denmark)

    Feizi, Amir; Österlund, Tobias; Petranovic, Dina

    2013-01-01

    The protein secretory machinery in Eukarya is involved in post-translational modification (PTMs) and sorting of the secretory and many transmembrane proteins. While the secretory machinery has been well-studied using classic reductionist approaches, a holistic view of its complex nature is lacking....... Here, we present the first genome-scale model for the yeast secretory machinery which captures the knowledge generated through more than 50 years of research. The model is based on the concept of a Protein Specific Information Matrix (PSIM: characterized by seven PTMs features). An algorithm...... was developed which mimics secretory machinery and assigns each secretory protein to a particular secretory class that determines the set of PTMs and transport steps specific to each protein. Protein abundances were integrated with the model in order to gain system level estimation of the metabolic demands...

  18. Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

    Science.gov (United States)

    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.

  19. Form and function in the trypanosomal secretory pathway.

    Science.gov (United States)

    Silverman, Jason S; Bangs, James D

    2012-08-01

    Recent advances in secretory biology of African trypanosomes reveal both similarities and striking differences with other model eukaryotic organisms. Secretion is streamlined for rapid and selective transport of the major cargo, VSG. Selectivity in the early and post-Golgi compartments is dependent on glycosylphosphatidyl inositol anchors. Streamlining includes reduced organellar abundance, and close association of ER exit sites with Golgi and with unique flagellar cytoskeletal elements that govern organellar replication and segregation. These elements include a novel centrin containing bilobe structure. Innate signals for post-Golgi sorting of biosynthetic lysosomal cargo trafficking have been defined, as have pathways for both biosynthetic and endocytic trafficking to the lysosome. Less well-defined secretory organelles such as the multivesicular body and acidocalcisomes are receiving closer scrutiny. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Unconventional secretion of FABP4 by endosomes and secretory lysosomes.

    Science.gov (United States)

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

    2017-12-06

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

  1. Isolation of intact sub-dermal secretory cavities from Eucalyptus

    Directory of Open Access Journals (Sweden)

    Goodger Jason QD

    2010-09-01

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

  2. Glutamine on intestinal secretory immunoglobulin A secretion: A mechanistic perspective

    OpenAIRE

    Wenkai Ren; Kai Wang; jie yin; shuai chen; gang liu; bie tan; Guoyao Wu; Fuller Warren Bazer; Yunayi Peng; Yulong Yin

    2016-01-01

    Secretory IgA (SIgA) is one important line of defense in intestinal mucosal surface to protect the intestinal epithelium from enteric toxins and pathogenic microorganisms. Multiple factors, such as intestinal microbiota, intestinal cytokines and nutrients, are highly involved in intestinal production of SIgA. Recently, glutamine has been shown to affect intestinal SIgA production, however, the underlying mechanism by which glutamine promotes intestinal SIgA secretion remains to know. Here, we...

  3. Widespread Occurrence of Expressed Fungal Secretory Peroxidases in Forest Soils

    OpenAIRE

    Kellner, Harald; Luis, Patricia; Pecyna, Marek J.; Barbi, Florian; Kapturska, Danuta; Krüger, Dirk; Zak, Donald R.; Marmeisse, Roland; Vandenbol, Micheline; Hofrichter, Martin

    2014-01-01

    Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g....

  4. Control of LH secretory-burst frequency and interpulse-interval regularity in women.

    Science.gov (United States)

    Keenan, Daniel M; Evans, William S; Veldhuis, Johannes D

    2003-11-01

    Hypothalamic neurons generate discrete bursts of gonadotropin-releasing hormone (GnRH) and thereby pulses of luteinizing hormone (LH) at randomly timed intervals centered on a probabilistic mean frequency. We tested the hypothesis that physiological mechanisms govern not only the number but also the stochastic dispersion of the GnRH/LH pulse-renewal process in humans; for example, in young women in the early (EF) and late (LF) follicular and midluteal (ML) phases of the menstrual cycle (n = 18) and in postmenopausal individuals (PM, n = 16). To this end, we quantify stochastic interpulse variability by way of the order-independent, two-parameter Weibull renewal process (Keenan DM and Veldhuis J. Am J Physiol Regul Integr Comp Physiol 281: R1917-R1924, 2001) and the sequence-specific, model-free approximate-entropy statistic (ApEn) (Pincus SM. Proc Natl Acad Sci USA 88: 2297-2301, 1991). Statistical testing unveiled 1) reduced probabilistic mean LH secretory-burst frequency (lower lambda of the Weibull distribution) in ML compared with each of EF, LF, and PM (P renewal variability, and secretory-waveform evolution.

  5. Immunolocalization of an enterotoxic glycoprotein exoantigen on the secretory organelles of Cryptosporidium parvum sporozoites

    Directory of Open Access Journals (Sweden)

    El-Shewy K.A.

    2004-06-01

    Full Text Available In this study, the fine ultrastructures of the secretory organelles of C. parvum sporozoites were demonstrated using transmission electron microscopy (TEM. Meanwhile, a previously identified enterotoxic 18-20 kDa copro-antigen (18-20 kDa CCA, associated with cryptosporidiosis in both human and calves, was isolated and immunolocalized on C. parvum sporozoites. Using immunoelectron microscopy and anti-18-20 kDa monospecific antibody demonstrated marked existence of the 18-20 kDa CCA on the apical organelles and at the trilaminar pellicles. An anterior extrusion of this protein was demonstrated around the excysted and released sporozoites. However, non excysted sporozoites did not show this protein. Affinity blotting, with biotinylated jacalin, demonstrated the O-linked oligosaccharide moiety of this protein. The potential role of this protein in the host cell invasion and/or gliding motility remains unelucidated. However, its enterotoxicity, location and secretory nature suggest that it may be a target for neutralization or invasion inhibition of Cryptosporidium.

  6. Quantification of secretory leukocyte protease inhibitor (SLPI) in oral gargle specimens collected using mouthwash

    Science.gov (United States)

    Pierce Campbell, Christine M.; Guan, Wei; Sprung, Robert; Koomen, John M.; O’Keefe, Michael T.; Ingles, Donna J.; Abrahamsen, Martha; Giuliano, Anna R.

    2014-01-01

    Background Secretory leukocyte protease inhibitor (SLPI) is an innate immunity-associated protein known to inhibit HIV transmission, and is thought to inhibit a variety of infectious agents, including human papillomaviruses (HPVs). We aimed to optimize an established ELISA-based SLPI quantification assay for use with oral gargle specimens collected using mouthwash, and to assess preliminary associations with age, smoking status, and alcohol intake. Methods Oral gargle supernatants from 50 individuals were used to optimize the Human SLPI Quantikine ELISA Kit. Sample suitability was assessed and quality control analyses were conducted. Results Salivary SLPI was successfully recovered from oral gargles with low intra-assay and high inter-individual variability. Initial measurements showed that salivary SLPI varied considerably across individuals, and that SLPI was inversely associated with age. Conclusions This optimized assay can be used to examine the role of SLPI in the acquisition of oral HPV and other infections. PMID:24140751

  7. Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis

    Science.gov (United States)

    Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-01-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research. PMID:24967841

  8. Souffle/Spastizin controls secretory vesicle maturation during zebrafish oogenesis.

    Directory of Open Access Journals (Sweden)

    Palsamy Kanagaraj

    2014-06-01

    Full Text Available During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP gene SPASTIZIN (SPG15. We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.

  9. Biochemical analysis of secretory trafficking in mammalian cells.

    Science.gov (United States)

    Roboti, Peristera; Witkos, Tomasz M; Lowe, Martin

    2013-01-01

    Protein trafficking within the secretory pathway of mammalian cells is amenable to analysis by biochemical methods. This can be achieved by monitoring posttranslational modifications that occur naturally within the secretory pathway, or by measuring the delivery of cargo to the cell surface or extracellular medium. These approaches can be combined with additional manipulations such as specific temperature blocks that permit analysis of distinct trafficking steps. Biochemical analysis is advantageous in that it permits both a sensitive and quantitative measure of trafficking along the pathway. The methods discussed in this chapter permit the analysis of trafficking of both endogenous cargo proteins and ectopically expressed model cargos, which can be followed using either Western blotting or metabolic pulse-chase approaches. These methods are relatively straightforward and suitable for use in most modern cell biology laboratories. In addition to the well-established methods that we describe here in detail, we also refer to the development of more recent tailored approaches that add further to the arsenal of tools that can be used to assess trafficking in the secretory pathway. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Unremitting Cell Proliferation in the Secretory Phase of Eutopic Endometriosis

    Science.gov (United States)

    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

  11. Postpartum breast involution reveals regression of secretory lobules mediated by tissue-remodeling

    Science.gov (United States)

    2014-01-01

    Introduction A postpartum diagnosis of breast cancer is an independent predictor of metastases, however the reason is unknown. In rodents, the window of postpartum mammary gland involution promotes tumor progression, suggesting a role for breast involution in the poor prognosis of human postpartum breast cancers. Rodent mammary gland involution is characterized by the programmed elimination of the secretory lobules laid down in preparation for lactation. This tissue involution process involves massive epithelial cell death, stromal remodeling, and immune cell infiltration with similarities to microenvironments present during wound healing and tumor progression. Here, we characterize breast tissue from premenopausal women with known reproductive histories to determine the extent, duration and cellular mechanisms of postpartum lobular involution in women. Methods Adjacent normal breast tissues from premenopausal women (n = 183) aged 20 to 45 years, grouped by reproductive categories of nulliparous, pregnant and lactating, and by time since last delivery were evaluated histologically and by special stain for lobular area, lobular type composition, apoptosis and immune cell infiltration using computer assisted quantitative methods. Results Human nulliparous glands were composed dominantly of small (approximately 10 acini per lobule) and medium (approximately 35 acini per lobule) sized lobules. With pregnancy and lactation, a >10 fold increase in breast epithelial area was observed compared to nulliparous cases, and lactating glands were dominated by mature lobules (>100 acini per lobule) with secretory morphology. Significant losses in mammary epithelial area and mature lobule phenotypes were observed within 12 months postpartum. By 18 months postpartum, lobular area content and lobule composition were indistinguishable from nulliparous cases, data consistent with postpartum involution facilitating regression of the secretory lobules developed in preparation for

  12. Immunohistochemical study on the secretory host defense system with lysozyme and secretory phospholipase A2 throughout rat respiratory tract.

    Science.gov (United States)

    Masuda, Natsumi; Mantani, Youhei; Yoshitomi, Chiaki; Yuasa, Hideto; Nishida, Miho; Arai, Masaya; Kawano, Junichi; Yokoyama, Toshifumi; Hoshi, Nobuhiko; Kitagawa, Hiroshi

    2017-12-08

    The host defense system with lysozyme and secretory phospholipase A2 (sPLA2) was immunohistochemically investigated in rat respiratory tract under healthy conditions. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NC) and a small number of goblet cells (GC) were immunopositive for lysozyme and sPLA2. A few acinar cells and almost all epithelial cells of intercalated ducts were immunopositive for both bactericidal substances in the nasal glands. In the laryngeal and tracheal epithelia, few NC and GC were immunopositive for both bactericidal substances. In the laryngeal and tracheal glands, a few acinar cells and most ductal epithelial cells were immunopositive for both bactericidal substances. In extra-pulmonary bronchus, small numbers of NC and GC were immunopositive for lysozyme and sPLA2, whereas few NC and no GC were immunopositive in the intra-pulmonary bronchus. No secretory source of either bactericidal substance was located in the bronchioles. In the alveolus, many glandular epithelial cells and alveolar macrophages were immunopositive for lysozyme but immunonegative for sPLA2. Moreover, lysozyme and sPLA2 were detected in the mucus layer and in the periciliary layer from the nose to the extra-pulmonary bronchus. These findings suggest that secretory sources of lysozyme and sPLA2 are distributed in almost all the respiratory tract. Their secretory products are probably transported to the pharynx and contribute to form the first line of defense against inhaled bacteria throughout the respiratory tract.

  13. Increased insulin secretory capacity but decreased insulin sensitivity after correction of iron overload by phlebotomy in hereditary haemochromatosis.

    Science.gov (United States)

    Abraham, D; Rogers, J; Gault, P; Kushner, J P; McClain, D A

    2006-11-01

    We recently demonstrated that humans with hereditary haemochromatosis have decreased insulin secretory capacity with a compensatory increase in insulin sensitivity. We therefore determined how these measures change after correction of tissue iron overload. Five non-diabetic subjects who had been studied previously at the time of initial diagnosis by means of the OGTT and frequently sampled intravenous glucose tolerance tests (FSIVGTT) underwent phlebotomy to normalise their serum ferritin. After normalisation of ferritin they were studied again (33+/-4 months after the initial studies) by OGTT and FSIVGTT. Normalisation of tissue iron stores resulted in an average 1.8-fold increase in the integrated area under the insulin curve during OGTT (pdisposition index (AIRgxSi) was unchanged (5% increase, p=0.90). BMI and fasting glucose were unchanged. At the time of diagnosis of haemochromatosis, four of the subjects had IGT. After normalisation of ferritin, two achieved NGT and two remained with IGT, despite 2.5- and 3.7-fold increases in insulin secretory capacity. Insulin secretory capacity improves after normalisation of iron stores in subjects with hereditary haemochromatosis. Glucose tolerance status improves incompletely because of decreased insulin sensitivity after phlebotomy. We conclude that tissue iron levels are an important determinant of insulin secretion and insulin action.

  14. Inhibition of intestinal chloride secretion by piperine as a cellular basis for the anti-secretory effect of black peppers.

    Science.gov (United States)

    Pongkorpsakol, Pawin; Wongkrasant, Preedajit; Kumpun, Saowanee; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2015-10-01

    Piperine is the principal alkaloid in black peppers (Piper nigrum L.), which is a commonly included spice in anti-diarrheal formulations. Piperine has antispasmodic activities, but its anti-secretory effect is not known. Therefore, this study investigated the anti-secretory effect of piperine and its underlying mechanism. Piperine inhibited cAMP-mediated Cl- secretion in human intestinal epithelial (T84) cells, similar to black pepper extract. Intraluminal administration of piperine (2 μg/loop) suppressed cholera toxin-induced intestinal fluid accumulation by ∼85% in mice. The anti-secretory mechanism of piperine was investigated by evaluating its effects on the activity of transport proteins involved in cAMP-mediated Cl- secretion. Notably, piperine inhibited CFTR Cl- channel activity (IC50#8'6#10 μM) without affecting intracellular cAMP levels. The mechanisms of piperine-induced CFTR inhibition did not involve MRP4-mediated cAMP efflux, AMPK or TRPV1. Piperine also inhibited cAMP-activated basolateral K+ channels, but it had no effect on Na+-K+-Cl- cotransporters or Na+-K+ ATPases. Piperine suppressed Ca2+-activated Cl- channels (CaCC) without affecting intracellular Ca2+ concentrations or Ca2+-activated basolateral K+ channels. Collectively, this study indicates that the anti-secretory effect of piperine involves the inhibition of CFTR, CaCC and cAMP-activated basolateral K+ channels. Piperine represents a novel class of drug candidates for the treatment of diarrheal diseases caused by the intestinal hypersecretion of Cl-. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells

    DEFF Research Database (Denmark)

    Nyegaard, Steffen; Novakovic, Valerie A.; Rasmussen, Jan Trige

    2013-01-01

    Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2’s do not bind to plasma membranes of quies......Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2’s do not bind to plasma membranes...

  16. Secretory Phospholipase A(2) Activity toward Diverse Substrates

    DEFF Research Database (Denmark)

    Madsen, Jesper Jonasson; Linderoth, Lars; Subramanian, Arun Kumar

    2011-01-01

    We have studied secretory phospholipase A(2)-IIA (sPLA(2)) activity toward different phospholipid analogues by performing biophysical 1 characterizations and molecular dynamics simulations. The phospholipids were natural substrates, triple alkyl phospholipids, a prodrug anticancer etherlipid......, and an inverted ester. The latter were included to study head group-enzyme interactions. Our simulation results show that the lipids are optimally placed into the binding cleft and that water molecules can freely reach the active site through a well-defined pathway; both are indicative that these substrates...

  17. Secretory pattern and regulatory mechanism of growth hormone in cattle.

    Science.gov (United States)

    Kasuya, Etsuko

    2016-02-01

    The ultradian rhythm of growth hormone (GH) secretion has been known in several animal species for years and has recently been observed in cattle. Although the physiological significance of the rhythm is not yet fully understood, it appears essential for normal growth. In this review, previous studies concerning the GH secretory pattern in cattle, including its ultradian rhythm, are introduced and the regulatory mechanism is discussed on the basis of recent findings. © 2015 The Authors. Animal Science Journal published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Society of Animal Science.

  18. [Mammary analog secretory carcinoma of the parotid gland].

    Science.gov (United States)

    Guérin, Maxime; Diedhiou, Abdoulaye; Nallet, Emmanuel; Duflo, Suzy; Laé, Marick; Wassef, Michel

    2014-10-01

    Mammary analog secretory carcinoma (MASC) of the parotid gland is a rare and recently described lesion. We report the case of a 46-year-old man with a tumor of the parotid gland which was carried to the diagnosis of MASC. Diagnostic was confirmed by highlighting the ETV6-NTRK3 gene translocation. However, some morphologic and immunohistochemical features are suggestive of this entity. This carcinoma should be distinguished from its main differential diagnoses: acinic cell carcinoma and low grade cribriform cystadenocarcinoma. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  19. Cysteine-rich domain 1 of CD40 mediates receptor self-assembly

    NARCIS (Netherlands)

    Smulski, C.R.; Beyrath, J.D.; Decossas, M.; Chekkat, N.; Wolff, P.; Estieu-Gionnet, K.; Guichard, G.; Speiser, D.; Schneider, P.; Fournel, S.

    2013-01-01

    The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of

  20. New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

    Science.gov (United States)

    Jung, Woongsic; Campbell, Robert L; Gwak, Yunho; Kim, Jong Im; Davies, Peter L; Jin, EonSeon

    2016-01-01

    Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

  1. The conserved scavenger receptor cysteine-rich superfamily in therapy and diagnosis

    DEFF Research Database (Denmark)

    Martínez, Vanesa Gabriela; Moestrup, Søren Kragh; Holmskov, Uffe

    2011-01-01

    members of the SRCR-SF, but also of the sequence versatility of the SRCR domains. Indeed, involvement of SRCR-SF members in quite different functions, such as pathogen recognition, modulation of the immune response, epithelial homeostasis, stem cell biology, and tumor development, have all been described...... expansion, now up to more than 30 members. The study of these members is attracting growing interest, which parallels that in innate immunity. No unifying function has been described to date for the SRCR domains, this being the result of the limited knowledge still available on the physiology of most...

  2. New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

    Directory of Open Access Journals (Sweden)

    Woongsic Jung

    Full Text Available Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

  3. Engineering a uniquely reactive thiol into a cysteine-rich peptide.

    Science.gov (United States)

    Shimony, E; Sun, T; Kolmakova-Partensky, L; Miller, C

    1994-04-01

    Cysteine mutagenesis for the purpose of chemical labelling was applied to the K+ channel neurotoxin charybdotoxin, a 37-residue peptide with six functionally essential cysteines. An additional 'spinster cysteine' was introduced at a position far away in space from the toxin's known interaction surface where it contacts its K+ channel receptor. Despite the presence of the extra unpaired cysteine residue, the toxin still folds efficiently and may be labelled by fluorescent and radioactive reagents to give a functionally competent toxin.

  4. Unfolding Thermodynamics of Cysteine-Rich Proteins and Molecular Thermal-Adaptation of Marine Ciliates

    Directory of Open Access Journals (Sweden)

    Giorgia Cazzolli

    2013-11-01

    Full Text Available Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative study of the thermal unfolding of disulfide-rich protein pheromones produced by these ciliates. Recent circular dichroism (CD measurements have shown that the two psychrophilic (E. nobilii and mesophilic (E. raikovi protein families are characterized by very different melting temperatures, despite their close structural homology. The enhanced thermal stability of the E. raikovi pheromones is realized notwithstanding the fact that these proteins form, as a rule, a smaller number of disulfide bonds. We perform Monte Carlo (MC simulations in a structure-based coarse-grained (CG model to show that the higher stability of the E. raikovi pheromones is due to the lower locality of the disulfide bonds, which yields a lower entropy increase in the unfolding process. Our study suggests that the higher stability of the mesophilic E. raikovi phermones is not mainly due to the presence of a strongly hydrophobic core, as it was proposed in the literature. In addition, we argue that the molecular adaptation of these ciliates may have occurred from cold to warm, and not from warm to cold. To provide a testable prediction, we identify a point-mutation of an E. nobilii pheromone that should lead to an unfolding temperature typical of that of E. raikovi pheromones.

  5. Identification of secretory odontoblasts using DMP1-GFP transgenic mice.

    Science.gov (United States)

    Balic, Anamaria; Mina, Mina

    2011-04-01

    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. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. Secretory structures of Ipomoea asarifolia: anatomy and histochemistry

    Directory of Open Access Journals (Sweden)

    Fabiano M. Martins

    2012-02-01

    Full Text Available Ipomoea asarifolia (Desr. Roem. & Schult., Convolvulaceae, is a weed that infests agricultural areas and is toxic to cattle. In spite of its toxicity, the leaves of this plant are used in traditional remedies in the state of Bahia, Brazil. The present work describes the leaf anatomy of I. asarifolia and characterizes the exudates of its secretory structures. The leaves have a unistratified epidermis composed of ordinary cells with straight to slightly sinuous anticlinal walls and thin cuticles. Paracytic stomata are found on both surfaces of the leaves at the same level as the ordinary epidermal cells. Trichomes producing polysaccharide secretions occur on the petiole and leaf blade and are considered colleters. The mesophyll is dorsiventral and the vascular bundle of the central vein is bicollateral. Two opposed nectaries occur on the petiole near the leaf blade. Each nectary is composed of a small canal with internal ramifications and numerous secretory trichomes. The laticiferous glands are articulated, not anastomosed, and are composed of large diameter cells with thin cell walls. The secretions of the laticiferous glands are lipidic.

  7. Optimization of Pathogenetic Treatment of Secretory Diarrhea in Infants

    Directory of Open Access Journals (Sweden)

    O.K. Koloskova

    2014-02-01

    Full Text Available The aim of the research was to access clinical efficacy of oral rehydration therapy using III generation solutions in the treatment of secretory diarrhea in infants. To achieve this aim, on the basis of infectious box unit (enteric infections of regional clinical hospital (Chernivtsi we examined 116 infants, randomly selected, with acute gastroenteritis, who admitted to the hospital with signs of exycosis due to secretory diarrhea. Among examined patients, 73 (67.5 % children with the purpose of oral rehydration therapy received rehydration solutions, and 35 (32.4 % patients received other rehydration solutions. Monitoring of the dynamics of patients’ state enabled to state that, when we used III generation mixture as a main component of oral rehydration therapy, rate of positive dynamics in terms of clinical status of patients was significantly faster, in particular, body temperature, frequency and nature of bowel movements normalized significantly earlier, vomiting disappeared. In children treated with rehydration solutions, compared with patients receiving other rehydration solutions, odds ratio to confine only oral rehydration was 3.7 (95% CI 0.4–38.9 with an absolute risk to avoid the need for infusion therapy — 11 %.

  8. Non-secretory multiple myeloma: from biology to clinical management

    Directory of Open Access Journals (Sweden)

    Dupuis MM

    2016-12-01

    Full Text Available Megan Murray Dupuis,1 Sascha A Tuchman2 1Department of Medicine, Duke University, Durham, 2Division of Hematology/Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Abstract: Multiple myeloma (MM is the second most common hematologic malignancy in the US. It is typically characterized by production of large amounts of defective immunoglobulin (Ig. Diagnosing MM and monitoring treatment response, including eventual relapse, are largely based on sequential measurements of Ig. However, a small subset of MM called non-secretory multiple myeloma (NSMM produces no detectable Ig. This subset of true NSMM has become even smaller over time, as the advent of the serum free light chain assay has resulted in the majority of NSMM patients being recategorized as light-chain MM – that is, MM cells that produce only the light-chain component of Ig. True forms of NSMM, meaning MM that secretes no monoclonal proteins whatsoever, constitute a distinct entity that is reviewed; definition of NSMM using current detection methods, discuss the biology underpinning NSMM development, and share recommendations for how NSMM should be managed clinically with respect to detection, treatment, and monitoring. Keywords: multiple myeloma, non-secretory, immunoglobulin

  9. Widespread occurrence of expressed fungal secretory peroxidases in forest soils.

    Science.gov (United States)

    Kellner, Harald; Luis, Patricia; Pecyna, Marek J; Barbi, Florian; Kapturska, Danuta; Krüger, Dirk; Zak, Donald R; Marmeisse, Roland; Vandenbol, Micheline; Hofrichter, Martin

    2014-01-01

    Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp.), which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation.

  10. Widespread occurrence of expressed fungal secretory peroxidases in forest soils.

    Directory of Open Access Journals (Sweden)

    Harald Kellner

    Full Text Available Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase, dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase. Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp., which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation.

  11. Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

    Science.gov (United States)

    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

  12. Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms.

    Science.gov (United States)

    Hogue, Ian B; Scherer, Julian; Enquist, Lynn W

    2016-06-07

    Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based on total internal reflection fluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway. The alphaherpesviruses, including the important human pathogens herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), are among the few viruses that have evolved to exploit the mammalian nervous system. These viruses typically cause mild recurrent herpetic or zosteriform lesions but can also cause debilitating herpes encephalitis, more frequently in very young, old, immunocompromised, or nonnatural hosts. Importantly, many of the molecular and cellular mechanisms of viral assembly and egress remain unclear. This study addresses the trafficking of viral glycoproteins to the plasma membrane, exocytosis of light particles, and exocytosis of virions. Trafficking of glycoproteins affects immune evasion and pathogenesis and may precede virus particle assembly. The release of light

  13. Volume Density, Distribution, and Ultrastructure of Secretory and Basolateral Membranes and Mitochondria Predict Parietal Cell Secretory (Dysfunction

    Directory of Open Access Journals (Sweden)

    Marian L. Miller

    2010-01-01

    Full Text Available Acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking. Histologically, consensus defines acid secretion as the ratio of the volume density (Vd of canalicular and apical membranes (CAMs to tubulovesicular (TV membranes, a value which varies widely under normal conditions. Examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion. Vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion. We confirmed that CAM to TV ratio fairly represented secretory activity in untreated and secretion-inhibited wild-type (WT mice and in NHE2−/− mice as well, though the response was significantly attenuated in the latter. However, high CAM to TV ratios wrongly posed as active acid secretion in AE2−/−, GHKA−/−, and NHE4−/− mice. Achlorhydric genotypes also had a significantly higher Vd of basolateral membrane than WT mice, and reduced Vd of mitochondria and canaliculi. The Vd of mitochondria, and ratio of the Vd of basolateral membranes/Vd of mitochondria were preferred predictors of the level of acid secretion. Alterations in acid secretion, then, cause significant changes not only in the Vd of secretory membranes but also in mitochondria and basolateral membranes.

  14. Volume density, distribution, and ultrastructure of secretory and basolateral membranes and mitochondria predict parietal cell secretory (dys)function.

    Science.gov (United States)

    Miller, Marian L; Andringa, Anastasia; Zavros, Yana; Bradford, Emily M; Shull, Gary E

    2010-01-01

    Acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking. Histologically, consensus defines acid secretion as the ratio of the volume density (Vd) of canalicular and apical membranes (CAMs) to tubulovesicular (TV) membranes, a value which varies widely under normal conditions. Examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion. Vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion. We confirmed that CAM to TV ratio fairly represented secretory activity in untreated and secretion-inhibited wild-type (WT) mice and in NHE2-/- mice as well, though the response was significantly attenuated in the latter. However, high CAM to TV ratios wrongly posed as active acid secretion in AE2-/-, GHKAalpha-/-, and NHE4-/- mice. Achlorhydric genotypes also had a significantly higher Vd of basolateral membrane than WT mice, and reduced Vd of mitochondria and canaliculi. The Vd of mitochondria, and ratio of the Vd of basolateral membranes/Vd of mitochondria were preferred predictors of the level of acid secretion. Alterations in acid secretion, then, cause significant changes not only in the Vd of secretory membranes but also in mitochondria and basolateral membranes.

  15. Isolation and identification of java race amniotic membrane secretory leukocyte protease inhibitor gene

    Directory of Open Access Journals (Sweden)

    Elly Munadziroh

    2008-09-01

    Full Text Available Background: Secretory leukocyte protease inhibitor (SLPI has been found to facilitate epithelialization, maintain a normal epithelial phenotype, reduce inflammation, secrete growth factors such as IL-4, IL-6, IL-10, EGF, FGF, TGF, HGFand 2-microbulin. SLPI is serine protease inhibitor, which found in secretions such as whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears, amniotic fluid and amniotic membrane. Impaired healing states are characterized by excessive proteolysis and oftenbacterial infection, leading to the hypothesis that SLPI may have a role in the healing process in oral inflammation and contributes to tissue repair in oral mucosa. The oral wound healing response is impaired in the SLPI sufficient mice since matrix synthesis and collagen deposition delayed. The objective of this research is to isolate and identify the amniotic membrane of Java Race SLPI Gene. Methods: SLPI RNA was isolated from Java Race amniotic membrane and the cDNA was amplified by polymerase chain reaction (PCR. Result: Through sequence analyses, SLPI cDNA was 530 nucleotide in length with a predicted molecular mass about 12 kDa. The nucleotide sequence showed that human SLPI from sample was 98% identical with human SLPI from gene bank. PCR analysis revealed that the mRNA of SLPI was highly expressed in the amniotic membrane from Java Race sample. Conclusion: it is demonstrated that human SLPI are highly conserved in sequence content as compared to the human SLPI from gene.

  16. Low levels of anti-secretory factor in placenta are associated with preterm birth and inflammation.

    Science.gov (United States)

    Gustafsson, Anna M; Fransson, Emma; Dubicke, Aurelija; Hjelmstedt, Anna K; Ekman-Ordeberg, Gunvor; Silfverdal, Sven-Arne; Lange, Stefan; Jennische, Eva; Bohlin, Kajsa

    2018-03-01

    Anti-secretory factor is a protein that regulates secretory and inflammatory processes and preterm birth is associated with inflammation. Therefore, our hypothesis was that anti-secretory factor might play a role in immune reactivity and homeostasis during pregnancy. Following spontaneous onset of labor and preterm or term delivery, placenta biopsies were collected. The levels of anti-secretory factor and markers of inflammation (CD68, CD163) and vascularization (CD34, smooth muscle actin) were analyzed by immunohistochemistry. The 61 placental biopsies included 31 preterm (preterm placentas exhibited lower levels of anti-secretory factor (p = 0.008) and larger numbers of CD68-positive cells (p Preterm placentas had blood vessel of smaller diameter (p = 0.036) indicative of immaturity. The level of interleukin-6 in cord blood was higher after very preterm than term birth, suggesting a fetal inflammatory response. The placenta level of anti-secretory factor was positively correlated to the length of gestation (p = 0.025) and negatively correlated to the levels of the inflammatory markers CD68 (p = 0.015) and CD163 (p = 0.028). Preterm delivery is associated with low levels of anti-secretory factor in placenta. Inflammation, a potential trigger of preterm birth, is more pronounced in the preterm placenta and inversely related to the placental level of anti-secretory factor, suggesting both a link and a potential target for intervention. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

  17. Increased biogenesis of glucagon-containing secretory granules and glucagon secretion in BIG3-knockout mice

    Directory of Open Access Journals (Sweden)

    Hongyu Li

    2015-03-01

    Conclusions: Together with our previous studies, the current data reveal a conserved role for BIG3 in regulating alpha- and beta-cell functions. We propose that BIG3 negatively regulates hormone production at the secretory granule biogenesis stage and that such regulatory mechanism may be used in secretory pathways of other endocrine cells.

  18. Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells

    DEFF Research Database (Denmark)

    Poulsen, Mona Dam; Hansen, Gert Helge; Dabelsteen, Erik

    1993-01-01

    labeling using a monoclonal glucagon antibody as the second primary antibody. These results show that DP IV is sorted to secretory granules in the pig pancreatic islet A-cells. Furthermore, this secretory granule enzyme, as opposed to intestinal brush border DP IV, is suggested to be a soluble protein...

  19. Mammary Analogue Secretory Carcinoma (MASC of the salivary gland: A new tumor entity

    Directory of Open Access Journals (Sweden)

    Ivan Damjanov

    2016-08-01

    Full Text Available Mammary analogue secretory carcinoma (MASC is a recently described low-grade malignant tumor of the salivary glands, biologically and morphologically equivalent to secretory breast carcinoma. We give a brief overview of this new entity, including morphological, immunohistochemical, molecular-genetic, clinical, epidemiologic features, differential diagnosis, and outcome results.

  20. Mammary Analogue Secretory Carcinoma (MASC) of the salivary gland: A new tumor entity.

    Science.gov (United States)

    Damjanov, Ivan; Skenderi, Faruk; Vranic, Semir

    2016-08-02

    Mammary analogue secretory carcinoma (MASC) is a recently described low-grade malignant tumor of the salivary glands, biologically and morphologically equivalent to secretory breast carcinoma. We give a brief overview of this new entity, including morphological, immunohistochemical, molecular-genetic, clinical, epidemiologic features, differential diagnosis, and outcome results.

  1. Internal secretory structures in stems of Silphium perfoliatum L.

    Directory of Open Access Journals (Sweden)

    Aneta Sulborska

    2013-04-01

    Full Text Available Silphium perfoliatum L. (Asteraceae is a North American perennial used as a medicinal, fodder, honey-bearing, and ornamental plant as well as for remediation of degraded soils. The location and structure of secretory reservoirs in the stem were examined with the use of light microscopy in cup plant stems. The stems were analysed at various heights (0-2 cm above the root collar, ½ of the stem length, and 0-2 cm below the stem apex/inflorescence and in three vegetation phases (vegetative phase, full bloom, withering. The plants belonged to the collection of the Department of Vegetable Crops and Medicinal Plants of the University of Life Sciences in Lublin. It was found that the secretory canals formed two rings: an external ring situated at the height of sclerenchymatous sheaths and bundle phloem, and the internal ring located in an immediate proximity of the xylem. Two external secretory reservoirs, one on each side of the bundle, were present at larger bundles. No internal reservoirs were formed in the proximity of these bundles. At smaller bundles, there were reservoirs of the internal verticil, but there were no external reservoirs. The canals of the external verticil (23-29 were more numerous that in the internal verticil (17-19. In both cases, the largest number of reservoirs was observed at half the length of the stem, and the lowest number was in the apical part. The less numerous internal canals were larger in comparison with the external reservoirs. Depending on the plant developmental stage and location in the stem, the diameter of the external ring canals was 49-91 μm in cross section, and that of the canals of the internal verticil was 52-101 μm. The analysis of the different vegetation phases in the cup plant demonstrated that the canals had the largest diameters and were the most abundant in the withering phase. The canals evolved through gradual separation of cells (of schizogenous origin. In the cross section they exhibited a

  2. File list: ALL.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: ALL.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: ALL.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. A novel role for the centrosomal protein, pericentrin, in regulation of insulin secretory vesicle docking in mouse pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Agata Jurczyk

    Full Text Available The centrosome is important for microtubule organization and cell cycle progression in animal cells. Recently, mutations in the centrosomal protein, pericentrin, have been linked to human microcephalic osteodysplastic primordial dwarfism (MOPD II, a rare genetic disease characterized by severe growth retardation and early onset of type 2 diabetes among other clinical manifestations. While the link between centrosomal and cell cycle defects may account for growth deficiencies, the mechanism linking pericentrin mutations with dysregulated glucose homeostasis and pre-pubertal onset of diabetes is unknown. In this report we observed abundant expression of pericentrin in quiescent pancreatic beta-cells of normal animals which led us to hypothesize that pericentrin may have a critical function in beta-cells distinct from its known role in regulating cell cycle progression. In addition to the typical centrosome localization, pericentrin was also enriched with secretory vesicles in the cytoplasm. Pericentrin overexpression in beta-cells resulted in aggregation of insulin-containing secretory vesicles with cytoplasmic, but not centrosomal, pericentriolar material and an increase in total levels of intracellular insulin. RNAi- mediated silencing of pericentrin in secretory beta-cells caused dysregulated secretory vesicle hypersecretion of insulin into the media. Together, these data suggest that pericentrin may regulate the intracellular distribution and secretion of insulin. Mice transplanted with pericentrin-depleted islets exhibited abnormal fasting hypoglycemia and inability to regulate blood glucose normally during a glucose challenge, which is consistent with our in vitro data. This previously unrecognized function for a centrosomal protein to mediate vesicle docking in secretory endocrine cells emphasizes the adaptability of these scaffolding proteins to regulate diverse cellular processes and identifies a novel target for modulating regulated

  6. Visualization of peptide secretory vesicles in living nerve cells.

    Science.gov (United States)

    Park, Joshua J; Loh, Y Peng

    2011-01-01

    Analysis of real-time movements of peptidergic vesicles in live neurons provides insight into molecular mechanism(s) supporting the activity-dependent secretion of neurotrophins and neuropeptides. We examined the effect of overexpression of exogenous peptides comprising of the cytoplasmic tail sequence of vesicular carboxypeptidase E (CPE), proposed to be involved in the mechanism of trafficking of peptidergic secretory vesicles, in live hippocampal neurons. E16 rat hippocampal neurons were transfected with the peptidergic vesicle markers, CPE C-terminally tagged with red or green fluorescent protein, or brain-derived neurotrophic factor (BDNF) tagged with green fluorescent protein, and grown on dishes specialized for real-time live cell visualization. Movements of peptidergic vesicles were imaged in a temperature-controlled chamber on a confocal inverted microscope and analyzed with respect to their velocity, displacement distance, and processivity.

  7. Heterogeneity of secretory granules of silent pituitary adenomas

    DEFF Research Database (Denmark)

    Holck, S; Wewer, U M; Albrechtsen, R

    1988-01-01

    Silent pituitary adenomas were compared with hormonally active tumors taking into account the size, number, and ultrastructural characteristics of secretory granules (SG). The study group (a total of 79 primary pituitary adenomas) comprised 27 silent, 21 growth hormone (GH)-producing-, 16 prolactin...... approximately 10 to 50% of the granules in each cell. These granules were not seen in hormonally active tumors and considered therefore diagnostic of silent pituitary adenomas....... (PRL)-producing-, 5 GH-PRL-producing- and 10 adrenocorticotropic hormone (ACTH)-producing adenomas. The SG of silent adenomas were significantly smaller than SG in endocrine active adenomas. All hormonally inactive tumors also contained small (mean, 94 nm) specific cytoplasmic granules, designated...

  8. Statistical analysis of the quantal basis of secretory granule formation.

    Science.gov (United States)

    Křepelová-Dror, Marika; Hammel, Ilan; Meilijson, Isaac

    2014-01-01

    The size distribution of vesicles exocytosed from secretory cells displays quantal nature, vesicle volume is periodic multi-modal, suggesting that these heterogeneous vesicles are aggregate sums of a variable number of homogeneous basic granules. Whether heterogeneity is a lumping-together artifact of the measurement or an inherent intra-cell feature of the vesicles is an unresolved question. Recent empirical evidence will be provided for the quantal nature of intra-cell vesicle volume, supporting the controversial paradigm of homotypic fusion: basic cytoplasmic granules fuse with each other to create heterogeneously sized vesicles. An EM-algorithm-based method is presented for the conversion of multi-modal to quantal data that provides as by-product estimates of means and variances of basic granule packaging. Copyright © 2013 Wiley Periodicals, Inc.

  9. Secretory expression of a heterologous nattokinase in Lactococcus lactis.

    Science.gov (United States)

    Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

    2007-05-01

    Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

  10. Mammary analog secretory carcinoma of thyroid: A case report.

    Science.gov (United States)

    Rupp, Aaron P; Bocklage, Thèrése J

    2017-01-01

    Mammary analog secretory carcinoma (MASC) is a recently described rare neoplasm that was first reported in the salivary gland with an associated ETV6-NTRK3 fusion. We present a case of MASC involving and presumably arising in the thyroid, which was originally diagnosed as papillary thyroid carcinoma on fine needle aspiration and surgical resection. The later correct diagnosis of MASC was confirmed by immunohistochemistry and molecular studies. The cytopathological features of MASC in the salivary gland are previously described; however, we present the first cytopathological description of MASC arising in the thyroid with the unique feature of prominent nuclear grooves. Differentiating MASC from overlapping features of cytopathologic mimics such as papillary thyroid carcinoma may carry crucial therapeutic implications. Diagn. Cytopathol. 2017;45:45-50. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Speed Controls in Translating Secretory Proteins in Eukaryotes - an Evolutionary Perspective

    Science.gov (United States)

    Mahlab, Shelly; Linial, Michal

    2014-01-01

    Protein translation is the most expensive operation in dividing cells from bacteria to humans. Therefore, managing the speed and allocation of resources is subject to tight control. From bacteria to humans, clusters of relatively rare tRNA codons at the N′-terminal of mRNAs have been implicated in attenuating the process of ribosome allocation, and consequently the translation rate in a broad range of organisms. The current interpretation of “slow” tRNA codons does not distinguish between protein translations mediated by free- or endoplasmic reticulum (ER)-bound ribosomes. We demonstrate that proteins translated by free- or ER-bound ribosomes exhibit different overall properties in terms of their translation efficiency and speed in yeast, fly, plant, worm, bovine and human. We note that only secreted or membranous proteins with a Signal peptide (SP) are specified by segments of “slow” tRNA at the N′-terminal, followed by abundant codons that are considered “fast.” Such profiles apply to 3100 proteins of the human proteome that are composed of secreted and signal peptide (SP)-assisted membranous proteins. Remarkably, the bulks of the proteins (12,000), or membranous proteins lacking SP (3400), do not have such a pattern. Alternation of “fast” and “slow” codons was found also in proteins that translocate to mitochondria through transit peptides (TP). The differential clusters of tRNA adapted codons is not restricted to the N′-terminal of transcripts. Specifically, Glycosylphosphatidylinositol (GPI)-anchored proteins are unified by clusters of low adapted tRNAs codons at the C′-termini. Furthermore, selection of amino acids types and specific codons was shown as the driving force which establishes the translation demands for the secretory proteome. We postulate that “hard-coded” signals within the secretory proteome assist the steps of protein maturation and folding. Specifically, “speed control” signals for delaying the translation

  12. Crofelemer, a novel agent for treatment of secretory diarrhea.

    Science.gov (United States)

    Crutchley, Rustin D; Miller, Jennifer; Garey, Kevin W

    2010-05-01

    To review the chemistry, pharmacology, pharmacokinetics, efficacy, and safety of crofelemer. A literature search using the terms SP-303, Provir, and crofelemer was performed with PubMed (up to April 2010), Google Scholar, and selected Ovid bibliography searches. Additional references from the bibliographies of articles included in the search, as well as company and Food and Drug Administration Web sites, were also assessed. English-language in vitro and clinical studies associated with the safety and efficacy of crofelemer were included. Crofelemer is a first-in-class agent that may be useful for different types of secretory diarrhea, since it prevents chloride and fluid secretion into the bowel by directly inhibiting 2 distinct intestinal chloride channels. Crofelemer significantly brought about faster symptom resolution in patients with traveler's diarrhea, along with lower rates of treatment failure compared to placebo-treated patients. In a post hoc analysis, crofelemer compared to placebo also appears to have reduced abnormal stool weight and frequency in patients with AIDS-associated diarrhea. In a third trial, crofelemer did not offer a significant benefit in improving stool consistency after 12 weeks of treatment in patients with diarrhea-predominant irritable bowel syndrome. However, a significant increase in pain-free days was noted in female patients. Preliminary studies also show that crofelemer may reduce watery stool output in patients with infectious diarrhea such as cholera. Oral crofelemer seemed to be well tolerated in clinical trials, with adverse effect profiles comparable to those with placebo. Crofelemer possesses a novel mechanism of action that shows promise in treating secretory diarrhea of several etiologies. However, results from further Phase 3 clinical trials are still needed in order to fully evaluate the efficacy and safety of this agent.

  13. Ancestry and evolution of a secretory pathway serpin

    Directory of Open Access Journals (Sweden)

    Ragg Hermann

    2008-09-01

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

  14. Effect of immune system imagery on secretory IgA.

    Science.gov (United States)

    Rider, M S; Achterberg, J; Lawlis, G F; Goven, A; Toledo, R; Butler, J R

    1990-12-01

    This study was an investigation of the effects of physiologically-oriented mental imagery on immune functioning. College students with normal medical histories were randomly selected to one of three groups. Subjects in Group 1 participated in short educational training on the production of secretory immunoglobulin A. They were then tested on salivary IgA, skin temperature, and the Profile of Mood States (POMS) before and after listening to a 17-minute tape of imagery instructions with specially composed background "entrainment" music designed to enhance imagery. Subjects in Group 2 (placebo controls) listened to the same music but received nor formal training on the immune system. Group 3 acted as a control and subjects were tested before and after 17 minutes of no activity. Treatment groups listened to their tapes at home on a bi-daily basis for six weeks. All groups were again tested at Weeks 3 and 6. Secretory IgA was analyzed using standard radial immunodiffusion techniques. Repeated measures analyses of variance with planned orthogonal contrasts were used to evaluate the data. Significant overall increases (p less than 0.05) were found between pre- and posttests for all three trials. Groups 1 and 2 combined (treatment groups) yielded significantly greater increases in sIgA over Group 3 (control) for all three trials. Group 1 (imagery) was significantly higher than Group 2 (music) in antibody production for Trials 2 and 3. Symptomatology, recorded by subjects at Weeks 3 and 6, was significantly lower for three symptoms (rapid heartbeat, breathing difficulty, and jaw clenching), favoring both treatment groups over the control group.

  15. Secretory immunoglobulin purification from whey by chromatographic techniques.

    Science.gov (United States)

    Matlschweiger, Alexander; Engelmaier, Hannah; Himmler, Gottfried; Hahn, Rainer

    2017-08-15

    Secretory immunoglobulins (SIg) are a major fraction of the mucosal immune system and represent potential drug candidates. So far, platform technologies for their purification do not exist. SIg from animal whey was used as a model to develop a simple, efficient and potentially generic chromatographic purification process. Several chromatographic stationary phases were tested. A combination of two anion-exchange steps resulted in the highest purity. The key step was the use of a small-porous anion exchanger operated in flow-through mode. Diffusion of SIg into the resin particles was significantly hindered, while the main impurities, IgG and serum albumin, were bound. In this step, initial purity was increased from 66% to 89% with a step yield of 88%. In a second anion-exchange step using giga-porous material, SIg was captured and purified by step or linear gradient elution to obtain fractions with purities >95%. For the step gradient elution step yield of highly pure SIg was 54%. Elution of SIgA and SIgM with a linear gradient resulted in a step yield of 56% and 35%, respectively. Overall yields for both anion exchange steps were 43% for the combination of flow-through and step elution mode. Combination of flow-through and linear gradient elution mode resulted in a yield of 44% for SIgA and 39% for SIgM. The proposed process allows the purification of biologically active SIg from animal whey in preparative scale. For future applications, the process can easily be adopted for purification of recombinant secretory immunoglobulin species. Copyright © 2017 Elsevier B.V. All rights reserved.

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  2. File list: DNS.Utr.10.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

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  3. File list: Unc.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

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  4. File list: DNS.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

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  13. File list: NoD.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 No description Uterus Fallopian tube... secretory epithelial cell http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  14. File list: Unc.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 Unclassified Uterus Fallopian tube... secretory epithelial cell http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  15. Translational regulation of viral secretory proteins by the 5' coding regions and a viral RNA-binding protein.

    Science.gov (United States)

    Nordholm, Johan; Petitou, Jeanne; Östbye, Henrik; da Silva, Diogo V; Dou, Dan; Wang, Hao; Daniels, Robert

    2017-08-07

    A primary function of 5' regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5' positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5' UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5' coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins. © 2017 Nordholm et al.

  16. Early Secretory Antigenic Target-6 Drives Matrix Metalloproteinase-10 Gene Expression and Secretion in Tuberculosis.

    Science.gov (United States)

    Brilha, Sara; Sathyamoorthy, Tarangini; Stuttaford, Laura H; Walker, Naomi F; Wilkinson, Robert J; Singh, Shivani; Moores, Rachel C; Elkington, Paul T; Friedland, Jon S

    2017-02-01

    Tuberculosis (TB) causes disease worldwide, and multidrug resistance is an increasing problem. Matrix metalloproteinases (MMPs), particularly the collagenase MMP-1, cause lung extracellular matrix destruction, which drives disease transmission and morbidity. The role in such tissue damage of the stromelysin MMP-10, a key activator of the collagenase MMP-1, was investigated in direct Mycobacterium tuberculosis (Mtb)-infected macrophages and in conditioned medium from Mtb-infected monocyte-stimulated cells. Mtb infection increased MMP-10 secretion from primary human macrophages 29-fold, whereas Mtb-infected monocytes increased secretion by 4.5-fold from pulmonary epithelial cells and 10.5-fold from fibroblasts. Inhibition of MMP-10 activity decreased collagen breakdown. In two independent cohorts of patients with TB from different continents, MMP-10 was increased in both induced sputum and bronchoalveolar lavage fluid compared with control subjects and patients with other respiratory diseases (both P < 0.05). Mtb drove 3.5-fold greater MMP-10 secretion from human macrophages than the vaccine strain bacillus Calmette-Guerin (P < 0.001), whereas both mycobacteria up-regulated TNF-α secretion equally. Using overlapping, short, linear peptides covering the sequence of early secretory antigenic target-6, a virulence factor secreted by Mtb, but not bacillus Calmette-Guerin, we found that stimulation of human macrophages with a single specific 15-amino acid peptide sequence drove threefold greater MMP-10 secretion than any other peptide (P < 0.001). Mtb-driven MMP-10 secretion was inhibited in a dose-dependent manner by p38 and extracellular signal-related kinase mitogen-activated protein kinase blockade (P < 0.001 and P < 0.01 respectively), but it was not affected by inhibition of NF-κB. In summary, Mtb activates inflammatory and stromal cells to secrete MMP-10, and this is partly driven by the virulence factor early secretory antigenic target-6

  17. Partitioning and Exocytosis of Secretory Granules during Division of PC12 Cells

    Directory of Open Access Journals (Sweden)

    Nickolay Vassilev Bukoreshtliev

    2012-01-01

    Full Text Available The biogenesis, maturation, and exocytosis of secretory granules in interphase cells have been well documented, whereas the distribution and exocytosis of these hormone-storing organelles during cell division have received little attention. By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells. During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules. During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge. Furthermore, by measuring exocytosis at the single granule level, we showed, that during all stages of cell division, secretory granules were competent for regulated exocytosis. In conclusion, our data shed new light on the complex molecular machinery of secretory granule redistribution during cell division, which facilitates their release from the F-actin-rich cortex and active transport along spindle microtubules.

  18. Changes of middle ear mucosa in secretory otitis media treated with ventilation tubes.

    Science.gov (United States)

    Kiroglu, F; Kaya, M; Ozşahinoglu, C; Soylu, L; Polat, S

    1990-01-01

    The present study was designed to obtain the precise ultrastructural status of the middle ear mucosa of patients to whom ventilation tubes were applied for the treatment of secretory otitis media (SOM). This study comprised 14 children with SOM aged 5-11. Normal epithelium obtained from otosclerotic patients as controls was composed of three main cell types; basal, ciliated, and secretory. Electron microscopic observations revealed that ciliated cells were almost completely absent in the epithelium of patients with SOM. Secretory cells, on the other hand, were noted to be increased in number with cytoplasmic features indicating active secretory phase; abundant secretory granules in these cells featured both serous and mucoid characteristics. Following the application of ventilation tubes, the micrographs exhibited a vast number of ciliated cells. Furthermore, the secretory cells were less conspicuous and the secretory granules were mostly serous in type. The ultrastructural findings of this investigation strongly suggest that the application of ventilation tubes in cases of SOM stimulates ciliated cell regeneration, inhibits secretion of mucoid material, and activates serous secretion; all of which eventually shorten the recovery period.

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

    Science.gov (United States)

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

    2017-07-01

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

  20. PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance

    DEFF Research Database (Denmark)

    Holst, Birgitte; Madsen, Kenneth L; Jansen, Anna M

    2013-01-01

    by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate...... for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis....

  1. Periodic activity of secretory glands of stomach in ulcer erosion of gastro-duodenal zone

    Directory of Open Access Journals (Sweden)

    A. I. Rudenko

    2005-12-01

    Full Text Available It was fixed, that development of atophanum-carbacholimun ulcer of the gastroduodenal zone invoked various changes of secretory activity of the stomach. The changes directly depend on a progress of pathological process. As this takes place the reaction of stomach secretory glands varies under the stimulation with histamine: the decrease of stomach secretory glands’ work capacity till 10th day and its increase after 10–15th day were observed. Disorders of the glands’ ultradian rhythms at initial stages of modeling of gastrointestinal nervous regulation disturbances testify to dependence of periodic activity of gastrointestinal tract on resistance of regulatory mechanisms correlation.

  2. Identification of annexin II as a novel secretory biomarker for breast cancer.

    Science.gov (United States)

    Jeon, You Rim; Kim, Sun Young; Lee, Eun Jeong; Kim, Yong Nyun; Noh, Dong-Young; Park, So Yeon; Moon, Aree

    2013-11-01

    Early prediction of metastatic breast cancer is important for improvement of prognosis and survival rate. The present study aimed to identify secreted protein biomarkers for detection of invasive breast cancer. To this end, we performed a comparative proteomic analysis by a combination of 2DE and MALDI-TOF MS analysis of conditioned media from invasive H-Ras MCF10A human breast epithelial cells and noninvasive MCF10A and N-Ras MCF10A cells. We identified a list of 25 proteins that were strongly detected in media of H-Ras MCF10A and focused on annexin II, which was shown to be involved in cell motility. Invasive triple-negative human breast carcinoma cells, Hs578T, and MDA-MB-231, showed increased levels of annexin II in media, demonstrating that secretion of annexin II correlated well with the invasive phenotype of cells. We demonstrated a crucial role of annexin II in breast cell invasion/migration and actin cytoskeleton reorganization required for filopodia formation. Annexin II levels in the plasma samples and breast cancer tissues of breast cancer patients were significantly higher than those of normal groups, providing a clinical relevance to our in vitro findings. Taken together, we identified annexin II as a novel secretory biomarker candidate for invasive breast cancer, especially estrogen receptor-negative breast cancer. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Human recombinant resistin protein displays a tendency to aggregate by forming intermolecular disulfide linkages.

    Science.gov (United States)

    Aruna, Battu; Ghosh, Sudip; Singh, Anil K; Mande, Shekhar C; Srinivas, V; Chauhan, Radha; Ehtesham, Nasreen Z

    2003-09-16

    Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein. The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but beta-mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from alpha-helical to beta-sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.

  4. A rare case of extensive ductal carcinoma in situ of the breast with secretory features

    Directory of Open Access Journals (Sweden)

    Kenichi Sugihara

    2012-10-01

    Full Text Available We report a very rare case of extensive ductal carcinoma in situ (DCIS of the breast with secretory features in a 30-year old Japanese woman. The patient presented with a nodule in the lower inner quadrant of the left breast measuring approximately 2-3 cm, accompanied by an irregular tumor shadow with segmental microcalcification on mammography. These findings suggested malignancy, and excisional biopsy was performed following core needle biopsy. Pathological diagnosis was that of DCIS with secretory features. A treatment plan of simple mastectomy and sentinel lymph node biopsy was chosen. Most previous reports have only described invasive secretory carcinoma of the breast. We have only been able to find 2 case reports of non-invasive secretory lesion in the English literature to date. Because the characteristics of this lesion are not widely known, we thought it important to share our findings.

  5. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    LENUS (Irish Health Repository)

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

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

    Science.gov (United States)

    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.

  7. Mammary analogue secretory carcinoma: update on a new diagnosis of salivary gland malignancy.

    Science.gov (United States)

    Sethi, Roshan; Kozin, Elliott; Remenschneider, Aaron; Meier, Josh; VanderLaan, Paul; Faquin, William; Deschler, Daniel; Frankenthaler, Robert

    2014-01-01

    To review the known histopathologic findings and clinical behavior of mammary analogue secretory carcinoma (MASC). PubMed. Literature search using the terms "Mammary analogue secretory carcinoma," "Mammary analog secretory carcinoma," and "MASC" to identify all relevant publications. MASC is an unusual and rare malignant salivary gland tumor first described in 2010. It shares histologic, immunohistochemical, and genetic features with secretory carcinoma of the breast. The clinical behavior of MASC ranges from slowly growing tumors that infrequently recur after surgical resection to aggressive tumors that cause widespread metastasis and death. Many cases of MASC were discovered in archived cases previously classified as acinic cell carcinoma, mucoepidermoid carcinoma, and adenocarcinoma not otherwise specified. MASC is a newly recognized variant of salivary gland malignancy. Further research is needed to better delineate its overall prevalence and to define an appropriate treatment algorithm for this new clinical entity. © 2013 The American Laryngological, Rhinological and Otological Society, Inc.

  8. Role of NBCe1 and AE2 in Secretory Ameloblasts

    Science.gov (United States)

    Paine, Michael L.; Snead, Malcolm L.; Wang, HongJun; Abuladze, Natalia; Pushkin, Alexander; Liu, Weixin; Kao, Li Yo; Wall, Susan M.; Kim, Young-Hee; Kurtz, Ira

    2008-01-01

    The H+/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, patients with mutations in the electrogenic sodium bicarbonate cotransporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. The present study aimed to understand the roles of AE2 and NBC1 in ameloblasts. The data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation. PMID:18362326

  9. Innate secretory antibodies protect against natural Salmonella typhimurium infection.

    Science.gov (United States)

    Wijburg, Odilia L C; Uren, Tania K; Simpfendorfer, Kim; Johansen, Finn-Eirik; Brandtzaeg, Per; Strugnell, Richard A

    2006-01-23

    The production of IgA is induced in an antigen-unspecific manner by commensal flora. These secretory antibodies (SAbs) may bind multiple antigens and are thought to eliminate commensal bacteria and self-antigens to avoid systemic recognition. In this study, we addressed the role of "innate" SAbs, i.e., those that are continuously produced in normal individuals, in protection against infection of the gastrointestinal tract. We used polymeric immunoglobulin receptor (pIgR-/-) knock-out mice, which are unable to bind and actively transport dimeric IgA and pentameric IgM to the mucosae, and examined the role of innate SAbs in protection against the invasive pathogen Salmonella typhimurium. In vitro experiments suggested that innate IgA in pIgR-/- serum bound S. typhimurium in a cross-reactive manner which inhibited epithelial cell invasion. Using a "natural" infection model, we demonstrated that pIgR-/- mice are profoundly sensitive to infection with S. typhimurium via the fecal-oral route and, moreover, shed more bacteria that readily infected other animals. These results imply an important evolutionary role for innate SAbs in protecting both the individual and the herd against infections, and suggest that the major role of SAbs may be to prevent the spread of microbial pathogens throughout the population, rather than protection of local mucosal surfaces.

  10. Academic stress, social support, and secretory immunoglobulin A.

    Science.gov (United States)

    Jemmott, J B; Magloire, K

    1988-11-01

    We examined the relation of academic stress and social support to salivary concentrations of secretory immunoglobulin A (S-IgA), an antibody class that plays an important role in mucosal defense against acute upper respiratory tract infections. We assayed whole, unstimulated saliva samples collected from 15 healthy undergraduates 5 days before their final exam period, during their exam period, and 14 days after their last final exam for S-IgA concentrations by single radial immunodiffusion. The students rated the university's general psychological climate as being more stressful during the exam period compared with the two other periods. Paralleling this, their salivary concentrations of S-IgA were lower during the exam period. Students who reported more adequate social support at the preexam period had consistently higher S-IgA levels than did their peers reporting less adequate social support. This latter finding is consonant with the social support direct effects hypothesis, which states that social support enhances health outcomes irrespective of whether the individual is exposed to stressful experiences.

  11. Correlation between CT and tympanogram in secretory otitis media

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Toshimitsu; Sakurai, Tokio; Taniguchi, Kazuhiko; Takahashi, Kuniaki (Iwaki-Kyoritsu General Hospital, Iwaki, Fukushima (Japan)); Ikeda, Katsuhisa; Kawamoto, Kazutomo

    1984-11-01

    In an attempt to evaluate the feasibility of the tympanometry in detecting the middle ear effusion (MEE) in secretory otitis media (SOM) in childhood, the findings of the computed tomography (CT) were evaluated whether they were compatible with that of tympanometry in 27 cases (51 ears) of SOM. Tympanometry (tympanogram, static compliance measurement and stapedial reflex test), pure tone audiometry and high resolution CT were performed sequentially, and the CT findings were compared with the results of the other tests. The conclusions obtained were summarized as follows. 1. Among the tests performed, tympanogram appeared to be the most reliable measure in detection of MEE. 2. Fifteen ears out of 16 with type B tympanograms and 6 ears out of 15 with type C/sub 2/ tympanograms, were diagnosed by CT as having MEE. MEE occupied the entire middile ear space in most ears with type B tympanograms. By contrast, in the ears with type C/sub 2/ tympanograms, air containing space of varying size were always observed even in the ears with MEE.

  12. Insulin sensitivity and secretory status of a healthy malay population.

    Science.gov (United States)

    Al-Mahmood, Abu Kholdun; Ismail, Aziz Al-Safi; Rashid, Faridah Abdul; Wan Bebakar, Wan Mohamad

    2006-07-01

    Insulin insensitivity is a common finding in several metabolic disorders including glucose intolerance, dyslipidemia, hyperuricemia and hypertension. Most of the previous studies on insulin sensitivity were performed on diabetic or obese population. So our knowledge about insulin sensitivity of healthy population remains limited. Rising prevalence of obesity, diabetes and metabolic syndrome is a serious issue in Malaysia and some other rapidly developing countries. So it is important to look at the insulin sensitivity status of healthy Malaysian subjects and to compare it in future with those of diabetic, obese or metabolic syndrome patients. In this study we sampled subjects who were independent of confounding factors such as obesity (including abdominal obesity), hypertension and glucose intolerance (diabetes, IGT or IFG) which may influence insulin sensitivity. Fasting plasma glucose, fasting insulin and lipid profile were determined. Insulin sensitivity and secretory status were calculated using the homeostasis model assessment (HOMA) software (HOMA%S, HOMA%B and HOMA-IR). The insulin sensitivity (HOMA%S) of healthy Malay subjects aged between 30-60 years was 155.17%, HOMA-IR was 1.05 and HOMA%B was 116.65% (values adjusted for age, sex, BMI and waist circumference). It was seen that non-obese Malaysians can prevent age related lowering of insulin sensitivity if they can retain their BMI within limit.

  13. Role of NBCe1 and AE2 in secretory ameloblasts.

    Science.gov (United States)

    Paine, M L; Snead, M L; Wang, H J; Abuladze, N; Pushkin, A; Liu, W; Kao, L Y; Wall, S M; Kim, Y-H; Kurtz, I

    2008-04-01

    The H(+)/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well-understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, persons with mutations in the electrogenic sodium bicarbonate co-transporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. In the present study, we aimed to understand the roles of AE2 and NBCe1 in ameloblasts. Analysis of the data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.

  14. Clinicopathological characterization of mammary analogue secretory carcinoma of salivary glands.

    Science.gov (United States)

    Chiosea, Simion I; Griffith, Christopher; Assaad, Adel; Seethala, Raja R

    2012-09-01

    Mammary analogue secretory carcinoma (MASC) is a recently described tumour with ETV6 translocation. We aimed to characterize the clinical significance of recognizing MASC. Thirty-six patients with MASC (27 identified retrospectively and nine prospectively) are presented. Historically, MASC mimicked other salivary tumours, as follows: 14 of 37 (37.8%) adenocarcinoma, not otherwise specified, 11 of 89 (12.4%) acinic cell carcinomas (AciCC), one of five (20%) mucin-producing signet ring adenocarcinomas, and one of 165 (0.6%) mucoepidermoid carcinomas. Demographically, MASC affected males more commonly (1.4:1). The average age at diagnosis was 45.7 years. Parotid gland was the most common site of involvement (26 of 36, 72.2%), although other head and neck sites, including the base of tongue, were affected. Of 18 patients with neck dissection, lymph node involvement was identified in four patients (four of 18, 22.2%). Survival analysis of MASC cases presented here, combined with those reported previously, revealed a mean disease-free survival for patients with MASC of 92 months [n = 29; 95% confidence interval (CI) 71-115 months], compared with a mean DFS of 121 months for patients with AciCC (n = 38; 95% CI 92-149, P = 0.43). Although perhaps slightly more aggressive, MASC clinical outcome mimics that of AciCC. © 2012 Blackwell Publishing Ltd.

  15. Multi-method analysis of calcium localization in the secretory ameloblast

    Energy Technology Data Exchange (ETDEWEB)

    Eisenmann, D.R.; Ashrafi, S.; Zaki, A.E.

    1982-12-01

    X-ray micro-analysis and electron energy loss analysis were used to confirm cytochemical localization by potassium pyro-antimonate of calcium in secretory ameloblasts. Neither intercellular calcium concentration between ameloblasts nor over-all calcium levels of the enamel organ were lowered during a period of inhibition of enamel mineralization by injected fluoride. Calcium concentrations in mitochondria and secretory granules were reduced.

  16. Therapeutic Effects of Mycobacterial Secretory Proteins Against Established Asthma in BALB/c Mice

    OpenAIRE

    Han, Eui-Ryoung; Choi, Inseon S.; Choi, Han-Gyu; Kim, Hwa-Jung

    2012-01-01

    Purpose Live/killed mycobacteria and culture supernatants can suppress asthmatic reactions. This study investigated whether mycobacterial secretory proteins have therapeutic effects on asthma. Methods Mycobacterium bovis bacille Calmette-Guérin (BCG; 2×105 CFUs) and mycobacterial secretory proteins (Ag85 complex, 38-kDa protein or MPB70; 4 or 20 µg) were administered intraperitoneally to female BALB/c mice with established airway hyperresponsiveness. One week after treatment, the mice underwe...

  17. Identification of Secretory Structure, Histochemistry and Phytochemical Compounds of Medicinal Plant Hyptis Capitata Jacq.

    OpenAIRE

    Rupa, Darius; Sulistyaningsih, Yohana Caecilia; Dorly, Dorly; Ratnadewi, Diah

    2017-01-01

    Hyptis capitata Jacq. (common name: Knobweed or False Ironwort) belongs to Lamiaceae family and is among known traditional medicine. The Anak Dalam Tribe of Jambi Province uses the leaves of H. capitata to cure external and internal wounds. This study was aimed at identifying and analyzing secretory structure, histochemistry and phytochemical content of H. capitata leaves. The results showed that H. capitata leaves have secretory structures in the form of peltate, capitate and uniseriate gla...

  18. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

    Science.gov (United States)

    Zhang, Xianjun; Zhao, Fei; Wu, Yiran; Yang, Jun; Han, Gye Won; Zhao, Suwen; Ishchenko, Andrii; Ye, Lintao; Lin, Xi; Ding, Kang; Dharmarajan, Venkatasubramanian; Griffin, Patrick R.; Gati, Cornelius; Nelson, Garrett; Hunter, Mark S.; Hanson, Michael A.; Cherezov, Vadim; Stevens, Raymond C.; Tan, Wenfu; Tao, Houchao; Xu, Fei

    2017-05-01

    The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

  19. A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting

    Directory of Open Access Journals (Sweden)

    So-Jung Kim

    2017-03-01

    Full Text Available Kelch-like ECH-associated protein 1 (keap1 is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a. Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC. The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential.

  20. The preliminary solution structure of human p8MTCP1, a protein encoded by the putative MTCP1 oncogene

    Science.gov (United States)

    Barthe, P.; Guignard, L.; Yang, Y.-S.; Chiche, L.; Strub, M.-P.; Hoh, F.; Stern, M.-H.; Roumestand, C.

    1998-02-01

    The NMR solution structure of the cystein-rich human p8 protein coded by the oncogene MTCP1 reveals an original scaffold consisting of three α-helices, two of them tightly held together by two disulphide bridges in an antiparallel α-hairpin. MTCP1 was found to be expressed in mature prolymphocytic leukemias. La structure RMN de la protéine humaine p8 en solution présente un repliement original en trois hélices α, deux d'entre elles étant étroitement maintenues dans une orientation antiparallèle par deux ponts disulfures. Cette protéine riche en cystéine est codée par l'oncogène MTCP1 qui est exprimé dans des leucémies prolymphocytaires de phénotype mature.

  1. [Yeast (Saccharomyces cerevisiae) secretory expression vector maintained stably in Pro3+ transformants in rich medium].

    Science.gov (United States)

    Xie, H Y; Tang, Y; Jiang, W D; He, H Y; Liu, M; Kuang, D R

    2000-01-01

    A yeast (Saccharomyces cerevisiae) secretory expression vector containing PRO3 gene was constructed (pCBy310). beta HCG(Human choriogonadotropin beta subunit)-cDNA was inserted into pCBy310 to form a recombinant plasmid pCBy314. Since yeast proline auxotroph will not survive in rich medium (YPD), YPD could be used as a selection pressure, and pCBy314 could be maintained mitotically stable in transformants of yeast Pro3- auxotroph (MB299-7A) in rich medium. At an improved, yet not optimized cultural condition, the expression of beta HCG in culture medium was 650 micrograms/L. Our results showed not only that YPD could be used as a selection medium, but also that yeast grew better in YPD so as to increase the gene expression product, and that the component of YPD was simple and cheap. Our data suggested that PRO genes might be used widely in constructing vectors to clone and express foreign genes in yeast so that rich medium can be used as a selection pressure for direct selection.

  2. Tattooing to "Toughen up": Tattoo experience and secretory immunoglobulin A.

    Science.gov (United States)

    Lynn, Christopher D; Dominguez, Johnna T; DeCaro, Jason A

    2016-09-10

    A costly signaling model suggests tattooing inoculates the immune system to heightened vigilance against stressors associated with soft tissue damage. We sought to investigate this "inoculation hypothesis" of tattooing as a costly honest signal of fitness. We hypothesized that the immune system habituates to the tattooing stressor in repeatedly tattooed individuals and that immune response to the stress of the tattooing process would correlate with lifetime tattoo experience. Participants were 24 women and 5 men (aged 18-47). We measured immune function using secretory immunoglobulin A (SIgA) and cortisol (sCORT) in saliva collected before and after tattoo sessions. We measured tattoo experience as a sum of number of tattoos, lifetime hours tattooed, years since first tattoo, percent of body covered, and number of tattoo sessions. We predicted an inverse relationship between SIgA and sCORT and less SIgA immunosuppression among those with more tattoo experience. We used hierarchical multiple regression to test for a main effect of tattoo experience on post-tattoo SIgA, controlling for pretest SIgA, tattoo session duration, body mass, and the interaction between tattoo experience and test session duration. The regression model was significant (P = 0.006) with a large effect size (r(2)  = 0.711) and significant and positive main (P = 0.03) and interaction effects (P = 0.014). Our data suggest that the body habituates over time to the tattooing stressor. It is possible that individuals with healthy immune systems heal faster, making them more likely to get multiple tattoos. Am. J. Hum. Biol. 28:603-609, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Secretory ribonucleases in the primitive ruminant chevrotain (Tragulus javanicus).

    Science.gov (United States)

    Breukelman, H J; Jekel, P A; Dubois, J Y; Mulder, P P; Warmels, H W; Beintema, J J

    2001-07-01

    Phylogenetic analyses of secretory ribonucleases or RNases 1 have shown that gene duplication events, giving rise to three paralogous genes (pancreatic, seminal and brain RNase), occurred during the evolution of ancestral ruminants. A higher number of paralogous sequences are present in chevrotain (Tragulus javanicus), the earliest diverged taxon within the ruminants. Two pancreatic RNase sequences were identified, one encoding the pancreatic enzyme, the other encoding a pseudogene. The identity of the pancreatic enzyme was confirmed by isolation of the protein and N-terminal sequence analysis. It is the most acidic pancreatic ribonuclease identified so far. Formation of the mature enzyme requires cleavage by signal peptidase of a peptide bond between two glutamic acid residues. The seminal-type RNase gene shows features of a pseudogene, like orthologous genes in other ruminants investigated with the exception of the bovine species. The brain-type RNase gene of chevrotain is expressed in brain tissue. A hybrid gene with a pancreatic-type N-terminal and a brain-type C-terminal sequence has been identified but nothing is known about its expression. Phylogenetic analysis of RNase 1 sequences of six ruminant, three other artiodactyl and two whale species support previous findings that two gene duplications occurred in a ruminant ancestor. Three distinct groups of pancreatic, seminal-type and brain-type RNases have been identified and within each group the chevrotain sequence it the first to diverge. In taxa with duplications of the RNase gene (ruminants and camels) the gene evolved at twice as fast than in taxa in which only one gene could be demonstrated; in ruminants there was an approximately fourfold increase directly after the duplications and then a slowing in evolutionary rate.

  4. [Clinicopathologic features of mammary analogue secretory carcinoma of salivary glands].

    Science.gov (United States)

    Zhang, X P; Ni, H; Wang, X; Chen, H; Shi, S S; Yu, B; Zhou, X J; Rao, Q

    2017-01-08

    Objective: To investigate the clinicopathological features of mammary analogue secretory carcinoma (MASC) of salivary glands, and its diagnosis, differential diagnosis, immunohistochemistry and molecular pathology. Methods: Seventeen cases of MASC were enrolled, with 9 cases of salivary acinar cell carcinoma and 18 cases of adenoid cystic carcinoma as control groups from Nanjing General Hospital from 1997 to 2014 were included in this retrospective study, combined with immunohistochemistry and molecular detection of ETV6-NTRK3 gene fusion. All cases were histologically reviewed with immunohistochemical staining (EnVision) for S-100 protein, SOX10, GATA3, CD117 expression in each group. Fluorescence in situ hybridization (FISH) was used to detect the ETV6-NTRK3 gene fusion. Results: The age of MASC patients ranged from 27 to 74 years with mean age of 47 and ratio of male and female was 4∶3. All cases showed infiltrative growth and diverse cytology and histology, including lobular (8 cases), cystic papillary (3 cases), cribriform mixed with papillary and glandular structures (6 cases) at various proportions. Some tumors of MASC also exhibited solid growth areas with occasional microcystic honeycombed pattern composed of small cysts merged into larger cysts resembling thyroid follicles. S-100 protein and SOX10 were strongly positive in all MASC cases (17/17). In addition, there was insignificant positivity for GATA3 (3/17) and CD117 (4/17). ETV6 gene fusion detection was informative in 12 MASC cases by FISH with 10 positive cases and 2 negative cases. Conclusions: Combined immunohistochemical positivity of S-100 protein, CD117 and SOX10 are useful in the diagnosis and differential diagnosis of MASC. FISH detection of ETV6-NTRK3 fusion offers an additional molecular diagnostic marker for the diagnosis.

  5. Evaluation of Humoral Immune Response against Somatic and Excretory-Secretory Antigens of Dicrocoelium Dendriticum in Infected Sheep by Western Blot

    Directory of Open Access Journals (Sweden)

    Mohammad Hosein Razi Jalali

    2013-12-01

    Full Text Available Introduction and objective: Dicrocoelium dendriticum is a worldwide spread parasite of liver, bile ducts and gallbladder of especially ruminants and humans as well. Identification of specific antigens is useful for early diagnosis of the infection. The goal of this study was the isolation and identification of excretory-secretory and somatic antigens from D. dendriticum by sodium dodecyl sulphate (SDS-PAGE and evaluation of humoral immune response against these antigens.   Methods: The parasites were collected and washed by phosphate buffered saline (PBS and supplemented by antibiotic for several times. For preparing somatic antigens, parasites were sonicated and centrifuged prior to collect supernatant. For preparing excretory-secretory antigens the viable parasites were transferred to the sterile medium. The samples were centrifuged and supernatants were collected. The sera of infected sheep with different infection degrees were collected too. Somatic and excretory-secretory proteins were isolated with SDS PAGE and stained with coomassie blue. Immunogenicity properties of the resulting proteins were determined using western blot analysis.   Results: The total extract of somatic antigens analyzed by SDS-PAGE revealed 21 proteins. In mild infection, bands of 130 KDa were immune dominant. In moderate infections 48, 80 and 130 KDa and in heavy infections 48, 60, 80, 130 KDa were detected as immune dominant bands. In excretory- secretory antigens seven bands of protein were detected. In mild infection 130 KDa, in moderate infection 100, 120 and 130 KDa and in heavy infection 45, 80, 85, 100, 120 and 130 KDa were immune dominant bands.   Conclusion: Probably the most immunogenic protein band during different degrees of infection was 130KDa that can be used for vaccination and inducing immunity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Faddy, Helen M.; Smart, Chanel E.; Xu, Ren; Lee, Genee Y.; Kenny, Paraic A.; Feng, Mingye; Rao, Rajini; Brown, Melissa A.; Bissell, Mina J.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    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 during 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.

  7. Multiphoton fluorescence lifetime imaging shows spatial segregation of secondary metabolites in Eucalyptus secretory cavities.

    Science.gov (United States)

    Heskes, A M; Lincoln, C N; Goodger, J Q D; Woodrow, I E; Smith, T A

    2012-07-01

    Multiphoton fluorescence lifetime imaging provides an excellent tool for imaging deep within plant tissues while providing a means to distinguish between fluorophores with high spatial and temporal resolution. Ideal candidates for the application of multiphoton fluorescence lifetime imaging to plants are the embedded secretory cavities found in numerous species because they house complex mixtures of secondary metabolites within extracellular lumina. Previous investigations of this type of structure have been restricted by the use of sectioned material resulting in the loss of lumen contents and often disorganization of the delicate secretory cells; thus it is not known if there is spatial segregation of secondary metabolites within these structures. In this paper, we apply multiphoton fluorescence lifetime imaging to investigate the spatial arrangement of metabolites within intact secretory cavities isolated from Eucalyptus polybractea R.T. Baker leaves. The secretory cavities of this species are abundant (up to 10 000 per leaf), large (up to 6 nL) and importantly house volatile essential oil rich in the monoterpene 1,8-cineole, together with an immiscible, non-volatile component comprised largely of autofluorescent oleuropeic acid glucose esters. We have been able to optically section into the lumina of secretory cavities to a depth of ∼80 μm, revealing a unique spatial organization of cavity metabolites whereby the non-volatile component forms a layer between the secretory cells lining the lumen and the essential oil. This finding could be indicative of a functional role of the non-volatile component in providing a protective region of low diffusivity between the secretory cells and potentially autotoxic essential oil. © 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  10. Histological and histochemical characterization of the secretory cells of Choeradoplana iheringi Graff, 1899 (Platyhelminthes: Tricladida: Terricola

    Directory of Open Access Journals (Sweden)

    S. A. de Souza

    Full Text Available The present study aims at providing a detailed description of the histology, as well as the first histochemical characterization, of the secretory cells of the epidermis, pharynx, and copulatory organs of Choeradoplana iheringi, in order to give further support to studies on the physiology of these organs. The secretory cells are distinguished on the basis of secretion morphology and its staining properties, using trichrome methods and histochemical reactions. Four cell types open through the epidermis of Ch. iheringi, three of them secreting basic protein and a fourth containing glycosaminoglycan mucins. The epidermal lining cells store glycogen. In the pharynx, four secretory cell types were distinguished. Two types produce glycoprotein, a third type secretes basic protein, and another one produces glycosaminoglycan mucins. In the male copulatory organs, the prostatic vesicle receives four secretory cell types containing basic protein, except for one type which produces glycoprotein. The two secretory cell types opening into the male atrium secrete, respectively, glycoprotein, and glycosaminoglycan mucins. In the female copulatory organs, the female atrium and its proximal diverticulum, the vagina, receive two types of secretory cells producing, respectively, basic protein and glycosaminoglycan mucins. Another secretory cell type constitutes the so-called shell glands which open into the common glandular duct, secreting basic protein. The lining cells of the male and female atria produce a mucous secretion containing glycosaminoglycans. In addition, the lining epithelium of the female atrium presents an apical secretion of a proteic nature. The occurrence of a kind of spermatophore is reported for the first time for a species of Choeradoplana. This structure is located in the male or female atria in different specimens, and characterized by erythrophil, xanthophil, and/or mixed secretions associated with sperm.

  11. Pharmacologic Comparison of Clinical Neutral Endopeptidase Inhibitors in a Rat Model of Acute Secretory Diarrhea.

    Science.gov (United States)

    Griggs, David W; Prinsen, Michael J; Oliva, Jonathan; Campbell, Mary A; Arnett, Stacy D; Tajfirouz, Deena; Ruminski, Peter G; Yu, Ying; Bond, Brian R; Ji, Yuhua; Neckermann, Georg; Choy, Robert K M; de Hostos, Eugenio; Meyers, Marvin J

    2016-05-01

    Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil-treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy. Copyright © 2016 The Author(s).

  12. Ionizing radiation effects on the secretory-stage ameloblasts and enamel organic extracellular matrix.

    Science.gov (United States)

    de Moraes Ramos-Perez, Flávia Maria; do Espírito Santo, Alexandre Ribeiro; da Cruz Perez, Danyel Elias; Novaes, Pedro Duarte; Bóscolo, Frab Norberto; Line, Sérgio Roberto Peres; de Almeida, Solange Maria

    2014-08-01

    This study assessed the effects of high doses of ionizing radiation on eruption rate, odontogenic region morphology, secretory-stage ameloblasts, and enamel organic extracellular matrix (EOECM) of rat maxillary incisors. For the study, 30 male rats were divided into three experimental groups: control (non-irradiated), irradiated by 15 Gy, and irradiated by 25 Gy. Irradiated groups received a single dose of 15 or 25 Gy of X-rays in the head and neck region. The maxillary incisor eruption rate was measured. Sections of 5-µm thickness of the maxillary incisor odontogenic regions were evaluated using bright field light microscopy. Ultrathin sections of secretory ameloblasts and their EOECM were analyzed by transmission electron microscopy (TEM). Irradiated groups showed significantly diminished eruption rate values at the 4th and at the 6th day after irradiation. Reduced optical retardation values were observed in the irradiated groups. The odontogenic region of maxillary incisors from irradiated rats exhibited altered and poorly organized preameloblasts. TEM showed degeneration areas in the secretory-stage EOECM and several autophagosomes in the secretory ameloblasts from irradiated animals. In conclusion, high radiation doses delay eruption and induce disturbances in secretory ameloblasts and EOECM of rat maxillary incisors. These findings may be associated with structural defects of mature enamel.

  13. Effect of Semen on Vaginal Fluid Cytokines and Secretory Leukocyte Protease Inhibitor

    Directory of Open Access Journals (Sweden)

    Kathy J. Agnew

    2008-01-01

    Methods: 138 pregnant women had vaginal fluid collected for Gram stain, acid phosphatase detection by colorimetric assay, and interleukin 1-Beta, interleukin-6, interleukin-8, and secretory leukocyte protease inhibitor measurement by enzyme immunoassay. Results for women with and without acid phosphatase were compared by Mann-Whitney test. Results: of 138 subjects, 28 (20% had acid phosphatase detected; of these, only 19 (68% reported recent intercourse and 3 (11% had sperm seen on Gram stain. There were no significant differences in proinflammatory cytokine concentrations; however, secretory leukocyte protease inhibitor concentrations were significantly higher among women with acid phosphatase. Conclusions: proinflammatory cytokine measurement does not appear to be affected by the presence of semen, but secretory leukocyte protease inhibitor is significantly higher when semen is present. Detection of semen by acid phosphatase was associated with higher vaginal SLPI concentrations, however, the presence of semen did not appear to influence vaginal proinflammatory cytokine concentrations.

  14. Radial secretory glia conserved in the postnatal vertebrate brain: a study in the rat.

    Science.gov (United States)

    Viehweg, J; Naumann, W W

    1996-10-01

    Secretory glial cells in the roof of the last diencephalic prosomer, ependymocytes and hypendymocytes, form the subcommissural organ. The cells of this complex were labelled immunocytochemically, using an antiserum against their specific secretory products. The study aims at the characterization of this cell type in the rat as an anatomical model situation. Radially oriented secretory glial cells remain after birth behind the posterior commissure in the mesencephalic aqueduct. At about postnatal day 10, the cell bodies descend into the conventional ependyma and at postnatal day 25 they assume a compact, rounded appearance. The secretory product they release is involved in the formation of Reissner's fiber. This differentiation in phenotype is not accompanied by a change of the intermediate filament expression. In the adult rat these cells had been labelled immunopositive for cytokeratins 8 and 18 as well as vimentin but not for glial fibrillary acidic protein. DiI-marking from the third ventricle and from the dorsal surface of the brain shows that the basal processes of ependymocytes and hypendymocytes project to the external and internal glial limiting membrane, respectively, through the commissural fiber bundles. Also the subependymal located hypendymocytes have apical processes with contacts to the cerebrospinal fluid. When this secretory cell population is studied with respect to cyto-architectonical changes during ontogeny the results lead to a new understanding of the subcommissural cells. They are not specialized ependymal cells in a regionally restricted and secondary differentiated ependymal area, but rather descendants of an ontogenetically ancient, specific type of radial glia. Characteristic features for all subcommissural cells are that they: (1) appear very early during ontogeny, (2) are derived from a radial oriented glial cell type, (3) carry at least one kinocilium, (4) possess an original intermediate filament pattern, (5) release a secretory

  15. A highly sensitive assay for monitoring the secretory pathway and ER stress.

    Directory of Open Access Journals (Sweden)

    Christian E Badr

    2007-06-01

    Full Text Available The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER stress in real-time based on the naturally secreted Gaussia luciferase (Gluc.An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP, a well established assay for monitoring of protein processing and ER stress in mammalian cells.The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced.

  16. Differential protein expression in the secretory fluids of maxillary sinusitis and maxillary retention cyst.

    Science.gov (United States)

    Kim, Soung Min; Eo, Mi Young; Cho, Yun Ju; Kim, Yeon Sook; Lee, Suk Keun

    2017-01-01

    Both maxillary sinusitis (MS) and maxillary retention cyst (MRC) involve the maxillary sinus and show similar clinical features. Clinically, differentiating between MS and MRC is sometimes difficult in asymptomatic patients, despite their quite different pathogenic behaviors. To identify differential protein expressions in the secretory fluids of MS and MRC, 25 cases of asymptomatic MS and 15 cases of asymptomatic MRC were examined pathologically in this study. All patients underwent routine endoscopic sinus surgery or modified Caldwell-Luc procedure and the sinus mucosal specimens obtained during these procedures with the approval of the Institutional Review Board. Their secretory fluids were analyzed via immunoprecipitation-based high-performance liquid chromatography (IP-HPLC) using 25 types of antiserum, including inflammatory cytokines, antimicrobial proteins, and mucosal protective proteins. In the histological examinations, MS and MRC showed similar features in the secretory columnar epithelial lining and thick submucosal connective tissue, both of which contained few inflammatory cells infiltrates. The IP-HPLC analysis revealed that TNFα, IL-1, -8, MMP-3, -10, α1-antitrypsin, cathepsin C, lysozyme, lactoferrin, β-defensin-1, -3, LL-37, mucocidin, and mucin-1 were more intensely expressed in MS than in MRC; whereas IgA, cystatin A, and proline-rich proteins were more strongly expressed in MRC than in MS. These data indicate that the secretory fluid of MS is indicative of a more robust inflammatory reaction to certain bacteria compared to that of MRC, while the secretory fluid of MRC contains more abundant mucosal protective proteins compared to that of MS. Taken together, the IP-HPLC analysis of MS and MRC secretory fluid revealed that MRC showed a weaker inflammatory reaction but a stronger mucosal protective function than MS.

  17. The secretory membrane system studied in real-time. Robert Feulgen Prize Lecture, 2001.

    Science.gov (United States)

    Lippincott-Schwartz, J

    2001-08-01

    The discovery and development of green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has revolutionized studies on protein localization and dynamics by allowing direct observation of a protein's life history and pathway in living cells, previously only deduced from genetic, biochemical, or immunolabeling studies. Applied to the secretory membrane system, which regulates delivery of newly synthesized proteins and lipids to the cell surface, GFP-based studies are providing important new insights into the maintenance and biogenesis of organelles, as well as the origin, pathway, and fate of secretory transport intermediates.

  18. Ca(2+)-independent fusion of secretory granules with phospholipase A2-treated plasma membranes in vitro.

    OpenAIRE

    Nagao, T.; Kubo, T.; Fujimoto, R.; Nishio, H; Takeuchi, T.; Hata, F.

    1995-01-01

    The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ fused wit...

  19. Synthesis of sn-1 functionalized phospholipids as substrates for secretory phospholipase A2

    DEFF Research Database (Denmark)

    Linderoth, Lars; Peters, Günther H.J.; Jørgensen, K.

    2007-01-01

    Secretory phospholipase A2 (sPLA2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids (1 and 2) with bulky allyl-substituen......Secretory phospholipase A2 (sPLA2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids (1 and 2) with bulky allyl...

  20. Large lipid-rich mammary analogue secretory carcinoma of parotid gland: An unusual case.

    Science.gov (United States)

    Joshi, Prashant; Mridha, Asit Ranjan; Singh, Shuchita; Kinra, Prateek; Ray, Ruma; Thakar, Alok

    2015-01-01

    Mammary analogue secretory carcinoma (MASC) of the salivary gland is a malignant tumor which bears morphologic, immunohistochemical and molecular features similar to those of mammary secretory carcinoma. The tumor is considered as a low-grade malignancy perhaps slightly more aggressive than acinic cell carcinoma. High-grade transformation with recurrences, regional nodal involvement, metastases, and cancer-related death has been reported in a few cases. We report an unusual case of large MASC of the parotid gland in a young patient without regional lymph node involvement. To the best of our knowledge till date such a large MASC of the salivary gland has not been reported in the English literature.

  1. Cytologic findings of mammary analogue secretory carcinoma arising in the thyroid.

    Science.gov (United States)

    Rodríguez-Urrego, Paula A; Dogan, Snjezana; Lin, Oscar

    2017-06-01

    Mammary analogue secretory carcinoma (MASC) of the salivary gland, first described by Skálová et al in 2010, is a tumor that morphologically and genetically resembles breast secretory carcinoma harboring ETV6-NTRK3 fusion gene. To date, only seven cases of primary thyroid MASC have been described. The overall findings are similar to those seen in the salivary gland counterpart including the ETV6-NTRK3 fusion gene. This is the second report describing the cytologic features of MASC at this primary location, which also showed a classical type papillary carcinoma component. Diagn. Cytopathol. 2017;45:552-556. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Bronchial Secretory Immunoglobulin A Deficiency Correlates With Airway Inflammation and Progression of Chronic Obstructive Pulmonary Disease

    Science.gov (United States)

    Cates, Justin M.; Lawson, William E.; Zaynagetdinov, Rinat; Milstone, Aaron P.; Massion, Pierre P.; Ocak, Sebahat; Ware, Lorraine B.; Lee, Jae Woo; Bowler, Russell P.; Kononov, Alexey V.; Randell, Scott H.; Blackwell, Timothy S.

    2011-01-01

    Rationale: Although airway inflammation can persist for years after smoking cessation in patients with chronic obstructive pulmonary disease (COPD), the mechanisms of persistent inflammation are largely unknown. Objectives: We investigated relationships between bronchial epithelial remodeling, polymeric immunoglobulin receptor (pIgR) expression, secretory IgA (SIgA), airway inflammation, and mural remodeling in COPD. Methods: Lung tissue specimens and bronchoalveolar lavage were obtained from lifetime nonsmokers and former smokers with or without COPD. Epithelial structural changes were quantified by morphometric analysis. Expression of pIgR was determined by immunostaining and real-time polymerase chain reaction. Immunohistochemistry was performed for IgA, CD4 and CD8 lymphocytes, and cytomegalovirus and Epstein-Barr virus antigens. Total IgA and SIgA were measured by ELISA and IgA transcytosis was studied using cultured human bronchial epithelial cells. Measurements and Main Results: Areas of bronchial mucosa covered by normal pseudostratified ciliated epithelium were characterized by pIgR expression with SIgA present on the mucosal surface. In contrast, areas of bronchial epithelial remodeling had reduced pIgR expression, localized SIgA deficiency, and increased CD4+ and CD8+ lymphocyte infiltration. In small airways (<2 mm), these changes were associated with presence of herpesvirus antigens, airway wall remodeling, and airflow limitation in patients with COPD. Patients with COPD had reduced SIgA in bronchoalveolar lavage. Air–liquid interface epithelial cell cultures revealed that complete epithelial differentiation was required for normal pIgR expression and IgA transcytosis. Conclusions: Our findings indicate that epithelial structural abnormalities lead to localized SIgA deficiency in COPD airways. Impaired mucosal immunity may contribute to persistent airway inflammation and progressive airway remodeling in COPD. PMID:21512171

  3. Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV-2 Microglial Cells.

    Science.gov (United States)

    Lee, Jinyoung; Kang, Jung-Mi; Kim, Tae Im; Kim, Jong-Hyun; Sohn, Hae-Jin; Na, Byoung-Kuk; Shin, Ho-Joon

    2017-03-01

    Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV-2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV-2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL-1α and TNF-α. NfESP-induced IL-1α and TNF-α expression in BV-2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP-induced IL-1α and TNF-α production in BV-2 cells were effectively downregulated by inhibition of NF-kB and AP-1. These results collectively suggest that NfESP stimulates BV-2 cells to release IL-1α and TNF-α via NF-kB- and AP-1-dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

  4. Redox-Dependent Calcium-Mediated Signaling Networks that Control the Senescence-Associated Secretory Phenotype

    Science.gov (United States)

    Chandrasekaran, Akshaya

    Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogenic potential. While senescent cells have lost the ability to divide, they remain metabolically active and adapt a deleterious senescence associated secretory phenotype (SASP) central to the progression of several age-associated disease pathologies. The SASP is mechanistically regulated by the pro-inflammatory cytokine interleukin-1 alpha (IL-1alpha) whose expression and activity is responsive to the senescence associated (SA) oxidant production and the accompanying disruption of calcium (Ca2+) homeostasis. Using primary IMR-90 human fetal lung fibroblasts as a model of replicative senescence, we explored the molecular underpinnings driving Ca2+ dysregulation in senescent cells. We establish that the redox-responsive Transient Receptor Potential TRPC6 channel is compromised due to desensitization owing to SA increases in steady state hydrogen peroxide (H2O2) production. SA dysregulation of Ca2+ is also accompanied by loss of response to H2O2-induced Ca2+ influx that can be rescued with catalase pre-treatments. Senescent cells are also insensitive to Ca2+ entry induced by hyperforin, a specific activator of TRPC6, that can be restored by catalase pre-treatments, further suggesting redox regulation of TRPC6 in senescence. Inhibition of TRPC6 channel activity restores the ability of senescent cells to respond to peroxide-induced Ca2+ in addition to suppressing SASP gene expression. Furthermore, mammalian target of rapamycin (mTOR) signaling regulates SASP by means of modulating TRPC6 channel expression. Together, our findings provide compelling evidence that redox and mTOR-mediated regulation of TRPC6 channel modulate SASP gene expression. Further, the gain-of-function mutation of TRPC6 has pathological implications in several chronic pathologies and renders it a viable target in age-associated diseases.

  5. Stage- and gender-specific proteomic analysis of Brugia malayi excretory-secretory products.

    Directory of Open Access Journals (Sweden)

    Yovany Moreno

    Full Text Available INTRODUCTION: While we lack a complete understanding of the molecular mechanisms by which parasites establish and achieve protection from host immune responses, it is accepted that many of these processes are mediated by products, primarily proteins, released from the parasite. Parasitic nematodes occur in different life stages and anatomical compartments within the host. Little is known about the composition and variability of products released at different developmental stages and their contribution to parasite survival and progression of the infection. METHODOLOGY/PRINCIPAL FINDINGS: To gain a deeper understanding on these aspects, we collected and analyzed through 1D-SDS PAGE and LC-MS/MS the Excretory-Secretory Products (ESP of adult female, adult male and microfilariae of the filarial nematode Brugia malayi, one of the etiological agents of human lymphatic filariasis. This proteomic analysis led to the identification of 228 proteins. The list includes 76 proteins with unknown function as well as also proteins with potential immunoregulatory properties, such as protease inhibitors, cytokine homologues and carbohydrate-binding proteins. Larval and adult ESP differed in composition. Only 32 proteins were shared between all three stages/genders. Consistent with this observation, different gene ontology profiles were associated with the different ESP. CONCLUSIONS/SIGNIFICANCE: A comparative analysis of the proteins released in vitro by different forms of a parasitic nematode dwelling in the same host is presented. The catalog of secreted proteins reflects different stage- and gender-specific related processes and different strategies of immune evasion, providing valuable insights on the contribution of each form of the parasite for establishing the host-parasite interaction.

  6. Secretory phospholipase A2-IIa upregulates HER/HER2-elicited signaling in lung cancer cells

    Science.gov (United States)

    DONG, ZHONGYUN; MELLER, JAROSLAW; SUCCOP, PAUL; WANG, JIANG; WIKENHEISER-BROKAMP, KATHRYN; STARNES, SANDRA; LU, SHAN

    2014-01-01

    Lung cancer is the leading cause of cancer death worldwide. There is an urgent need for early diagnostic tools and novel therapies in order to increase lung cancer survival. Secretory phospholipase A2 group IIa (sPLA2-IIa) is involved in inflammation, tumorigenesis and metastasis. We were the first to uncover that cancer cells secrete sPLA2-IIa. sPLA2-IIa is overexpressed in almost all specimens of human lung cancers examined and is significantly elevated in the plasma of lung cancer patients. High levels of plasma sPLA2-IIa are significantly associated with advanced stage and decreased overall cancer survival. In this study, we further showed that elevated HER/HER2-PI3K-Akt-NF-κB signaling contributes to sPLA2-IIa overexpression in lung cancer cells. sPLA2-IIa in turn phosphorylates and activates HER2 and HER3 in a time- and dose-dependent manner in lung cancer cells. The structure and sequence-based docking analysis revealed that sPLA2-IIa β hairpin shares structural similarity with the corresponding EGF hairpin. sPLA2-IIa forms an extensive interface with EGFR and brings the two lobes of EGFR into an active conformation. sPLA2-IIa also enhances the NF-κB promoter activity. Anti-sPLA2-IIa antibody, but not the small molecule sPLA2-IIa inhibitor LY315920, significantly inhibits sPLA2-IIa-induced activation of NF-κB promoter. Our findings support the notion that sPLA2-IIa functions as a ligand for the EGFR family of receptors leading to an elevated HER/HER2-elicited signaling. Plasma sPLA2-IIa can potentially serve as lung cancer biomarker and sPLA2-IIa is a potential therapeutic target against lung cancer. PMID:24913497

  7. Eicosanoid and cytokine production by human peritoneal macrophages : characterization and modulation

    NARCIS (Netherlands)

    W.M. Pruimboom (Wanda)

    1995-01-01

    textabstractThe human body has an extensive and specialized cellular network to defend against invading microorganisms, and to prevent the development and spread of neoplastic disease. The mononuclear phagocytic cells are important secretory cells that, through their receptors and secretory

  8. Comparative Assay of Glutathione S- Transferase (GSTs Activity of Excretory-Secretory Materials and Somatic Extract of Fasciola spp Parasites

    Directory of Open Access Journals (Sweden)

    Taghi Golmohamadi

    2010-11-01

    Full Text Available Fascioliasis is a worldwide parasitic disease in human and domestic animals. The causative agents of fascioliasis are Fasciola hepatica and Fasciola gigantica. In the recent years, fasciola resistance to drugs has been reported in the many of publications. Fasciola spp has detoxification system including GST enzyme which may be responsible for its resistance. Therefore , the aim of the study was to assay of GST enzyme activity in fasciola parasites. Fasciola gigantica and Fasciola hepatica helminths were collected from abattoir as a live and cultured in buffer media for 4 h at 37 °C. Excretory-Secretory products were collected and stored in -80◦C. F. gigantica and Fasciola hepatica were homogenized with homogenizing buffer in a glass homogenizer to prepare of somatic extract. Suspension was then centrifuged and supernatant was stored at -80°C. In order to assay the enzyme activity, excretory-secretory and somatic extracts in the form of cocktails (potassium phosphate buffer, reduced glutathione and 1-chloro-2,4-dinitrobenzene substrates were prepared and their absorbance recorded for 5 minutes at 340 nm. The total and specific GST activity of F. gigantica somatic and ES products were obtained as 2916.00, 272.01 micromole/minute and 1.33, 1.70 micromole/minute/mg protein, respectively. Fasciola hepatica also showed 2705.00, 276.86 micromole/minute and 1.33, 1.52 micromole/minute/mg protein, respectively. These results are important for analysis of parasite survival / resistance to drugs which use for treatment of fascioliasis.

  9. RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses

    Science.gov (United States)

    Panda, Debasis; Das, Anshuman; Dinh, Phat X.; Subramaniam, Sakthivel; Nayak, Debasis; Barrows, Nicholas J.; Pearson, James L.; Thompson, Jesse; Kelly, David L.; Ladunga, Istvan; Pattnaik, Asit K.

    2011-01-01

    Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genome-wide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of VSV infection, such as entry/uncoating, gene expression, and assembly/release, were identified. Depletion of the proteins of the coatomer complex I or its upstream effectors ARF1 or GBF1 led to detection of reduced levels of VSV RNA. Coatomer complex I was also required for infection of lymphocytic choriomeningitis virus and human parainfluenza virus type 3. These results highlight the evolutionarily conserved requirements for gene expression of diverse families of NS RNA viruses and demonstrate the involvement of host cell secretory pathway in the process. PMID:22065774

  10. File list: His.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

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  18. Localization of the noradrenaline transporter in rat adrenal medulla and PC12 cells: evidence for its association with secretory granules in PC12 cells.

    Science.gov (United States)

    Kippenberger, A G; Palmer, D J; Comer, A M; Lipski, J; Burton, L D; Christie, D L

    1999-09-01

    The noradrenaline transporter (NAT) is present in noradrenergic neurons and a few other specialized cells such as adrenal medullary chromaffin cells and the rat pheochromocytoma (PC12) cell line. We have raised antibodies to a 49-residue segment (NATM2) of the extracellular region (residues 184-232) of bovine NAT. Affinity-purified NATM2 antibodies specifically recognized an 80-kDa band in PC12 cell membranes by western blotting. Bands of a similar size were also detected in membranes from human neuroblastoma (SK-N-SH) cells expressing endogenous NAT and human embryonic kidney (HEK293) cells stably expressing bovine NAT. Immunocytochemistry of rat adrenal tissue showed that NAT staining was colocalized with tyrosine hydroxylase in medullary chromaffin cells. Most NAT immunoreactivity in rat adrenal chromaffin and PC12 cells was present in the cytoplasm and had a punctate appearance. Cell surface biotinylation experiments in PC12 cells confirmed that only a minor fraction of the NAT was present at the cell surface. Subcellular fractionation of PC12 cells showed that relatively little NAT colocalized with plasma membrane, synaptic-like microvesicles, recycling endosomes, or trans-Golgi vesicles. Most of the NAT was associated with [3H]noradrenaline-containing secretory granules. Following nerve growth factor treatment, NAT was localized to the growing tip of neurites. This distribution was similar to the secretory granule marker secretogranin I. We conclude that the majority of NAT is present intracellularly in secretory granules and suggest that NAT may undergo regulated trafficking in PC12 cells.

  19. Golgi apparatus: finally mechanics comes to play in the secretory pathway.

    Science.gov (United States)

    Egea, Gustavo; Serra-Peinado, Carla

    2014-08-18

    New findings report a mechanical role for actin in Golgi organization and vesicular trafficking. An elegant study uses optical tweezers and live-cell imaging to demonstrate the effects of a mechanical constraint on the dynamics of secretory membrane trafficking, combining physical experimental approaches with in cellulo studies of endomembranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Identification of the sorting signal motif within pro-opiomelanocortin for the regulated secretory pathway

    DEFF Research Database (Denmark)

    Cool, D R; Fenger, M; Snell, C R

    1995-01-01

    amino acid residues (Asp10-Leu11-Glu14-Leu1). Thus the sorting signal for POMC to the regulated secretory pathway appears to be encoded by a specific conformational motif comprised of a 13-amino acid amphipathic loop structure stabilized by a disulfide bridge, located at the NH2 terminus of the molecule....

  1. Morphological, histochemical and immunohistochemical characterization of secretory production of the ciliary glands in the porcine eyelid

    Directory of Open Access Journals (Sweden)

    T Yasui

    2009-06-01

    Full Text Available In addition to performing general histology and cytology of the ciliary glands of the miniature pig, we studied the localization of glycoconjugates and b-defensins in these glands with the use of carbohydrate histochemical and immunohistochemical methods. The secretory cells of the glands were equipped with non-homogeneous secretory granules, a welldeveloped Golgi apparatus and rough endoplasmic reticulum. The secretory epithelium and luminal secretion of the glands contained large amounts of neutral and acidic glycoconjugates with various saccharide residues (a-L-Fuc, b-DGal, a-D-GalNAc and sialic acid. The sebaceous glands and tarsal glands also exhibited positive reactions to most of the histochemical methods. Additionally, the antimicrobial peptide group of b-defensins was demonstrated to be products of the ciliary glands, as well as the sebaceous glands and tarsal glands. The results obtained are discussed with regard to the specific function of the ciliary glandular secretions. These secretory products may be related to the moistening and general protection of the skin surface of the eyelid and ocular surface.

  2. Disparate effects of p24alpha and p24delta on secretory protein transport and processing.

    Directory of Open Access Journals (Sweden)

    Jeroen R P M Strating

    Full Text Available BACKGROUND: The p24 family is thought to be somehow involved in endoplasmic reticulum (ER-to-Golgi protein transport. A subset of the p24 proteins (p24alpha(3, -beta(1, -gamma(3 and -delta(2 is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC. METHODOLOGY/PRINCIPAL FINDINGS: Here we find that transgene expression of p24alpha(3 or p24delta(2 specifically in the Xenopus melanotrope cells in both cases causes an effective displacement of the endogenous p24 proteins, resulting in severely distorted p24 systems and disparate melanotrope cell phenotypes. Transgene expression of p24alpha(3 greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24delta(2-transgenesis does not influence the overall ultrastructure of the cells nor POMC transport and cleavage, but affects the Golgi-based processes of POMC glycomaturation and sulfation. CONCLUSIONS/SIGNIFICANCE: Transgenic expression of two distinct p24 family members has disparate effects on secretory pathway functioning, illustrating the specificity and non-redundancy of our transgenic approach. We conclude that members of the p24 family furnish subcompartments of the secretory pathway with specific sets of machinery cargo to provide the proper microenvironments for efficient and correct secretory protein transport and processing.

  3. Polyamines are present in mast cell secretory granules and are important for granule homeostasis.

    Science.gov (United States)

    García-Faroldi, Gianni; Rodríguez, Carlos E; Urdiales, José L; Pérez-Pomares, José M; Dávila, José C; Pejler, Gunnar; Sánchez-Jiménez, Francisca; Fajardo, Ignacio

    2010-11-30

    Mast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG) present within the granules. Polyamines (putrescine, spermidine and spermine) are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be components of mast cell secretory granules. Spermidine was released by mouse bone marrow derived mast cells (BMMCs) after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with α-difluoromethylornithine (DFMO) caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased β-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system. Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles.

  4. Polyamines are present in mast cell secretory granules and are important for granule homeostasis.

    Directory of Open Access Journals (Sweden)

    Gianni García-Faroldi

    2010-11-01

    Full Text Available Mast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG present within the granules. Polyamines (putrescine, spermidine and spermine are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be components of mast cell secretory granules.Spermidine was released by mouse bone marrow derived mast cells (BMMCs after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with α-difluoromethylornithine (DFMO caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased β-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system.Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles.

  5. Resin secretory structures of Boswellia papyrifera and implications for frankincense yield.

    Science.gov (United States)

    Tolera, Motuma; Menger, David; Sass-Klaassen, Ute; Sterck, Frank J; Copini, Paul; Bongers, Frans

    2013-01-01

    Frankincense, a gum-resin, has been tapped from Boswellia papyrifera trees for centuries. Despite the intensive tapping and economic interest of B. papyrifera, information on the resin secretory structures, which are responsible for synthesis, storage and transport of frankincense, is virtually absent. This study describes the type, architecture and distribution of resin secretory structures of B. papyrifera and its relevance for the ecophysiology and economic use of the tree. The type and architecture of resin secretory structures present in bark and wood was investigated from transversal, tangential and radial sections of bark and wood samples. The diameter and density (number of resin canals mm(-2)) of axial resin canals were determined from digital images of thin sections across the different zones of inner bark. Resin canals form a three-dimensional network within the inner bark. Yet, the intact resin-conducting and producing network is on average limited to the inner 6·6 mm of the inner bark. Within the inner bark, the density of non-lignified axial resin canals decreases and the density of lignified resin canals increases from the vascular cambium towards the outer bark. In the wood, only radial resin canals were encountered. Frankincense tapping techniques can be improved based on knowledge of bark anatomy and distribution and architecture of resin secretory structures. The suggested new techniques will contribute to a more sustainable frankincense production that enhances the contribution of frankincense to rural livelihoods and the national economy.

  6. Resin secretory structures of Boswellia papyrifera and implications for frankincense yield

    NARCIS (Netherlands)

    Tolera, M.; Menger, D.; Sass, U.G.W.; Sterck, F.J.; Copini, P.; Bongers, F.

    2013-01-01

    Frankincense, a gum-resin, has been tapped from Boswellia papyrifera trees for centuries. Despite the intensive tapping and economic interest of B. papyrifera, information on the resin secretory structures, which are responsible for synthesis, storage and transport of frankincense, is virtually

  7. Immunomodulatory effects of excretory/secretory compounds from Contracaecum osculatum larvae in a zebrafish inflammation model

    DEFF Research Database (Denmark)

    Mehrdana, Foojan; Kania, Per Walter; Nazemi, Sasan

    2017-01-01

    Excretory/secretory (ES) compounds isolated from third-stage larvae of the anisakid nematode Contracaecum osculatum parasitizing liver of Baltic cod were investigated for effects on immune gene expression in a zebrafish LPS-induced inflammation model. ES products containing a series of proteins...

  8. Secretory phospholipase A2 potentiates glutamate-induced rat striatal neuronal cell death in vivo

    DEFF Research Database (Denmark)

    Kolko, M; Bruhn, T; Christensen, Thomas

    1999-01-01

    The secretory phospholipases A2 (sPLA2) OS2 (10, 20 and 50 pmol) or OS1, (50 pmol) purified from taipan snake Oxyuranus scutellatus scutellatus venom, and the excitatory amino acid glutamate (Glu) (2.5 and 5.0 micromol) were injected into the right striatum of male Wistar rats. Injection of 10 an...

  9. Synaptotagmin 1 Negatively Controls the Two Distinct Immune Secretory Pathways to Powdery Mildew Fungi in Arabidopsis.

    Science.gov (United States)

    Kim, Hyeran; Kwon, Hyeokjin; Kim, Soohong; Kim, Mi Kyung; Botella, Miguel A; Yun, Hye Sup; Kwon, Chian

    2016-06-01

    PEN1, one of the plasma membrane (PM) syntaxins, comprises an immune exocytic pathway by forming the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex with SNAP33 and VAMP721/722 in plants. Although this secretory pathway is also involved in plant growth and development, how plants control their exocytic activity is as yet poorly understood. Since constitutive PEN1 cycling between the PM and endocytosed vesicles is critical for its immune activity, we studied here the relationship of PEN1 to synaptotagmin 1 (SYT1) that is known to regulate endocytosis at the PM. Interestingly, syt1 plants showed enhanced disease resistance to the Arabidopsis-adapted Golovinomyces orontii fungus, and elevated protein but not transcript levels of PEN1 Calcium-dependent promotion of PEN1-SYT1 interaction suggests that SYT1 controls defense activities of the PEN1-associated secretory pathway by post-translationally modulating PEN1. Increased PEN1-SYT1 interaction and inhibited PEN1 SNARE complex induction by G. orontii additionally suggest that the adaption of phytopathogens to host plants might partly result from effective suppression of the PEN1-related secretory pathway. Further genetic analyses revealed that SYT1 also regulates the atypical peroxisomal myrosinase PEN2-associated secretory pathway. © 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.

  10. A highway for war and peace: the secretory pathway in plant-microbe interactions.

    Science.gov (United States)

    Wang, Dong; Dong, Xinnian

    2011-07-01

    Secretion of proteins and other molecules is the primary means by which a cell interacts with its surroundings. The overall organization of the secretory system is remarkably conserved among eukaryotes, and many of the components have been investigated in detail in animal models. Plant cells, because of their sessile lifestyle, are uniquely reliant on the secretory pathway to respond to changes in their environments, either abiotic, such as the absence of nutrients, or biotic, such as the presence of predators or pathogens. In particular, most plant pathogens are extracellular, which demands a robust and efficient host secretory system directed at the site of attack. Here, we present a summary of recent advances in our understanding of the molecular details of the secretory pathway during plant-microbe interactions. Secretion is required not only for the delivery of antimicrobial molecules, but also for the biogenesis of cell surface sensors to detect microbes. The deposition of extracellular material is important in the defense against classical bacterial pathogens as well as in the so-called 'non-host' resistance. Finally, boosting the protein secretion capacity is vital for avoiding infection as well as for achieving symbiosis, even though in the latter case, the microbes are engulfed in intracellular compartments. The emerging evidence indicates that secretion provides an essential interface between plant hosts and their associated microbial partners.

  11. Anti-diarrheal, anti-secretory, anti-spasmodic and antiulcer activities of

    African Journals Online (AJOL)

    In isolated tissue (rabbit jejunum), Am.Cr concentration-dependently (0.01 - 3.0 mg/mL) produced relaxation of K+ (80 mM)-induced and spontaneous contractions at ... anti-secretory, antispasmodic and anti-ulcer activities, probably mediated through dual mechanisms, including Ca2+ influx and PDE enzyme(s) inhibition.

  12. Secretory Phospholipase A2-IIA and Cardiovascular Disease: A Mendelian Randomization Study

    NARCIS (Netherlands)

    Holmes, Michael V.; Simon, Tabassome; Exeter, Holly J.; Folkersen, Lasse; Asselbergs, Folkert W.; Guardiola, Montse; Cooper, Jackie A.; Palmen, Jutta; Hubacek, Jaroslav A.; Carruthers, Kathryn F.; Horne, Benjamin D.; Brunisholz, Kimberly D.; Mega, Jessica L.; van Iperen, Erik P. A.; Li, Mingyao; Leusink, Maarten; Trompet, Stella; Verschuren, Jeffrey J. W.; Hovingh, G. Kees; Dehghan, Abbas; Nelson, Christopher P.; Kotti, Salma; Danchin, Nicolas; Scholz, Markus; Haase, Christiane L.; Rothenbacher, Dietrich; Swerdlow, Daniel I.; Kuchenbaecker, Karoline B.; Staines-Urias, Eleonora; Goel, Anuj; van 't Hooft, Ferdinand; Gertow, Karl; de Faire, Ulf; Panayiotou, Andrie G.; Tremoli, Elena; Baldassarre, Damiano; Veglia, Fabrizio; Holdt, Lesca M.; Beutner, Frank; Gansevoort, Ron T.; Navis, Gerjan J.; Mateo Leach, Irene; Breitling, Lutz P.; Brenner, Hermann; Thiery, Joachim; Dallmeier, Dhayana; Franco-Cereceda, Anders; Boer, Jolanda M. A.; Stephens, Jeffrey W.; Hofker, Marten H.; Tedgui, Alain; Hofman, Albert; Uitterlinden, André G.; Adamkova, Vera; Pitha, Jan; Onland-Moret, N. Charlotte; Cramer, Maarten J.; Nathoe, Hendrik M.; Spiering, Wilko; Klungel, Olaf H.; Kumari, Meena; Whincup, Peter H.; Morrow, David A.; Braund, Peter S.; Hall, Alistair S.; Olsson, Anders G.; Doevendans, Pieter A.; Trip, Mieke D.; Tobin, Martin D.; Hamsten, Anders; Watkins, Hugh; Koenig, Wolfgang; Nicolaides, Andrew N.; Teupser, Daniel; Day, Ian N. M.; Carlquist, John F.; Gaunt, Tom R.; Ford, Ian; Sattar, Naveed; Tsimikas, Sotirios; Schwartz, Gregory G.; Lawlor, Debbie A.; Morris, Richard W.; Sandhu, Manjinder S.; Poledne, Rudolf; Maitland-van der Zee, Anke H.; Khaw, Kay-Tee; Keating, Brendan J.; van der Harst, Pim; Price, Jackie F.; Mehta, Shamir R.; Yusuf, Salim; Witteman, Jaqueline C. M.; Franco, Oscar H.; Jukema, J. Wouter; de Knijff, Peter; Tybjaerg-Hansen, Anne; Rader, Daniel J.; Farrall, Martin; Samani, Nilesh J.; Kivimaki, Mika; Fox, Keith A. A.; Humphries, Steve E.; Anderson, Jeffrey L.; Boekholdt, S. Matthijs; Palmer, Tom M.; Eriksson, Per; Paré, Guillaume; Hingorani, Aroon D.; Sabatine, Marc S.; Mallat, Ziad; Casas, Juan P.; Talmud, Philippa J.

    2013-01-01

    This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease. Higher circulating levels of sPLA2-IIA mass or sPLA2 enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clear if this association is

  13. Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

    Directory of Open Access Journals (Sweden)

    Srijana Upadhyay

    2016-11-01

    Full Text Available Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs and nonribosomal peptide synthetases (NRPSs in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism.

  14. Molecular characterization and expression of two putative protective excretory secretory proteins of Haemonchus contortus

    NARCIS (Netherlands)

    Schallig, H. D.; van Leeuwen, M. A.; Verstrepen, B. E.; Cornelissen, A. W.

    1997-01-01

    It has been shown that vaccination with two low molecular mass excretory secretory (ES) antigens of 15 and 24 kDa, respectively, afforded a substantial degree of protection against Haemonchus contortus to sheep. In vitro cultivation of the parasite usually yields a limited amount of these proteins

  15. The secretory endometrial protein, placental protein 14, in women with ectopic gestation

    DEFF Research Database (Denmark)

    Ruge, S; Sørensen, Steen; Vejtorp, M

    1992-01-01

    OBJECTIVE: To determine the serum level of the secretory endometrial protein, placental protein 14 (PP14) and progesterone (P) in women with ectopic gestation. DESIGN: Blood samples were collected prospectively and preoperatively. Reference range was determined from a prospective population of 98...

  16. Anti-diarrheal, anti-secretory, anti-spasmodic and anti- ulcer ...

    African Journals Online (AJOL)

    ethanol-induced gastric ulceration in rats. In acute toxicity testing Am.Cr did not produce any mortality up to 5 g/kg dose. Conclusion: These results show that Acacia modesta possesses anti-diarrheal, anti-secretory, anti- spasmodic and anti-ulcer activities, probably mediated through dual mechanisms, including Ca2+ influx.

  17. Anti-ulcer and gastric anti-secretory activities of seed extract of ...

    African Journals Online (AJOL)

    Ethanol extract of Buchholzia coriacea seed was evaluated for anti-ulcer as well as anti-secretory activity in rats because of its use in Nigerian folk medicine as an anti-ulcer agent. Standard pharmacological methods were used to carry out phytochemical analysis of the plant. Quantitative phytochemical analysis of the ...

  18. Secretory phospholipase A(2) induces delayed neuronal COX-2 expression compared with glutamate

    DEFF Research Database (Denmark)

    Kolko, Miriam; Nielsen, Marianne; Bazan, Nicolas G

    2002-01-01

    Agonists of the binding site for secretory phospholipase A(2) (sPLA(2)) potentiate glutamate-induced neuronal cell death in primary cell cultures and in vivo (Kolko et al. [1996] J. Biol. Chem. 271:32722; Kolko et al. [1999] Neurosci. Lett. 274:167]. Here, we tested the hypothesis that COX-2...

  19. Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger

    NARCIS (Netherlands)

    Ngiam, C.; Jeenes, D.J.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Archer, D.B.

    2000-01-01

    Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it

  20. Differential testosterone secretory capacity of the testes of aggressive and nonaggressive house mice during ontogeny

    NARCIS (Netherlands)

    de Ruiter, Anne J H; Koolhaas, Jaap M; van Oortmerssen, Geert A; Bohus, Bela

    1992-01-01

    In this study, testosterone secretory capacity of testicular Leydig cells during ontogeny was determined in males of an aggressive and a nonaggressive genetic selection line of wild house mice. Neonates, 23-day-old prepubertals, and adult male mice were studied. A morphometric method was used to

  1. THE ROLE OF SECRETORY GRANULES IN RADIATION-INDUCED DYSFUNCTION OF RAT SALIVARY-GLANDS

    NARCIS (Netherlands)

    PETER, B; VANWAARDE, MAWH; VISSINK, A; SGRAVENMADE, EJ; KONINGS, AWT

    To investigate the possible role of secretory granules in radiation-induced salivary gland dysfunction, rats were pretreated with isoproterenol (5 mg/kg intraperitoneally) to degranulate salivary gland acini, At maximal depletion, salivary glands were locally irradiated with a single dose of 15 Gy

  2. ETV6 rearrangement in a case of mammary analogue secretory carcinoma of the skin.

    Science.gov (United States)

    Chang, Michael D; Arthur, Allison K; García, Joaquín J; Sukov, William R; Shon, Wonwoo

    2016-11-01

    Mammary analog secretory carcinoma of salivary glands is a relatively recently recognized entity that harbors the ETV6-NTRK3 fusion transcript. To date, only rare cases of mammary analog secretory carcinoma of the skin have been reported. A 57-year-old man presented with a 6.0 cm cystic mass in the axilla, involving the dermis and superficial subcutis. Microscopically, the tumor exhibited nodular aggregation of tubular and microcystic structures embedded in the dense fibrotic and hyalinized stroma. Characteristic 'colloid-like' eosinophilic secretory material was present within intraluminal spaces. Tumor cells were largely characterized by vesicular nuclei with inconspicuous nucleoli and pink vacuolated cytoplasm. With respect to immunohistochemistry, tumor cells were intensely positive for AE1/AE3, Cam 5.2, and CK7, whereas Ber-EP4 and CEA were completely negative. A dual color break-apart fluorescence in situ hybridization probe identified rearrangement of the ETV6 gene locus on chromosome 12. The patient is alive with no evidence of recurrent disease or metastasis 3 years after the initial surgery. In conclusion, we report a rare example of mammary analog secretory carcinoma of the skin with ETV6 rearrangement. Awareness of this unique cutaneous tumor and subsequent reporting of additional cases is necessary for better characterization of its completely clinicopathologic spectrum. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

    DEFF Research Database (Denmark)

    Klemm, Robin W; Ejsing, Christer S.; Surma, Michal A

    2009-01-01

    The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane ...

  4. PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance.

    Directory of Open Access Journals (Sweden)

    Birgitte Holst

    Full Text Available Secretory vesicles in endocrine cells store hormones such as growth hormone (GH and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs domain protein PICK1 (protein interacting with C kinase 1 as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of

  5. Effect of nicotine on exocytotic pancreatic secretory response: role of calcium signaling

    Directory of Open Access Journals (Sweden)

    Chowdhury Parimal

    2013-01-01

    Full Text Available Abstract Background Nicotine is a risk factor for pancreatitis resulting in loss of pancreatic enzyme secretion. The aim of this study was to evaluate the mechanisms of nicotine-induced secretory response measured in primary pancreatic acinar cells isolated from Male Sprague Dawley rats. The study examines the role of calcium signaling in the mechanism of the enhanced secretory response observed with nicotine exposure. Methods Isolated and purified pancreatic acinar cells were subjected to a nicotine exposure at a dose of 100 μM for 6 minutes and then stimulated with cholecystokinin (CCK for 30 min. The cell’s secretory response was measured by the percent of amylase released from the cells in the incubation medium Calcium receptor antagonists, inositol trisphosphate (IP3 receptor blockers, mitogen activated protein kinase inhibitors and specific nicotinic receptor antagonists were used to confirm the involvement of calcium in this process. Results Nicotine exposure induced enhanced secretory response in primary cells. These responses remained unaffected by mitogen activated protein kinases (MAPK’s inhibitors. The effects, however, have been completely abolished by nicotinic receptor antagonist, calcium channel receptor antagonists and inositol trisphosphate (IP3 receptor blockers. Conclusions The data suggest that calcium activated events regulating the exocytotic secretion are affected by nicotine as shown by enhanced functional response which is inhibited by specific antagonists… The results implicate the role of nicotine in the mobilization of both intra- and extracellular calcium in the regulation of stimulus-secretory response of enzyme secretion in this cell system. We conclude that nicotine plays an important role in promoting enhanced calcium levels inside the acinar cell.

  6. Tracheobronchial epithelium of the sheep: IV. Lectin histochemical characterization of secretory epithelial cells.

    Science.gov (United States)

    Mariassy, A T; Plopper, C G; St George, J A; Wilson, D W

    1988-09-01

    Conventional histochemical characterization of the mucus secretory apparatus is often difficult to reconcile with the biochemical analysis of respiratory secretions. This study was designed to examine the secretory glycoconjugates in airways using lectins with biochemically defined affinities for main sugar residues of mucus. We used five biotinylated lectins--DBA (Dolichos biflorus) and SBA (Glycine max) for N-acetyl galactosamine (galNAc), BSA I (Bandeiraea simplicifolia) and PNA (Arachis hypogea) for galactose (gal), and UEA I (Ulex europeus)--for detection of fucose (fuc) in HgCl2-fixed, paraffin-embedded, serially sectioned trachea, lobar and segmental bronchi and bronchioles of nine sheep. Lectins selectively localized the carbohydrate residues in luminal secretions, on epithelial cell surfaces, and in secretory cells. In proximal airways, the major carbohydrate residues in luminal secretions, cell surfaces, goblet cells, and glands were fuc and gal-NAc. PNA reacted mainly with apical granules of less than 10% of goblet cells, and gal residues were only detected in some of the mucous cells and on basolateral cell surfaces. Distal airways contained sparse secretion in the lumen, mucous cells contained weakly reactive fuc and gal-NAc, and the epithelial surfaces of Clara cells contained gal. Sugars abundant in the airway secretions were also the major component of cells in glands. We conclude that there is a correlation between specific sugar residues in secretory cells, glycocalyx, and luminal secretions in proximal and distal airways. This suggests that lectins may be used to obtain information about airway secretory cell composition from respiratory secretions.

  7. Excretory/secretory proteases and mechanical movement of Anisakis pegreffii infective larvae in the penetration of BALB/c mice gastrointestine

    Directory of Open Access Journals (Sweden)

    June-Der Lee

    2017-12-01

    Full Text Available Anisakiasis is a human parasitic disease caused by infection with the infective larvae of Anisakis. Accidental infection in humans causes the gastrointestinal pathophysiological effects of mechanical tissue damage by migrating larvae. The mechanism of the infective larval invasion and migration is suspected to involve larval excretory/secretory proteases and motility. This study demonstrates the penetration rate of the infective larvae of Anisakis pegreffii in mouse gastrointestine depends on the time after infection, and that only 15% of larvae remain in the gastrointestinal tract 3 h after infection. Strong activities of matrix metalloproteinases (MMPs and serine proteases, especially plasmin, were found in the excretory/secretory products of A. pegreffii; these can be inhibited by ONO-4817 and phenylmethylsulfonyl fluoride, respectively. The protease activity was also significantly decreased in another 1 h of cultivation of larvae in fresh 0.9% normal saline (NS after previous cultivation for 48 h in NS. The motility scores of larvae were significantly lower after 48 h of cultivation in NS. The penetration rate of A. pegreffii larvae in the gastrointestine of infected mice sequentially were 90% in the freshly prepared, 68% in serine protease inhibited, 55% in MMPs inhibited larvae, and 16% in larvae cultivated in NS for 48 h. Therefore, this study demonstrates that MMPs and serine proteases excreted and secreted by A. pegreffii and the mechanical movement of infective larvae participate in the penetration of the gastrointestine of mice after infection.

  8. [Recognition of excretory/secretory antigens of Anisakis type I and evolution of IgE in experimentally infected rats].

    Science.gov (United States)

    Gómez-Mateos, Magdalena; Valero-López, Adela; de la Rubia-Nieto, Teresa; Romero-López, María Del Carmen; Díaz-Sáez, Victoriano

    2014-10-01

    Anisakis spp., during parasitism, release excretory-secretory antigens that, in contact with the human immune system, can trigger a hypersensitivity response mediated by IgE, causing various allergic symptoms. To evaluate the IgE response in Wistar rats after infection with L3 larvae of the parasite Anisakis spp. Some determining factors involved in the technique have been improved in this work, such as: the concentration of polyacrylamide used in the preparation of the gels, the antigen concentration used, and the temperature required for denaturation of proteins. Immune responses (Ag-Ab) observed by the immunoblotting technique showed a greater intensity with serum obtained after reinfection, which have recognized proteins that may correspond to the major antigen Ani s 1 and other polypeptides of interest in the diagnosis of human anisakiasis. This paper concludes that immunoblotting is a useful technique to detect IgE antibodies against Anisakis proteins. Copyright © 2013 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  9. Regulation of basal resistance by a powdery mildew-induced cysteine-rich receptor-like protein kinase in barley

    DEFF Research Database (Denmark)

    Rayapuram, Cbgowda; Jensen, Michael K; Maiser, Fabian

    2012-01-01

    -gene-mediated resistance. Silencing of HvCRK1 phenocopied the effective penetration resistance found in mlo-resistant barley plants, and the possible link between HvCRK1 and MLO was substantiated by the fact that HvCRK1 induction on Bgh inoculation was dependent on Mlo. Finally, using both experimental and insilico...... approaches, we demonstrated that HvCRK1 localizes to the ER of barley cells. The negative effect on basal resistance against Bgh and the functional aspects of MLO- and ER-localized HvCRK1 signalling on Bgh inoculation are discussed....

  10. Convergent solid-phase and solution approaches in the synthesis of the cysteine-rich Mdm2 RING finger domain.

    Science.gov (United States)

    Vasileiou, Zoe; Barlos, Kostas; Gatos, Dimitrios

    2009-12-01

    The RING finger domain of the Mdm2, located at the C-terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48-residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid-phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C-terminus as the amino component. Best results were achieved using solution condensation where the N-component was applied with the C-terminal carboxyl group left unprotected. The developed method is well suited for large-scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid-phase and solution synthesis. (c) 2009 European Peptide Society and John Wiley & Sons, Ltd.

  11. Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins

    Directory of Open Access Journals (Sweden)

    Gordon W. Irvine

    2017-04-01

    Full Text Available Structural information regarding metallothioneins (MTs has been hard to come by due to its highly dynamic nature in the absence of metal-thiolate cluster formation and crystallization difficulties. Thus, typical spectroscopic methods for structural determination are limited in their usefulness when applied to MTs. Mass spectrometric methods have revolutionized our understanding of protein dynamics, structure, and folding. Recently, advances have been made in residue modification mass spectrometry in order to probe the hard-to-characterize structure of apo- and partially metalated MTs. By using different cysteine specific alkylation reagents, time dependent electrospray ionization mass spectrometry (ESI-MS, and step-wise “snapshot” ESI-MS, we are beginning to understand the dynamics of the conformers of apo-MT and related species. In this review we highlight recent papers that use these and similar techniques for structure elucidation and attempt to explain in a concise manner the data interpretations of these complex methods. We expect increasing resolution in our picture of the structural conformations of metal-free MTs as these techniques are more widely adopted and combined with other promising tools for structural elucidation.

  12. MASEP gamma knife radiosurgery for secretory pituitary adenomas: experience in 347 consecutive cases

    Directory of Open Access Journals (Sweden)

    Yuan Shubin

    2009-03-01

    Full Text Available Abstract Background Secretory pituitary adenomas are very common brain tumors. Historically, the treatment armamentarium for secretory pituitary adenomas included neurosurgery, medical management, and fractionated radiotherapy. In recent years, MASEP gamma knife radiosurgery (MASEP GKRS has emerged as an important treatment modality in the management of secretory pituitary adenomas. The goal of this research is to define accurately the efficacy, safety, complications, and role of MASEP GKRS for treatment of secretory pituitary adenomas. Methods Between 1997 and 2007 a total of 347 patients with secretory pituitary adenomas treated with MASEP GKRS and with at least 60 months of follow-up data were identified. In 47 of these patients some form of prior treatment such as transsphenoidal resection, or craniotomy and resection had been conducted. The others were deemed ineligible for microsurgery because of body health or private choice, and MASEP GKRS served as the primary treatment modality. Endocrinological, ophthalmological, and neuroradiological responses were evaluated. Results MASEP GKRS was tolerated well in these patients under the follow-up period ranged from 60 to 90 months; acute radioreaction was rare and 17 patients had transient headaches with no clinical significance. Late radioreaction was noted in 1 patient and consisted of consistent headache. Of the 68 patients with adrenocorticotropic hormone-secreting(ACTH adenomas, 89.7% showed tumor volume decrease or remain unchanged and 27.9% experienced normalization of hormone level. Of the 176 patients with prolactinomas, 23.3% had normalization of hormone level and 90.3% showed tumor volume decrease or remain unchanged. Of the 103 patients with growth hormone-secreting(GH adenomas, 95.1% experienced tumor volume decrease or remain unchanged and 36.9% showed normalization of hormone level. Conclusion MASEP GKRS is safe and effective in treating secretory pituitary adenomas. None of the

  13. Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation.

    Science.gov (United States)

    Upadhyay, Srijana; Xu, Xinping; Lin, Xiaorong

    2016-11-22

    Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered "atypical" secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism. Subcellular compartmentalization is increasingly recognized as an important aspect of fungal secondary metabolism. It facilitates sequential enzymatic reactions, provides

  14. Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca{sup 2+}-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B., E-mail: korn@mail.ibch.ru [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Dmitriev, Ruslan I.; Kostina, Maria B. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Korneenko, Tatyana V. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shakhparonov, Mikhail I. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Full-length secretory pathway Ca-ATPase (SPCA2) cloned from rat duodenum. Black-Right-Pointing-Pointer ATP2C2 gene (encoding SPCA2) exists only in genomes of Tetrapoda. Black-Right-Pointing-Pointer Rat and pig SPCA2 are expressed in intestines, lung and some secretory glands. Black-Right-Pointing-Pointer Subcellular localization of SPCA2 may depend on tissue type. Black-Right-Pointing-Pointer In rat duodenum, SPCA2 is localized in plasma membrane-associated compartments. -- Abstract: Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2

  15. Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of a excretory-secretory antigen-fructose-1,6-bisphosphatase.

    Science.gov (United States)

    Zheng, Minghui; Hu, Kunhua; Liu, Wei; Hu, Xuchu; Hu, Fengyu; Huang, Lisi; Wang, Peng; Hu, Yue; Huang, Yan; Li, Wenfang; Liang, Chi; Yin, Xingfeng; He, Qingyu; Yu, Xinbing

    2011-09-01

    Clonorchis sinensis is a food-borne zoonotic parasite that resides in bile ducts and causes clonorchiasis, which may result in cholelithiasis, cholecystitis, hepatic fibrosis, and liver tumors. Although total excretory secretory products (ESP) of C. sinensis adults induce hepatic fibrosis in vivo in rats, the causative mechanism is not well understood. To study components of the ESP, C. sinensis culture medium was collected and analyzed using shotgun LC-MS/MS. We identified a total of 110 proteins, including glycometabolic enzymes (such as fructose-1,6-bisphosphatase (FBPase) and enolase), detoxification enzymes (such as glutamate dehydrogenase, dihydrolipoamide dehydrogenase and cathepsin B endopeptidase), and a number of RAB family proteins. To identify a potential causative agent for hepatic fibrosis, we expressed and purified a recombinant FBPase, a 1,041-bp gene product that encodes a 41.7-kDa protein with prototypical FBPase domains and that can form a tetramer with a molecular mass of 166.8 kDa. In addition, we found that FBPase is an antigen present in the ESP and in circulation. Immunofluorescence showed that FBPase localizes to the intestinal cecum and vitellarium in C. sinensis adults. Our results describe the components of the excretory secretory products from C. sinensis adult worms and suggest that FBPase may be an important antigen present in the ESP of C. sinensis and may lay the foundation for additional studies on the development of clonorchiasis-associated hepatic fibrosis.

  16. Comparison of Excretory-Secretory and Somatic Antigens of Ornithobilharzia turkestanicum in Agar Gel Diffusion Test

    Directory of Open Access Journals (Sweden)

    H Miranzadeh

    2008-12-01

    Full Text Available Background: Ornithobilharziosis as one of the parasitic infections may give rise to serious economic problems in animal husbandry. The Aim of the study was to prepare and compare the somatic and excretory-secretory (ES antigens of O. tur­kestanicum in gel diffusion test. Methods: Excretory-secretory (ES and somatic antigens of Ornithobilharzia turkestanicum were prepared from collected worms from mesentric blood vessels of infected sheep. The laboratory bred rabbits were immunized with antigens and then antisera were prepared. The reaction of antigens and antisera was observed in gel diffusion test. Results: ES antigens of this species showed positive reaction with antisera raised against ES and also somatic antigens. Somatic antigens also showed positive reaction with antisera raised against somatic and also ES antigens. Conclusion: The antigenicity of O. turkestanicum ES and somatic antigens is the same in gel diffusion test.

  17. Planar cell polarity protein localization in the secretory ameloblasts of rat incisors.

    Science.gov (United States)

    Nishikawa, Sumio; Kawamoto, Tadafumi

    2012-05-01

    The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes' processes of inner enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin and β-catenin were localized. frizzled-3 was also localized in differentiating inner enamel epithelial cells. Vang11 was localized sparsely in differentiating preameloblasts and extensively at the cell boundary of stratum intermedium. Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively. These results suggest the involvement of planar cell polarity proteins in odontogenesis.

  18. Large lipid-rich mammary analogue secretory carcinoma of parotid gland: An unusual case

    Directory of Open Access Journals (Sweden)

    Prashant Joshi

    2015-01-01

    Full Text Available Mammary analogue secretory carcinoma (MASC of the salivary gland is a malignant tumor which bears morphologic, immunohistochemical and molecular features similar to those of mammary secretory carcinoma. The tumor is considered as a low-grade malignancy perhaps slightly more aggressive than acinic cell carcinoma. High-grade transformation with recurrences, regional nodal involvement, metastases, and cancer-related death has been reported in a few cases. We report an unusual case of large MASC of the parotid gland in a young patient without regional lymph node involvement. To the best of our knowledge till date such a large MASC of the salivary gland has not been reported in the English literature.

  19. Mammary Analogue Secretory Carcinoma of Parotid Gland in a Teenage Boy.

    Science.gov (United States)

    Mahmood, Humera; Fatima, Hadia; Faheem, Mohammad

    2017-09-01

    Mammary analogue secretory carcinoma (MASC) is a rare salivary gland malignancy that exhibits resemblance with secretory carcinoma of the breast (SC) due to the presence of ETV6-NTRK3 gene fusion, formerly classified as acinic cell carcinoma. It is a slowly growing painless tumor that exhibits wide range of clinical behavior. This tumor typically affects middle aged people. Less than 100 such cases have been reported in the literature so far with only 8 case reports of patients less than 19 years of age. Very little is known about the clinical management of such cases. We hereby report a case of 15-year boy who had been managed for MASC of left parotid gland with exision.

  20. Cytopathological features of mammary analogue secretory carcinoma--review of literature.

    Science.gov (United States)

    Takeda, Maiko; Kasai, Takahiko; Morita, Kohei; Takeuchi, Mao; Nishikawa, Takeshi; Yamashita, Akinori; Mikami, Shinji; Hosoi, Hiroshi; Ohbayashi, Chiho

    2015-02-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor that morphologically resembles mammary secretory carcinoma and carries the identical ETV6-NTRK3 fusion gene. We report a surgical resected case of MASC in the parotid gland of a 41-year-old man. The cytological smears of a preoperative fine-needle aspiration showed many sheets and crowded clusters of monotonous epithelioid cells with mild atypia, suggestive of monomorphic tumor. Histologically, the tumor was composed of cuboidal cells with follicular, tubular, and solid structures, reminiscent of acinic cell carcinoma of follicular variant, which had been previously classified. This case had ETV6-NTRK3 fusion gene transcript confirmed by fluorescence in situ hybridization and reverse transcription polymerase chain reaction. In the cytological and histopathological diagnosis of monomorphic tumor of salivary gland, MASC needs to be taken into consideration as a differential diagnosis. Further immunohistochemical and gene analyses are needed in diagnosis of MASC. © 2014 Wiley Periodicals, Inc.

  1. Mammary analogue secretory carcinoma of the salivary glands: a diagnostic dilemma.

    Science.gov (United States)

    Hindocha, N; Wilson, M H; Pring, M; Hughes, C W; Thomas, S J

    2017-04-01

    Mammary analogue secretory carcinoma (MASC) is a recently identified salivary gland neoplasm that can mimic other salivary gland tumours such as acinic cell carcinoma and cystadenocarcinoma. It is distinguished from these by differences in immunohistochemical profile and the identification of an ETV6-NTRK3 translocation (12;15)(p13;q25), which is also found in secretory carcinomas of the breast. Previous publications have suggested that MASC tumours have similar biological behaviour to acinic cell carcinoma. We report two cases of MASC that affected the upper lip, and showed an infiltrative and locally aggressive growth pattern that required several operations to ensure clearance of microscopic tumour cells. Copyright © 2016 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  2. Arginine-specific gingipains from Porphyromonas gingivalis deprive protective functions of secretory leucocyte protease inhibitor in periodontal tissue

    National Research Council Canada - National Science Library

    Into, T; Inomata, M; Kanno, Y; Matsuyama, T; Machigashira, M; Izumi, Y; Imamura, T; Nakashima, M; Noguchi, T; Matsushita, K

    2006-01-01

    .... In this study, we found that the expression of secretory leucocyte protease inhibitor (SLPI), an endogenous inhibitor for neutrophil-derived proteases, was reduced in gingival tissues with chronic periodontitis associated with P...

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

    Science.gov (United States)

    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.

  4. An Alternative Terminal Step of the General Secretory Pathway in Staphylococcus aureus.

    Science.gov (United States)

    Craney, Arryn; Dix, Melissa M; Adhikary, Ramkrishna; Cravatt, Benjamin F; Romesberg, Floyd E

    2015-08-18

    Type I signal peptidase (SPase) is essential for viability in wild-type bacteria because the terminal step of the bacterial general secretory pathway requires its proteolytic activity to release proteins from their membrane-bound N-terminal leader sequences after translocation across the cytoplasmic membrane. Here, we identify the Staphylococcus aureus operon ayrRABC (SA0337 to SA0340) and show that once released from repression by AyrR, the protein products AyrABC together confer resistance to the SPase inhibitor arylomycin M131 by providing an alternate and novel method of releasing translocated proteins. Thus, the derepression of ayrRABC allows cells to bypass the essentiality of SPase. We demonstrate that AyrABC functionally complements SPase by mediating the processing of the normally secreted proteins, albeit in some cases with reduced efficiency and either without cleavage or via cleavage at a site N-terminal to the canonical SPase cleavage site. Thus, ayrRABC encodes a secretion stress-inducible alternate terminal step of the general secretory pathway. IMPORTANCE : Addressing proteins for proper localization within or outside a cell in both eukaryotes and prokaryotes is often accomplished with intrinsic signals which mediate membrane translocation and which ultimately must be removed. The canonical enzyme responsible for the removal of translocation signals is bacterial type I signal peptidase (SPase), which functions at the terminal step of the general secretory pathway and is thus essential in wild-type bacteria. Here, we identify a four-gene operon in S. aureus that encodes an alternate terminal step of the general secretory pathway and thus makes SPase nonessential. The results have important implications for protein secretion in bacteria and potentially for protein trafficking in prokaryotes and eukaryotes in general. Copyright © 2015 Craney et al.

  5. Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

    Science.gov (United States)

    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

  6. Spatial partitioning of secretory cargo from Golgi resident proteins in live cells

    Directory of Open Access Journals (Sweden)

    White Jamie

    2001-10-01

    Full Text Available Abstract Background To maintain organelle integrity, resident proteins must segregate from itinerant cargo during secretory transport. However, Golgi resident enzymes must have intimate access to secretory cargo in order to carry out glycosylation reactions. The amount of cargo and associated membrane may be significant compared to the amount of Golgi membrane and resident protein, but upon Golgi exit, cargo and resident are efficiently sorted. How this occurs in live cells is not known. Results We observed partitioning of the fluorescent Golgi resident T2-CFP and fluorescent cargo proteins VSVG3-YFP or VSVG3-SP-YFP upon Golgi exit after a synchronous pulse of cargo was released from the ER. Golgi elements remained stable in overall size, shape and relative position as cargo emptied. Cargo segregated from resident rapidly by blebbing into micron-sized domains that contained little or no detectable resident protein and that appeared to be continuous with the parent Golgi element. Post-Golgi transport carriers (TCs exited repeatedly from these domains. Alternatively, entire cargo domains exited Golgi elements, forming large TCs that fused directly with the plasma membrane. However, domain formation did not appear to be an absolute prerequisite for TC exit, since TCs also exited directly from Golgi elements in the absence of large domains. Quantitative cargo-specific photobleaching experiments revealed transfer of cargo between Golgi regions, but no discrete intra-Golgi TCs were observed. Conclusions Our results establish domain formation via rapid lateral partitioning as a general cellular strategy for segregating different transmembrane proteins along the secretory pathway and provide a framework for consideration of molecular mechanisms of secretory transport.

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

    Science.gov (United States)

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

    2005-12-01

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

  8. Secretory carcinoma in a 79-year-old woman: an exceptionally rare type of breast carcinoma

    Directory of Open Access Journals (Sweden)

    Nelson Montalvo

    2016-12-01

    Full Text Available Secretory breast carcinoma is an exceptionally rare mammary gland neoplasia described mainly in adult females and children of both sexes, and very rarely in the elderly. It has particular histopathological and immunohistochemical features and a favorable prognosis. We report the case of a 79-year-old Hispanic woman with a palpable breast mass. Currently, the patient is disease free after a follow- up period of 6 years without local recurrence or axillary lymph-nodes nor distant metastases.

  9. Distribution of immunoglobulin G antibody secretory cells in small intestine of Bactrian camels (Camelus bactrianus)

    OpenAIRE

    Zhang, Wang-Dong; WANG, Wen-hui; Jia, Shuai

    2015-01-01

    Background To explore the morphological evidence of immunoglobulin G (IgG) participating in intestinal mucosal immunity, 8 healthy adult Bactrian camels used. First, IgG was successfully isolated from their serum and rabbit antibody against Bactrian camels IgG was prepared. The IgG antibody secretory cells (ASCs) in small intestine were particularly observed through immumohistochemical staining, then after were analyzed by statistical methods. Results The results showed that the IgG ASCs were...

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

    Science.gov (United States)

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

    2005-01-01

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

  11. Clinicopathologic and molecular characterization of mammary analogue secretory carcinoma of salivary gland origin.

    Science.gov (United States)

    Baghai, F; Yazdani, F; Etebarian, A; Garajei, A; Skalova, A

    2017-09-01

    Mammary analogue secretory carcinoma (MASC) is a newly recognized salivary gland tumor that harbors a characteristic balanced chromosomal translocation t (12; 15) (p13; q25) resulting in an ETV6-NTRK3 fusion gene. Retrospective study of 111 salivary gland carcinomas revealed 37 cases with secretory features and growth patterns resembling secretory carcinoma of breast. These 37 cases were originally diagnosed as acinic cell carcinoma, adenocarcinoma not otherwise specified and cystadenocarcinoma. Positive immunostaining for S-100 protein and mammaglobin, followed by detection of ETV6 gene rearrangement by FISH and/or ETV6-NTRK3 fusion transcript by RT-PCR were used to identify MASCs. In the cohort of 37 salivary carcinomas with secretory features we have identified 10 cases of MASC. All 10 MASCs were positive for mammaglobin, S-100 protein and SOX10, while staining for DOG1 and p63 protein were mostly absent. In 7/10 cases, both FISH and RT-PCR were positive while three remaining cases showed break of ETV6 gene by FISH analysis and the RT-PCR was negative. Clinical follow-up data were obtained in 6 out of 10 patients with MASC. In 3 patients cervical lymph node metastases developed, one patient with high grade transformed MASC died with multiple distant bone metastases, and local recurrence was observed in three patients. Our clinicopathological data are in keeping with previous studies; in most cases, MASC is a low-grade malignancy with overall favorable prognosis. In rare cases, however, MASC with high-grade transformation may behave aggressively, and these patients could benefit from targeted biological treatment using tyrosine kinase inhibitors. Copyright © 2017 Elsevier GmbH. All rights reserved.

  12. Do Neural Cells Communicate with Endothelial Cells via Secretory Exosomes and Microvesicles?

    Directory of Open Access Journals (Sweden)

    Neil R. Smalheiser

    2009-01-01

    Full Text Available Neurons, glial, cells, and brain tumor cells tissues release small vesicles (secretory exosomes and microvesicles, which may represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance.

  13. Alteration in endometrial proteins during early- and mid-secretory phases of the cycle in women with unexplained infertility.

    Science.gov (United States)

    Manohar, Murli; Khan, Huma; Sirohi, Vijay Kumar; Das, Vinita; Agarwal, Anjoo; Pandey, Amita; Siddiqui, Waseem Ahmad; Dwivedi, Anila

    2014-01-01

    Compromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2) to mid-secretory phase (LH+7), in women with unexplained infertility. 2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8) and mid-secretory (n = 8) phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4) and mid-secretory (n = 4) phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4) and mid-secretory (n = 4) phase endometrium of fertile women. Nine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1), Cofilin-1 and RAN GTP-binding nuclear protein (Ran) were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women. De-regulation of the expression of Sorcin

  14. Alteration in endometrial proteins during early- and mid-secretory phases of the cycle in women with unexplained infertility.

    Directory of Open Access Journals (Sweden)

    Murli Manohar

    Full Text Available BACKGROUND: Compromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2 to mid-secretory phase (LH+7, in women with unexplained infertility. METHODS: 2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8 and mid-secretory (n = 8 phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4 and mid-secretory (n = 4 phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4 and mid-secretory (n = 4 phase endometrium of fertile women. RESULTS: Nine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1, Cofilin-1 and RAN GTP-binding nuclear protein (Ran were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women

  15. Homocysteine augments BK channel activity and decreases exocytosis of secretory granules in rat GH3 cells.

    Science.gov (United States)

    Gaifullina, Aisylu S; Yakovlev, Aleksey V; Mustafina, Alsu N; Weiger, Thomas M; Hermann, Anton; Sitdikova, Guzel F

    2016-10-01

    In this study, we investigated the effects of L-homocysteine (Hcy) on maxi calcium-activated potassium (BK) channels and on exocytosis of secretory granules in GH3 rat pituitary-derived cells. A major finding of our study indicates that short-term application of Hcy increased the open probability of oxidized BK channels in inside-out recordings. Whole-cell recordings show that extracellular Hcy also augmented BK currents during long-term application. Furthermore, Hcy decreased the exocytosis of secretory granules. This decrease was partially prevented by the BK channel inhibitor paxilline and fully prevented by N-acetylcysteine, a reactive oxygen species scavenger. Taken together, our data show that elevation of cellular Hcy level induces oxidative stress, increases BK channel activity, and decreases exocytosis of secretory granules. These findings may provide insight into some of the developmental impairments and neurotoxicity associated with Hyperhomocysteinemia (HHcy), a disease arising due to abnormally elevated levels of Hcy in the plasma. © 2016 Federation of European Biochemical Societies.

  16. MicroRNA expression profiling of eutopic secretory endometrium in women with versus without endometriosis.

    Science.gov (United States)

    Burney, R O; Hamilton, A E; Aghajanova, L; Vo, K C; Nezhat, C N; Lessey, B A; Giudice, L C

    2009-10-01

    Endometriosis is a common gynecologic disorder characterized by pain and infertility. In addition to estrogen dependence, progesterone resistance is an emerging feature of this disorder. Specifically, a delayed transition from the proliferative to secretory phase as evidenced by dysregulation of progesterone target genes and maintenance of a proliferative molecular fingerprint in the early secretory endometrium (ESE) has been reported. MicroRNAs (miRNAs) are small noncoding RNAs that collectively represent a novel class of regulators of gene expression. In an effort to investigate further the observed progesterone resistance in the ESE of women with endometriosis, we conducted array-based, global miRNA profiling. We report distinct miRNA expression profiles in the ESE of women with versus without endometriosis in a subset of samples previously used in global gene expression analysis. Specifically, the miR-9 and miR-34 miRNA families evidenced dysregulation. Integration of the miRNA and gene expression profiles provides unique insights into the molecular basis of this enigmatic disorder and, possibly, the regulation of the proliferative phenotype during the early secretory phase of the menstrual cycle in affected women.

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

    Energy Technology Data Exchange (ETDEWEB)

    Somanath, Sangeeta [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Partridge, Christopher J. [Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Churchill Hospital, University of Oxford, Oxford, OX3 7LJ (United Kingdom); Marshall, Catriona [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Rowe, Tony [CSL Limited, 45 Poplar Road, Parkville, Victoria 3052 (Australia); Turner, Mark D., E-mail: mark.turner@ntu.ac.uk [Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, NG11 8NS (United Kingdom)

    2016-04-29

    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. 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.

  18. Secretory Duct Structure and Phytochemistry Compounds of Yellow Latex in Mangosteen Fruit

    Directory of Open Access Journals (Sweden)

    DORLY

    2008-09-01

    Full Text Available Yellow latex is the main problem in mangosteen agribusiness, because it is one factor lowering the fruit quality. The structure of yellow latex secretory ducts in the flower and fruit as well as in the root, stem and leaf of mangosteen (Garcinia mangostana L. seedling and the qualitative phytochemistry of yellow latex were studied. The ducts were branched, canal-like type. They were found in the exocarp, mesocarp, endocarp, aril of the fruit, flower, stem, and leaf. In the fruit, the biggest diameter of the secretory ducts was found in the endocarp. There were continuous secretory ducts from fruit stalk to the fruit. Ultrastructural observation showed that the ducts surrounded by specific epithelial cells, which were living cells containing dense cytoplasm with plastid, mitochondria and golgi apparatus organelles. The qualitative test indicated that the yellow latex collected from stem bark, outer part of fruit, young fruit pericarp, mature aril and young aril contained terpenoid, flavonoid and tannin, but not alkaloid, saponin and steroid, except in the young aril containing the steroid.

  19. Bothrops jararaca venom gland secretory cells in culture: Effects of noradrenaline on toxin production and secretion.

    Science.gov (United States)

    Viana, Luciana Godoy; Valente, Richard Hemmi; Heluany, Cíntia Scucuglia; Souza-Imberg, Andreia; Luna, Milene Schmidt; Perales, Jonas; Yamanouye, Norma

    2017-07-01

    Primary culture of snake venom gland secretory cells could be a good model to study the mechanism(s) of toxin(s) production. These cells can produce and secrete venom to the medium with a hemorrhagic activity comparable to that induced by venom collected from snakes. Production of new venom is triggered by the sympathetic outflow, through the release of noradrenaline, but the importance of this neurotransmitter on toxin synthesis has not been addressed. This work led to the identification and comparison of the toxin panel produced by cultured secretory cells, during a 12-day time-course analysis, as well as to the effects of noradrenaline on the process. The results showed that in our culture model the synthesis of new toxins is asynchronous, mimicking data previously published from proteomic analyses of venom glands harvested from animal experimentation. Furthermore, noradrenaline did regulate the synthesis and/or secretion of venom toxins over the analyzed period. Finally, we demonstrated that snake venom metalloproteinases present in these cultured cells secretome were mostly in their zymogen forms; consequently, processing occurs after secretion to the gland lumen. Overall, the data support the use of venom gland secretory cells as a reliable model to investigate the mechanism(s) of toxin(s) synthesis and secretion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells.

    Science.gov (United States)

    Cesaratto, Francesca; López-Requena, Alejandro; Burrone, Oscar R; Petris, Gianluca

    2015-10-20

    Tobacco etch virus protease (TEVp) is a unique endopeptidase with stringent substrate specificity. TEVp has been widely used as a purified protein for in vitro applications, but also as a biological tool directly expressing it in living cells. To adapt the protease to diverse applications, several TEVp mutants with different stability and enzymatic properties have been reported. Herein we describe the development of a novel engineered TEVp mutant designed to be active in the secretory pathway. While wild type TEVp targeted to the secretory pathway of mammalian cells is synthetized as an N-glycosylated and catalytically inactive enzyme, a TEVp mutant with selected mutations at two verified N-glycosylation sites and at an exposed cysteine was highly efficient. This mutant was very active in the endoplasmic reticulum (ER) of living cells and can be used as a biotechnological tool to cleave proteins within the secretory pathway. As an immediate practical application we report the expression of a complete functional monoclonal antibody expressed from a single polypeptide, which was cleaved by our TEVp mutant into the two antibody chains and secreted as an assembled and functional molecule. In addition, we show active TEVp mutants lacking auto-cleavage activity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Ultrastructure and immunohistochemical characterization of proteins concerned with the secretory machinery in goat ceruminous glands

    Directory of Open Access Journals (Sweden)

    Tadashi Yasui

    2017-08-01

    Full Text Available The expression of soluble N-ethyl-maleimide sensitive fusion attachment protein receptor (SNARE proteins in apocrine glands has not been fully elucidated. In addition to performing ultrastructural observation of the ceruminous glands in goats, our study focuses on the demonstration of β-defensins, SNARE proteins and Rab3D in these glands with the use of immunohistochemical methods. The secretory cells were equipped with two types of vesicles, Golgi apparatus and abundant rough endoplasmic reticulum (ER. Additionally, in some of them, the characteristic concentric structures composed of rough ER were observed in their circum- and infranuclear parts. The expression of phosphorylated inositol requiring enzyme 1a was also detected. These findings may indicate their ability to produce numerous secretory proteins and the maintenance of homeostasis in the glandular cells. Furthermore, β-defensins were demonstrated as products of the ceruminous glands. The present investigation also revealed the presence of SNARE proteins and Rab3D. It is suggested that these proteins are concerned with the secretory machinery of this gland type.

  2. A pediatric case of mammary analogue secretory carcinoma within the parotid.

    Science.gov (United States)

    Quattlebaum, S Craig; Roby, Brianne; Dishop, Megan K; Said, M Sherif; Chan, Kenny

    2015-01-01

    Mammary analogue secretory carcinoma (MASC) is a recently described entity in the differential diagnosis of salivary gland tumors. It is notable for a characteristic t(12;15)(p13;q25) translocation that results in a unique fusion protein, ETV6-NTRK3. While several studies have retrospectively identified this translocation in cases previously diagnosed as a different salivary malignancy, there have been relatively few cases where this translocation was identified on initial pathology results, and fewer still in a pediatric population. We present a case of a 15 year old female with a slowly enlarging, painless, left facial mass. MRI demonstrated a cystic mass extending into the deep lobe of the parotid, and she underwent parotidectomy. The tumor cells stained positive for S100 and CK19. ETV6 translocation was present, confirming the diagnosis. Mammary analogue secretory carcinoma is a recently described tumor of the salivary glands, which often masquerades as more common primary salivary gland tumors and cysts. More research is needed to characterize the typical behavior of this neoplasm and the optimal treatment regimen. With identification of its characteristic translocation, mammary analogue secretory carcinoma can be easily differentiated from its more prevalent counterparts, and should therefore remain within the differential of the pathologist and head and neck surgeon. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  4. The class V myosin motor, myosin 5c, localizes to mature secretory vesicles and facilitates exocytosis in lacrimal acini.

    Science.gov (United States)

    Marchelletta, Ronald R; Jacobs, Damon T; Schechter, Joel E; Cheney, Richard E; Hamm-Alvarez, Sarah F

    2008-07-01

    We investigated the role of the actin-based myosin motor, myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of green fluorescent protein (GFP) fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant (P < or = 0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGAC to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGAC. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFP-Myo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles.

  5. Secretory Aspartyl Proteinases Cause Vaginitis and Can Mediate Vaginitis Caused by Candida albicans in Mice.

    Science.gov (United States)

    Pericolini, Eva; Gabrielli, Elena; Amacker, Mario; Kasper, Lydia; Roselletti, Elena; Luciano, Eugenio; Sabbatini, Samuele; Kaeser, Matthias; Moser, Christian; Hube, Bernhard; Vecchiarelli, Anna; Cassone, Antonio

    2015-06-02

    Vaginal inflammation (vaginitis) is the most common disease caused by the human-pathogenic fungus Candida albicans. Secretory aspartyl proteinases (Sap) are major virulence traits of C. albicans that have been suggested to play a role in vaginitis. To dissect the mechanisms by which Sap play this role, Sap2, a dominantly expressed member of the Sap family and a putative constituent of an anti-Candida vaccine, was used. Injection of full-length Sap2 into the mouse vagina caused local neutrophil influx and accumulation of the inflammasome-dependent interleukin-1β (IL-1β) but not of inflammasome-independent tumor necrosis factor alpha. Sap2 could be replaced by other Sap, while no inflammation was induced by the vaccine antigen, the N-terminal-truncated, enzymatically inactive tSap2. Anti-Sap2 antibodies, in particular Fab from a human combinatorial antibody library, inhibited or abolished the inflammatory response, provided the antibodies were able, like the Sap inhibitor Pepstatin A, to inhibit Sap enzyme activity. The same antibodies and Pepstatin A also inhibited neutrophil influx and cytokine production stimulated by C. albicans intravaginal injection, and a mutant strain lacking SAP1, SAP2, and SAP3 was unable to cause vaginal inflammation. Sap2 induced expression of activated caspase-1 in murine and human vaginal epithelial cells. Caspase-1 inhibition downregulated IL-1β and IL-18 production by vaginal epithelial cells, and blockade of the IL-1β receptor strongly reduced neutrophil influx. Overall, the data suggest that some Sap, particularly Sap2, are proinflammatory proteins in vivo and can mediate the inflammasome-dependent, acute inflammatory response of vaginal epithelial cells to C. albicans. These findings support the notion that vaccine-induced or passively administered anti-Sap antibodies could contribute to control vaginitis. Candidal vaginitis is an acute inflammatory disease that affects many women of fertile age, with no definitive cure and, in

  6. Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway.

    Science.gov (United States)

    Gutiérrez-Martínez, Enric; Fernández-Ulibarri, Inés; Lázaro-Diéguez, Francisco; Johannes, Ludger; Pyne, Susan; Sarri, Elisabet; Egea, Gustavo

    2013-06-15

    The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER-Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER-Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

  7. Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria

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    Eric W. Rogier

    2014-04-01

    Full Text Available Antibodies of the secretory IgA (SIgA class comprise the first line of antigen-specific immune defense, preventing access of commensal and pathogenic microorganisms and their secreted products into the body proper. In addition to preventing infection, SIgA shapes the composition of the gut microbiome. SIgA is transported across intestinal epithelial cells into gut secretions by the polymeric immunoglobulin receptor (pIgR. The epithelial surface is protected by a thick network of mucus, which is composed of a dense, sterile inner layer and a loose outer layer that is colonized by commensal bacteria. Immunofluorescence microscopy of mouse and human colon tissues demonstrated that the SIgA co-localizes with gut bacteria in the outer mucus layer. Using mice genetically deficient for pIgR and/or mucin-2 (Muc2, the major glycoprotein of intestinal mucus, we found that Muc2 but not SIgA was necessary for excluding gut bacteria from the inner mucus layer in the colon. Our findings support a model whereby SIgA is anchored in the outer layer of colonic mucus through combined interactions with mucin proteins and gut bacteria, thus providing immune protection against pathogens while maintaining a mutually beneficial relationship with commensals.

  8. Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria.

    Science.gov (United States)

    Rogier, Eric W; Frantz, Aubrey L; Bruno, Maria E C; Kaetzel, Charlotte S

    2014-04-29

    Antibodies of the secretory IgA (SIgA) class comprise the first line of antigen-specific immune defense, preventing access of commensal and pathogenic microorganisms and their secreted products into the body proper. In addition to preventing infection, SIgA shapes the composition of the gut microbiome. SIgA is transported across intestinal epithelial cells into gut secretions by the polymeric immunoglobulin receptor (pIgR). The epithelial surface is protected by a thick network of mucus, which is composed of a dense, sterile inner layer and a loose outer layer that is colonized by commensal bacteria. Immunofluorescence microscopy of mouse and human colon tissues demonstrated that the SIgA co-localizes with gut bacteria in the outer mucus layer. Using mice genetically deficient for pIgR and/or mucin-2 (Muc2, the major glycoprotein of intestinal mucus), we found that Muc2 but not SIgA was necessary for excluding gut bacteria from the inner mucus layer in the colon. Our findings support a model whereby SIgA is anchored in the outer layer of colonic mucus through combined interactions with mucin proteins and gut bacteria, thus providing immune protection against pathogens while maintaining a mutually beneficial relationship with commensals.

  9. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

    Energy Technology Data Exchange (ETDEWEB)

    Copp& #233; , Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith

    2008-10-24

    Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

  10. Immunochromatographic assay using gold nanoparticles for measuring salivary secretory IgA in dogs as a stress marker

    Directory of Open Access Journals (Sweden)

    Aki Takahashi, Shigeru Uchiyama, Yuya Kato, Teruko Yuhi, Hiromi Ushijima, Makoto Takezaki, Toshihiro Tominaga, Yoshiko Moriyama, Kunio Takeda, Toshiro Miyahara and Naoki Nagatani

    2009-01-01

    Full Text Available The concentration of salivary secretory immunoglobulin A (sIgA is a well-known stress marker for humans. The concentration of salivary sIgA in dogs has also been reported as a useful stress marker. In addition, salivary sIgA in dogs has been used to determine the adaptive ability of dogs for further training. There are conventional procedures based on enzyme-linked immunosorbent assay (ELISA for measuring salivary sIgA in dogs. However, ELISA requires long assay time, complicated operations and is costly. In the present study, we developed an immunochromatographic assay for measuring salivary sIgA in dogs using a dilution buffer containing a non-ionic surfactant. We determined 2500-fold dilution as the optimum condition for dog saliva using a phosphate buffer (50 mM, pH 7.2 containing non-ionic surfactant (3 wt% Tween 20. The results obtained from the saliva samples of three dogs using immunochromatographic assay were compared with those obtained from ELISA. It was found that the immunochromatographic assay is applicable to judge the change in salivary sIgA in each dog. The immunochromatographic assay for salivary sIgA in dogs is a promising tool, which should soon become commercially available for predicting a dog's psychological condition and estimating adaptive ability for training as guide or police dogs.

  11. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

    DEFF Research Database (Denmark)

    Wewer, U M; Gerecke, D R; Durkin, M E

    1994-01-01

    Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... beta 2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human beta 2 chain is predicted to have all of the seven structural domains typical of the beta chains of laminin, including the short cysteine-rich alpha region. The amino acid sequence of human beta 2...... chain showed 86.1% sequence identity to the rat beta 2 chain, 50.0% to the human beta 1 chain, and 36.3% to the human beta 3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the beta 2 chain of laminin purified from human...

  12. Therapeutic potential of larval excretory/secretory proteins of the pig whipworm Trichuris suis in allergic disease.

    Science.gov (United States)

    Ebner, F; Hepworth, M R; Rausch, S; Janek, K; Niewienda, A; Kühl, A; Henklein, P; Lucius, R; Hamelmann, E; Hartmann, S

    2014-11-01

    Gastrointestinal nematodes are currently being evaluated as a novel therapeutic in the treatment of chronic human inflammatory disorders, due to their unique ability to induce immunoregulatory pathways in their hosts. In particular, administration of ova from the pig whipworm Trichuris suis (T. suis; TSO) has been proposed for the treatment of allergic, inflammatory and autoimmune disorders. Despite these advances, the biological pathways through which TSO therapy modulates the host immune system in the context of human disease remain undefined. We characterized the dominant proteins present in the excretory/secretory (E/S) products of first-stage (L1) T. suis larvae (Ts E/S) using LC-MS/MS analysis and examined the immunosuppressive properties of whole larval Ts E/S in vitro and in a murine model of allergic airway disease. Administration of larval Ts E/S proteins in vivo during the allergen sensitization phase was sufficient to suppress airway hyperreactivity, bronchiolar inflammatory infiltrate and allergen-specific IgE production. Three proteins in larval Ts E/S were unambiguously identified. The immunomodulatory function of larval Ts E/S was found to be partially dependent on the immunoregulatory cytokine IL-10. Taken together, these data demonstrate that the released proteins of larval T. suis have significant immunomodulatory capacities and efficiently dampen allergic airway hyperreactivity. Thus, the therapeutic potential of defined larval E/S proteins should be exploited for the treatment of human allergic disorders. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Identification of Pathways in Liver Repair Potentially Targeted by Secretory Proteins from Human Mesenchymal Stem Cells

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    Sandra Winkler

    2016-07-01

    Full Text Available Background: The beneficial impact of mesenchymal stem cells (MSC on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1 increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor signalling pathways as relevant networks. Relationships to transforming growth factor β (TGF-β and hypoxia-inducible factor 1-α (HIF1-α signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

  14. Human secretory phospholipase A(2), group IB in normal eyes and in eye diseases

    DEFF Research Database (Denmark)

    Kolko, Miriam; Prause, Jan U; Bazan, Nicolas G

    2007-01-01

    , retinitis pigmentosa and glaucoma were evaluated. RESULTS: Expression of hGIB was found in various cells of the eye. The most abundant expression was found in retinal pigment epithelium (RPE) cells, the inner photoreceptor segments, ganglion cells and the corneal endothelium. We explored diseases involving...... a downregulation of hGIB in migrating RPE cells and in diseased corneal endothelium....

  15. Human calumenin localizes to the secretory pathway and is secreted to the medium

    DEFF Research Database (Denmark)

    Vorum, H; Hager, H; Christensen, Birgitte Mønster

    1999-01-01

    Calumenin belongs to a family of multiple EF-hand proteins that include reticulocalbin, ERC-55, and Cab45. Reticulocalbin and ERC-55 localize to the ER due to a C-terminal HDEL retrieval signal. Cab45 contains a HEEF C-terminal sequence and is localized to the Golgi apparatus. The murine homologu......, suggesting that the glycosylated protein has been further modified in the Golgi apparatus and secreted to the medium. Udgivelsesdato: 1999-May-1...

  16. Human buccal epithelium acquires microbial hyporesponsiveness at birth, a role for secretory leukocyte protease inhibitor

    NARCIS (Netherlands)

    Menckeberg, Celia L; Hol, Jeroen; Simons-Oosterhuis, Ytje; Raatgeep, H Rolien C; de Ruiter, Lilian F; Lindenbergh-Kortleve, Dicky J; Korteland-van Male, Anita M; El Aidy, Sahar; van Lierop, Pieter P E; Kleerebezem, Michiel; Groeneweg, Michael; Kraal, Georg; Elink-Schuurman, Beatrix E; de Jongste, Johan C; Nieuwenhuis, Edward E S; Samsom, Janneke N

    OBJECTIVE: Repetitive interaction with microbial stimuli renders epithelial cells (ECs) hyporesponsive to microbial stimulation. Previously, we have reported that buccal ECs from a subset of paediatric patients with Crohn's disease are not hyporesponsive and spontaneously released chemokines. We now

  17. Human buccal epithelium acquires microbial hyporesponsiveness at birth, a role for secretory leukocyte protease inhibitor

    NARCIS (Netherlands)

    Menckeberg, C.L.; Hol, J.; Simons-Oosterhuis, Y.; Raatgeep, H.R.; Ruiter, de L.F.; Lindenbergh-Kortleve, D.J.; Korteland-van Male, A.M.; Aidy, El S.F.; Lierop, van P.P.E.; Kleerebezem, M.; Groeneweg, M.; Kraal, G.; Elink-Schuurman, B.E.; Jongste, de J.C.; Nieuwenhuis, E.E.S.; Samsom, J.N.

    2015-01-01

    Objective Repetitive interaction with microbial stimuli renders epithelial cells (ECs) hyporesponsive to microbial stimulation. Previously, we have reported that buccal ECs from a subset of paediatric patients with Crohn's disease are not hyporesponsive and spontaneously released chemokines. We now

  18. Role of secretory granules in inositol 1,4,5-trisphosphate-dependent Ca(2+) signaling: from phytoplankton to mammals.

    Science.gov (United States)

    Yoo, Seung Hyun

    2011-08-01

    The majority of secretory cell calcium is stored in secretory granules that serve as the major IP(3)-dependent intracellular Ca(2+) store. Even in unicellular phytoplankton secretory granules are responsible for the IP(3)-induced Ca(2+) release that triggers exocytosis. The number of secretory granules in the cell is directly related not only to the magnitude of IP(3)-induced Ca(2+) release, which accounts for the majority of the IP(3)-induced cytoplasmic Ca(2+) release in neuroendocrine cells, but also to the IP(3) sensitivity of the cytoplasmic IP(3) receptor (IP(3)R)/Ca(2+) channels. Moreover, secretory granules contain the highest IP(3)R concentrations and the largest amounts of IP(3)Rs in any subcellular organelles in neuroendocrine cells. Secretory granules from phytoplankton to mammals contain large amounts of polyanionic molecules, chromogranins being the major molecules in mammals, in addition to acidic intragranular pH and high Ca(2+) concentrations. The polyanionic molecules undergo pH- and Ca(2+)-dependent conformational changes that serve as a molecular basis for condensation-decondensation phase transitions of the intragranular matrix. Likewise, chromogranins undergo pH- and Ca(2+)-dependent conformational changes with increased exposure of the structure and increased interactions with Ca(2+) and other granule components at acidic pH. The unique physico-chemical properties of polyanionic molecules appear to be at the center of biogenesis, and physiological functions of secretory granules in living organisms from primitive to advanced species. 2010 Elsevier Ltd. All rights reserved.

  19. Secretory glands in cercaria of the neuropathogenic schistosome Trichobilharzia regenti - ultrastructural characterization, 3-D modelling, volume and pH estimations

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    Koberna Karel

    2011-08-01

    Full Text Available Abstract Background Cercariae of schistosomes employ bioactive molecules for penetration into their hosts. These are released from specialized unicellular glands upon stimuli from host skin. The glands were previously well-described in the human pathogen Schistosoma mansoni. As bird schistosomes can also penetrate human skin and cause cercarial dermatitis, our aim was to characterize the architecture and ultrastructure of glands in the neurotropic bird schistosome Trichobilharzia regenti and compare it with S. mansoni. In the context of different histolytic enzymes used by these two species, we focused also on the estimations of gland volumes and pH in T. regenti. Results The architecture and 3-D models of two types of acetabular penetration glands, their ducts and of the head gland are shown here. We characterized secretory vesicles in all three gland types by means of TEM and confirmed accuracy of the models obtained by confocal microscopy. The results of two independent approaches showed that the glands occupy ca. one third of cercarial body volume (postacetabular glands ca. 15%, circumacetabular 12% and head gland 6%. The inner environment within the two types of acetabular glands differed significantly as evidenced by dissimilar ability to bind fluorescent markers and by pH value which was higher in circumacetabular (7.44 than in postacetabular (7.08 glands. Conclusions As far as we know, this is the first presentation of a 3-D model of cercarial glands and the first exact estimation of the volumes of the three gland types in schistosomes. Our comparisons between T. regenti and S. mansoni implied that the architecture and ultrastructure of the glands is most likely conserved within the family. Only minor variations were found between the two species. It seems that the differences in molecular composition have no effect on general appearance of the secretory cells in TEM. Fluorescent markers employed in this study, distinguishing between

  20. Delta1 expression, cell cycle exit, and commitment to a specific secretory fate coincide within a few hours in the mouse intestinal stem cell system.

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    Despina Stamataki

    Full Text Available The stem cells of the small intestine are multipotent: they give rise, via transit-amplifying cell divisions, to large numbers of columnar absorptive cells mixed with much smaller numbers of three different classes of secretory cells--mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, and bactericide-secreting Paneth cells. Notch signaling is known to control commitment to a secretory fate, but why are the secretory cells such a small fraction of the population, and how does the diversity of secretory cell types arise? Using the mouse as our model organism, we find that secretory cells, and only secretory cells, pass through a phase of strong expression of the Notch ligand Delta1 (Dll1. Onset of this Dll1 expression coincides with a block to further cell division and is followed in much less than a cell cycle time by expression of Neurog3--a marker of enteroendocrine fate--or Gfi1--a marker of goblet or Paneth cell fate. By conditional knock-out of Dll1, we confirm that Delta-Notch signaling controls secretory commitment through lateral inhibition. We infer that cells stop dividing as they become committed to a secretory fate, while their neighbors continue dividing, explaining the final excess of absorptive over secretory cells. Our data rule out schemes in which cells first become committed to be secretory, and then diversify through subsequent cell divisions. A simple mathematical model shows how, instead, Notch signaling may simultaneously govern the commitment to be secretory and the choice between alternative modes of secretory differentiation.

  1. Secretory IgA (SIgA: designed for antimicrobial defence

    Directory of Open Access Journals (Sweden)

    Per eBrandtzaeg

    2013-08-01

    Full Text Available 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 stimulated by antigens that target the mucosae. 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 defensce and changes in their intestinal microbiota. In the gut, induction of B cells occurs in gut-associated lymphoid tissue (GALT, particularly the Peyer’s patches and isolated lymphoid follicles, but also in mesenteric lymph nodes. Plasma cell 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 (NALT 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.

  2. Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Bongkun; Kang, Soon-Suk [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Kang, Sang-Wook [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Min, Bon-Hong [Department of Pharmacology, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Lee, Eun-Jin; Song, Da-Hyun; Kim, Sang-Min [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Song, Youngsup [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Yoon, Seung-Yong [Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Chang, Eun-Ju, E-mail: ejchang@amc.seoul.kr [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of)

    2014-07-18

    Highlights: • We describe the expression and secretion of clusterin in osteoclasts. • Endogenous clusterin deficiency does not affect osteoclast formation. • Exogenous treatment with secretory clusterin decreases osteoclast differentiation. • Secretory clusterin attenuates osteoclast precursor cell proliferation by inhibiting M-CSF-mediated ERK activation. - Abstract: Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU{sup −/−} mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

  3. Ca(2+)-independent fusion of secretory granules with phospholipase A2-treated plasma membranes in vitro.

    Science.gov (United States)

    Nagao, T; Kubo, T; Fujimoto, R; Nishio, H; Takeuchi, T; Hata, F

    1995-04-15

    The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ fused with the granules within 30 s, and induced amylase release by reacting with the membranes of granules, whereas without this pretreatment they had no significant effect. The fusion process accompanied by amylase release was induced in the presence of 10 mM EGTA, and therefore was apparently Ca(2+)-independent. On the other hand, the presence of EGTA or 100 microM quinacrine, an inhibitor of PLA2, during treatment of plasma membranes with PLA2 inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Arachidonic acid and linoleic acid were released from the plasma membranes during the PLA2 treatment. The presence of albumin, an adsorbent of fatty acids, during the treatment also inhibited the activity. Pretreatment of the membranes with arachidonic acid or linoleic acid did not have any effect, but the presence of exogenously added arachidonic acid during PLA2 treatment enhanced the membrane-fusion-inducing effect of PLA2. Pretreatment of the membranes with lysophosphatidylcholine induced fusogenic activity. These findings suggest that the conformational change in the plasma-membrane phospholipids induced by PLA2 and the presence of arachidonic acid or linoleic acid produced by PLA2 are important in the process of fusion of secretory granules with the plasma membranes of rat parotid acinar cells and that the fusion process itself is independent of Ca2+.

  4. Mammary Analogue Secretory Carcinoma (MASC) of salivary gland in four Mexican patients

    Science.gov (United States)

    Mosqueda-Taylor, Adalberto; Domínguez-Malagón, Hugo; Michal, Michal

    2015-01-01

    The Clinco-pathological, immunohistochemical and molecular findings of four cases of Mammary Analogue Secretory Carcinoma (MASC) of salivary glands found in Mexico are described. The cases were extracted from 253 salivary gland tumors from a single institution in Mexico City. The 85 candidates for initial selection were: low grade mucoepidermoid carcinoma (MEC) (N=70 ), acinic cell cancinoma (AciCC) (N=14), papillary cystadenocarcinoma (N=1), and adenocarcinoma NOS (N=0). Tumors with some histological features consistent with MASC (N= 17, 6.7%) were studied by immunohistochemistry for mammaglobin, STAT5, and S-100 protein and four cases were positive (1.5%), thus the diagnosis of MASC was established, and these were submitted for molecular studies for ETV6-NTRK3. Fusion gene was demonstrated in three cases, two had been erroneously diagnosed as poorly granulated AciCC, and one as low grade MEC with microcystic pattern. Female gender predominated (3:1); one occurred in the parotid, two in minor salivary glands and one in the submaxillary gland; infiltrating borders, atypical mitosis and lymph node metastases were seen in the parotideal tumor. Two patients with major salivary gland tumors are alive and well at 10 and 20 months respectively, the two patients with minor salivary gland tumors are lost. It can be concluded that is important to think in MASC in poorly granulated AciCC and low grade MEC with microcystic pattern. Immunohistochemisty studies confirm the diagnosis, preferentially supported by molecular studies. MASC may follow aggressive behavior or transform into a high grade neoplasm. Key words:Acinic cell carcinoma, ETV6-NTRK3, Mammary Analogue Secretory Carcinoma, secretory breast carcinoma. PMID:25481229

  5. The Ultrastructure of Secretory Cells of the Islets of Langerhans in South American Catfish Rhamdia quelen

    Directory of Open Access Journals (Sweden)

    Laura Luchini

    2015-01-01

    Full Text Available The present work shows that a detailed description of the ultrastructure of the secretory cells of the South American catfish Rhamdia quelen pancreatic islets is presented. Evidence is offered to support the contention that the α-granules consist of a central and an outer portion of different electron densities and solubilities, that the δ-cells are most probably morphologically altered but viable α-cells, and that the β-granules possibly possess a repeating substructure and may therefore represent an intracellular crystalline storage form of insulin.

  6. Reversal by EGTA of the enhanced secretory responsiveness of mast cells due to treatment with ouabain

    DEFF Research Database (Denmark)

    Johansen, Torben; Knudsen, T; Bertelsen, Niels Haldor

    1990-01-01

    The effect of EGTA on the enhancement by ouabain of compound 48/80-induced secretion from mast cells was compared with the effect on the Na(+)-K+ pump activity. The time-dependent secretory enhancement by ouabain was blocked by addition of EGTA to the cell suspension concomitantly with the addition...... of ouabain, and EGTA caused a large increase in the pump activity. Addition of 10 microM EGTA to ouabain-treated cells stopped but did not reverse the enhancement. The experiments show that the effect of ouabain was due to changes in a calcium pool utilized in compound 48/80-induced secretion following...

  7. [Unilateral exophthalmos as the debut of a non-secretory multiple myeloma].

    Science.gov (United States)

    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.

  8. An abundantly secreted glycoprotein from Drosophila melanogaster is related to mammalian secretory proteins produced in rheumatoid tissues and by activated macrophages.

    Science.gov (United States)

    Kirkpatrick, R B; Matico, R E; McNulty, D E; Strickler, J E; Rosenberg, M

    1995-02-14

    An abundantly secreted 47-kDa glycoprotein, DS47, was purified from Drosophila melanogaster (Dm) Schneider line-2 cells, a line exhibiting macrophage-like properties. DS47 is also secreted from several Dm cell lines resembling S2 but not from lines that are morphologically distinct. A cDNA cline was isolated from an S2 cell cDNA library using oligodeoxyribonucleotide probes based on the DS47 amino acid (aa) sequence and found to encode a novel secretory glycoprotein of 452 aa. Analysis of DS47 protein production and mRNA expression during fly development indicates that both are present throughout the entire Dm life cycle, suggesting that DS47 may be important at all developmental stages. In larvae, the DS47 message is made in the fat body and by hemocytes, and secreted into the hemolymph. DS47 is related to a human cartilage glycoprotein, HC gp-39, that is secreted from cell types associated with the arthritic joint, such as synovial cells and activated macrophages. Interestingly, the HC gp-39 message is most readily detected in the human liver, an organ that is somewhat analogous to the Dm fat body. DS47 also shares homology to a mouse secretory glycoprotein, YM-1, identified in activated macrophages. These homologies extend to the chitinase gene family and include a conserved cysteine aa motif, as well as two blocks of aa within the enzymatic active site, although neither DS-47 nor HC gp-39 exhibit chitinase activity. Potential functions of this conserved protein family are discussed.

  9. Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis

    DEFF Research Database (Denmark)

    Carobbio, Stefania; Frigerio, Francesca; Rubi, Blanca

    2009-01-01

    Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not bee...... weight gain was preserved. The results demonstrate that GDH is essential for the full development of the secretory response in beta-cells. However, maximal secretory capacity is not required for maintenance of glucose homeostasis in normo-caloric conditions.......Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been...... tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin...

  10. In vitro permeation of β-lactam antibiotics across rat jejunum and its correlation with oral bioavailability in humans

    Science.gov (United States)

    Hiroshi, Saitoh; Aungst, Bruce J; Tohyama, Masashi; Hatakeyama, Yuko; Ohwada, Keiko; Kobayashi, Michiya; Fujisaki, Hiroko; Miyazaki, Katsumi

    2002-01-01

    Aims To investigate the correlation between in vitro permeation of 11 β-lactam antibiotics across rat jejunum and their oral bioavailability in humans. Methods The absorptive and secretory permeation across rat jejunum was evaluated and apparent permeability coefficients (Papp) were determined. Results A steep, sigmoid-type curve was obtained for the relationship between Papp in the absorptive permeation and human oral bioavailability. When the ratios of Papp in the absorptive direction to Papp in the secretory direction were plotted against human oral bioavailability, a much improved correlation was obtained (r = 0.98, P ceftibuten and cephalexin from the absorptive to the secretory direction. Conclusions For 11 β-lactam antibiotics rat intestinal permeation correlated well with human oral bioavailability, especially when corrected for secretory transport. PMID:12392595

  11. Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

    Science.gov (United States)

    Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

    2015-06-01

    Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

  12. Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells

    DEFF Research Database (Denmark)

    Olsen, Hervør L; Hoy, Marianne; Zhang, Wei

    2003-01-01

    Insulin secretion is controlled by the beta cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends...... on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] stimulated exocytosis by promoting the priming of secretory...... granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological...

  13. Extracellular magnesium decreases the secretory response of rat peritoneal mast cells to compound 48/80 in vitro

    DEFF Research Database (Denmark)

    Bertelsen, Niels Haldor; Johansen, Torben

    1991-01-01

    Exposure of rat peritoneal mast cells to magnesium in the absence of extracellular calcium resulted in a time- and dose-dependent decrease in the secretory response induced by compound 48/80. The decrease was prevented by a low extracellular concentration of calcium. Furthermore, the decreased...... and the secretory stimulus. A dose-dependent decrease in antigen induced histamine secretion that was reversed by calcium was also observed. Exposure of the mast cells to magnesium for 15 min resulted in a parallel decrease in histamine secretion and in the cellular content of 45Ca2+. These observations suggest...... secretory responsiveness was dose-dependently restored by the addition of calcium to the cells simultaneously with compound 48/80. Preincubation with magnesium also inhibited antigen-induced histamine secretion in a dose-dependent manner. This was reversed by the simultaneous addition of calcium...

  14. Complexin Activates Exocytosis of Distinct Secretory Vesicles Controlled by Different Synaptotagmins

    Science.gov (United States)

    Cao, Peng; Yang, Xiaofei; Südhof, Thomas C.

    2013-01-01

    Complexins are SNARE-complex binding proteins essential for the Ca2+-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. Here we show that in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a co-factor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, our data indicate that complexins are essential for activation of multiple types of Ca2+-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin-adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved. PMID:23345244

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

    Science.gov (United States)

    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

  16. Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter.

    Science.gov (United States)

    Hong, Seung-Pyo; Seip, John; Walters-Pollak, Dana; Rupert, Ross; Jackson, Raymond; Xue, Zhixiong; Zhu, Quinn

    2012-02-01

    Oleaginous yeast Yarrowia lipolytica is an important host for the production of lipid-derived compounds or heterologous proteins. Selection of strong promoters and effective expression systems is critical for heterologous protein secretion. To search for a strong promoter in Y. lipolytica, activities of FBA1, TDH1 and GPM1 promoters were compared to that of TEF1 promoter by constructing GUS reporter fusions. The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively. The FBA1IN promoter (FBA1 sequence of -826 to +169) containing an intron (+64 to +165) showed five-fold higher expression than the FBA1 promoter (-831 to -1). The transcriptional enhancement by the 5'-region within the FBA1 gene was confirmed by GPM1::FBA1 chimeric promoter construction. Using the strong FBA1IN promoter, four different S. cerevisiae SUC2 expression cassettes were tested for the SUC+ phenotype in Y. lipolytica. Functional invertase secretion was facilitated by the Xpr2 prepro-region with an additional 13 amino acids of mature Xpr2, or by the native Suc2 signal sequence. However, these two secretory signals in tandem, or the mature Suc2 with no secretory signal, did not direct secretion of functional invertase. Unlike previously reported Y. lipolytica SUC+ strains, our engineered stains secreted most of invertase into the medium. Copyright © 2011 John Wiley & Sons, Ltd.

  17. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J

    2011-02-01

    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 ± 2.6 and 38.8 ± 6.7% (n=16; P≤0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  18. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J

    2012-02-01

    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 +\\/- 2.6 and 38.8 +\\/- 6.7% (n=16; P<\\/=0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  19. Lactation defect with impaired secretory activation in AEBP1-null mice.

    Directory of Open Access Journals (Sweden)

    Lei Zhang

    Full Text Available Adipocyte enhancer binding protein 1 (AEBP1 is a multifunctional protein that negatively regulates the tumor suppressor PTEN and IκBα, the inhibitor of NF-κB, through protein-protein interaction, thereby promoting cell survival and inflammation. Mice homozygous for a disrupted AEBP1 gene developed to term but showed defects in growth after birth. AEBP1(-/- females display lactation defect, which results in the death of 100% of the litters nursed by AEBP1(-/- dams. Mammary gland development during pregnancy appears normal in AEBP1(-/- dams; however these mice exhibit expansion of the luminal space and the appearance of large cytoplasmic lipid droplets (CLDs in the mammary epithelial cells at late pregnancy and parturition, which is a clear sign of failed secretory activation, and accumulation of milk proteins in the mammary gland, presumably reflecting milk stasis following failed secretory activation. Eventually, AEBP1(-/- mammary gland rapidly undergoes involution at postpartum. Stromal restoration of AEBP1 expression by transplanting wild-type bone marrow (BM cells is sufficient to rescue the mammary gland defect. Our studies suggest that AEBP1 is critical in the maintenance of normal tissue architecture and function of the mammary gland tissue and controls stromal-epithelial crosstalk in mammary gland development.

  20. Localization of oleuropeyl glucose esters and a flavanone to secretory cavities of Myrtaceae.

    Science.gov (United States)

    Heskes, Allison M; Goodger, Jason Q D; Tsegay, Sammi; Quach, Tim; Williams, Spencer J; Woodrow, Ian E

    2012-01-01

    We report the widespread occurrence of structurally diverse oleuropeyl glucose esters, including the new diester eucaglobulin B, localized specifically to the essential oil secretory cavities of myrtaceous species. Clear taxonomic patterns in the composition of cavity extracts within the genus Eucalyptus are shown with species from subgenus Symphyomyrtus dominated by oleuropeyl glucose esters and species from subgenus Eucalyptus dominated instead by the flavanone, pinocembrin. We also examined the intra-species occurrence of oleuropeyl glucose esters by quantifying the abundant constituents cuniloside B and froggattiside A in trees from two populations of Eucalyptus polybractea R.T. Baker. All trees contained both compounds, which were positively correlated with total essential oil concentration. This apparent ubiquity of oleuropeyl glucose esters at both intra- and inter-specific levels in Eucalyptus is indicative of important physiological or ecological functions. The significance of their prevalence and the sequestration of these esters and also pinocembrin to the extracellular domain of secretory cavities is discussed in light of their potential biological activities and our findings that they are spatially segregated to the exterior of cavity lumina. The localization of oleuropeyl glucose esters to a specific and isolatable tissue type has the potential to aid in future elucidation of function and biosynthesis.

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

    Science.gov (United States)

    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.

  2. Pasteurella multocida toxin: Targeting mast cell secretory granules during kiss-and-run secretion.

    Science.gov (United States)

    Danielsen, Elisabeth M; Christiansen, Nina; Danielsen, E Michael

    2016-02-01

    Pasteurella multocida toxin (PMT), a virulence factor of the pathogenic Gram-negative bacterium P. multocida, is a 146 kDa protein belonging to the A-B class of toxins. Once inside a target cell, the A domain deamidates the α-subunit of heterotrimeric G-proteins, thereby activating downstream signaling cascades. However, little is known about how PMT selects and enters its cellular targets. We therefore studied PMT binding and uptake in porcine cultured intestinal mucosal explants to identify susceptible cells in the epithelium and underlying lamina propria. In comparison with Vibrio cholera B-subunit, a well-known enterotoxin taken up by receptor-mediated endocytosis, PMT binding to the epithelial brush border was scarce, and no uptake into enterocytes was detected by 2h, implying that none of the glycolipids in the brush border are a functional receptor for PMT. However, in the lamina propria, PMT distinctly accumulated in the secretory granules of mast cells. This also occurred at 4 °C, ruling out endocytosis, but suggestive of uptake via pores that connect the granules to the cell surface. Mast cell granules are known to secrete their contents by a "kiss-and-run" mechanism, and we propose that PMT may exploit this secretory mechanism to gain entry into this particular cell type. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Expression of Clara cell secretory protein in experimental otitis media in the rat.

    Science.gov (United States)

    Kim, Seo Jin; Jung, Hak Hyun

    2005-01-01

    These results suggest that CCSP is upregulated in OME and may play a protective role in the pathogenesis of OME. Clara cell secretory protein (CCSP) is an abundant 16-kDa homodimeric protein and is secreted by non-ciliated secretory epithelial cells in the lung. It has an important protective role against the intrapulmonary inflammatory process. The aim of this study was to investigate the expression of CCSP in endotoxin-induced otitis media with effusion (OME) in the rat. We instilled endotoxin and saline (control) into the middle ear cavity of the rat. Middle ear mucosa were taken at 0, 1, 3, 6 and 12 h and 1, 3, 7 and 14 days, and the expression of both CCSP mRNA and protein were then evaluated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. RT-PCR revealed that the expression of CCSP was first identified at 1 h after endotoxin instillation, was dramatically increased between 1 h and Day 1, with maximal expression at 12 h, and then decreased after Day 3. The expression pattern of CCSP protein identified by means of Western blotting was similar to the CCSP mRNA patterns observed using RT-PCR. Expression of CCSP at both mRNA and protein levels was not detected in either normal middle ear mucosa or saline-instilled middle ear mucosa. Immunohistochemistry revealed that some epithelial cells in the middle ear mucosa were stained.

  4. Localization of oleuropeyl glucose esters and a flavanone to secretory cavities of Myrtaceae.

    Directory of Open Access Journals (Sweden)

    Allison M Heskes

    Full Text Available We report the widespread occurrence of structurally diverse oleuropeyl glucose esters, including the new diester eucaglobulin B, localized specifically to the essential oil secretory cavities of myrtaceous species. Clear taxonomic patterns in the composition of cavity extracts within the genus Eucalyptus are shown with species from subgenus Symphyomyrtus dominated by oleuropeyl glucose esters and species from subgenus Eucalyptus dominated instead by the flavanone, pinocembrin. We also examined the intra-species occurrence of oleuropeyl glucose esters by quantifying the abundant constituents cuniloside B and froggattiside A in trees from two populations of Eucalyptus polybractea R.T. Baker. All trees contained both compounds, which were positively correlated with total essential oil concentration. This apparent ubiquity of oleuropeyl glucose esters at both intra- and inter-specific levels in Eucalyptus is indicative of important physiological or ecological functions. The significance of their prevalence and the sequestration of these esters and also pinocembrin to the extracellular domain of secretory cavities is discussed in light of their potential biological activities and our findings that they are spatially segregated to the exterior of cavity lumina. The localization of oleuropeyl glucose esters to a specific and isolatable tissue type has the potential to aid in future elucidation of function and biosynthesis.

  5. Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms

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    Ian B. Hogue

    2016-06-01

    Full Text Available Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV exocytosis, based on total internal reflection fluorescence (TIRF microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i trafficking of viral glycoproteins to the plasma membrane, (ii exocytosis of viral light particles, and (iii exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway.

  6. Mapping dynamic protein interactions to insulin secretory granule behavior with TIRF-FRET.

    Science.gov (United States)

    Lam, Alice D; Ismail, Sahar; Wu, Ray; Yizhar, Ofer; Passmore, Daniel R; Ernst, Stephen A; Stuenkel, Edward L

    2010-08-09

    Biological processes are governed by extensive networks of dynamic molecular interactions. Yet, establishing a spatial and temporal map of these interactions and their direct relationship to specific cell functions has remained a challenge. Here, we implement sensitized emission Förster resonance energy transfer (FRET) stoichiometry under total internal reflection fluorescence (TIRF) microscopy. We demonstrate through quantitative analysis and modeling that evanescent fields must be precisely matched between FRET excitation wavelengths to isolate dynamic interactions between bimolecular FRET pairs that are not entirely membrane-delimited. We then use TIRF-FRET to monitor the behavior of individual insulin-containing secretory granules at the plasma membrane of living cells, while simultaneously tracking the dynamic interaction between the GTPase Rab27A and its effector Slp4A, on those same granules. Notably, insulin granules that underwent exocytosis demonstrated a specific increase in Rab27A-GTP/Slp4A FRET in the 5 s before membrane fusion, which coincided temporally with an increase in granule displacement and mobility. These results demonstrate an initial spatiotemporal mapping of a dynamic protein-protein interaction on individual secretory granules that is linked to a specific granule behavior in living cells. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. A systematic review including an additional pediatric case report: Pediatric cases of mammary analogue secretory carcinoma.

    Science.gov (United States)

    Ngouajio, Amanda L; Drejet, Sarah M; Phillips, D Ryan; Summerlin, Don-John; Dahl, John P

    2017-09-01

    Mammary Analogue Secretory Carcinoma (MASC) is a newly characterized salivary gland carcinoma resembling secretory carcinoma of the breast. Prior to being described, MASC was most commonly misdiagnosed as Acinic Cell Carcinoma. Though MASC is predominantly an adult neoplasm, cases have been reported in the pediatric population. Reporting and summarizing of known cases is imperative to understand the prognosis and clinical behavior of MASC. EVIDENCE REVIEW: Web of Science, Medline, EMBASE, and The Cochrane Library were searched for studies that included pediatric cases of MASC. Data on clinical presentation, diagnosis and management, and pathology were collected from all pediatric cases. CONCLUSIONS AND RELEVANCE: Since the first case of MASC in the pediatric population was described in 2011, only 12 cases, including this one, have been described in the literature. With this paucity of information, much remains unknown regarding this new pathologic diagnosis. The collection of clinical outcomes data of children with MASC is needed to better understand the behavior of this malignancy as well as determine optimal treatment regimens. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Aspiration cytology of mammary analogue secretory carcinoma of the salivary gland.

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    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.

  9. Finding and characterizing mammary analogue secretory carcinoma of the salivary gland.

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    Jung, Min Jung; Song, Joon Seon; Kim, Sang Yoon; Nam, Soon Yuhl; Roh, Jong-Lyel; Choi, Seung-Ho; Kim, Sung-Bae; Cho, Kyung-Ja

    2013-02-01

    A new tumor entity of the salivary glands, mammary analogue secretory carcinoma (MASC) with ETV6-NTRK3 translocation, has recently been proposed. MASC was originally diagnosed as adenocarcinoma, not otherwise specified (ANOS), or acinic cell carcinoma (AciCC) by the current World Health Organization classification. We aimed to identify MASC cases by molecular tests, and to characterize their clinical, histological, and immunohistochemical features. Thirty cases of MASC candidates were selected after review of 196 salivary gland tumors, and subjected to break-apart ETV6 fluorescence in situ hybridization (FISH), and immunohistochemical study for S100 protein, gross cystic disease fluid protein 15, DOG1, estrogen receptor, and progesterone receptor. Valid FISH results were obtained in 23 cases, and 13 positive cases were retrieved. MASCs were histologically varied, and the most frequent features observed in 10 cases were low-grade papillary/cystic/glandular patterns, intraluminal secretory materials, ovoid/wrinkled nuclei, and relatively abundant granular eosinophilic cytoplasms, corresponding to papillary-cystic or follicular types of AciCC. All cases showed diffuse immunopositivity for S100 protein. Three cases developed recurrences, but all patients remained alive. MASC could be a molecularly well-defined salivary gland neoplasm, encompassing some portions of AciCC and ANOS, but its histological spectrum and clinical implication require further investigation.

  10. Mammary analogue secretory carcinoma of salivary glands: diagnostic pitfall with distinct immunohistochemical profile and molecular features.

    Science.gov (United States)

    Bissinger, Oliver; Götz, Carolin; Kolk, Andreas; Bier, Henning A; Agaimy, Abbas; Frenzel, Henning; Perner, Sven; Ribbat-Idel, Julika; Wolff, Klaus Dietrich; Weichert, Wilko; Mogler, Caroline

    2017-10-03

    Mammary analogue secretory carcinoma (MASC) is a newly defined entity among salivary gland malignancies which has just been established in the 4(th) edition of the WHO classification of head and neck tumors. MASC (synonym: secretory carcinoma) are characterized by a specific rearangement of the ETV6 gene locus. Here, we present a series of 3 MASC cases including clinical data with follow-up for up to 26 months. All tumours immunhistochemically displayed strong positivity for cytokeratin 7, and mammaglobin, focal positivity for S100, cytokeratin 5/6 and muc-4. In contrast, immunhistochemical stainings against cytokeratin 14, hormon receptors, Her2/neu, androgen receptor and prostate-specific antigen were consistently negative. FISH analysis showed translocation of the ETV6 gene locus in the majority of tumour cell nuclei. During clinical follow-up, no local relapse or metastasis was detected. As these carcinomas are clinically and radiologically indistinguishable from other salivary gland tumours and as therapeutic approaches and prognosis might differ, we need to be able to diagnose MASC correctly.

  11. WNT5A inhibits human dental papilla cell proliferation and migration

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    Peng, L. [West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan (China); State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, Sichuan (China); Ye, L.; Dong, G.; Ren, L.B.; Wang, C.L.; Xu, P. [West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan (China); Zhou, X.D., E-mail: pl_huaxi@yahoo.com.cn [West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan (China); State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, Sichuan (China)

    2009-12-18

    WNT proteins are a large family of cysteine-rich secreted molecules that are linked to both canonical and non-canonical signal pathways, and have been implicated in oncogenesis and tissue development. Canonical WNT proteins have been proven to play critical roles in tooth development, while little is known about the role of non-canonical WNT proteins such as WNT5A. In this study, WNT5A was localized to human dental papilla tissue and human dental papilla cells (HDPCs) cultured in vitro, using immunochemistry and RT-PCR. Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of WNT5A on HDPCs. The BrdU incorporation assay, the MTT assay and flow cytometric analysis showed that over-expression of Wnt5a strongly inhibited the proliferation of HDPCs in vitro. Wound healing and transwell migration assays indicated that over-expression of WNT5A reduced migration of HDPCs. In conclusion, our results showed that WNT5A negatively regulates both proliferation and migration of HDPCs, suggesting its important role in odontogenesis via controlling the HDPCs.

  12. The unique ultrastructure of secretory membranes in gastric parietal cells depends upon the presence of H+, K+ -ATPase.

    Science.gov (United States)

    Miller, Marian L; Judd, Louise M; Van Driel, Ian R; Andringa, Anastasia; Flagella, Michael; Bell, Sheila M; Schultheis, Patrick J; Spicer, Zachary; Shull, Gary E

    2002-09-01

    Ion transporters play a central role in gastric acid secretion. To determine whether some of these transporters are necessary for the normal ultrastructure of secretory membranes in gastric parietal cells, mice lacking transporters for H+, K+, Cl-, and Na+ were examined for alterations in volume density (Vd) of basolateral, apical, tubulovesicular and canalicular membranes, microvillar dimensions, membrane flexibility, and ultrastructure. In mice lacking Na+/H+ exchanger 1 (NHE1) or the Na+-K+-2Cl- cotransporter (NKCC1), the ultrastructure and Vd of secretory membranes and the secretory canalicular to tubulovesicular membrane ratio (SC/TV), a morphological correlate of secretory activity, were similar to those of wild-type mice. In mice lacking Na+/H+ exchanger 2 (NHE2) or gastric H+, K+ -ATPase alpha- or beta-subunits, the SC/TV ratio and Vd of secretory membranes were decreased, though canaliculi were often dilated. In H+, K+ -ATPase-deficient parietal cells, canalicular folds were decreased, normally abundant tubulovesicles were replaced with a few rigid round vesicles, and microvilli were sparse, stiff and short, in contrast to the long and flexible microvilli in wild-type cells. In addition, microvilli of the H+, K+ -ATPase-deficient parietal cells had centrally bundled F-actin filaments, unlike the microvilli of wild-type cells, in which actin filaments were peripherally positioned concentric to the plasmalemma. Data showed that the absence of H+, K+ -ATPase produced fundamental changes in parietal cell membrane ultrastructure, suggesting that the pump provides an essential link between the membranes and F-actin, critical to the gross architecture and suppleness of the secretory membranes.

  13. Mycobacterial excretory secretory-31 protein with serine protease and lipase activities: An immunogen and drug target against tuberculosis infection.

    Science.gov (United States)

    Harinath, Bhaskar C

    2016-12-01

    Tuberculosis (TB) has been declared as a global emergency by the World Health Organization in 1993 and still remains one of the world's biggest threats. Worldwide, 9.6 million people have been estimated to have fallen ill with TB in 2014: 5.4 million men, 3.2 million women, and 1.0 million children. To reduce this burden, detection and treatment gaps must be addressed and new tools developed (Global TB report 2015). Seroreactivity of the purified excretory secretory (ES) antigens ES-31, ES-43, ES-41, and ES-6 have been assessed in pulmonary TB (fresh, relapse, chronic, and latent), extrapulmonary TB, and in human immunodeficiency virus-TB coinfection. Analysis of immune response to these purified antigens by indirect and sandwich enzyme-linked immunosorbent assay (ELISA) using sensitive penicillinase enzyme-immuno assay, showed ES-31 antigen as having good diagnostic potential in pulmonary TB and in certain groups of extrapulmonary TB, in particular tuberculous lymphadenopathy, tuberculous meningitis, whereas ES-41 was found to be more seroreactive in abdominal and bone and joint TB. ES-43 antigen was primarily recognized by serum antibodies in relapse cases, while ES-6 was useful in contacts. Antigen assay was found to be more sensitive than antibody-based assay for detecting TB with human immunodeficiency virus coinfection. Immunomonitoring for the presence of antigens in TB patients under antitubercular treatment showed that ES-31 antigen assay was useful in determining the effectiveness of therapy and the patient's compliance. User-friendly peroxidase ELISA has been standardized for the detection of circulating mycobacterial ES-31 serine protease (free antigen and immune-complexed antigen) with 70-75% sensitivity and 90% specificity and with a limit of detection of antigen at 1ng/2μL (0.5μg/mL serum). In-house developed SEVA TB ELISA assay using a cocktail of antigens (ES-31+EST-6) and a cocktail of specific antibodies is being routinely done for screening of

  14. Inhalation Forms of Ambroxol in the Therapy of Respiratory Tract Secretory-Evacuation Disorders in Acute Bronchitis in Children

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    L.S. Ovcharenko

    2014-04-01

    Full Text Available The mucoactive therapy of children aged 1 to 5 years with acute bronchitis using drug Lasolvan solution for oral and inhalation use or syrup for oral use Lasolvan is highly effective and safe method for management of respiratory tract secretory-evacuation disorders. Given the chance of inhalation therapy, effective treatment of choice, while maintaining a high safety profile, is inhalation of Lasolvan solution for oral and inhalation use by nebulizer. More rapid decline in the clinical severity of acute bronchitis in children treated with inhaled Lasolvan associated with accelerated recovery of disturbed secretory-evacuation mechanisms of bronchial tree.

  15. Transcriptome profiles of the protoscoleces of Echinococcus granulosus reveal that excretory-secretory products are essential to metabolic adaptation.

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    Wei Pan

    2014-12-01

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

  16. Proteomic analysis of the excretory-secretory products from larval stages of Ascaris suum reveals high abundance of glycosyl hydrolases.

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    Tao Wang

    Full Text Available BACKGROUND: Ascaris lumbricoides and Ascaris suum are socioeconomically important and widespread parasites of humans and pigs, respectively. The excretory-secretory (ES molecules produced and presented at the parasite-host interface during the different phases of tissue invasion and migration are likely to play critical roles in the induction and development of protective immune and other host responses. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to identify the ES proteins of the different larval stages (L3-egg, L3-lung and L4 by LC-MS/MS. In total, 106 different proteins were identified, 20 in L3-egg, 45 in L3-lung stage and 58 in L4. Although most of the proteins identified were stage-specific, 15 were identified in the ES products of at least two stages. Two proteins, i.e. a 14-3-3-like protein and a serpin-like protein, were present in the ES products from the three different larval stages investigated. Interestingly, a comparison of ES products from L4 with those of L3-egg and L3-lung showed an abundance of metabolic enzymes, particularly glycosyl hydrolases. Further study indicated that most of these glycolytic enzymes were transcriptionally upregulated from L4 onwards, with a peak in the adult stage, particularly in intestinal tissue. This was also confirmed by enzymatic assays, showing the highest glycosidase activity in protein extracts from adult worms gut. CONCLUSIONS/SIGNIFICANCE: The present proteomic analysis provides important information on the host-parasite interaction and the biology of the migratory stages of A. suum. In particular, the high transcriptional upregulation of glycosyl hydrolases from the L4 stage onwards reveals that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine.

  17. Proteomic analysis of the excretory-secretory products from larval stages of Ascaris suum reveals high abundance of glycosyl hydrolases.

    Science.gov (United States)

    Wang, Tao; Van Steendam, Katleen; Dhaenens, Maarten; Vlaminck, Johnny; Deforce, Dieter; Jex, Aaron R; Gasser, Robin B; Geldhof, Peter

    2013-01-01

    Ascaris lumbricoides and Ascaris suum are socioeconomically important and widespread parasites of humans and pigs, respectively. The excretory-secretory (ES) molecules produced and presented at the parasite-host interface during the different phases of tissue invasion and migration are likely to play critical roles in the induction and development of protective immune and other host responses. The aim of this study was to identify the ES proteins of the different larval stages (L3-egg, L3-lung and L4) by LC-MS/MS. In total, 106 different proteins were identified, 20 in L3-egg, 45 in L3-lung stage and 58 in L4. Although most of the proteins identified were stage-specific, 15 were identified in the ES products of at least two stages. Two proteins, i.e. a 14-3-3-like protein and a serpin-like protein, were present in the ES products from the three different larval stages investigated. Interestingly, a comparison of ES products from L4 with those of L3-egg and L3-lung showed an abundance of metabolic enzymes, particularly glycosyl hydrolases. Further study indicated that most of these glycolytic enzymes were transcriptionally upregulated from L4 onwards, with a peak in the adult stage, particularly in intestinal tissue. This was also confirmed by enzymatic assays, showing the highest glycosidase activity in protein extracts from adult worms gut. The present proteomic analysis provides important information on the host-parasite interaction and the biology of the migratory stages of A. suum. In particular, the high transcriptional upregulation of glycosyl hydrolases from the L4 stage onwards reveals that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine.

  18. Nilotinib Does Not Alter the Secretory Functions of Carotid Artery Endothelial Cells in a Prothrombotic or Antithrombotic Fashion.

    Science.gov (United States)

    Katgı, Abdullah; Sevindik, Ömür Gökmen; Gökbulut, Aysun Adan; Özsan, Güner Hayri; Yüksel, Faize; Solmaz, Şerife Medeni; Alacacıoğlu, İnci; Özcan, Mehmet Ali; Demirkan, Fatih; Baran, Yusuf; Pişkin, Özden

    2015-10-01

    There have been concerns about the possible prothrombotic effects of nilotinib, especially in patients having cardiovascular risk factors. The potential mechanism behind the increased risk of thromboembolic events is still not clear. In this study, we aimed to evaluate possible harmful effects of nilotinib on endothelial cells. To this aim, we examined proliferative capacity and secretory functions of healthy human carotid artery endothelial cells (HCtAECs) in response to nilotinib. 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation method was used to determine antiproliferative effects of nilotinib on HCtAECs. The HCtAECs were incubated with 5, 10, and 100 nmol/L doses of nilotinib for 72 hours. Then, in order to assess the endothelial function, levels of nitric oxide (NO), von Willebrand factor (vWF), tissue plasminogen activator, plasminogen activator inhibitor 1 (PAI-1), and endothelin 1 (ET-1) were evaluated using enzyme-linked immunosorbent assay from tissue culture supernatants. There were slight but statistically significant decreases in cell proliferation in response to nilotinib. Nilotinib increased the secretion of t-PA, PAI-1, and vWF in a dose-dependent manner when compared with the untreated control group. The ET-1 secretion was lower in 5 nmol/L and higher in 10 and 100 nmol/L nilotinib-treated cells as compared to untreated cells. Regarding NO secretion, lower levels were observed in 5 and 10 nmol/L, and higher levels were detected in 100 nmol/L nilotinib-treated cells as compared to untreated control group cells. Considering the results obtained in our study, nilotinib does not affect the functions of endothelial cells either in a prothrombotic or an antithrombotic fashion, despite a dose-dependent decline in cell viability. © The Author(s) 2014.

  19. βIII spectrin regulates the structural integrity and the secretory protein transport of the Golgi complex.

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    Salcedo-Sicilia, Laia; Granell, Susana; Jovic, Marko; Sicart, Adrià; Mato, Eugenia; Johannes, Ludger; Balla, Tamas; Egea, Gustavo

    2013-01-25

    A spectrin-based cytoskeleton is associated with endomembranes, including the Golgi complex and cytoplasmic vesicles, but its role remains poorly understood. Using new generated antibodies to specific peptide sequences of the human βIII spectrin, we here show its distribution in the Golgi complex, where it is enriched in the trans-Golgi and trans-Golgi network. The use of a drug-inducible enzymatic assay that depletes the Golgi-associated pool of PI4P as well as the expression of PH domains of Golgi proteins that specifically recognize this phosphoinositide both displaced βIII spectrin from the Golgi. However, the interference with actin dynamics using actin toxins did not affect the localization of βIII spectrin to Golgi membranes. Depletion of βIII spectrin using siRNA technology and the microinjection of anti-βIII spectrin antibodies into the cytoplasm lead to the fragmentation of the Golgi. At ultrastructural level, Golgi fragments showed swollen distal Golgi cisternae and vesicular structures. Using a variety of protein transport assays, we show that the endoplasmic reticulum-to-Golgi and post-Golgi protein transports were impaired in βIII spectrin-depleted cells. However, the internalization of the Shiga toxin subunit B to the endoplasmic reticulum was unaffected. We state that βIII spectrin constitutes a major skeletal component of distal Golgi compartments, where it is necessary to maintain its structural integrity and secretory activity, and unlike actin, PI4P appears to be highly relevant for the association of βIII spectrin the Golgi complex.

  20. Gastrin upregulates the prosurvival factor secretory clusterin in adenocarcinoma cells and in oxyntic mucosa of hypergastrinemic rats.

    Science.gov (United States)

    Fjeldbo, Christina Sæten; Bakke, Ingunn; Erlandsen, Sten Even; Holmseth, Jannicke; Lægreid, Astrid; Sandvik, Arne K; Thommesen, Liv; Bruland, Torunn

    2012-01-01

    We show that the gastric hormone gastrin induces the expression of the prosurvival secretory clusterin (sCLU) in rat adenocarcinoma cells. Clusterin mRNA was still upregulated in the presence of the protein synthesis inhibitor cycloheximide, although at a lower level. This indicates that gastrin induces clusterin transcription independently of de novo protein synthesis but requires de novo protein synthesis of signal transduction pathway components to achieve maximal expression level. Luciferase reporter assay indicates that the AP-1 transcription factor complex is involved in gastrin-mediated activation of the clusterin promoter. Gastrin-induced clusterin expression and subsequent secretion is dependent on sustained treatment, because removal of gastrin after 1-2 h abolished the response. Neutralization of secreted clusterin by a specific antibody abolished the antiapoptotic effect of gastrin on serum starvation-induced apoptosis, suggesting that extracellular clusterin is involved in gastrin-mediated inhibition of apoptosis. The clusterin response to gastrin was validated in vivo in hypergastrinemic rats, showing increased clusterin expression in the oxyntic mucosa, as well as higher levels of clusterin in plasma. In normal rat oxyntic mucosa, clusterin protein was strongly expressed in chromogranin A-immunoreactive neuroendocrine cells, of which the main cell type was the histidine decarboxylase-immunoreactive enterochromaffin-like (ECL) cell. The association of clusterin with neuroendocrine differentiation was further confirmed in human gastric ECL carcinoids. Interestingly, in hypergastrinemic rats, clusterin-immunoreactive cells formed distinct groups of diverse cells at the base of many glands. Our results suggest that clusterin may contribute to gastrin's growth-promoting effect on the oxyntic mucosa.

  1. Salivary secretory leukocyte protease inhibitor (SLPI) and head and neck cancer: The Cancer Prevention Study II Nutrition Cohort

    Science.gov (United States)

    Campbell, Christine M. Pierce; Giuliano, Anna R.; Torres, B. Nelson; O'Keefe, Michael T.; Ingles, Donna J.; Anderson, Rebecca L.; Teras, Lauren R.; Gapstur, Susan M.

    2016-01-01

    Objectives Secretory leukocyte protease inhibitor (SLPI) is an innate-immunity protein displaying antimicrobial and anti-inflammatory properties that is found in high concentrations in saliva. The role of extracellular salivary SLPI in head and neck squamous cell carcinoma (HNSCC) remains unclear. Thus, we aimed to evaluate the association between SLPI and HNSCC risk in the Cancer Prevention Study II Nutrition Cohort. Materials and Methods Among 53,180 men and women with no history of cancer who provided an oral rinse between 2001 and 2002, 60 were subsequently diagnosed with incident HNSCC between specimen collection and June 2009. In this nested case-control study, archived oral supernatants were evaluated using the Human SLPI Quantikine ELISA Kit for all 60 cases and 180 controls individually matched on gender, race, date of birth, and date of oral rinse collection. Conditional logistic regression was used to estimate HNSCC risk. Results Overall, pre-diagnostic salivary SLPI was associated with a non-statistically significant higher risk of HNSCC (OR=1.6, 95% CI=0.9–3.0). Among never smokers, high SLPI was associated with a non-statistically significant lower risk (OR=0.5, 95% CI=0.1–1.9), whereas among ever smokers, high SLPI was associated with a statistically significant higher risk (OR=2.1, 95% CI=1.0–4.3) of HNSCC, compared to low SLPI. Conclusion While results from this study suggest that higher concentrations of salivary SLPI might increase the risk of HNSCC among ever smokers, more research is needed to verify these findings and define the mechanisms by which SLPI and smoking influence the etiology of HNSCC. PMID:27016010

  2. Murine leukemia virus spreading in mice impaired in the biogenesis of secretory lysosomes and Ca2+-regulated exocytosis.

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    Wai-Tsing Chan

    2008-07-01

    Full Text Available Retroviruses have been observed to bud intracellularly into multivesicular bodies (MVB, in addition to the plasma membrane. Release from MVB is thought to occur by Ca(2+-regulated fusion with the plasma membrane.To address the role of the MVB pathway in replication of the murine leukemia virus (MLV we took advantage of mouse models for the Hermansky-Pudlak syndrome (HPS and Griscelli syndrome. In humans, these disorders are characterized by hypopigmentation and immunological alterations that are caused by defects in the biogenesis and trafficking of MVBs and other lysosome related organelles. Neonatal mice for these disease models lacking functional AP-3, Rab27A and BLOC factors were infected with Moloney MLV and the spread of virus into bone marrow, spleen and thymus was monitored. We found a moderate reduction in MLV infection levels in most mutant mice, which differed by less than two-fold compared to wild-type mice. In vitro, MLV release form bone-marrow derived macrophages was slightly enhanced. Finally, we found no evidence for a Ca(2+-regulated release pathway in vitro. Furthermore, MLV replication was only moderately affected in mice lacking Synaptotagmin VII, a Ca(2+-sensor regulating lysosome fusion with the plasma membrane.Given that MLV spreading in mice depends on multiple rounds of replication even moderate reduction of virus release at the cellular level would accumulate and lead to a significant effect over time. Thus our in vivo and in vitro data collectively argue against an essential role for a MVB- and secretory lysosome-mediated pathway in the egress of MLV.

  3. Export of a Cysteine-Free Misfolded Secretory Protein from the Endoplasmic Reticulum for Degradation Requires Interaction with Protein Disulfide Isomerase

    Science.gov (United States)

    Gillece, Pauline; Luz, José Manuel; Lennarz, William J.; de la Cruz, Francisco Javier; Römisch, Karin

    1999-01-01

    Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane. PMID:10613903

  4. Unmasking MASC: bringing to light the unique morphologic, immunohistochemical and genetic features of the newly recognized mammary analogue secretory carcinoma of salivary glands.

    Science.gov (United States)

    Bishop, Justin A

    2013-03-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland neoplasm that is characterized by its striking morphologic and molecular similarities to secretory carcinoma of the breast. This review highlights the characteristic clinical, histologic, immunophenotypic, and molecular features of MASC, and draws attention to the differential diagnosis of this increasingly recognized tumor.

  5. [Clinical importance of tympanometry in the diagnosis of chronic secretory otitis].

    Science.gov (United States)

    Spremo, S; Markić, Z; Kurbalija

    1998-01-01

    Secretory otitis media is defined as a fluid in the middle ear without signs or symptoms of infection. As the aetiology and pathogenesis of the disease are unknown, and as it affects children aged from 3 to 12 years, treatment procedures proposed for management of secretory otitis media, are not uniform. Some authors [1, 4, 6] consider that functional or mechanical obstructions of the Eustachian tube could provoke secretory otitis. The purpose of the treatment is to remove exudate from the middle ear and appropriately ventilate it for a longer period. That could instantly normalize the hearing and exclude the appearance of late complications of secretory otitis. Although the disease could heal spontaneously, the treatment should be performed immediately for preventing sequelae of secretory otitis. The aim of the study was to evaluate possible aetiologic factors of secretory otitis in our population, and to evaluate results of lympanometry in children with exudate in the middle ear. There were 65 children, aged from 3 to 12 years (Table 1), who complained of deafness and were examined at the ORL Department in Banja Luka. The clinical examination revealed the integrity and color of tympanic membrane, scars, adhesions and atrophic areas. Audiometry and tympanometry had been performed in addition. Patients who proved to have exudate in the middle ear received nasal decongestants and mucolitics during three months, and were evaluated every three weeks by audiometry and tympanometry. Pathologic findings in the nose and epipharynx were the most common findings: enlarged adenoids in 38 (58%) patients, hypetrophic rhinitis in 15 (23%) and allergic rhinitis in 5 (8%) patients. Frequent relapses of middle ear infection in the first three years of life were found in 26 (40%) patients and early first attacks in the first year of life in 15 (23%) patients (Table 2). Premature onset (15%) and allergy (21%) had also been frequently found. Results of tympanometry and audiometry are

  6. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

    Directory of Open Access Journals (Sweden)

    Klatt Stephan

    2012-07-01

    Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

  7. Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

    Science.gov (United States)

    Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

    2014-01-01

    The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.

  8. Effects of excretory/secretory products from Anisakis simplex (Nematoda) on immune gene expression in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Bahlool, Qusay Zuhair Mohammad; Skovgaard, Alf; Kania, Per Walter

    2013-01-01

    Excretory/secretory (ES) products are molecules produced by parasitic nematodes, including larval Anisakis simplex, a parasite occurring in numerous marine fish hosts. The effects of these substances on host physiology have not been fully described. The present work elucidates the influence of ES...

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

    Science.gov (United States)

    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.

  10. Use of cloned excretory/secretory low-molecular-weight proteins of Cooperia oncophora in a serological assay

    NARCIS (Netherlands)

    Poot, J.; Kooyman, F. N.; Dop, P. Y.; Schallig, H. D.; Eysker, M.; Cornelissen, A. W.

    1997-01-01

    The potential of Cooperia oncophora excretory/secretory (ES) proteins as antigens in a serological assay which aims to establish exposure levels in cattle was assessed. ES proteins were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis and immunoblotting. The N-terminal domains

  11. Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites.

    Science.gov (United States)

    Lefèbvre, Fabien; Prouzet-Mauléon, Valérie; Hugues, Michel; Crouzet, Marc; Vieillemard, Aurélie; McCusker, Derek; Thoraval, Didier; Doignon, François

    2012-05-01

    Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P(2) production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

  12. Expression and location of mRNAs encoding multiple forms of secretory phospholipase A2 in the rat retina

    DEFF Research Database (Denmark)

    Kolko, Miriam; Christoffersen, Nanna R; Barreiro, Sebastian G

    2004-01-01

    Low-molecular-weight secretory phospholipases A(2) (sPLA(2)s) are a subgroup of PLA(2)s, which are secreted, bind to receptors, and may act as intercellular signaling modulators. At least 10 different groups have been characterized in mammals, and there is expanding evidence of the significance...

  13. Lactoferrin and secretory IgA in the bronchoalveolar lavage fluid from patients with a stable asthma

    NARCIS (Netherlands)

    van de Graaf, E. A.; Out, T. A.; Kobesen, A.; Jansen, H. M.

    1991-01-01

    We have measured lactoferrin and secretory IgA (sIgA) in the unconcentrated bronchoalveolar lavage fluid (BALF) from nonsmoking healthy volunteers (n = 10) and nonsmoking patients with stable asthma (n = 14). The median concentrations and the ranges of lactoferrin were controls, 0.13 mg/L (0.01-0.43

  14. Mammary analogue secretory carcinoma of salivary gland origin: an update and expanded morphologic and immunohistochemical spectrum of recently described entity.

    Science.gov (United States)

    Skalova, Alena

    2013-07-01

    Mammary analogue secretory carcinoma of salivary gland origin (MASC) is a recently described tumor with ETV6 translocation. Akin to secretory breast cancer, MASC expresses S-100 protein, mammaglobin, vimentin, and harbors a t(12;15) (p13;q25) translocation which leads to ETV6-NTRK3 fusion product. Histologically, MASC displays a lobulated growth pattern and is often composed of microcystic, tubular, and solid structures with abundant eosinophilic homogeneous or bubbly secretions. Colloid-like secretory material stains positive for periodic acid-Schiff (PAS) with and without diastase and for Alcian blue. The cells of MASC are devoid of PAS-positive secretory zymogen granules. These features help to exclude the most important differential diagnostic considerations, namely acinic cell carcinoma, low-grade cribriform cystadenocarcinoma, cystadenocarcinoma (not otherwise specified), and low-grade mucoepidermoid carcinoma. To date the presence of the ETV6-NTRK3 fusion gene has not been demonstrated in any other salivary gland tumor than MASC. It is likely that MASC is more common than currently recognized and with further studies, the clinical need for molecular studies of the ETV6-NTRK3 fusion may diminish. However, molecular testing is recommended at this time to arrive at the diagnosis of MASC.

  15. Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.

    Directory of Open Access Journals (Sweden)

    Kerry Routenberg Love

    Full Text Available Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

  16. The Senescence-Associated Secretory Phenotype: Critical Effector in Skin Cancer and Aging

    Science.gov (United States)

    Ghosh, Kanad; Capell, Brian C.

    2017-01-01

    Cellular senescence, a state of stable cell cycle arrest in response to cellular stress, is an indispensable mechanism to counter tumorigenesis by halting the proliferation of damaged cells. However, through the secretion of an array of diverse cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP), senescent cells can paradoxically promote carcinogenesis. Consistent with this, removal of senescent cells delays the onset of cancer and prolongs lifespan in vivo, potentially in part through SASP reduction. In this review, we consider the evidence for the SASP and “SASP-like” inflammation in driving skin carcinogenesis, emphasizing how further understanding of both the roles and mechanisms of SASP expression may offer new targets for skin cancer prevention and therapy. PMID:27543988

  17. The Senescence-Associated Secretory Phenotype: Critical Effector in Skin Cancer and Aging.

    Science.gov (United States)

    Ghosh, Kanad; Capell, Brian C

    2016-11-01

    Cellular senescence, a state of stable cell cycle arrest in response to cellular stress, is an indispensable mechanism to counter tumorigenesis by halting the proliferation of damaged cells. However, through the secretion of an array of diverse cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP), senescent cells can paradoxically promote carcinogenesis. Consistent with this, removal of senescent cells delays the onset of cancer and prolongs lifespan in vivo, potentially in part through SASP reduction. In this review, we consider the evidence for the SASP and "SASP-like" inflammation in driving skin carcinogenesis, emphasizing how further understanding of both the roles and mechanisms of SASP expression may offer new targets for skin cancer prevention and therapy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  18. On psychobiology in psychoanalysis - salivary cortisol and secretory IgA as psychoanalytic process parameters

    Science.gov (United States)

    Euler, Sebastian; Schimpf, Heinrich; Hennig, Jürgen; Brosig, Burkhard

    2005-01-01

    This study investigates the psychobiological impact of psychoanalysis in its four-hour setting. During a period of five weeks, 20 subsequent hours of psychoanalysis were evaluated, involving two patients and their analysts. Before and after each session, saliva samples were taken and analysed for cortisol (sCortisol) and secretory immunoglobuline A (sIgA). Four time-series (n=80 observations) resulted and were evaluated by "Pooled Time Series Analysis" (PTSA) for significant level changes and setting-mediated rhythms. Over all sessions, sCortisol levels were reduced and sIgA secretion augmented parallel to the analytic work. In one analytic dyad a significant rhythm within the four-hour setting was observed with an increase of sCortisol in sessions 2 and 3 of the week. Psychoanalysis may, therefore, have some psychobiological impact on patients and analysts alike and may modulate immunological and endocrinological processes. PMID:19742067

  19. The secretory endometrial protein, placental protein 14, in women with ectopic gestation

    DEFF Research Database (Denmark)

    Ruge, S; Sørensen, Steen; Vejtorp, M

    1992-01-01

    women with uncomplicated pregnancies and normal outcome. SETTING: The women were admitted to a university hospital. PATIENTS: Fifty-nine women with laparoscopically verified ectopic pregnancy entered the study. INTERVENTION: At the time of diagnosis PP14 and P were measured. MAIN OUTCOME MEASURE: After......OBJECTIVE: To determine the serum level of the secretory endometrial protein, placental protein 14 (PP14) and progesterone (P) in women with ectopic gestation. DESIGN: Blood samples were collected prospectively and preoperatively. Reference range was determined from a prospective population of 98...... observing the low serum levels of PP14 and P, a correlation analysis was made and compared with the findings in normally pregnant women. RESULTS: A significant positive correlation was found between the level of PP14 and P (P less than 0.00002), not found in normal intrauterine pregnancies. CONCLUSIONS...

  20. Comparison of ion transport by cultured secretory and absorptive canine airway epithelia

    DEFF Research Database (Denmark)

    Boucher, R C; Larsen, Erik Hviid

    1988-01-01

    The use of primary cell culture techniques to predict the function of native respiratory epithelia was tested in studies of dog airway epithelia. Epithelial cells from Cl- secretory (tracheal) and Na+ absorptive (bronchial) airway regions were isolated by enzymatic digestion, plated on collagen...... matrices, and maintained in serum-free, hormone-supplemented media. Transepithelial and intracellular studies showed that both the tracheal and bronchial culture preparations exhibited bioelectric parameters quantitatively similar to those of intact tissues. Similar to the native tissue, the tracheal...... preparation exhibited an equivalent short-circuit circuit (Ieq) that was sensitive to inhibitors of Cl- transport (bumetanide, diphenylamine carboxylic acid) but was insensitive to an inhibitor of Na+ transport, amiloride. In contrast, the bronchial preparation, like the native tissue, exhibited an Ieq...