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Sample records for seca2-dependent secretory protein

  1. Suppressor analysis reveals a role for SecY in the SecA2-dependent protein export pathway of Mycobacteria.

    Science.gov (United States)

    Ligon, Lauren S; Rigel, Nathan W; Romanchuk, Artur; Jones, Corbin D; Braunstein, Miriam

    2013-10-01

    All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins.

  2. 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...

  3. 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.

  4. 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.

  5. 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.

  6. 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 ...

  7. 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...

  8. 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...

  9. 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

  10. 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...

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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...

  17. Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona.

    Science.gov (United States)

    Hayashi, Yuya; Miclaus, Teodora; Engelmann, Péter; Autrup, Herman; Sutherland, Duncan S; Scott-Fordsmand, Janeck J

    2016-01-01

    Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affect the corona composition, the extent to which nanoparticles influence the cells' protein secretion profile is of remarkable interest that has rarely acquired attention. Here, we have probed transcriptional responses of E. fetida coelomocytes to the representative nanosilver NM-300K (15 nm) in a time-dependent manner (2, 4, 8 and 24 h at a low-cytotoxic concentration), and examined the implication of the temporal changes in transcriptional profiles of secretory proteins with a particular reference to that of lysenin. NM-300K was accumulated in/at the cells and lysenin was, after transient induction, gradually suppressed over time indicating a negative feedback cycle. This may limit further enrichment of lysenin in the corona and thereby decrease the lysenin-assisted uptake of the nanoparticles. Other differentially expressed genes were those involved in metal stress (likewise in AgNO3-stressed cells) and in Toll-like receptor (TLR) signaling. This offers an intriguing perspective of the nanosilver pathophysiology in earthworms, in which the conserved pattern recognition receptor TLRs may play an effector role.

  18. tor/mouse secretory phospholipase A2 proteins

    Indian Academy of Sciences (India)

    Unknown

    amino acid protein with a 21 amino acid long signal pep- tide. A 100 kDa cell .... Site-directed mutagenesis of EF/sPLA2. 491 air dried and reconstituted in 1 M acetic acid. The acid- soluble proteins were dialysed against 0⋅1 M acetic acid at 4ºC. ... wtEF produced mutant GE1, mutant GE2 and wild type. EF protein ...

  19. 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.

  20. 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

  1. Machine Learning of Protein Interactions in Fungal Secretory Pathways.

    Science.gov (United States)

    Kludas, Jana; Arvas, Mikko; Castillo, Sandra; Pakula, Tiina; Oja, Merja; Brouard, Céline; Jäntti, Jussi; Penttilä, Merja; Rousu, Juho

    2016-01-01

    In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker's yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities.

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

    Directory of Open Access Journals (Sweden)

    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.

  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. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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

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

    Science.gov (United States)

    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.

  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. 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).

  13. Comparative proteome analysis of secretory proteins from pathogenic and nonpathogenic Listeria species.

    Science.gov (United States)

    Trost, Matthias; Wehmhöner, Dirk; Kärst, Uwe; Dieterich, Guido; Wehland, Jürgen; Jänsch, Lothar

    2005-04-01

    Extracellular proteins of bacterial pathogens play a crucial role in the infection of the host. Here we present the first comprehensive validation of the secretory subproteome of the Gram positive pathogen Listeria monocytogenes using predictive bioinformatic and experimental proteomic approaches. The previous original signal peptide (SP) prediction (Glaser et al., Science 2001, 294, 849-852) has been greatly improved by an in-depth analysis using seven different bioinformatic tools. Subsequent careful classification of the resulting data gives a probability dependent annotation of 121 putatively secreted proteins of which 45 are novel. Complementary proteomic analysis using both two-dimensional gel electrophoresis/matrix assisted laser desorption/ionization mass spectrometry and high performance liquid chromatography/electrospray ionization-mass spectrometry has identified 105 proteins in the culture supernatant of L. monocytogenes. Among these, we were able to detect all the currently known virulence factors with an SP showing the importance of this subproteome and demonstrating the reliability of the techniques used. The comparison between the L. monocytogenes wildtype and the nonpathogenic species Listeria innocua was performed to reveal proteins probably involved in pathogenicity and/or the adaptation to their respective lifestyles. In addition to the eight known virulence factors, all of which have no orthologous genes in L. innocua, eight additional proteins have been identified that exhibit the typical key feature defining the known listerial virulence factors. Further significant differences between the two species are evident in the group of cell wall and secretory proteins that warrant further study. Our investigation clearly demonstrates that the major difference between the pathogenic and nonpathogenic species, noted in the comparative genome analysis, manifests itself strongest in the secretome.

  14. 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.

  15. 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

  16. 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....

  17. 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

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

    Directory of Open Access Journals (Sweden)

    Maria Francesca Mossuto

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

  19. 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.

  20. 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.

  1. Apocrine Secretion in Drosophila Salivary Glands: Subcellular Origin, Dynamics, and Identification of Secretory Proteins

    Science.gov (United States)

    Farkaš, Robert; Ďatková, Zuzana; Mentelová, Lucia; Löw, Péter; Beňová-Liszeková, Denisa; Beňo, Milan; Sass, Miklós; Řehulka, Pavel; Řehulková, Helena; Raška, Otakar; Kováčik, Lubomír; Šmigová, Jana; Raška, Ivan; Mechler, Bernard M.

    2014-01-01

    In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal β-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity. PMID:24732043

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Altered synthesis of some secretory proteins in pancreatic lobules isolated from streptozotocin-induced diabetic rats

    Energy Technology Data Exchange (ETDEWEB)

    Duan, R.D.; Erlanson-Albertsson, C. (Univ. of Lund (Sweden))

    1990-03-01

    The in vitro incorporation of (35S)cysteine into lipase, colipase, amylase, procarboxypeptidase A and B, and the serine proteases and total proteins was studied in pancreatic lobules isolated from normal and diabetic rats with or without insulin treatment. The incorporation of (35S)cysteine into total proteins was 65% greater in pancreatic lobules from diabetic animals than from normal rats. The increased incorporation was partly reversed by insulin treatment (2 U/100 g/day for 5 days) of diabetic rats. The relative rates of biosynthesis for amylase and the procarboxypeptidases in diabetic pancreatic lobules were decreased by 75 and 25%, respectively, after 1 h of incubation, while those for lipase, colipase, and the serine proteases were increased by 90, 85, and 35%, respectively. The absolute rates of synthesis for these enzymes changed in the same direction as the relative rates in diabetic lobules, except that for the procarboxypeptidases, which did not change. The changed rates of biosynthesis for the pancreatic enzymes were reversed by insulin treatment of the diabetic rats. Kinetic studies showed that the incorporation of (35S)cysteine into amylase, lipase, and colipase was linear until up to 2 h of incubation in normal pancreatic lobules, while in the diabetic lobules the incorporation into lipase and colipase was accelerated, reaching a plateau level already after 1 h of incubation. It is concluded that the biosynthesis of pancreatic secretory proteins in diabetic rats is greatly changed both in terms of quantity and kinetics.

  4. Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.

    Science.gov (United States)

    Fujita-Yoshigaki, Junko; Matsuki-Fukushima, Miwako; Yokoyama, Megumi; Katsumata-Kato, Osamu

    2013-11-15

    The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a β-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.

  5. Bacterial lipid modification of proteins requires appropriate secretory signals even for expression - implications for biogenesis and protein engineering.

    Science.gov (United States)

    Kumar, Subramani; Balamurali, M M; Sankaran, Krishnan

    2014-09-01

    Sec- and Tat-mediated bacterial lipid modification of proteins are important posttranslational processes owing to their vital roles in cellular functions, membrane targeting and biotechnological applications like ELISA, biosensor, adjuvant-free vaccines, liposomal drug delivery etc. However a better understanding of the tight coupling of secretory and lipid modification machineries and the processes associated will help unravel this essential biological event and utilize it for engineering applications. Further, there is a need for a systematic and convincing investigation into membrane targeting, solubilization and ease-of-purification of engineered lipoproteins to facilitate scientists in readily applying this new protein engineering tool. Therefore, in this study, we have investigated systematically recombinant expression, translocation, solubilization and purification of three White Spot Syndrome Viral (WSSV) proteins, ICP11, VP28 and VP281. Our study shows that the lipid modification and secretion processes are tightly coupled to the extent that mismatch between folding kinetics and signal sequence of target proteins could lead to transcriptional-translational uncoupling or aborted translation. The proteins expressed as lipoproteins through Tat-pathway were targeted to the inner membrane achieving considerable enrichment. These His-tagged proteins were then purified to apparent homogeneity in detergent-free form using single-step Immobilized Metal Affinity Chromatography. This study has interesting findings in lipoprotein biogenesis enhancing the scope of this unique post-translational protein engineering tool for obtaining pure detergent-free, membrane or hydrophobic surface-associating diagnostic targets and vaccine candidates for WSSV.

  6. Club cell secretory protein improves survival in a murine obliterative bronchiolitis model.

    Science.gov (United States)

    Wendt, Christine; Tram, Kevin; Price, Andrew; England, Kristen; Stiehm, Andrew; Panoskaltsis-Mortari, Angela

    2013-11-01

    Club cell secretory protein (CCSP) is an indirect phospholipase A2 inhibitor with some immunosuppressive and antiproliferative properties that is expressed in bronchiolar Club cells. In our murine bone marrow transplant (BMT) model of obliterative bronchiolitis (OB), CCSP is diminished; however, its role is unknown. To determine the role of CCSP, B6 wild-type (WT) or CCSP-deficient (CCSP(-/-)) mice were lethally conditioned and given allogeneic bone marrow with a sublethal dose of allogeneic splenic T cells to induce OB. We found that CCSP(-/-) mice demonstrated a higher mortality following BMT-induced OB compared with WT mice. Mice were analyzed 60 days post-BMT for protein expression, pulmonary function, and histology. CCSP levels were reduced in WT mice with BMT-induced OB, and lower levels correlated to decreased lung compliance. CCSP(-/-) had a higher degree of injury and fibrosis as measured by hydroxy proline, along with an increased lung resistance and the inflammatory markers, leukotriene B4 and CXCL1. Replacement with recombinant intravenous CCSP partially reversed the weight loss and improved survival in the CCSP(-/-) mice. In addition, CCSP replacement improved histology and decreased inflammatory cells and markers. These findings indicate that CCSP has a regulatory role in OB and may have potential as a preventive therapy.

  7. 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].

  8. 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.

  9. 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.

  10. Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona

    DEFF Research Database (Denmark)

    Hayashi, Yuya; Miclaus, Teodora; Engelmann, Péter

    2016-01-01

    Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affect...... the corona composition, the extent to which nanoparticles influence the cells’ protein secretion profile is of remarkable interest that has rarely acquired attention. Here, we have probed transcriptional responses of E. fetida coelomocytes to the representative nanosilver NM-300K (15 nm) in a time...... suppressed over time indicating a negative feedback cycle. This may limit further enrichment of lysenin in the corona and thereby decrease the lysenin-assisted uptake of the nanoparticles. Other differentially expressed genes were those involved in metal stress (likewise in AgNO3-stressed cells) and in Toll...

  11. Secretory proteins are delivered to the septin-organized penetration interface during root infection by Verticillium dahliae

    Science.gov (United States)

    Zhou, Ting-Ting; Zhao, Yun-Long

    2017-01-01

    Successful infection of the host requires secretion of effector proteins to evade or suppress plant immunity. Secretion of effectors in root-infecting fungal pathogens, however, remains unexplored. We previously reported that Verticillium dahliae, a root-infecting phytopathogenic fungus, develops a penetration peg from a hyphopodium to infect cotton roots. In this study, we report that a septin ring, requiring VdSep5, partitions the hyphopodium and the invasive hypha and form the specialized fungus-host interface. The mutant strain, VdΔnoxb, in which NADPH oxidase B (VdNoxB) is deleted, impaired formation of the septin ring at the hyphal neck, indicating that NADPH oxidases regulate septin ring organization. Using GFP tagging and live cell imaging, we observed that several signal peptide containing secreted proteins showed ring signal accumulation/secretion at the penetration interface surrounding the hyphal neck. Targeted mutation for VdSep5 reduced the delivery rate of secretory proteins to the penetration interface. Blocking the secretory pathway by disrupting the vesicular trafficking factors, VdSec22 and VdSyn8, or the exocyst subunit, VdExo70, also arrested delivery of the secreted proteins inside the hyphopodium. Reduced virulence was observed when cotton roots were infected with VdΔsep5, VdΔsec22, VdΔsyn8 and VdΔexo70 mutants compared to infection with the isogenic wild-type V592. Taken together, our data demonstrate that the hyphal neck is an important site for protein secretion during plant root infection, and that the multiple secretory routes are involved in the secretion. PMID:28282450

  12. Surfactant protein B inhibits secretory phospholipase A2 hydrolysis of surfactant phospholipids

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    Grier, Bonnie L.; Waite, B. Moseley; Veldhuizen, Ruud A.; Possmayer, Fred; Yao, Li-Juan; Seeds, Michael C.

    2012-01-01

    Hydrolysis of surfactant phospholipids (PL) by secretory phospholipases A2 (sPLA2) contributes to surfactant damage in inflammatory airway diseases such as acute lung injury/acute respiratory distress syndrome. We and others have reported that each sPLA2 exhibits specificity in hydrolyzing different PLs in pulmonary surfactant and that the presence of hydrophilic surfactant protein A (SP-A) alters sPLA2-mediated hydrolysis. This report tests the hypothesis that hydrophobic SP-B also inhibits sPLA2-mediated surfactant hydrolysis. Three surfactant preparations were used containing varied amounts of SP-B and radiolabeled tracers of phosphatidylcholine (PC) or phosphatidylglycerol (PG): 1) washed ovine surfactant (OS) (pre- and postorganic extraction) compared with Survanta (protein poor), 2) Survanta supplemented with purified bovine SP-B (1–5%, wt/wt), and 3) a mixture of dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (DPPC:POPC:POPG, 40:40:20) prepared as vesicles and monomolecular films in the presence or absence of SP-B. Hydrolysis of PG and PC by Group IB sPLA2 (PLA2G1A) was significantly lower in the extracted OS, which contains SP-B, compared with Survanta (P = 0.005), which is SP-B poor. Hydrolysis of PG and PC in nonextracted OS, which contains all SPs, was lower than both Survanta and extracted OS. When Survanta was supplemented with 1% SP-B, PG and PC hydrolysis by PLA2G1B was significantly lower (P hydrolysis by both PLA2G1B and Group IIA sPLA2 (PLA2G2A). In films, PLA2G1B hydrolyzed surfactant PL monolayers at surface pressures ≤30 mN/m (P hydrolysis can occur. These results suggest the hydrophobic SP, SP-B, protects alveolar surfactant PL from hydrolysis mediated by multiple sPLA2 in both vesicles (alveolar subphase) and monomolecular films (air-liquid interface). PMID:22037357

  13. Personal exposure to fine particulate matter, lung function and serum club cell secretory protein (Clara).

    Science.gov (United States)

    Wang, Cuicui; Cai, Jing; Chen, Renjie; Shi, Jingjin; Yang, Changyuan; Li, Huichu; Lin, Zhijing; Meng, Xia; Liu, Cong; Niu, Yue; Xia, Yongjie; Zhao, Zhuohui; Li, Weihua; Kan, Haidong

    2017-06-01

    The underlying mechanisms about the association between ambient fine particulate matter (PM 2.5 ) and lung function were unclear. Few epidemiological studies have evaluated the potential mediating effects of serum club cell secretory protein (Clara) (CC16), a biomarker of pulmonary epithelium integrity. To evaluate the short-term effect of personal PM 2.5 exposure on lung function and to explore the potential mediating role of CC16 in this effect. We enrolled 36 healthy, nonsmoking college students for a panel study in Shanghai, China from December 17, 2014 to July 11, 2015. We measured personal and real-time exposure to PM 2.5 for 72 h preceding each of four rounds of health examinations, including lung function test and serum CC16 measurement. We used linear mixed-effect models to examine the effects of PM 2.5 on lung function and CC16 over various lag times. Furthermore, we analyzed the mediating effect of CC16 in the association between PM 2.5 and lung function. Average PM 2.5 exposure ranged from 36 to 52 μg/m3 across different lag periods. PM 2.5 exposure was negatively associated with lung function and positively associated with serum CC16 concentration. The effect of PM 2.5 on CC16 occurred earlier than that on lung function. For instance, an interquartile range (IQR) increase in 0-2 h average exposure to PM 2.5 was significantly associated with a 4.84% increase in serum CC16; and an IQR increase in 3-6 h average exposure to PM 2.5 was significantly associated with a 1.08% decrease in 1-sec forced expiratory volume. These effects lasted up to 24 h after exposure. Increased serum CC16 contributed 3.9%-36.3% of the association between PM 2.5 and impaired lung function. Acute exposure to PM 2.5 might induce an immediate decrease in lung function by virtue of the loss of pulmonary epithelium integrity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. High abundance of Serine/Threonine-rich regions predicted to be hyper-O-glycosylated in the secretory proteins coded by eight fungal genomes

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    González Mario

    2012-09-01

    Full Text Available Abstract Background O-glycosylation of secretory proteins has been found to be an important factor in fungal biology and virulence. It consists in the addition of short glycosidic chains to Ser or Thr residues in the protein backbone via O-glycosidic bonds. Secretory proteins in fungi frequently display Ser/Thr rich regions that could be sites of extensive O-glycosylation. We have analyzed in silico the complete sets of putatively secretory proteins coded by eight fungal genomes (Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago maydis, Aspergillus nidulans, Neurospora crassa, Trichoderma reesei, and Saccharomyces cerevisiae in search of Ser/Thr-rich regions as well as regions predicted to be highly O-glycosylated by NetOGlyc (http://www.cbs.dtu.dk. Results By comparison with experimental data, NetOGlyc was found to overestimate the number of O-glycosylation sites in fungi by a factor of 1.5, but to be quite reliable in the prediction of highly O-glycosylated regions. About half of secretory proteins have at least one Ser/Thr-rich region, with a Ser/Thr content of at least 40% over an average length of 40 amino acids. Most secretory proteins in filamentous fungi were predicted to be O-glycosylated, sometimes in dozens or even hundreds of sites. Residues predicted to be O-glycosylated have a tendency to be grouped together forming hyper-O-glycosylated regions of varying length. Conclusions About one fourth of secretory fungal proteins were predicted to have at least one hyper-O-glycosylated region, which consists of 45 amino acids on average and displays at least one O-glycosylated Ser or Thr every four residues. These putative highly O-glycosylated regions can be found anywhere along the proteins but have a slight tendency to be at either one of the two ends.

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

    Science.gov (United States)

    2014-01-01

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

  16. The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

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    Mehnert, Martin; Sommermeyer, Franziska; Berger, Maren; Kumar Lakshmipathy, Sathish; Gauss, Robert; Aebi, Markus; Jarosch, Ernst; Sommer, Thomas

    2015-01-01

    Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1. PMID:25428985

  17. ACCUMULATION OF RECOMBINANT FUSION PROTEINSECRETORY ANALOG OF Ag85B AND ESAT6 MYCOBACTERIUM TUBERCULOSIS PROTEINS – IN TRANSGENIC Lemna minor L. PLANTS

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    A.A.Peterson

    2015-10-01

    Full Text Available Determination of the presence of the recombinant fusion protein (ESAT6-Ag85B(ΔTMD-6His and its accumulation level in duckweed plants (Lemna minor L. was the aim of the research. ESAT6 and Ag85B are secretory proteins of Mycobacterium tuberculosis and are considered as potential candidates for development of new vaccine against tuberculosis (TB. Transgenic duckweed plants were obtained previously by Agrobacterium rhizogenes-mediated transformation and possessed fusion gene sequence esxA-fbpBΔTMD. Specific polyclonal antibodies were produced in immunized mice to identify levels of accumulation of TB antigens in plants. Recombinant antigen used for mice immunization was obtained in our laboratory by expression in E. coli. Western blot analysis revealed the recombinant tuberculosis antigen ESAT6-Ag85B(ΔTMD-6His in extracts from transgenic L. minor plants. The level of accumulation of the protein corresponds to 0.4-0.5 µg protein per 1 g of fresh weight of plant. Additionally, the accumulation of recombinant protein was investigated in lyophilized transgenic plants after 1.5 year storage. Duckweed plants accumulating a recombinant analogue of M. tuberculosis secretory proteins can be used for development of plant-based edible vaccines.

  18. Analyses of advanced rice anther transcriptomes reveal global tapetum secretory functions and potential proteins for lipid exine formation.

    Science.gov (United States)

    Huang, Ming-Der; Wei, Fu-Jin; Wu, Cheng-Cheih; Hsing, Yue-Ie Caroline; Huang, Anthony H C

    2009-02-01

    The anthers in flowers perform important functions in sexual reproduction. Several recent studies used microarrays to study anther transcriptomes to explore genes controlling anther development. To analyze the secretion and other functions of the tapetum, we produced transcriptomes of anthers of rice (Oryza sativa subsp. japonica) at six progressive developmental stages and pollen with sequencing-by-synthesis technology. The transcriptomes included at least 18,000 unique transcripts, about 25% of which had antisense transcripts. In silico anther-minus-pollen subtraction produced transcripts largely unique to the tapetum; these transcripts include all the reported tapetum-specific transcripts of orthologs in other species. The differential developmental profiles of the transcripts and their antisense transcripts signify extensive regulation of gene expression in the anther, especially the tapetum, during development. The transcriptomes were used to dissect two major cell/biochemical functions of the tapetum. First, we categorized and charted the developmental profiles of all transcripts encoding secretory proteins present in the cellular exterior; these transcripts represent about 12% and 30% of the those transcripts having more than 100 and 1,000 transcripts per million, respectively. Second, we successfully selected from hundreds of transcripts several transcripts encoding potential proteins for lipid exine synthesis during early anther development. These proteins include cytochrome P450, acyltransferases, and lipid transfer proteins in our hypothesized mechanism of exine synthesis in and export from the tapetum. Putative functioning of these proteins in exine formation is consistent with proteins and metabolites detected in the anther locule fluid obtained by micropipetting.

  19. Extracellular Vesicles from Parasitic Helminths Contain Specific Excretory/Secretory Proteins and Are Internalized in Intestinal Host Cells

    Science.gov (United States)

    Marcilla, Antonio; Trelis, María; Cortés, Alba; Sotillo, Javier; Cantalapiedra, Fernando; Minguez, María Teresa; Valero, María Luz; Sánchez del Pino, Manuel Mateo; Muñoz-Antoli, Carla; Toledo, Rafael; Bernal, Dolores

    2012-01-01

    The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30–100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents. PMID:23029346

  20. Immunocytochemical localization of secretory phospholipase A(2)-like protein in the pituitary gland and surrounding tissue of the bullfrog, Rana catesbeiana.

    Science.gov (United States)

    Yaoi, Y; Kikuyama, S; Hayashi, H; Hanaoka, Y; Sakai, M; Tanaka, S

    2001-05-01

    Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A(2) (PLA(2)) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA(2)-like protein were observed predominately in these secretory granules. These findings support the view that this PLA(2)-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631-637, 2001)

  1. Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness.

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    Xiao-Yang Wang

    Full Text Available Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+ cells. Moreover, CCSP(+ cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs in terminal bronchioles (TBs. We conclude that CCSP(+ cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

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

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    Kosalková Katarina

    2012-01-01

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

  3. Dirofilaria immitis exhibits sex- and stage-specific differences in excretory/secretory miRNA and protein profiles.

    Science.gov (United States)

    Tritten, Lucienne; Clarke, Damian; Timmins, Scott; McTier, Tom; Geary, Timothy G

    2016-12-15

    The canine heartworm Dirofilaria immitis releases excretory/secretory molecules into its host and in culture. We report analyses of the types, amounts and stage-dependence of microRNAs and proteins found in D. immitis culture media recovered after incubating 800,000 microfilariae for 6days, 500L3 and 500L4 for 7days, as well as 40 adult females and 40 adult males for 48h, all separately. In addition, the presence of exosome-like particles was established by nanoparticle tracking analysis. Our results are in concordance with the D. immitis molecules previously detected in dog blood and in culture medium, but add additional insight into the sex- and stage-specificity of these processes. Of 131 miRNA candidates analyzed, none of the most abundant sequences was exclusively associated with one stage. Several isoforms of the nematode miR-100 family, miR-279, miR-71, were highly represented and overlapped substantially with the profile of heartworm miRNAs described from infected dog blood. lin-4 was primarily associated with males. We also report 4, 27 and 72 proteins in media from microfilariae, females and males, respectively. The only protein in common to all samples was actin, and only 9/88 proteins with a gene ontology description had not been reported in other studies of filarial secretomes. Exosomal proteins were well represented, dominated by cytoskeletal proteins, metabolic enzymes, zeta polypeptide, and chaperones. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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    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

  5. Characterization of the DMAE-modified juvenile excretory-secretory protein Juv-p120 of Litomosoides sigmodontis.

    Science.gov (United States)

    Wagner, Ulrike; Hirzmann, Jörg; Hintz, Martin; Beck, Ewald; Geyer, Rudolf; Hobom, Gerd; Taubert, Anja; Zahner, Horst

    2011-04-01

    Juv-p120 is an excretory-secretory 160 kDa glycoprotein of juvenile female Litomosoides sigmodontis and exhibits features typical for mucins. 50% of its molecular mass is attributed to posttranslational modifications with the unusual substituent dimethylaminoethanol (DMAE). By that Juv-p120 corresponds to the surface proteins of the microfilarial sheath, Shp3 and Shp3a. The secreted protein consists of 697 amino acids, organized in two different domains of repeat elements separated by a stretch of polar residues. The N-terminal domain shows fourteen P/S/T/F-rich repeat elements highly modified with phospho-DMAE substituted O-glycans confering a negative charge to the protein. The C-terminal domain is extremely rich in glutamine (35%) and leucine (25%) in less organized repeats and may play a role in oligomerization of Juv-p120 monomers. A protein family with a similar Q/L-rich region and conserved core promoter region was identified in Brugia malayi by homology screening and in Wuchereria bancrofti and Loa loa by database similarity search. One of the Q/L-rich proteins in each genus has an extended S/T-rich region and due to this feature is supposed to be a putative Juv-p120 ortholog. The corresponding modification of Juv-p120 and the microfilarial sheath surface antigens Shp3/3a explains the appearance of anti-sheath antibodies before the release of microfilariae. The function of Juv-p120 is unknown. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Therapeutic potential of larval excretory/secretory proteins of the pig whipworm Trichuris suis in allergic disease.

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    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.

  7. Diagnosis of Fasciola infection by SDS–PAGE eluted excretory secretory (ES protein fractions using dot-ELISA

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    M.A. Sabry

    2014-12-01

    Full Text Available Fascioliasis is now recognized as an emerging zoonotic disease in Egypt. Diagnosis in suspected patients still needs some degree of accuracy. In the present study, three Fasciola gigantica execratory secretory (ES protein bands of molecular weight (MW ranging from 14 to 20 kDa, 25 to 32 kDa and 45 to 65 kDa were eluted after fractionation of the parasite antigen using SDS–PAGE. The extracted kDa protein bands were concentrated and evaluated in diagnosis of Fasciola infection. Moreover the level of their cross reaction with other parasitic infections in infected and suspected patients of known parasite eggs/gram stool was evaluated using the dot-ELISA technique. Protein bands in the range of 14–20 kDa and that of 25–32 kDa were markedly specific and sensitive in diagnosis of different levels of anti-Fasciola antibodies (Ab in sera of infected cases. These two groups of bands were able to exclude cross-reaction between anti-Fasciola Ab and other parasites recorded in stool of selected patients suffering from Schistosoma mansoni, Ascaris, and Giardia, either in single or mixed conditions with Fasciola eggs. While that of 45–65 kDa appeared less specific than the other previously mentioned bands. Protein bands in the range of 25–32 kDa appeared more sensitive than the other protein bands in detection of anti-Fasciola Ab at higher serum dilutions. The Dot-ELISA technique was proved to be more economic and easy in application. The dotted very small amount of antigens can be stored in a freezer and used at request in diagnosis of large numbers of samples.

  8. BPI Fold-Containing Family A Member 2/Parotid Secretory Protein Is an Early Biomarker of AKI.

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    Kota, Satya K; Pernicone, Elizabeth; Leaf, David E; Stillman, Isaac E; Waikar, Sushrut S; Kota, Savithri Balasubramanian

    2017-12-01

    AKI is a major cause of morbidity and mortality and an important contributor to the development and progression of CKD. Molecular biomarkers that improve the detection and prognostication of AKI are therefore required. We assessed the utility as such of BPI fold-containing family A member 2 (BPIFA2), also known as parotid secretory protein, which we identified via a multiplex quantitative proteomics screen of acutely injured murine kidneys. In physiologic conditions, BPIFA2 is expressed specifically in the parotid glands and is abundant in salivary secretions. In our study, AKI induced Bpifa2 expression in the kidneys of mice within 3 hours. Furthermore, we detected BPIFA2 protein in plasma and urine in these models as early as 6 hours after injury. However, renal injury did not induce the expression of Bpifa2 in mice lacking Nur77 , an immediate early gene expressed in the kidneys during AKI. Notably, patients with AKI had higher blood and urine levels of BPIFA2 than did healthy individuals. Together, our results reveal that BPIFA2 is a potential early biomarker of AKI. Copyright © 2017 by the American Society of Nephrology.

  9. Proteomic Analysis of Trichinella spiralis Adult Worm Excretory-Secretory Proteins Recognized by Sera of Patients with Early Trichinellosis

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    Zhong Q. Wang

    2017-05-01

    Full Text Available The most commonly used serodiagnostic antigens for trichinellosis are the excretory-secretory (ES antigens from T. spiralis muscle larvae (ML, but the specific antibodies against the ML ES antigens are usually negative during early stage of Trichinella infection. The recent studies demonstrated that T. spiralis adult worm (AW antigens were recognized by mouse or swine infection sera on Western blot as early as 7–15 days post-infection (dpi, the AW antigens might contain the early diagnostic markers for trichinellosis. The purpose of this study was to screen early diagnostic antigens in T. spiralis AW ES proteins recognized by sera of early patients with trichinellosis. T. spiralis AW were collected at 72 h post-infection (hpi, and their ES antigens were analyzed by SDS-PAGE and Western blot. Our results showed that 5 protein bands (55, 48–50, 45, 44, and 36 kDa were recognized by sera of early patients with trichinellosis collected at 19 dpi, and were subjected to shotgun LC–MS/MS and bioinformatics analyses. A total of 185 proteins were identified from T. spiralis protein database, of which 116 (67.2% proteins had molecular weights of 30∼60 kDa, and 125 (67.6% proteins with pI 4–7. Bioinformatic analyses showed that the identified proteins have a wide diversity of biological functions (binding of nucleotides, proteins, ions, carbohydrates, and lipids; hydrolase, transferase, and oxidoreductase, etc.. Several enzymes (e.g., adult-specific DNase II, serine protease and serine protease inhibitor could be the invasion-related proteins and early diagnostic markers for trichinellosis. Moreover, recombinant T. spiralis serine protease (rTsSP-ZH68 was expressed in E. coli and its antigenicity was analyzed by Western blot with the early infection sera. The rTsSP-ZH68 was recognized by sera of infected mice at 8–10 dpi and sera of early patients with trichinellosis at 19 dpi. T. spiralis AW proteins identified in this study, especially serine

  10. A secretory system for bacterial production of high-profile protein targets

    DEFF Research Database (Denmark)

    Kotzsch, Alexander; Vernet, Erik; Hammarström, Martin

    2011-01-01

    Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To impr...

  11. Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.

    Science.gov (United States)

    Claes, Katrien; Vandewalle, Kristof; Laukens, Bram; Laeremans, Toon; Vosters, Olivier; Langer, Ingrid; Parmentier, Marc; Steyaert, Jan; Callewaert, Nico

    2016-10-21

    Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in Pichia pastoris can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based N-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in P. pastoris from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2-3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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.

  14. Lipopolysaccharide-binding protein: localization in secretory granules of Paneth cells in the mouse small intestine

    DEFF Research Database (Denmark)

    Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte

    2009-01-01

    in closer detail the synthesis and storage of LBP in the intestinal mucosal epithelium, we performed an immunolocalization of LBP in mouse small intestine. By immunofluorescence microscopy, an antibody recognizing the 58-60 kDa protein of LBP distinctly labeled a small population of cells located deep......Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host's response to endotoxin and mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including the enterocyte-like cell line Caco-2. To study...... together with other proteins acting in the innate immune response of the gut, such as lysozyme, defensins and intelectin....

  15. Immunoproteomic Analysis of the Excretory-Secretory Proteins from Spirometra Mansoni Sparganum

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    Zhong Quan Wang

    2013-09-01

    Full Text Available Background: Sparganosis is caused by the invasion of Spirometra sparganum into various tissues/organs. Subcutaneous sparganosis can be diagnosed by biopsy, while visceral/cerebral sparganosis is not easy to be diagnosed. The diagnosis de­pends largely on the detection of specific anti-sparganum antibodies. The specific­ity of the ELISA could be increased by using S. mansoni sparganum excretory–secre­tory (ES antigens, but it also had the cross-reactions with sera of patients with cysticercosis or paragonimiasis. The aim of this study was to identify early specific diagnostic antigens in S. mansoni sparganum ES proteins.Methods: The sparganum ES proteins were analyzed by two-dimensional electrophore­sis (2-DE and Western blot probed with early sera from infected mice at 14 days post-infection. The immunoreactive protein spots were characterized by MALDI-TOF/ TOF-MS.Results: A total of approximately 149 proteins spots were detected with isoelectric point (pI varying from 3 to 7.5 and molecular weight from 20 to 115 kDa and seven protein spots with molecular weight of 23-31 kDa were recognized by the infection sera. Three of seven spots were successfully identified and characterized as the same S. mansoni protein (cysteine protease, and the proteins of other 4 spots were not included in the databases.Conclusion: The cysteine protease from S. mansoni ES proteins recognized by early infection sera might be the early diagnostic antigens for sparganosis.

  16. 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

    patients suspected of TB attending Kasturba Hospital-a tertiary health care center. The characterization of ES-31 antigen protein showed that ES-31 is a 31-kDa protein antigen with zinc containing serine protease as well as lipase activities and shown to be a chymotrypsin-like protein which has the catalytic triad responsible for both the activities. Addition of serine protease inhibitors: (1) pefabloc; (2) 3,4 dichloroisocoumarin; (3) phenyl methyl sulfonyl fluoride (53-76%); and metallo-protease inhibitors: (1) EDTA; (2) 1,10 phenanthroline (46-61%), lipase inhibitor, orlistat (61%), or anti-ES-31 serine protease antibody (89%) inhibited the Mycobacterium tuberculosis (MTB) H37Ra growth in axenic culture which is further confirmed by a decreased amount of ES-31 protein secreted in the culture filtrate. The importance of excretory secretory ES-31 protein for the survival of MTB H37Ra and H37Rv bacilli has been shown by 77% and 78% growth inhibition in macrophage culture by protease inhibitor pefabloc and was further confirmed by the enhancement of growth of TB bacilli in the presence of ES-31. Inhibition of ES-31 leads to the growth inhibition of MTB bacilli, suggesting that ES-31 is important for entry and multiplication of bacilli and an important drug target for exploring new drugs for TB based on protease and lipase activities of ES-31 protein. Copyright © 2016.

  17. βIII spectrin regulates the structural integrity and the secretory protein transport of the Golgi complex.

    Science.gov (United States)

    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.

  18. liver-enriched gene 1a and 1b encode novel secretory proteins essential for normal liver development in zebrafish.

    Directory of Open Access Journals (Sweden)

    Changqing Chang

    Full Text Available liver-enriched gene 1 (leg1 is a liver-enriched gene in zebrafish and encodes a novel protein. Our preliminary data suggested that Leg1 is probably involved in early liver development. However, no detailed characterization of Leg1 has been reported thus far. We undertook both bioinformatic and experimental approaches to study leg1 gene structure and its role in early liver development. We found that Leg1 identifies a new conserved protein superfamily featured by the presence of domain of unknown function 781 (DUF781. There are two copies of leg1 in zebrafish, namely leg1a and leg1b. Both leg1a and leg1b are expressed in the larvae and adult liver with leg1a being the predominant form. Knockdown of Leg1a or Leg1b by their respective morpholinos specifically targeting their 5'-UTR each resulted in a small liver phenotype, demonstrating that both Leg1a and Leg1b are important for early liver development. Meanwhile, we found that injection of leg1-ATG(MO, a morpholino which can simultaneously block the translation of Leg1a and Leg1b, caused not only a small liver phenotype but hypoplastic exocrine pancreas and intestinal tube as well. Further examination of leg1-ATG(MO morphants with early endoderm markers and early hepatic markers revealed that although depletion of total Leg1 does not alter the hepatic and pancreatic fate of the endoderm cells, it leads to cell cycle arrest that results in growth retardation of liver, exocrine pancreas and intestine. Finally, we proved that Leg1 is a secretory protein. This intrigued us to propose that Leg1 might act as a novel secreted regulator that is essential for liver and other digestive organ development in zebrafish.

  19. 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.

  20. Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Dileep Tiwari

    2017-04-01

    Conclusion: Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application.

  1. 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.

  2. Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production.

    Science.gov (United States)

    Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2015-12-01

    Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Aphid Gel Saliva: Sheath Structure, Protein Composition and Secretory Dependence on Stylet-Tip Milieu

    Science.gov (United States)

    Will, Torsten; Steckbauer, Kathrin; Hardt, Martin; van Bel, Aart J. E.

    2012-01-01

    In order to separate and analyze saliva types secreted during stylet propagation and feeding, aphids were fed on artificial diets. Gel saliva was deposited as chains of droplets onto Parafilm membranes covering the diets into which watery saliva was secreted. Saliva compounds collected from the diet fluid were separated by SDS-PAGE, while non-soluble gel saliva deposits were processed in a novel manner prior to protein separation by SDS-PAGE. Soluble (watery saliva) and non-soluble (gel saliva) protein fractions were significantly different. To test the effect of the stylet milieu on saliva secretion, aphids were fed on various diets. Hardening of gel saliva is strongly oxygen-dependent, probably owing to formation of sulfide bridges by oxidation of sulphydryl groups. Surface texture of gel saliva deposits is less pronounced under low-oxygen conditions and disappears in dithiothreitol containing diet. Using diets mimicking sieve-element sap and cell-wall fluid respectively showed that the soluble protein fraction was almost exclusively secreted in sieve elements while non-soluble fraction was preferentially secreted at cell wall conditions. This indicates that aphids are able to adapt salivary secretion in dependence of the stylet milieu. PMID:23056521

  4. Structures of Azemiops feae venom phospholipases and cys-rich-secretory protein and implications for taxonomy and toxinology.

    Science.gov (United States)

    Tsai, Inn-Ho; Wang, Ying-Ming; Huang, Kai-Fa

    2016-05-01

    The Azemiops snakes are pit-less and phylogenetically located at the Crotalinae and Viperinae divergence. cDNAs encoding five Azemiops venom phospholipase (sPLA2) molecules were cloned and sequenced; their signal-peptides were similar to those of crotalid sPLA2s. Based on their calculated pI-values and residue-49 substitutions, they were designated as Af-E6, Af-N49a, Af-N49a1, Af-N49a2, and Af-N49b, respectively. The first three isoforms, comprising 3-4% of the venom proteins, were purified by reversed-phase HPLC. Af-E6 is catalytically active and has >80% sequence-similarity to other Glu(6)-PLA2 (a pitviper venom-marker). Results of phylogenetic analyses reveal that acidic Af-N49a and Af-N49a1 are rather unique and loosely linked with crotalid PLA2s, while Af-N49b is related to the viperid PLA2s with Ser(1) substitution. Notably, the Asn(49)-substitutions in these molecules imply catalytic-independent mechanisms. The 3D-models of Af-E6 and Af-N49a have surface electropotential maps similar to each other and to those of antiplatelet PLA2s, while the Af-N49b model is similar to basic and myotoxic sPLA2 molecules. From Azemiops feae and four other Viperidae, we cloned five novel Cys-rich secretory proteins (CRISPs). Azemiops CRISP and natriuretic-peptide precursors share more sequence similarities with those of crotalid venoms than with viperid venoms, further supporting the theory that Azemiops are sister taxons to pit vipers, especially Tropedolaemus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Use of cloned excretory/secretory low-molecular-weight proteins of Cooperia oncophora in a serological assay.

    Science.gov (United States)

    Poot, J; Kooyman, F N; Dop, P Y; Schallig, H D; Eysker, M; Cornelissen, A W

    1997-07-01

    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 of two ES proteins were sequenced, and the corresponding cDNAs were cloned. Two cDNAs, designated CoES14.0 and CoES14.2, were expressed in Escherichia coli. The recombinant proteins were tested in an indirect enzyme-linked immunosorbent assay (ELISA) in which crude worm antigen (CWA) was used as a reference standard. In total, 67 reference serum samples were used: 27 negative serum samples, 29 C. oncophora-specific serum samples, 7 Dictyocaulus viviparus-specific serum samples, and 4 Ostertagia ostertagi-specific serum samples. This showed respective sensitivities and specificities of 17 and 84%, 0 and 100%, and 100 and 100% by the ELISAs with the three different types of proteins (CWA, CoES14.0, and CoES14.2, respectively). Since the CoES14.2 ELISA had the best sensitivity and specificity with reference sera, its specificity was further validated in an antigen inhibition ELISA. In this assay CoES14.2 and CWA preparations of C. oncophora, Cooperia curticei, O. ostertagi, Nematodirus helvetianus, Fasciola hepatica, D. viviparus, Haemonchus placei, and Trichostrongylus colubriformus were used as competitor antigens. This experiment showed that only the homologous antigens C. oncophora CWA and CoEs14.2 resulted in 100% inhibition. The CWA preparations of all other nematodes did not affect the ELISA, even if concentrations of 250 times the 50% inhibitory concentration of C. oncophora CWA were used. These results indicate that CoES14.2 does not share cross-reactive epitopes with heterologous CWAs. Finally, we tested the CoES14.2 ELISA with sequential serum samples from naturally infected groups of animals. The optical density values that were obtained correlated well with

  6. Identification of Pathways in Liver Repair Potentially Targeted by Secretory Proteins from Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    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.

  7. Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis.

    Science.gov (United States)

    Tiwari, Dileep; Haque, Shafiul; Tiwari, Ram P; Jawed, Arshad; Govender, Thavendran; Kruger, Hendrik G

    2017-04-01

    A rapid and efficient diagnostic test was developed for the detection of Mycobacterium tuberculosis antigens in serum samples of active tuberculosis (TB) and extrapulmonary TB patients via a liposomal agglutination-based method. A rapid card test has been developed to facilitate the recognition of high-affinity binding rabbit raised purified culture filtrate protein antibodies coupled on the surface of activated liposomal preparation. In the presence of TB antigens, the polyclonal antibodies bound to the liposomal particles demonstrate a visible agglutination reaction. The developed assay was simple, rapid, reliable, sensitive, and specific as a diagnostic test for the detection of antigens in serum samples of clinically confirmed cases of TB within 4-5 minutes' duration. The test was evaluated at different hospitals, medical colleges, and pathology centers, and involved 1483 participants. This investigation was conducted to detect the presence of these antigens during the period of active growth of the microorganism in serum samples for pulmonary TB and processed tissue biopsy for other extrapulmonary TB. Results obtained using this test were compared with acid-fast bacilli smear and culture results. Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application. Copyright © 2015. Published by Elsevier B.V.

  8. Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis.

    Directory of Open Access Journals (Sweden)

    Nikhil R Gandasi

    Full Text Available Fluorescent proteins (FPs have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo- and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo- or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release.

  9. The CREC family, a novel family of multiple EF-hand, low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells

    DEFF Research Database (Denmark)

    Honoré, B; Vorum, H

    2000-01-01

    The CREC family consists of a number of recently discovered multiple (up to seven) EF-hand proteins that localise to the secretory pathway of mammalian cells. At present, the family includes reticulocalbin, ERC-55/TCBP-49/E6BP, Cab45, calumenin and crocalbin/CBP-50. Similar proteins are found...... in quite diverse invertebrate organisms such as DCB-45 and SCF in Drosophila melanogaster, SCF in Bombyx mori, CCB-39 in Caenorhabditis elegans and Pfs40/PfERC in Plasmodium falciparum. The Ca(2+) affinity is rather low with dissociation constants around 10(-4)-10(-3) M. The proteins may participate in Ca...

  10. A secretory protein of necrotrophic fungus Sclerotinia sclerotiorum that suppresses host resistance.

    Science.gov (United States)

    Zhu, Wenjun; Wei, Wei; Fu, Yanping; Cheng, Jiasen; Xie, Jiatao; Li, Guoqing; Yi, Xianhong; Kang, Zhensheng; Dickman, Martin B; Jiang, Daohong

    2013-01-01

    SSITL (SS1G_14133) of Sclerotinia sclerotiorum encodes a protein with 302 amino acid residues including a signal peptide, its secretion property was confirmed with immunolocalization and immunofluorescence techniques. SSITL was classified in the integrin alpha N-terminal domain superfamily, and its 3D structure is similar to those of human integrin α4-subunit and a fungal integrin-like protein. When S. sclerotiorum was inoculated to its host, high expression of SSITL was detected during the initial stages of infection (1.5-3.0 hpi). Targeted silencing of SSITL resulted in a significant reduction in virulence; on the other hand, inoculation of SSITL silenced transformant A10 initiated strong and rapid defense response in Arabidopsis, the highest expressions of defense genes PDF1.2 and PR-1 appeared at 3 hpi which was 9 hr earlier than that time when plants were inoculated with the wild-type strain of S. sclerotiorum. Systemic resistance induced by A10 was detected by analysis of the expression of PDF1.2 and PR-1, and confirmed following inoculation with Botrytis cinerea. A10 induced much larger lesions on Arabidopsis mutant ein2 and jar1, and slightly larger lesions on mutant pad4 and NahG in comparison with the wild-type plants. Furthermore, both transient and constitutive expression of SSITL in Arabidopsis suppressed the expression of PDF1.2 and led to be more susceptible to A10 and the wild-type strain of S. sclerotiorum and B. cinerea. Our results suggested that SSITL is an effector possibly and plays significant role in the suppression of jasmonic/ethylene (JA/ET) signal pathway mediated resistance at the early stage of infection.

  11. A secretory protein of necrotrophic fungus Sclerotinia sclerotiorum that suppresses host resistance.

    Directory of Open Access Journals (Sweden)

    Wenjun Zhu

    Full Text Available SSITL (SS1G_14133 of Sclerotinia sclerotiorum encodes a protein with 302 amino acid residues including a signal peptide, its secretion property was confirmed with immunolocalization and immunofluorescence techniques. SSITL was classified in the integrin alpha N-terminal domain superfamily, and its 3D structure is similar to those of human integrin α4-subunit and a fungal integrin-like protein. When S. sclerotiorum was inoculated to its host, high expression of SSITL was detected during the initial stages of infection (1.5-3.0 hpi. Targeted silencing of SSITL resulted in a significant reduction in virulence; on the other hand, inoculation of SSITL silenced transformant A10 initiated strong and rapid defense response in Arabidopsis, the highest expressions of defense genes PDF1.2 and PR-1 appeared at 3 hpi which was 9 hr earlier than that time when plants were inoculated with the wild-type strain of S. sclerotiorum. Systemic resistance induced by A10 was detected by analysis of the expression of PDF1.2 and PR-1, and confirmed following inoculation with Botrytis cinerea. A10 induced much larger lesions on Arabidopsis mutant ein2 and jar1, and slightly larger lesions on mutant pad4 and NahG in comparison with the wild-type plants. Furthermore, both transient and constitutive expression of SSITL in Arabidopsis suppressed the expression of PDF1.2 and led to be more susceptible to A10 and the wild-type strain of S. sclerotiorum and B. cinerea. Our results suggested that SSITL is an effector possibly and plays significant role in the suppression of jasmonic/ethylene (JA/ET signal pathway mediated resistance at the early stage of infection.

  12. Ancylostoma ceylanicum Excretory-Secretory Protein 2 Adopts a Netrin-Like Fold and Defines a Novel Family of Nematode Proteins

    Energy Technology Data Exchange (ETDEWEB)

    K Kucera; L Harrison; M Cappello; Y Modis

    2011-12-31

    Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory-secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 {angstrom} resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinant AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2's netrin-likefold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.

  13. Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures.

    Science.gov (United States)

    Vanz, Ana Letícia; Nimtz, Manfred; Rinas, Ursula

    2014-02-13

    Pichia pastoris is a popular yeast preferably employed for secretory protein production. Secretion is not always efficient and endoplasmic retention of proteins with aberrant folding properties, or when produced at exaggerated rates, can occur. In these cases production usually leads to an unfolded protein response (UPR) and the induction of the endoplasmic reticulum associated degradation (ERAD). P. pastoris is nowadays also an established host for secretory insulin precursor (IP) production, though little is known about the impact of IP production on the host cell physiology, in particular under industrially relevant production conditions. Here, we evaluate the cellular response to aox1 promoter-controlled, secretory IP production in controlled fed-batch processes using a proteome profiling approach. Cells were first grown in a batch procedure using a defined medium with a high glycerol concentration. After glycerol depletion IP production was initiated by methanol addition which was kept constant through continuous methanol feeding. The most prominent changes of the intracellular proteome after the onset of methanol feeding were related to the enzymes of central carbon metabolism. In particular, the enzymes of the methanol dissimilatory pathway - virtually absent in the glycerol batch phase - dominated the proteome during the methanol fed-batch phase. Unexpectedly, a strong decrease of UPR and ERAD related proteins was also observed during methanol-induced IP production. Compared to non-producing control strains grown under identical conditions the UPR down-regulation was less pronounced indicating that IP production elicits a detectable but non prominent UPR response which is repressed by the general culture condition-dependent UPR down-regulation after the shift from glycerol to methanol. The passage of IP through the secretory pathway using an optimized IP vector and growing the strain at fed-batch conditions with a high initial glycerol concentration does

  14. Inhibition of the activity of pro-inflammatory secretory phospholipase A2 by acute phase proteins

    Directory of Open Access Journals (Sweden)

    W. Pruzanski

    1996-01-01

    Full Text Available Pro-Inflammatory non-pancreatic phospholipase A2 (sPLA2 is markedly over-expressed in acute systemic and chronic local inflammatory processes. Since in acute phase reaction sPLA2 is often over-expressed simultaneously with acute phase proteins (APP, it is important to determine whether APP interacts with sPLA2. We tested ten APPs for interaction with sPLA2 using as a substrate multilamellar Hposomes composed either of PC:Lyso PC or PE:Lyso PE. Using PC:Lyso PC substrate, CRP, lactoferrin and SAP were found to inhibit sPLA2 activity with an IC50 of 25 μg/ml, 7.5 μg/ml and 50 μg/ml, respectively, corresponding to 0.21 μM, 0.1 μM and 0.21 μM respectively. Using PE:Lyso PE substrate only SAP was inhibitory, with an IC50 of 10 μg/ml (0.04 μM. Phosphorylcholine abolished the inhibitory activity of CRP but not of SAP or lactoferrin. Addition of phosphorylethanolamine or of excess calcium had no effect on the inhibitory activity of APP. Limulin, lysozyme, transferrin, β2-microglobulin, α2-macroglobulin, human and bovine albumins had no effect on sPLA2 activity. Therefore neither the structure of pentraxins, or ironbinding, bacteriostatic property or amyloidogenic property preclude whether APP modulates sPLA2 activity. Inhibition of pro-inflammatory sPLA2 by APP may be one of the protective mechanisms of the acute phase reaction.

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

    Science.gov (United States)

    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

  16. The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells.

    Science.gov (United States)

    Cruz-García, David; Díaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R; Gracia-Navarro, Francisco; Castaño, Justo P; Montero-Hadjadje, Maité; Tonon, Marie-Christine; Vaudry, Hubert; Anouar, Youssef; Vázquez-Martínez, Rafael; Malagón, María M

    2012-04-15

    Golgi-associated long coiled-coil proteins, often referred to as golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1 (neuroendocrine long coiled-coil protein 1). The present study shows that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K+-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture, but enhanced K+-stimulated hormone secretion in PC12 cells. In all, the results of the present study identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells.

  17. Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC-ESI-MS/MS and LC-MALDI-TOF/TOF.

    Science.gov (United States)

    Ujang, Jorim Anak; Kwan, Soon Hong; Ismail, Mohd Nazri; Lim, Boon Huat; Noordin, Rahmah; Othman, Nurulhasanah

    2016-01-01

    Excretory-secretory (ES) proteins of E. histolytica are thought to play important roles in the host invasion, metabolism, and defence. Elucidation of the types and functions of E. histolytica ES proteins can further our understanding of the disease pathogenesis. Thus, the aim of this study is to use proteomics approach to better understand the complex ES proteins of the protozoa. E. histolytica ES proteins were prepared by culturing the trophozoites in protein-free medium. The ES proteins were identified using two mass spectrometry tools, namely, LC-ESI-MS/MS and LC-MALDI-TOF/TOF. The identified proteins were then classified according to their biological processes, molecular functions, and cellular components using the Panther classification system (PantherDB). A complementary list of 219 proteins was identified; this comprised 201 proteins detected by LC-ESI-MS/MS and 107 proteins by LC-MALDI-TOF/TOF. Of the 219 proteins, 89 were identified by both mass-spectrometry systems, while 112 and 18 proteins were detected exclusively by LC-ESI-MS/MS and LC-MALDI-TOF/TOF respectively. Biological protein functional analysis using PantherDB showed that 27% of the proteins were involved in metabolic processes. Using molecular functional and cellular component analyses, 35% of the proteins were found to be involved in catalytic activity, and 21% were associated with the cell parts. This study showed that complementary use of LC-ESI-MS/MS and LC-MALDI-TOF/TOF has improved the identification of ES proteins. The results have increased our understanding of the types of proteins excreted/secreted by the amoeba and provided further evidence of the involvement of ES proteins in intestinal colonisation and evasion of the host immune system, as well as in encystation and excystation of the parasite.

  18. The CREC family, a novel family of multiple EF-hand, low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells

    DEFF Research Database (Denmark)

    Honoré, B; Vorum, H

    2000-01-01

    The CREC family consists of a number of recently discovered multiple (up to seven) EF-hand proteins that localise to the secretory pathway of mammalian cells. At present, the family includes reticulocalbin, ERC-55/TCBP-49/E6BP, Cab45, calumenin and crocalbin/CBP-50. Similar proteins are found...... in quite diverse invertebrate organisms such as DCB-45 and SCF in Drosophila melanogaster, SCF in Bombyx mori, CCB-39 in Caenorhabditis elegans and Pfs40/PfERC in Plasmodium falciparum. The Ca(2+) affinity is rather low with dissociation constants around 10(-4)-10(-3) M. The proteins may participate in Ca......(2+)-regulated activities. Recent evidence has been obtained that some CREC family members are involved in pathological activities such as malignant cell transformation, mediation of the toxic effects of snake venom toxins and putative participation in amyloid formation. Udgivelsesdato: 2000-Jan-21...

  19. Differential expression patterns of the tight junction-associated proteins occludin and claudins in secretory and mature ameloblasts in mouse incisor.

    Science.gov (United States)

    Hata, Masaki; Kawamoto, Tadafumi; Kawai, Mariko; Yamamoto, Toshio

    2010-06-01

    Tight junctions (TJs) function primarily as a barrier against paracellular transport between epithelial cells and are composed mainly of occludin (OLD) and claudins (CLDs). The CLD family consists of 24 members that show tissue- or cell-specific expression. Ameloblasts, which originate from the oral epithelium, form enamel, and enamel proteins and minerals are transported across the ameloblastic layer during amelogenesis. We immunohistochemically examined the distribution patterns of TJs in ameloblasts by observing the expression patterns of OLD and CLDs (CLD-1 to CLD-10). Secretory ameloblasts contained OLD and CLD-1, -8, and -9 at the distal end of the cell. In mature ameloblasts, OLD and CLD-1, -6, -7, -8, -9, and -10 were present mainly at both the distal and proximal ends of the cell, regardless of whether the ameloblasts were ruffle-ended or smooth-ended. Mature ameloblasts in which only the proximal ends were stained for OLD and CLDs were also found. These results indicate that the expression patterns of CLDs and the distribution patterns of TJs change drastically between the secretory and mature ameloblast stages, suggesting that these patterns reflect the different functions of these cells, specifically in the transport of proteins and ions for enamel formation.

  20. Autoradiographic investigation of the effect of 1-hydroxyethylidene-1, 1-bisphosphonate on matrix protein synthesis and secretion by secretory ameloblasts in rat incisors.

    Science.gov (United States)

    Fuangtharnthip, P; Yamada, Y; Takagi, Y; Ohya, K

    2000-06-01

    Seven daily subcutaneous injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) can induce enamel hypoplasia. Several enamel-free zones were observed along the crown-analogue side of rat incisors during the secretory stage of amelogenesis. Ameloblasts related to the enamel-free zones lay directly on the abnormally non-mineralized mantle dentine, whereas the adjacent ameloblasts, which were forming the enamel matrix layer, were associated with the region where mineralization of dentine was proceeding. The further purpose of this study was to investigate the synthetic and secretory activity of these two groups of ameloblasts and to trace the fate of the radioactively labelled proteins. [(3)H]-proline was administered to Wistar rats 12 h after the last injection of HEBP. Light-microscopic autoradiography was performed. Quantitative analysis indicated that the ameloblasts of the enamel-forming zones in the drug-treated group showed a distribution pattern of silver grains similar to that of the controls. The ameloblasts of the enamel-free zones also demonstrated incorporation of [(3)H]-proline at the same level. There was some labelling over the non-mineralized mantle dentine, which was supposed to indicate the penetration of ameloblast products. From these results, it is concluded that HEBP does not affect the ameloblast activity in protein synthesis. The complete failure of enamel-layer formation in some specific regions is probably due to the failure in protein secretion and protein deposition. This study provides additional evidence that the mineralization of dentine is an essential factor in successful enamel matrix secretion and deposition.

  1. Protein kinase C delta localizes to secretory lysosomes in CD8+ CTL and directly mediates TCR signals leading to granule exocytosis-mediated cytotoxicity.

    Science.gov (United States)

    Ma, Jennifer S Y; Haydar, Tarik F; Radoja, Sasa

    2008-10-01

    Lytic granule exocytosis is the major effector function used by CD8(+) CTL in response to intracellular pathogens and tumors. Despite recent progress in the field, two important aspects of this cytotoxic mechanism remain poorly understood. First, TCR-signaling pathway(s) that selectively induces granule exocytosis in CTL has not been defined to date. Second, it is unclear how Ag receptor-induced signals are converted into mobilization of lytic granules. We recently demonstrated that protein kinase C delta (PKC delta) selectively regulates TCR-induced lytic granule polarization in mouse CD8(+) CTL. To better understand how PKC delta facilitates granule movement, here we studied dynamics of intracellular localization of PKC delta in living CD8(+) CTL. Strikingly, we found that PKC delta localizes to the secretory lysosomes and polarizes toward immunological synapse during the process of target cell killing. Also, biochemical and structure-function studies demonstrated that upon TCR ligation, PKC delta becomes rapidly phosphorylated on the activation loop and regulates granule exocytosis in a kinase-dependent manner. Altogether, our current studies provide new insights concerning the regulation of TCR-induced lytic granule exocytosis by revealing novel intracellular localization of PKC delta, providing the first example of colocalization of a kinase with secretory lysosomes in CD8(+) CTL and demonstrating that PKC delta directly transduces TCR signals leading to polarized granule secretion.

  2. Species-Specific Antibody Responses to the Recombinant 53-Kilodalton Excretory and Secretory Proteins in Mice Infected with Trichinella spp.▿

    Science.gov (United States)

    Nagano, Isao; Wu, Zhiliang; Takahashi, Yuzo

    2008-01-01

    The 53-kDa proteins in larval excretory and secretory (E-S) products were expressed from five Trichinella species (T. spiralis, T. britovi, T. nativa, T. pseudospiralis, and T. papuae), using the Escherichia coli expression system, and the antibody responses to the 53-kDa recombinant proteins in mice infected with Trichinella spp. were analyzed by Western blotting. The 53-kDa protein is conserved among the five Trichinella species, with >60% similarity in amino acid sequences. The 53-kDa recombinant proteins of T. spiralis and T. pseudospiralis reacted to sera from mice infected with T. spiralis and T. pseudospiralis at 8 days postinfection (p.i.), respectively. An antibody against the 53-kDa recombinant protein of T. spiralis recognized the 53-kDa protein in the crude extracts from adult worms and 30-day p.i. muscle larvae and E-S products from muscle larvae of T. spiralis but did not recognize any proteins from T. pseudospiralis. The sera from the mice infected with T. spiralis strongly reacted with the 53-kDa recombinant protein of T. spiralis but did not react with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, and T. papuae. Similarly, the sera from mice infected with T. britovi, T. nativa, T. pseudospiralis, or T. papuae strongly reacted with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, or T. papuae, respectively. These results showed that the 53-kDa recombinant proteins provide early and species-specific antibody responses in mice infected with Trichinella spp. PMID:18184826

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

    Science.gov (United States)

    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.

  4. Group X secretory phospholipase A2 regulates the expression of steroidogenic acute regulatory protein (StAR) in mouse adrenal glands.

    Science.gov (United States)

    Shridas, Preetha; Bailey, William M; Boyanovsky, Boris B; Oslund, Rob C; Gelb, Michael H; Webb, Nancy R

    2010-06-25

    We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A(2) (GX KO). These mice have approximately 80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A(2) (sPLA(2)), but not a catalytically inactive mutant form of GX sPLA(2), significantly reduced steroid production 30-40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA(2) inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA(2)-overexpressing Y1 cells, ruling out a role for this sPLA(2) receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was approximately 2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA(2). Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA(2) antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA(2) is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.

  5. 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.

  6. 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

  7. Immunosuppressive PAS-1 is an excretory/secretory protein released by larval and adult worms of the ascarid nematode Ascaris suum.

    Science.gov (United States)

    Antunes, M F P; Titz, T O; Batista, I F C; Marques-Porto, R; Oliveira, C F; Alves de Araujo, C A; Macedo-Soares, M F

    2015-05-01

    Helminths use several strategies to evade and/or modify the host immune response, including suppression or inactivation of the host antigen-specific response. Several helminth immunomodulatory molecules have been identified. Our studies have focused on immunosuppression induced by the roundworm Ascaris suum and an A. suum-derived protein named protein 1 from A. suum (PAS-1). Here we assessed whether PAS-1 is an excretory/secretory (E/S) protein and whether it can suppress lipopolysaccharide-induced inflammation. Larvae from infective eggs were cultured in unsupplemented Dulbecco's modified Eagle medium (DMEM) for 2 weeks. PAS-1 was then measured in the culture supernatants and in adult A. suum body fluid at different time points by enzyme-linked immunosorbent assay (ELISA) with the monoclonal antibody MAIP-1. Secreted PAS-1 was detected in both larval culture supernatant and adult body fluid. It suppressed lipopolysaccharide (LPS)-induced leucocyte migration and pro-inflammatory cytokine production, and stimulated interleukin (IL)-10 secretion, indicating that larval and adult secreted PAS-1 suppresses inflammation in this model. Moreover, the anti-inflammatory activity of PAS-1 was abolished by treatment with MAIP-1, a PAS-1-specific monoclonal antibody, confirming the crucial role of PAS-1 in suppressing LPS-induced inflammation. These findings demonstrate that PAS-1 is an E/S protein with anti-inflammatory properties likely to be attributable to IL-10 production.

  8. Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

    Science.gov (United States)

    Rahimpour, Azam; Vaziri, Behrouz; Moazzami, Reza; Nematollahi, Leila; Barkhordari, Farzaneh; Kokabee, Leila; Adeli, Ahmad; Mahboudi, Fereidoun

    2013-08-01

    Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

  9. 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.

  10. [Effect of excretory/secretory protein ofTrichinella spiralisadult worm on CLP-induced sepsis in mice].

    Science.gov (United States)

    Xiao-di, Yang; Wen-Xin, He; Qiang, Fang; Di, Song; Qi, Wu; Xiao-Li, Wang; Nan, Li; Qi, Qi; Yong-Kun, Wan; Hui, Zhang; Rui, Zhou; Xing-Zhi, Chen; Mu-Lin, Liu; Hui-Hui, Li; Liang, Chu

    2016-05-25

    To observe the effect of excretory/secretory products from Trichinella spiralis adult worms (AES) on cecal ligation and puncture (CLP) -induced sepsis in mice. Forty-eight BALB/c mice were randomly divided into 3 groups:a sham operation group (PBS + sham group, Group A), a CLP-induced sepsis group (PBS+CLP group, Group B) and an AES treatment group (AES+CLP group, Group C). The mice of each group were intraperitoneally injected with 25 μg of AES or PBS only as a control in a total volume of 200 μl. Eight mice from each group were selected randomly for survival analysis of 96 hours. The other 8 mice in each group were observed for pathological changes in the lung, liver and kidney tissues by HE staining 12 h after CLP, and then determined for the detection of cytokines including TNF-α, IL-1β, IL-6, IL-10 and TGF-β in the sera by ELISA. The difference among the survival rates of mice in the 3 groups was statistically significant ( χ 2 = 21.16, P CLP. Compared with the mice in group B, the survival rate of those in Group C (70%) increased significantly ( P CLP in mice.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  14. Novel secretory protein Ss-Caf1 of the plant-pathogenic fungus Sclerotinia sclerotiorum is required for host penetration and normal sclerotial development.

    Science.gov (United States)

    Xiao, Xueqiong; Xie, Jiatao; Cheng, Jiasen; Li, Guoqing; Yi, Xianhong; Jiang, Daohong; Fu, Yanping

    2014-01-01

    To decipher the mechanism of pathogenicity in Sclerotinia sclerotiorum, a pathogenicity-defective mutant, Sunf-MT6, was isolated from a T-DNA insertional library. Sunf-MT6 could not form compound appressorium and failed to induce lesions on leaves of rapeseed though it could produce more oxalic acid than the wild-type strain. However, it could enter into host tissues via wounds and cause typical necrotic lesions. Furthermore, Sunf-MT6 produced fewer but larger sclerotia than the wild-type strain Sunf-M. A gene, named Ss-caf1, was disrupted by T-DNA insertion in Sunf-MT6. Gene complementation and knockdown experiments confirmed that the disruption of Ss-caf1 was responsible for the phenotypic changes of Sunf-MT6. Ss-caf1 encodes a secretory protein with a putative Ca(2+)-binding EF-hand motif. High expression levels of Ss-caf1 were observed at an early stage of compound appressorium formation and in immature sclerotia. Expression of Ss-caf1 without signal peptides in Nicotiana benthamiana via Tobacco rattle virus-based vectors elicited cell death. These results suggest that Ss-caf1 plays an important role in compound appressorium formation and sclerotial development of S. sclerotiorum. In addition, Ss-Caf1 has the potential to interact with certain host proteins or unknown substances in host cells, resulting in subsequent host cell death.

  15. Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Schalén, Martin; Anyaogu, Diana Chinyere; Hoof, Jakob Blæsbjerg

    2016-01-01

    Background: The considerable capacity of filamentous fungi for the secretion of proteins is the basis for multi-billion dollar industries producing enzymes and proteins with therapeutic value. The stepwise pathway from translation to secretion is therefore well studied, and genes playing major ro...... results indicate that increased expression may be a way for the cell to slow down secretion in order to cope with the increased protein load. By constructing a secretion reporter strain, the study demonstrates a robust way to study the secretion pathway in filamentous fungi....

  16. Mycobacterium tuberculosis secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex inhibit lipopolysaccharide-induced NF-kappaB transactivation by downregulation of reactive oxidative species (ROS) production.

    Science.gov (United States)

    Ganguly, Niladri; Giang, Pham H; Gupta, Chitra; Basu, Sandip K; Siddiqui, Imran; Salunke, Dinakar M; Sharma, Pawan

    2008-01-01

    Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people annually and is considered one of the most successful intracellular pathogens to persist inside the host macrophage. Recent studies have implicated the role of RD-1 region of Mtb genome in the mycobacterial pathogenesis. The role of RD-1-encoded secretory proteins of Mtb in modulation of macrophage function has not been investigated in detail. Here we show that RD-1 encoded two major secretory proteins, namely, culture filtrate protein-10 kDa (CFP-10) and early secreted antigenic target-6 kDa (ESAT-6), and their 1:1 CFP-10:ESAT6 complex inhibit production of reactive oxidative species (ROS) in RAW264.7 cells. These proteins also downregulated the bacterial lipopolysaccharide (LPS)-induced ROS production, which, in turn, downregulated LPS-induced nuclear factor-kappaB (NF-kappaB) p65 DNA-binding activity, as well as inhibited the NF-kappaB-dependent reporter gene (chloramphenicol acetyl transferase) expression in the treated macrophages. Moreover, addition of N-acetyl cysteine, which is a scavenger of ROS, also inhibited LPS-induced reporter gene expression by scavenging the ROS, thereby preventing NF-kappaB transactivation. These studies indicate that the secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex of Mtb can inhibit LPS-induced NF-kappaB-dependent gene expression via downregulation of ROS production.

  17. Disruption of gene expression rhythms in mice lacking secretory vesicle proteins IA-2 and IA-2β

    OpenAIRE

    Punia, Sohan; Rumery, Kyle K.; Yu, Elizabeth A.; Christopher M Lambert; Notkins, Abner L.; David R Weaver

    2012-01-01

    Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molec...

  18. 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...

  19. 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.

  20. Proteoglycans from Boswellia serrata Roxb. and B. carteri Birdw. and identification of a proteolytic plant basic secretory protein

    DEFF Research Database (Denmark)

    Herrmann, Andreas; König, Simone; Lechtenberg, Matthias

    2012-01-01

    groups: (i) Hyp-/Ser-rich extensins with O-glycosidic attached arabinan side chains; (ii) Modified extensins, with arabinogalactosylated side chains containing GlA and 4-O-Me-GlcA; (iii) Glycoproteins with N-glycosidic side chains containing higher amounts of Fuc, Man and GluNH(2,) featuring a 200 k......Water-soluble high molecular weight compounds were isolated in yields of 21-22% from the oleogum of Boswellia serrata and B. carteri. Using anion exchange chromatography and gel permeation chromatography, different proteoglycans were purified and characterized, leading to four principally different......D metalloproteinase that has been de novo sequenced and is described for the first time; (iv) Type II arabinogalactans-proteins. Significant differences between the gums from the two species were observed in the protein content (6% vs 22%), offering the possibility of a quick differentiation of gums from both species...

  1. The influence of gestation and mechanical ventilation on serum clara cell secretory protein (CC10) concentrations in ventilated and nonventilated newborn infants.

    Science.gov (United States)

    Loughran-Fowlds, Alison; Oei, Julee; Wang, He; Xu, Hongxiu; Wimalasundera, Neil; Egan, Claire; Henry, Richard; Lui, Kei

    2006-07-01

    Clara cell secretory protein (CC10) is an important anti-inflammatory mediator in the adult lung, but its role in newborn pulmonary protection is uncertain. We examined the early postnatal behavior of CC10 in newborn serum and tracheal fluid and hypothesized that CC10 production is positively influenced by gestation. Blood from 165 infants from the first, third/fourth, and seventh days of life (gestational ages: 23-29 wk, 30-36 wk, >36 wk) and tracheal fluid (TF) from the first day of life from 32 ventilated infants were analyzed for CC10. Surfactant proteins A (SPA) and B (SPB) were also analyzed from the blood of a subgroup of infants. Serum CC10 on day 1 was highest in term infants (69.4 ng/mL), followed by moderately preterm (55.8 ng/mL), and then extremely preterm infants (median 42.1 ng/mL). Term infants also had higher tracheal fluid CC10 than preterm infants. (20.152 ng/mL versus 882 ng/mL). Mechanical ventilation increased serum CC10 only in moderately preterm infants, and only on d 1 [68.4 ng/mL versus 42.1 ng/mL (nonventilated moderately preterm infants)]. Serum CC10 decreased progressively by the end of the first week in all infants, in contrast to SPA and SPB, which increased. Our results show that CC10 is detectable in the blood of newborn infants and that a production surge occurs at birth. This surge is more pronounced in term infants and may confer them with superior extrauterine pulmonary protection compared with preterm infants.

  2. Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress.

    Directory of Open Access Journals (Sweden)

    Christine R Collins

    2013-05-01

    Full Text Available The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV. Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

  3. A 24 kDa Excretory-Secretory Protein of Anisakis simplex Larvae Could Elicit Allergic Airway Inflammation in Mice

    Science.gov (United States)

    Park, Hye-Kyung; Cho, Min Kyoung; Park, Mi Kyung; Kang, Shin Ae; Kim, Yun Seong; Kim, Ki Uk; Lee, Min Ki; Ock, Mee Sun; Cha, Hee Jae

    2011-01-01

    We have reported that a 24 kDa protein (22U homologous; As22U) of Anisakis simplex larvae could elicit several Th2-related chemokine gene expressions in the intestinal epithelial cell line which means that As22U may play a role as an allergen. In order to determine the contribution of As22U to allergic reactions, we treated mice with 6 times intra-nasal application of recombinant As22U (rAs22U). In the group challenged with rAs22U and ovalbumin (OVA), the number of eosinophils in the bronchial alveolar lavage fluid (BALF) was significantly increased, as compared to the group receiving only OVA. In addition, mice treated with rAs22U and OVA showed significantly increased airway hyperresponsiveness. Thus, severe inflammation around the airway and immune cell recruitment was observed in mice treated with rAs22U plus OVA. The levels of IL-4, IL-5, and IL-13 cytokines in the BALF increased significantly after treatment with rAs22U and OVA. Similarly, the levels of anti-OVA specific IgE and IgG1 increased in mice treated with rAs22U and OVA, compared to those treated only with OVA. The Gro-α (CXCL1) gene expression in mouse lung epithelial cells increased instantly after treatment with rAs22U, and allergy-specific chemokines eotaxin (CCL11) and thymus-and-activation-regulated-chemokine (CCL17) gene expressions significantly increased at 6 hr after treatment. In conclusion, rAs22U may induce airway allergic inflammation, as the result of enhanced Th2 and Th17 responses. PMID:22355204

  4. Liver-Enriched Gene 1, a Glycosylated Secretory Protein, Binds to FGFR and Mediates an Anti-stress Pathway to Protect Liver Development in Zebrafish.

    Directory of Open Access Journals (Sweden)

    Minjie Hu

    2016-02-01

    Full Text Available Unlike mammals and birds, teleost fish undergo external embryogenesis, and therefore their embryos are constantly challenged by stresses from their living environment. These stresses, when becoming too harsh, will cause arrest of cell proliferation, abnormal cell death or senescence. Such organisms have to evolve a sophisticated anti-stress mechanism to protect the process of embryogenesis/organogenesis. However, very few signaling molecule(s mediating such activity have been identified. liver-enriched gene 1 (leg1 is an uncharacterized gene that encodes a novel secretory protein containing a single domain DUF781 (domain of unknown function 781 that is well conserved in vertebrates. In the zebrafish genome, there are two copies of leg1, namely leg1a and leg1b. leg1a and leg1b are closely linked on chromosome 20 and share high homology, but are differentially expressed. In this report, we generated two leg1a mutant alleles using the TALEN technique, then characterized liver development in the mutants. We show that a leg1a mutant exhibits a stress-dependent small liver phenotype that can be prevented by chemicals blocking the production of reactive oxygen species. Further studies reveal that Leg1a binds to FGFR3 and mediates a novel anti-stress pathway to protect liver development through enhancing Erk activity. More importantly, we show that the binding of Leg1a to FGFR relies on the glycosylation at the 70th asparagine (Asn(70 or N(70, and mutating the Asn(70 to Ala(70 compromised Leg1's function in liver development. Therefore, Leg1 plays a unique role in protecting liver development under different stress conditions by serving as a secreted signaling molecule/modulator.

  5. 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.

  6. Differentiation in neuroblastoma: diffusion-limited hypoxia induces neuro-endocrine secretory protein 55 and other markers of a chromaffin phenotype.

    Directory of Open Access Journals (Sweden)

    Fredrik Hedborg

    2010-09-01

    Full Text Available Neuroblastoma is a childhood malignancy of sympathetic embryonal origin. A high potential for differentiation is a hallmark of neuroblastoma cells. We have previously presented data to suggest that in situ differentiation in tumors frequently proceeds along the chromaffin lineage and that decreased oxygen (hypoxia plays a role in this. Here we explore the utility of Neuro-Endocrine Secretory Protein 55 (NESP55, a novel member of the chromogranin family, as a marker for this process.Immunohistochemical analyses and in situ hybridizations were performed on human fetal tissues, mouse xenografts of human neuroblastoma cell lines, and on specimens of human neuroblastoma/ganglioneuroma. Effects of anaerobic exposure on gene expression by cultured neuroblastoma cells was analyzed with quantitative real-time PCR. Fetal sympathetic nervous system expression of NESP55 was shown to be specific for chromaffin cell types. In experimental and clinical neuroblastoma NESP55 immunoreactivity was specific for regions of chronic hypoxia. NESP55 expression also correlated strikingly with morphological evidence of differentiation and with other chromaffin-specific patterns of gene expression, including IGF2 and HIF2α. Anaerobic culture of five neuroblastoma cell lines resulted in an 18.9-fold mean up-regulation of NESP55.The data confirms that chronic tumor hypoxia is a key microenvironmental factor for neuroblastoma cell differentiation, causing induction of chromaffin features and NESP55 provides a reliable marker for this neuronal to neuroendocrine transition. The hypoxia-induced phenotype is the predominant form of differentiation in stroma-poor tumors, while in stroma-rich tumors the chromaffin phenotype coexists with ganglion cell-like differentiation. The findings provide new insights into the biological diversity which is a striking feature of this group of tumors.

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

    Science.gov (United States)

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

    2010-06-16

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

  8. 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.

  9. Isolation of a cDNA clone for spinach lipid transfer protein and evidence that the protein is synthesized by the secretory pathway

    Energy Technology Data Exchange (ETDEWEB)

    Bernhard, W.R.; Thoma, S.; Botella, J.; Somerville, C.R. (Michigan State Univ., East Lansing (United States))

    1991-01-01

    A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the cytoplasm, the proposed role of this protein in catalyzing lipid transfer between intracellular membranes is in doubt. Although the lipid transfer protein is one of the most abundant proteins in leaf cells, the results of genomic Southern analysis were consistent with the presence of only one gene. Analysis of the level of mRNA by Northern blotting indicated that the transcript was several-fold more abundant than an actin transcript in leaf and petiole tissue, but was present in roots at less than 1% of the level in petioles.

  10. Concentration of Endogenous Secretory Receptor for Advanced Glycation End Products and Matrix Gla Protein in Controlled and Uncontrolled Type 2 Diabetes Mellitus Patients

    Directory of Open Access Journals (Sweden)

    Dwi Yuniati Daulay

    2013-04-01

    Full Text Available BACKGROUND: Advanced glycation end products (AGE and their receptor (RAGE system play an important role in the development of diabetic vascular complications. Recently, an endogenous secretory RAGE (esRAGE has been identified as a novel splice variant, which lacks the transmembrane domain and is secreted in human sera. Interestingly, it was reported that esRAGE binds AGE ligands and neutralizes AGE actions. Many studies have reported that diabetes mellitus correlates with vascular calcification event and increases progressively in uncontrolled diabetes. Matrix Gla Protein (MGP is known to act as an inhibitor in vascular calcification. The aim of this study was to observe progress of vascular calcification in uncontrolled diabetes patient by biochemical markers MGP as inhibitor in vascular calcification, via mechanism of AGEs. METHODS: This study was an observational study with cross sectional design on adult type 2 diabetic male patients who were defined by the 2011 Indonesian diabetes mellitus consensus criteria. RESULTS: The results of this study showed that there was a positive significant correlation between esRAGE and HbA1C (r=0.651, p=0.009, and negative correlation between MGP and HbA1C (r=-0.465, p=0.081 in controlled diabetes group. In uncontrolled diabetes group there was a positive significant correlation between MGP and HbA1C (r=0.350, p=0.023, despite the fact esRAGE showed no significant correlation with HbA1C. There was no significant difference in level of esRAGE and MGP in controlled and uncontrolled diabetes group, but MGP showed lower level in uncontrolled diabetes group, contrary to esRAGE that had higher concentration. CONCLUSIONS: In diabetes condition, complications of vascular calcification are caused by the mechanism of increased AGE formation represented by esRAGE. In diabetes control it is very important to keep the blood vessels from complications caused by vascular calcification. KEYWORDS: type 2 diabetes mellitus

  11. Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p ▿

    Science.gov (United States)

    Basso, Luiz R.; Gast, Charles E.; Mao, Yuxin; Wong, Brian

    2010-01-01

    A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [3H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [3H]fluconazole than did PGVs from empty-vector controls. The Kms (expressed in micromolar concentrations) and Vmaxs (expressed in picomoles per milligram of protein per minute), respectively, for [3H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [3H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [3H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [3H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC50s; expressed as proportions of the [3H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi. PMID:20348384

  12. Listeria-vectored vaccine expressing the Mycobacterium tuberculosis 30 kDa major secretory protein via the constitutively active prfA* regulon boosts BCG efficacy against tuberculosis.

    Science.gov (United States)

    Jia, Qingmei; Dillon, Barbara Jane; Masleša-Galić, Saša; Horwitz, Marcus A

    2017-06-19

    A potent vaccine against tuberculosis, one of the world's deadliest diseases, is needed to enhance the immunity of people worldwide, most of whom have been vaccinated with the partially effective BCG vaccine. Here we investigate novel live attenuated recombinant Listeria monocytogenes (rLm) vaccines expressing the Mycobacterium tuberculosis (Mtb) 30 kDa major secretory protein (r30/Ag85B) (rLm30) as heterologous booster vaccines in animals primed with BCG. Using three attenuated Lm vectors, rLm ΔactA (LmI), rLm ΔactA ΔinlB (LmII), and rLm ΔactA ΔinlBprfA* (LmIII), we constructed five rLm30 vaccine candidates expressing the r30 linked in-frame to the Lm Listeriolycin O signal sequence and driven by the hly promoter (h30) or linked in-frame to the ActA N-terminus and driven by the actA promoter (a30). All five rLm30 vaccines secreted r30 in broth and macrophages; while rLm expressing r30 via a constitutively active prfA* regulon (rLmIII/a30) expressed the greatest amount of r30 in broth culture, all five rLm vaccines expressed equivalent amounts of r30 in infected macrophages. In comparative studies, boosting BCG-immunized mice with rLmIII/a30 induced the strongest antigen-specific T-cell responses, including splenic and lung polyfunctional CD4+ T-cells expressing the three cytokines of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-2 (IL-2) (P vaccines were generally more potent booster vaccines than r30 in adjuvant and a recombinant adenovirus vaccine expressing r30. In a setting in which BCG alone was highly immunoprotective, boosting mice with rLmIII/a30, the most potent of the vaccines, significantly enhanced protection against aerosolized Mtb (P <0.01). Copyright © 2017 American Society for Microbiology.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Secretory structure and histochemistry test of some Zingiberaceae plants

    Science.gov (United States)

    Indriyani, Serafinah

    2017-11-01

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

  18. 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

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

    Science.gov (United States)

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

    2014-01-01

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

  20. 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...

  1. 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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. 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.

  3. [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.

  4. Unintended molecular interactions in transgenic plants expressing clinically useful proteins: the case of bovine aprotinin traveling the potato leaf cell secretory pathway.

    Science.gov (United States)

    Badri, M Amine; Rivard, Daniel; Coenen, Karine; Michaud, Dominique

    2009-02-01

    We assessed the impact of subcellular targeting on the heterologous expression of a clinically useful protease inhibitor, bovine aprotinin, in leaves of potato, Solanum tuberosum. Transgenic potato lines targeting aprotinin to the cytosol, the ER or the apoplast were first generated, and then assessed for their ability to accumulate the recombinant protein. On-chip detection and quantitation of aprotinin variants by SELDI TOF MS showed the inhibitor to be absent in the cytosol, but present under different forms in the ER and the apoplast. No visible phenotypic effects of aprotinin were observed for the transgenic lines, but aprotinin retention in the ER was associated with a significant decrease of leaf soluble protein content. A 2-D gel assessment of control and transgenic lines revealed a possible link between this altered protein content and the down-regulation of proteins implicated in protein synthesis and maturation. These observations, supported by complementary 2-DE analyses with potato lines targeting aprotinin to the apoplast, suggest an aprotinin-mediated feedback in planta negatively altering protein anabolism. From a practical viewpoint, these data illustrate the importance of taking into account not only the characteristics of recombinant proteins expressed in heterologous environments, but also their possible effects on protein accumulation in the host plant factory.

  5. The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal-recognition particle in Escherichia coli.

    NARCIS (Netherlands)

    Adams, H; Scottie, P.A.; Cock, H.; Luirink, S.; Tommassen, J.

    2002-01-01

    Signal sequences often contain α-helix-destabilizing amino acids within the hydrophobic core. In the precursor of the Escherichia coli outer-membrane protein PhoE, the glycine residue at position -10 (Gly

  6. The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells

    National Research Council Canada - National Science Library

    Cruz-García, David; Díaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R; Gracia-Navarro, Francisco; Castaño, Justo P; Montero-Hadjadje, Maité; Tonon, Marie-Christine; Vaudry, Hubert; Anouar, Youssef; Vázquez-Martínez, Rafael; Malagón, María M

    2012-01-01

    .... In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1...

  7. Activity of Specific Lipid-regulated ADP Ribosylation Factor-GTPase–activating Proteins Is Required for Sec14p-dependent Golgi Secretory Function in Yeast

    OpenAIRE

    Yanagisawa, Lora L.; Marchena, Jennifer; Xie, Zhigang; Li, Xinmin; Poon, Pak P.; Singer, Richard A.; Johnston, Gerald C.; Randazzo, Paul A.; Bankaitis, Vytas A.

    2002-01-01

    Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events. This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism. In addition to these “bypass Sec14p” mutations, Sec14p-independent Golgi function requires phospholipase D activity. The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the p...

  8. 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

  9. 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

  10. A 28-fold increase in secretory protein synthesis is associated with DNA puff activity in the salivary gland of Bradysia hygida (Diptera, Sciaridae

    Directory of Open Access Journals (Sweden)

    de-Almeida J.C.

    1997-01-01

    Full Text Available When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that these polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7, C5, C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time (Spradling AC and Mahowald AP (1980 Proceedings of the National Academy of Sciences, USA, 77: 1096-1100.

  11. Identification of LZP gene from Mus musculus and Rattus norvegicus coding for a novel liver-specific ZP domain-containing secretory protein.

    Science.gov (United States)

    Xu, Zhi-Gang; Du, Jian-Jun; Cui, Shu-Jian; Wang, Zhi-Qin; Huo, Ke-Ke; Li, Yu-Yang; Han, Ze-Guang

    2004-04-01

    Zona pellucida (ZP) domain has been recognized in a number of receptor-like eukaryotic glycoproteins, which involved in many important biological processes, such as signal transduction, development, differentiation and so on. Here we report the identification of Mus musculus and Rattus norvegicus orthologues of Homo sapiens LZP gene which codes for a novel ZP domain-containing protein. Sequence analysis revealed that human, rat and mouse LZP proteins are highly conserved. Mouse LZP gene has two transcripts, 2.4 and 2.8 KB long respectively, coding for identical protein. Mouse LZP mRNA is expressed specifically in hepatocytes. Our data also showed that mouse LZP localizes mostly on nuclear envelope, and at the same time, it can be secreted into blood in a truncated form.

  12. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Q. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Xiong, J. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Lu, J. [Office of Medical Education, Training Department, Second Military Medical University, Shanghai (China); Xu, S. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Li, Y. [State Food and Drug Administration of China,Huangdao Branch, Qingdao (China); Zhong, X.P.; Gao, G.K. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Liu, H.Q. [2Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China)

    2012-06-22

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  13. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Directory of Open Access Journals (Sweden)

    Q. Sun

    2012-10-01

    Full Text Available The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3 can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  14. 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...

  15. 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.

  16. Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory-secretory proteins and their biochemical properties.

    Science.gov (United States)

    Lee, Jinyoung; Kim, Jong-Hyun; Sohn, Hae-Jin; Yang, Hee-Jong; Na, Byoung-Kuk; Chwae, Yong-Joon; Park, Sun; Kim, Kyongmin; Shin, Ho-Joon

    2014-08-01

    Naegleria fowleri causes a lethal primary amoebic meningoencephalitis (PAM) in humans and experimental animals, which leads to death within 7-14 days. Cysteine proteases of parasites play key roles in nutrient uptake, excystment/encystment, host tissue invasion, and immune evasion. In this study, we cloned N. fowleri cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes from our cDNA library of N. fowleri. The full-length sequences of genes were 1,038 and 939 bp (encoded 345 and 313 amino acids), and molecular weights were 38.4 and 34 kDa, respectively. Also, nfcpb and nfcpb-L showed a 56 and 46 % identity to Naegleria gruberi cathepsin B and cathepsin B-like enzyme, respectively. Recombinant NfCPB (rNfCPB) and NfCPB-L (rNfCPB-L) proteins were expressed by the pEX5-NT/TOPO vector that was transformed into Escherichia coli BL21, and they showed 38.4 and 34 kDa bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using their respective antibodies. Proteolytic activity of refolded rNfCPB and rNfCPB-L was maximum at a pH of 4.5, and the most effective substrate was Z-LR-MCA. rNfCPB and rNfCPB-L showed proteolytic activity for several proteins such as IgA, IgG, IgM, collagen, fibronectin, hemoglobin, and albumin. These results suggested that NfCPB and NfCPB-L cysteine protease are important components of the N. fowleri ESP, and they may play important roles in host tissue invasion and immune evasion as pathogens that cause N. fowleri PAM.

  17. 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.

  18. 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....

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. A comparative immunohistochemistry study of diagnostic tools in salivary gland tumors: usefulness of mammaglobin, gross cystic disease fluid protein 15, and p63 cytoplasmic staining for the diagnosis of mammary analog secretory carcinoma?

    Science.gov (United States)

    Projetti, Fabrice; Lacroix-Triki, Magali; Serrano, Elie; Vergez, Sebastien; Barres, Béatrice Herbault; Meilleroux, Julie; Delisle, Marie-Bernadette; Uro-Coste, Emmanuelle

    2015-04-01

    Mammary analog secretory carcinoma (MASC) of the salivary gland has been recently described according to morphological, immunohistochemical, and molecular (ETV6-NTRK3 translocation) similarities with the mammary secretory carcinoma. The most important differential diagnostic considerations of MASC are low-grade adenocarcinoma not otherwise specified (NOS), cystadenocarcinoma, and acinic cell carcinoma (AciCC). These tumors may share an overlapping morphology with MASC, and additional immunohistochemical studies are required to reinforce the diagnosis. Mammaglobin, GCDFP-15, and p63 staining have been reported in MASC. Our study was designed to check the specificity of these antibodies in MASC compared to other frequent tumors of salivary glands. A series of 62 salivary gland tumors [10 MASCs, 5 adenocarcinomas NOS and 2 cystadenocarcinomas with MASC features and without ETV6 rearrangement, one low-grade cribriform cystadenocarcinoma (LGCCC), 9 AciCCs, 10 MECs, 10 adenoid cystic carcinomas (AdeCCs), 5 polymorphous low-grade adenocarcinomas (PLGAs), and 10 pleomorphic adenomas (PAs)] was analyzed by immunohistochemistry with mammaglobin, GCDFP-15, and p63 antibodies. Positivity for mammaglobin was observed in all MASCs, cystadenocarcinomas, LGCCC, and PLGAs, in some adenocarcinomas NOS, PAs, and MECs, rarely in AciCCs and never in AdeCCs. Positivity for GCDFP-15 was observed in most of the tumor types except in AdeCCs. Interestingly, cytoplasmic positivity for p63 was observed in most of MASCs and PLGAs while rarely in adenocarcinomas NOS and PAs, and never in the other tumor types. Our study revealed the usefulness of mammaglobin and p63 cytoplasmic staining to define which tumors are worth to be screened for ETV6 rearrangement. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. 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.

  10. 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.

  11. 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

  12. 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...

  13. 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 ...

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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...

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Structural Similarities and Differences between Two Functionally Distinct SecA Proteins, Mycobacterium tuberculosis SecA1 and SecA2.

    Science.gov (United States)

    Swanson, Stephanie; Ioerger, Thomas R; Rigel, Nathan W; Miller, Brittany K; Braunstein, Miriam; Sacchettini, James C

    2015-12-14

    While SecA is the ATPase component of the major bacterial secretory (Sec) system, mycobacteria and some Gram-positive pathogens have a second paralog, SecA2. In bacteria with two SecA paralogs, each SecA is functionally distinct, and they cannot compensate for one another. Compared to SecA1, SecA2 exports a distinct and smaller set of substrates, some of which have roles in virulence. In the mycobacterial system, some SecA2-dependent substrates lack a signal peptide, while others contain a signal peptide but possess features in the mature protein that necessitate a role for SecA2 in their export. It is unclear how SecA2 functions in protein export, and one open question is whether SecA2 works with the canonical SecYEG channel to export proteins. In this study, we report the structure of Mycobacterium tuberculosis SecA2 (MtbSecA2), which is the first structure of any SecA2 protein. A high level of structural similarity is observed between SecA2 and SecA1. The major structural difference is the absence of the helical wing domain, which is likely to play a role in how MtbSecA2 recognizes its unique substrates. Importantly, structural features critical to the interaction between SecA1 and SecYEG are preserved in SecA2. Furthermore, suppressor mutations of a dominant-negative secA2 mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG or the translocating polypeptide substrate. These results support a model in which the mycobacterial SecA2 works with SecYEG. SecA2 is a paralog of SecA1, which is the ATPase of the canonical bacterial Sec secretion system. SecA2 has a nonredundant function with SecA1, and SecA2 exports a distinct and smaller set of substrates than SecA1. This work reports the crystal structure of SecA2 of Mycobacterium tuberculosis (the first SecA2 structure reported for any organism). Many of the structural features of SecA1 are conserved in the SecA2 structure, including putative

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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...

  19. 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

  20. 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.

  1. 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 ...

  2. 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.

  3. 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....

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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...

  10. Proteomic analysis of adult Ascaris suum fluid compartments and secretory products.

    Science.gov (United States)

    Chehayeb, James F; Robertson, Alan P; Martin, Richard J; Geary, Timothy G

    2014-06-01

    Strategies employed by parasites to establish infections are poorly understood. The host-parasite interface is maintained through a molecular dialog that, among other roles, protects parasites from host immune responses. Parasite excretory/secretory products (ESP) play major roles in this process. Understanding the biology of protein secretion by parasites and their associated functional processes will enhance our understanding of the roles of ESP in host-parasite interactions. ESP was collected after culturing 10 adult female Ascaris suum. Perienteric fluid (PE) and uterine fluid (UF) were collected directly from adult females by dissection. Using SDS-PAGE coupled with LC-MS/MS, we identified 175, 308 and 274 proteins in ESP, PE and UF, respectively. Although many proteins were shared among the samples, the protein composition of ESP was distinct from PE and UF, whereas PE and UF were highly similar. The distribution of gene ontology (GO) terms for proteins in ESP, PE and UF supports this claim. Comparison of ESP composition in A. suum, Brugia malayi and Heligmosoides polygyrus showed that proteins found in UF were also secreted by males and by larval stages of other species, suggesting that multiple routes of secretion may be used for homologous proteins. ESP composition of nematodes is both phylogeny- and niche-dependent. Analysis of the protein composition of A. suum ESP and UF leads to the conclusion that the excretory-secretory apparatus and uterus are separate routes for protein release. Proteins detected in ESP have distinct patterns of biological functions compared to those in UF. PE is likely to serve as the source of the majority of proteins in UF. This analysis expands our knowledge of the biology of protein secretion from nematodes and will inform new studies on the function of secreted proteins in the orchestration of host-parasite interactions.

  11. 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.

  12. 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.

  13. Identification of CTLA2A, DEFB29, WFDC15B, SERPINA1F and MUP19 as Novel Tissue-Specific Secretory Factors in Mouse.

    Directory of Open Access Journals (Sweden)

    Jibin Zhang

    Full Text Available Secretory factors in animals play an important role in communication between different cells, tissues and organs. Especially, the secretory factors with specific expression in one tissue may reflect important functions and unique status of that tissue in an organism. In this study, we identified potential tissue-specific secretory factors in the fat, muscle, heart, lung, kidney and liver in the mouse by analyzing microarray data from NCBI's Gene Expression Omnibus (GEO public repository and searching and predicting their subcellular location in GeneCards and WoLF PSORT, and then confirmed tissue-specific expression of the genes using semi-quantitative PCR reactions. With this approach, we confirmed 11 lung, 7 liver, 2 heart, 1 heart and muscle, 7 kidney and 2 adipose and liver-specific secretory factors. Among these genes, 1 lung-specific gene--CTLA2A (cytotoxic T lymphocyte-associated protein 2 alpha, 3 kidney-specific genes--SERPINA1F (serpin peptidase inhibitor, Clade A, member 1F, WFDC15B (WAP four-disulfide core domain 15B and DEFB29 (defensin beta 29 and 1 liver-specific gene--MUP19 (major urinary protein 19 have not been reported as secretory factors. These genes were tagged with hemagglutinin at the 3'end and then transiently transfected to HEK293 cells. Through protein detection in cell lysate and media using Western blotting, we verified secretion of the 5 genes and predicted the potential pathways in which they may participate in the specific tissue through data analysis of GEO profiles. In addition, alternative splicing was detected in transcripts of CTLA2A and SERPINA1F and the corresponding proteins were found not to be secreted in cell culture media. Identification of novel secretory factors through the current study provides a new platform to explore novel secretory factors and a general direction for further study of these genes in the future.

  14. [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.

  15. 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...

  16. 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.

  17. 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.

  18. 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.

  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. Endoplasmic reticulum stress sensing in the unfolded protein response

    National Research Council Canada - National Science Library

    Gardner, Brooke M; Pincus, David; Gotthardt, Katja; Gallagher, Ciara M; Walter, Peter

    2013-01-01

    Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation...

  1. 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.

  2. SNARE protein mediated secretory events in porcine fertilization

    NARCIS (Netherlands)

    Tsai, P.S.|info:eu-repo/dai/nl/304834742

    2010-01-01

    Fertilization is an event that involves highly dynamic intra- and intercellular processes which enable the merge of two gametes with the aim to ultimately form a new organism. Preparative changes are required in both gametes prior to fertilization since freshly ejaculated sperm cells and germinal

  3. 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

  4. Direct imaging of RAB27B-enriched secretory vesicle biogenesis in lacrimal acinar cells reveals origins on a nascent vesicle budding site.

    Directory of Open Access Journals (Sweden)

    Lilian Chiang

    Full Text Available This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the "nascent vesicle site," from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150(Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules.

  5. Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae

    DEFF Research Database (Denmark)

    Liu, Lifang; Feizi, Amir; Osterlund, Tobias

    2014-01-01

    to the poorly annotated proteome. Results: Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely...

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis.

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    Irfan Prabudiansyah

    Full Text Available The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers.

  11. 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

  12. Finding and characterizing mammary analogue secretory carcinoma of the salivary gland.

    Science.gov (United States)

    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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Protein translocation across the endoplasmic reticulum membrane in cold-adapted organisms

    NARCIS (Netherlands)

    Römisch, Karin; Collie, Nicola; Soto, Nelyn; Logue, James; Lindsay, Margaret; Scheper, Wiep; Cheng, Chi-Hing C.

    2003-01-01

    Secretory proteins enter the secretory pathway by translocation across the membrane of the endoplasmic reticulum (ER) via a channel formed primarily by the Sec61 protein. Protein translocation is highly temperature dependent in mesophilic organisms. We asked whether the protein translocation

  19. 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.

  20. 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.

  1. ADF/cofilin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1.

    Science.gov (United States)

    von Blume, Julia; Alleaume, Anne-Marie; Cantero-Recasens, Gerard; Curwin, Amy; Carreras-Sureda, Amado; Zimmermann, Timo; van Galen, Josse; Wakana, Yuichi; Valverde, Miguel Angel; Malhotra, Vivek

    2011-05-17

    Actin-severing proteins ADF/cofilin are required for the sorting of secretory cargo at the trans-Golgi network (TGN) in mammalian cells. How do these cytoplasmic proteins interact with the cargoes in the lumen of the TGN? Put simply, how are these two sets of proteins connected across the TGN membrane? Mass spectrometry of cofilin1 immunoprecipitated from HeLa cells revealed the presence of actin and the Ca(2+) ATPase SPCA1. Moreover, cofilin1 was localized to the TGN and bound to SPCA1 via dynamic actin. SPCA1 knockdown, like ADF/cofilin1 knockdown, inhibited Ca(2+) uptake into the TGN and caused missorting of secretory cargo. These defects were rescued by the overexpression of the TGN-localized SPCA1. We propose that ADF/cofilin-dependent severing of actin filaments exposes and promotes the activation of SPCA1, which pumps Ca(2+) into the lumen of the TGN for the sorting of the class of secretory cargo that binds Ca(2+). Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease.

    Science.gov (United States)

    Hook, Vivian; Toneff, Thomas; Bogyo, Matthew; Greenbaum, Doron; Medzihradszky, Katalin F; Neveu, John; Lane, William; Hook, Gregory; Reisine, Terry

    2005-09-01

    The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.

  3. 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.

  4. 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.

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

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    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

  6. 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

  7. Combined inhibition of p97 and the proteasome causes lethal disruption of the secretory apparatus in multiple myeloma cells.

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    Holger W Auner

    Full Text Available Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER-associated degradation (ERAD and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48 has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

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

    Science.gov (United States)

    Hood-DeGrenier, Jennifer K.

    2008-01-01

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

  9. 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....

  10. 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 ...

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

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

    Science.gov (United States)

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

    2017-01-01

    Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. PMID:28100639

  18. 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

  19. 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

  20. Establishing human lacrimal gland cultures with secretory function.

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    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

  1. 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.

  2. 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

  3. Immunoproteomic analysis of the excretory-secretory products of Trichinella pseudospiralis adult worms and newborn larvae

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    Yang Wang

    2017-11-01

    Full Text Available Abstract Background The nematode Trichinella pseudospiralis is an intracellular parasite of mammalian skeletal muscle cells and exists in a non-encapsulated form. Previous studies demonstrated that T. pseudospiralis could induce a lower host inflammatory response. Excretory-secretory (ES proteins as the most important products of host-parasite interaction may play the main functional role in alleviating host inflammation. However, the ES products of T. pseudospiralis early stage are still unknown. The identification of the ES products of the early stage facilitates the understanding of the molecular mechanisms of the immunomodulation and may help finding early diagnostic markers. Results In this study, we used two-dimensional gel electrophoresis (2-DE-based western blotting coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS/MS to separate and identify the T. pseudospiralis adult worms ES products immunoreaction-positive proteins. In total, 400 protein spots were separated by 2-DE. Twenty-eight protein spots were successfully identified using the sera from infected pigs and were characterized to correlate with 12 different proteins of T. pseudospiralis, including adult-specific DNase II-10, poly-cysteine and histidine-tailed protein isoform 2, serine protease, serine/threonine-protein kinase ULK3, enolase, putative venom allergen 5, chymotrypsin-like elastase family member 1, uncharacterized protein, peptidase inhibitor 16, death-associated protein 1, deoxyribonuclease II superfamily and golgin-45. Bioinformatic analyses showed that the identified proteins have a wide diversity of molecular functions, especially deoxyribonuclease II (DNase II activity and serine-type endopeptidase activity. Conclusions Early candidate antigens from the ES proteins of T. pseudospiralis have been screened and identified. Our results suggest these proteins may play key roles during the T. pseudospiralis

  4. 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.

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

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    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.

  6. 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.

  7. 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...

  8. 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....

  9. Peptide aptamers expressed in the secretory pathway interfere with cellular PrPSc formation.

    Science.gov (United States)

    Gilch, Sabine; Kehler, Claudia; Schätzl, Hermann M

    2007-08-10

    Prion diseases are rare and obligatory fatal neurodegenerative disorders caused by the accumulation of a misfolded isoform (PrPSc) of the host-encoded prion protein (PrPc). Prophylactic and therapeutic regimens against prion diseases are very limited. To extend such strategies we selected peptide aptamers binding to PrP from a combinatorial peptide library presented on the Escherichia coli thioredoxin A (trxA) protein as a scaffold. In a yeast two-hybrid screen employing full-length murine PrP (aa 23-231) as a bait we identified three peptide aptamers that reproducibly bind to PrP. Treatment of prion-infected cells with recombinantly expressed aptamers added to the culture medium abolished PrPSc conversion with an IC50 between 350 and 700 nM. For expression in eukaryotic cells, peptide aptamers were fused to an N-terminal signal peptide for entry of the secretory pathway. The C terminus was modified by a glycosyl-phosphatidyl-inositol-(GPI) anchoring signal, a KDEL retention motif and the transmembrane and cytosolic domain of LAMP-I, respectively. These peptide aptamers retained their binding properties to PrPc and, depending on peptide sequence and C-terminal modification, interfered with endogenous PrPSc conversion upon expression in prion-infected cells. Notably, infection of cell cultures could be prevented by expression of KDEL peptide aptamers. For the first time, we show that trxA-based peptide aptamers can be targeted to the secretory pathway, thereby not losing the affinity for their target protein. Beside their inhibitory effect on prion conversion, these molecules could be used as fundament for rational drug design.

  10. 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

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

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    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.

  12. Unraveling the mystery of cancer by secretory microRNA: Horizontal microRNA transfer between living cells

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    Nobuyoshi eKosaka

    2012-01-01

    Full Text Available microRNAs (miRNAs have been identified as a fine-tuner in a wide array of biological processes, including development, organogenesis, metabolism, and homeostasis. Deregulation of miRNA leads to the disease status, especially cancer. This occurs through a variety of mechanisms, such as genetic alterations, epigenetic regulation, or altered expression of transcription factors, which target miRNAs. Recently, it was discovered that extracellular miRNAs circulate in the blood of both healthy and diseased patients. Most of the circulating miRNAs are included in RNA-binding proteins, apoptotic bodies, microvesicles, and exosomes. However, the secretory mechanism and biological function, as well as the meaning of the existence of extracellular miRNAs, remain largely unclear. In this article, we summarize the latest and most significant discoveries of original research on secretory miRNA involvement in many aspects of physiological and pathological conditions, with a special focus on cancer. In addition, we discuss a new aspect of cancer research that is revealed by the emergence of secretory miRNA.

  13. Triclabendazole (Anthelmintic Drug Effects on the Excretory- Secretory Proteome of Fasciola hepatica in Two Dimension Electrophoresis Gel.

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    Ashkan Faridi

    2014-06-01

    Full Text Available The aim of this study was to evaluate the protein spots of excretory - secretory products of Fasciola hepatica using two dimension electrophoresis method in the presence and absence of triclabendazole drug which can be considered to detect the target protein of the drug.F. hepatica parasites were collected from infected cattle livers, divided in two groups and cultivated in RPMI 1640 medium. First group was treated with triclabendazole (TCBZ and second group considered as control. The excretory-secretory (ES products of each group were separated and total protein determined by Bradford method. To provide proteome spots, the ES proteins were precipitated and two dimension electrophoresis (2-DE gel prepared. Protein amounts of two groups were compared using the statistical t-test and protein spots from 2-DE in test and control groups were also statistically analyzed. The protein spots of gels were identified by using protein database.The t-test showed a significant increase of total proteins in treated group (P0.05. Cathepsin L- protein (MW 36.7 pH 5.34, 14-3-3 epsilon 2 isoform (MW 28.2 pH 5.36, Cathepsin L1D (MW 36.5 pH 5.8 and Cathepsin L1D (MW 36.6 pH 6.26 were identified in test group.It seems that, these results can be considered to determine the proteins which the drug acts as a target on them.

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

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    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. 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.

  16. 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

  17. Calcineurin is universally involved in vesicle endocytosis at neuronal and non-neuronal secretory cells

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    Wu, Xin-Sheng; Zhang, Zhen; Zhao, Wei-Dong; Wang, Dongsheng; Luo, Fujun; Wu, Ling-Gang

    2014-01-01

    Calcium influx triggers and accelerates endocytosis in nerve terminals and non-neuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. At three preparations where controversies arose, including large calyx-type synapses, conventional cerebellar synapses and neuroendocrine chromaffin cells containing large dense-core vesicles, we reported that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell types, developmental stages, or endocytic forms (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at relatively small calcium influx, but did not inhibit endocytosis at large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the controversies in pharmacological studies. We therefore suggest including calcineurin as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis. PMID:24835995

  18. Shedding light on the role of lipid flippases in the secretory pathway

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura

    flippases, play an essential role in this transport process. We have recently characterized several members of the P4 subfamily of P-type ATPases as prime candidate lipid flippases in the secretory pathway of several eukaryotic cells. Our studies in yeast, plants and mammalian cells uncovered......A fundamental feature of eukaryotic cells is the presence of distinct organelles surrounded by lipid bilayers. Assembly and maintenance of the various organellar membranes requires translocation of lipids from one leaflet of the bilayer to the other. Specific membrane proteins, termed lipid...... biophysical approaches based on giant vesicles and several advanced bioimaging methods. The limitations and future perspectives of these techniques for the characterization of lipid translocases will be discussed in the light of our recent results....

  19. 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.

  20. Mammary analog secretory carcinoma of the parotid gland: A case report and literature review.

    Science.gov (United States)

    Balanzá, Ricardo; Arrangoiz, Rodrigo; Cordera, Fernando; Muñoz, Manuel; Luque-de-León, Enrique; Moreno, Eduardo; Toledo, Carlos; González, Edgar

    2015-01-01

    Background Mammary analog secretory carcinoma (MASC) was first described in 2010 by Skálová et al. This entity shares morphologic and immunohistochemical features with the secretory carcinoma (SC) of the breast. MASC usually presents as an asymptomatic mass in the parotid gland and predominantly affects men. This tumor is considered a low-grade carcinoma but has the potential for high-grade transformation. We report one MASC case and a review of world literature. Case report A 66-year-old male patient presented because he noticed a mass of approximately 3 × 3 cm on the right pre-auricular region. Physical examination demonstrated a 3 × 3.5 cm, firm, fixed, non-tender mass in the right pre-auricular region. An MRI of the head and neck showed an ovoid heterogeneous lesion, dependent of the right parotid gland of 27 × 28 mm. We performed a superficial parotidectomy with identification and preservation of the facial nerve. The immunophenotype was positive for epithelial membrane antigen (EMA), CK8/18, vimentin, S-100 protein, and mammoglobin. No further surgical interventions or adjuvant therapies were needed. The patient will have a close follow up. Conclusion The presence of t(12;15) (p13;q25) translocation which results in the ETV6-NTRK3 gene fusion or positive immunochemical studies for STAT5, mammoglobin and S100 protein, are necessary to confirm the diagnosis of MASC. MASC treatment should mimic the management of other low-grade malignant salivary gland neoplasms. The inhibition of ETV6-NTRK3 gene fusion could be used as treatment in the future. PMID:26496413

  1. Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells.

    Science.gov (United States)

    Taraska, Justin W; Perrais, David; Ohara-Imaizumi, Mica; Nagamatsu, Shinya; Almers, Wolfhard

    2003-02-18

    Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuro-peptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH(4)Cl. We conclude that most granules reseal in <10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.

  2. Cutis laxa: intersection of elastic fiber biogenesis, TGFβ signaling, the secretory pathway and metabolism.

    Science.gov (United States)

    Urban, Zsolt; Davis, Elaine C

    2014-01-01

    Cutis laxa (CL), a disease characterized by redundant and inelastic skin, displays extensive locus heterogeneity. Together with geroderma osteodysplasticum and arterial tortuosity syndrome, which show phenotypic overlap with CL, eleven CL-related genes have been identified to date, which encode proteins within 3 groups. Elastin, fibulin-4, fibulin-5 and latent transforming growth factor-β-binding protein 4 are secreted proteins which form elastic fibers and are involved in the sequestration and subsequent activation of transforming growth factor-β (TGFβ). Proteins within the second group, localized to the secretory pathway, perform transport and membrane trafficking functions necessary for the modification and secretion of elastic fiber components. Key proteins include a subunit of the vacuolar-type proton pump, which ensures the efficient secretion of tropoelastin, the precursor or elastin. A copper transporter is required for the activity of lysyl oxidases, which crosslink collagen and elastin. A Rab6-interacting goglin recruits kinesin motors to Golgi-vesicles facilitating the transport from the Golgi to the plasma membrane. The Rab and Ras interactor 2 regulates the activity of Rab5, a small guanosine triphosphatase essential for the endocytosis of various cell surface receptors, including integrins. Proteins of the third group related to CL perform metabolic functions within the mitochondria, inhibiting the accumulation of reactive oxygen species. Two of these proteins catalyze subsequent steps in the conversion of glutamate to proline. The third transports dehydroascorbate into mitochondria. Recent studies on CL-related proteins highlight the intricate connections among membrane trafficking, metabolism, extracellular matrix assembly, and TGFβ signaling. © 2013 Elsevier B.V. All rights reserved.

  3. 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.

  4. 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...

  5. 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...

  6. 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.

  7. [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.

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

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    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.

  9. The effect of metformin on monocyte secretory function in simvastatin-treated patients with impaired fasting glucose.

    Science.gov (United States)

    Krysiak, Robert; Okopien, Bogusław

    2013-01-01

    This study was designed to investigate whether metformin affects monocyte secretory function in patients with impaired fasting glucose receiving chronic statin therapy. The study included 48 patients with impaired fasting glucose treated for at least three months with simvastatin (40 mg daily). These patients were randomized to either metformin (3 g daily) or placebo, which was administered together with simvastatin for 90 days. Plasma lipids, glucose homeostasis markers, monocyte cytokine release and plasma C-reactive protein levels were determined before randomization and at the end of the treatment. Compared to placebo, metformin reduced monocyte release of tumor necrosis factor-α, interleukin-1β, interleukin-6, monocyte chemoattractant protein-1 and interleukin-8, as well as decreased plasma C-reactive protein levels, which were accompanied by an improvement in insulin sensitivity. The obtained results suggest that metformin may inhibit monocyte secretory function and reduce systemic inflammation in statin-treated patients with prediabetes. Impaired fasting glucose patients with high cardiovascular risk may receive the greatest benefits from concomitant treatment with a statin and metformin. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. An overview of conventional and novel routes of protein secretion.

    Science.gov (United States)

    Colman, A

    1991-04-01

    The purpose of this overview is to demonstrate the new complexities that have been revealed in secretory pathways. It is clear that in some areas (e.g. ATP-driven translocators), mechanisms have been conserved between bacteria and higher eukaryotic cells, while certain new processes involve modified secretory routes (e.g. antigen presentation) which will be restricted to eukaryotic cells. It is generally believed that in the evolution of living systems, the development of membrane-delimited compartments was crucial, and that insertion of proteins into membranes allowed more control of the passage of molecules through the membrane. It has been suggested that the first secretory proteins were in fact membrane bound, and extracellular proteolysis was responsible for the release of the first soluble secretory proteins. If so, it is interesting to note that the process still exists today and Pandiella [259-262] has described how the release of the growth factor transforming growth factor-alpha (TGF alpha) from mammalian cells is a case of regulated surface proteolysis. Finally, it is clear to see that as regards secretory pathways, we should keep an open mind as to which routes are utilized and which are not, and indeed, which routes exist and which do not. It appears that if two secretory organelles exist then some protein will be shown to move between them. Even the cytosol can provide a reservoir for secretory proteins or peptides. Perhaps in time S. S. Rothman will be thought of as the Nostradamus of protein secretion.

  11. 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.

  12. 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 %.

  13. 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.

  14. 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.

  15. Development and characterization of monoclonal antibodies against excretory-secretory antigens of Fasciola gigantica.

    Science.gov (United States)

    Viyanant, V; Upatham, E S; Sobhon, P; Krailas, D; Ardseungnoen, P; Anatawara, S

    1997-01-01

    Monoclonal antibodies (MAbs) directed against Fasciola gigantica excretory-secretory (ES) antigens were developed from BALB/c mice. Four were selected for further study, from the panel of hybridomas. The antigen specificities of these MAbs were characterized and localized by enzyme-linked immunoeletrotransfer blot (EITB) and immunoperoxidase technique. The target epitopes of these MAbs are 66 kDa protein (MAb 2D10), 66 and 27-26 kDa proteins (MAbs 5D10 and 4F5) and 27-26 kDa protein (MAb 2D9). MAb 2D9 reacted to the antigenic components of the luminal content and epithelial cell lining the cecum, whereas MAb 2D10 reacted specifically to the antigens of the tegument and surface membrane. It was found that all MAbs cross-reacted to various degrees with the antigens extracted from Schistosoma mansoni, S. mekongi, S. spindale and Paramphistomum spp. However, when MAbs were diluted to 1:100 or 1:400 significant reduction of their cross-reactivities was observed.

  16. 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...

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 All antigens Uterus Fallopian tube secret...hg19/assembled/ALL.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 All antigens Uterus Fallopian tube...hg19/assembled/ALL.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 All antigens Uterus Fallopian tube...hg19/assembled/ALL.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  6. 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.

  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. Triclabendazole Effect on Protease Enzyme Activity in the Excretory- Secretory Products of Fasciola hepatica in Vitro.

    Directory of Open Access Journals (Sweden)

    Yosef Shrifi

    2014-03-01

    Full Text Available Fasciola hepatica is one of the most important helminthes parasites and triclabendazole (TCBZ is routinely used for treatment of infected people and animals. Secreted protease enzymes by the F. hepatica plays a critical role in the invasion, migration, nutrition and the survival of parasite and are key targets for novel drugs and vaccines. The aim of study was to determine the protease activity of excretory- secretory products (ESP of F. hepatica in the presence of TCBZ anthelmintic.F. hepatica helminthes were collected and cultured within RPMI 1640 [TCBZ treated (test and untreated (control] for 6 h at 37 °C. ESP of treated and control were collected, centrifuged and supernatants were stored at -20°C. Protein concentrations were measured according to Bradford method. Protease enzymes activities of ESP samples were estimated by using sigma's non-specific protease activity assay. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE.Mean protein concentrations in control and treated of ESP samples were determined 196.1 ±14.52 and 376.4 ±28.20 μg/ml, respectively. Mean protease enzymes activities in control and treated were 0.37 ±0.1 and 0.089 ±0.03 U/ml, respectively. Significant difference between proteins concentrations and protease enzymes activities of two groups was observed (P<0.05. SDS-PAGE showed different patterns of protein bands between treated and control samples.The TCBZ reduced secreted protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues.

  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. 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.

  14. 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.

  15. Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

    DEFF Research Database (Denmark)

    Vester-Christensen, Malene Bech; Abou Hachem, Maher; Næsted, Henrik

    2010-01-01

    Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch ...... catalytic coefficient, k(cat,app)/K-m,K-app = 488 +/- 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed alpha-, beta-, and gamma-cyclodextrin binding to LD with K-d of 27.2, 0.70, and 34.7 mu M, respectively.......Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch...... biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha...

  16. An alternate technique for isolation of Toxocara canis excretory-secretory antigens

    Directory of Open Access Journals (Sweden)

    Cristiane Maria Colli

    2011-03-01

    Full Text Available The aim of the present study was to test the effectiveness of a sausage-casing membrane for dialysis of Toxocara excretory-secretory antigens (TES. The protein concentrated by the tested membrane was compared with that obtained using a Sigma commercial membrane, as were the protein fractions found by polyacrylamide gel electrophoresis. Standard positive and negative serum samples were evaluated in an ELISA immunoassay, and equivalent data were obtained in all steps, indicating that the sausage-casing membrane is efficient, besides being less expensive to process.O objetivo do presente estudo foi testar a eficácia de uma membrana utilizada para o preparo de embutidos, na obtenção do antígeno de excreção e secreção de Toxocara (TES. A concentração protéica foi comparada com a obtida com a membrana Sigma tanto quanto as frações protéicas separadas por eletroforese em gel de poliacrilamida. Amostras de soros padrão positivo e negativo foram avaliadas no teste imunoenzimático ELISA. Dados equivalentes foram observados em todas as etapas, sugerindo que a membrana possa ser utilizada para diálise por ser eficiente e de menor custo no preparo do antígeno.

  17. 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.

  18. 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.

  19. Morphology in conjunction with immunohistochemistry is sufficient for the diagnosis of mammary analogue secretory carcinoma.

    Science.gov (United States)

    Shah, Akeesha A; Wenig, Bruce M; LeGallo, Robin D; Mills, Stacey E; Stelow, Edward B

    2015-03-01

    The recently described mammary analogue secretory carcinoma (MASC) is a low-grade salivary gland malignancy that harbors the recurrent cytogenetic abnormality t(12;15) (p13;q25) ETV6-NTRK3. Confirmation of this is currently considered the gold standard for diagnosis. Some have postulated that morphology together with supporting immunohistochemistry is sufficient to diagnose MASC. In this study we retrospectively review a series of 19 MASCs diagnosed based on histology in conjunction with immunohistochemistry; subsequently we performed in situ hybridization using an ETV6 break-apart probe. Immunohistochemistry for S100 protein and mammaglobin as well as fluorescence in situ hybridization using the Vysis ETV6 Dual Color Break-Apart FISH Probe Kit were performed on all cases. The 19 cases were from 12 females and 7 males with ages ranging from 16 to 76 years (mean = 45 years). Sixteen cases were from the parotid gland, 1 case was from a periparotid lymph node and 2 cases were from the submandibular gland. All 19 cases demonstrated moderate to strong expression of S100 protein. Eighteen cases demonstrated strong, diffuse expression of mammaglobin, while one case had only rare tumor cells that strongly expressed mammaglobin. Eighteen of 19 cases (95 %) demonstrated the ETV6 rearrangement by fluorescence in situ hybridization. Given that morphology together with immunohistochemistry is highly correlated with the ETV6 gene rearrangement, we conclude that molecular confirmation is not required to diagnose MASC.

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  14. File list: NoD.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Utr.50.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.50.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  15. File list: Oth.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 TFs and others Uterus Fallopian tube... secretory epithelial cell http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  16. File list: Oth.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 TFs and others Uterus Fallopian tube... secretory epithelial cell http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  17. 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 ...

  18. 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 ...

  19. Uptake of ascorbic acid by freshly isolated cells and secretory granules from the intermediate lobe of ox hypophyses

    DEFF Research Database (Denmark)

    Zhou, A; Matsumoto, T; Farver, O

    1990-01-01

    Mechanically isolated cells from the intermediate lobe of ox hypophyses contained 40.6 +/- 3.7 nmol mg-1 protein (mean +/- SE, n = 5) of ascorbic acid. They accumulated radioactivity time dependently, on incubation with L-[14C]ascorbic acid in ionic medium dominated by NaCl. No definite saturation...... of uptake occurred when mechanically isolated cells were incubated with increasing ascorbic acid concentrations up to 0.6 mM. But if such cells were purified on a Percoll gradient, a clear saturation of uptake could be observed. Acetylsalicylic acid reduced the uptake markedly. When cells loaded with L-[14C]ascorbic...... sodium-dependent. Phloridzin inhibited uptake. Secretory granules from pars intermedia contained 40.0 +/- 3.8 nmol mg-1 protein of ascorbic acid (mean +/- SE, n = 3) and could accumulate L-[14C]ascorbic acid rapidly in a KCl-dominated medium. The uptake was not saturable with ascorbic acid concentration...

  20. 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.

  1. 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.

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

    Science.gov (United States)

    Hur, Yong Suk; Yoo, Seung Hyun

    2015-01-01

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

  3. Feature-based prediction of non-classical and leaderless protein secretion

    DEFF Research Database (Denmark)

    Bendtsen, Jannick Dyrløv; Jensen, Lars Juhl; Blom, Nikolaj

    2004-01-01

    We present a sequence-based method, SecretomeP, for the prediction of mammalian secretory proteins targeted to the non-classical secretory pathway, i.e. proteins without an N-terminal signal peptide. So far only a limited number of proteins have been shown experimentally to enter the non......-classical secretory pathway. These are mainly fibroblast growth factors, interleukins and galectins found in the extracellular matrix. We have discovered that certain pathway-independent features are shared among secreted proteins. The method presented here is also capable of predicting (signal peptide......-containing) secretory proteins where only the mature part of the protein has been annotated or cases where the signal peptide remains uncleaved. By scanning the entire human proteome we identified new proteins potentially undergoing non-classical secretion. Predictions can be made at http://www.cbs.dtu.dk/services/SecretomeP....

  4. Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae.

    Science.gov (United States)

    Liu, Lifang; Feizi, Amir; Österlund, Tobias; Hjort, Carsten; Nielsen, Jens

    2014-06-24

    The koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome. Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three α-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase. In combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus.

  5. Therapeutic effect of soluble worm protein acting as immune regulatory on colitis

    Directory of Open Access Journals (Sweden)

    Agustina Tri Endharti

    2017-01-01

    Conclusions: Excretory-secretory 55 kDa protein could reduce inflammation and have potential therapy. H. polygyrus excretory-secretory 55 kDa was the soluble factor that may help in the development of novel treatments to cure colitis.

  6. The exocyst subunit Sec3 is regulated by a protein quality control pathway

    DEFF Research Database (Denmark)

    Kampmeyer, Caroline; Karakostova, Antonina; Schenstrøm, Signe Marie

    2017-01-01

    Exocytosis involves fusion of secretory vesicles with the plasma membrane, thereby delivering membrane proteins to the cell surface and releasing material into the extracellular space. The tethering of the secretory vesicles before membrane fusion is mediated by the exocyst, an essential...

  7. 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.

  8. Increased expression and activity of group IIA and X secretory phospholipase A2 in peritumoral versus central colon carcinoma tissue

    DEFF Research Database (Denmark)

    Tribler, Line; Jensen, Lotte T; Jørgensen, Kent

    2007-01-01

    Secretory phospholipase A2 (sPLA2) type IIA and X was analyzed in tumors from 22 patients with colon adenocarcinomas in order to determine the involvement and activity of sPLA2 in colon cancer. Evaluation of immunoreactive sPLA2 IIA by Western blotting showed a significantly higher level in the p......Secretory phospholipase A2 (sPLA2) type IIA and X was analyzed in tumors from 22 patients with colon adenocarcinomas in order to determine the involvement and activity of sPLA2 in colon cancer. Evaluation of immunoreactive sPLA2 IIA by Western blotting showed a significantly higher level...... in the periphery of the tumors, compared to central tumor regions. Increased levels of sPLA2 IIA protein correlated with a two-fold increase in sPLA2 enzymatic activity in the peripheral regions compared to central regions. Nineteen out of 22 tumors showed high levels of sPLA2 IIA, whereas 7 out of the 22 tumors...

  9. [Western blot technique standardization for specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes].

    Science.gov (United States)

    Escalante, Hermes; Jara, César; Davelois, Kelly; Iglesias, Miguel; Benites, Adderly; Espinoza, Renzo

    2014-01-01

    Evaluate the effectiveness of Western Blot for the specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes. Antigens were obtained after twenty hours of incubation in Eagle’s Minimum Essential Medium, which were prepared at a protein concentration of 0.2 ug/uL to be faced with 10 mL pool of serum from patients with Chagas disease and a conjugated anti-IgG labeled with peroxidase. The presence of the following antigens was observed: 10, 12, 14, 15, 19, 20, 23, 26, 30, 33, 36, 40, 42, 46, 58, 63, 69, 91, 100, and 112 kDa; of which antigens of 10, 12, 14, 15, 19, 20, 23, and 26 kDa were considered to be specific using pools of serum from patients with other parasitosis and serum from people with no parasites. The sensitivity of the technique was assessed using individual serum from 65 patients with Chagas disease; and the specificity with serum from 40 patients with other parasitosis, and serums from five people who did not have parasites. The technique has a sensitivity of 95.4% in the detection of one to eight specific bands, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 93.7%. Western Blot technique with excretory-secretory antigens of T. cruzi epimastigotes is effective in the diagnosis of Chagas disease in Peru; therefore, it can be used as a confirmatory test.

  10. Impact of senescence-associated secretory phenotype and its potential as a therapeutic target for senescence-associated diseases.

    Science.gov (United States)

    Watanabe, Sugiko; Kawamoto, Shimpei; Ohtani, Naoko; Hara, Eiji

    2017-04-01

    "Cellular senescence" is a state in which cells undergo irreversible cell cycle arrest in response to a variety of cellular stresses. Once cells senesce, they are strongly resistant to any mitogens, including oncogenic stimuli. Therefore, cellular senescence has been assumed to be a potent anticancer mechanism. Although irreversible cell-cycle arrest is traditionally considered the major characteristic of senescent cells, recent studies have revealed some additional functions. Most noteworthy is the increased secretion of various secretory proteins, such as inflammatory cytokines, chemokines, growth factors, and MMPs, into the surrounding extracellular fluid. These newly recognized senescent phenotypes, termed senescence-associated secretory phenotypes (SASPs), reportedly contribute to tumor suppression, wound healing, embryonic development, and even tumorigenesis promotion. Thus, SASPs appear to be beneficial or deleterious, depending on the biological context. As senescent cells are known to accumulate during the aging process in vivo, it is quite possible that their accumulation in aged tissues promotes age-associated functional decline and various diseases, including cancers, at least to some extent. Here, we focus on and discuss the functional and regulatory network of SASPs toward opening up new possibilities for controlling aging and aging-associated diseases. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  11. Protein export by the mycobacterial SecA2 system is determined by the preprotein mature domain.

    Science.gov (United States)

    Feltcher, Meghan E; Gibbons, Henry S; Ligon, Lauren S; Braunstein, Miriam

    2013-02-01

    At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles "housekeeping" export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mosley, C.A.; Taupenot, L.; Biswas, N.; Taulane, J.P.; Olson, N.H.; Vaingankar, S.M.; Wen, G.; Schork, N.J.; Ziegler, M.G.; Mahata, S.K.; O' Connor, D.T.

    2009-06-03

    The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein 'packing' in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a

  13. 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

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

    Science.gov (United States)

    Roboti, Peristera; Sato, Keisuke; Lowe, Martin

    2015-04-15

    Golgins are coiled-coil proteins that participate in membrane-tethering events at the Golgi complex. Golgin-mediated tethering is thought to be important for vesicular trafficking and Golgi organization. However, the degree to which individual golgins contribute to these processes is poorly defined, and it has been proposed that golgins act in a largely redundant manner. Previous studies on the golgin GMAP-210 (also known as TRIP11), which is mutated in the rare skeletal disorder achondrogenesis type 1A, have yielded conflicting results regarding its involvement in trafficking. Here, we re-investigated the trafficking role of GMAP-210, and found that it is indeed required for efficient trafficking in the secretory pathway. GMAP-210 acts at both the endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) and Golgi complex during anterograde trafficking, and is also required for retrograde trafficking to the ER. Using co-depletion experiments, we also found that GMAP-210 acts in a partially redundant manner with the golgin GM130 to ensure efficient anterograde cargo delivery to the cis-Golgi. In summary, our results indicate a role for GMAP-210 in several trafficking steps at the ER-Golgi interface, some of which are partially redundant with another golgin, namely GM130 (also known as GOLGA2). © 2015. Published by The Company of Biologists Ltd.

  15. SIRT1 suppresses the senescence-associated secretory phenotype through epigenetic gene regulation.

    Directory of Open Access Journals (Sweden)

    Tomohisa Hayakawa

    Full Text Available Senescent cells develop a pro-inflammatory response termed the senescence-associated secretory phenotype (SASP. As many SASP components affect surrounding cells and alter their microenvironment, SASP may be a key phenomenon in linking cellular senesence with individual aging and age-related diseases. We herein demonstrated that the expression of Sirtuin1 (SIRT1 was decreased and the expression of SASP components was reciprocally increased during cellular senescence. The mRNAs and proteins of SASP components, such as IL-6 and IL-8, quickly accumulated in SIRT1-depleted cells, and the levels of these factors were also higher than those in control cells, indicating that SIRT1 negatively regulated the expression of SASP factors at the transcriptional level. SIRT1 bound to the promoter regions of IL-8 and IL-6, but dissociated from them during cellular senescence. The acetylation of Histone H3 (K9 and H4 (K16 of the IL-8 and IL-6 promoter regions gradually increased during cellular senescence. In SIRT1-depleted cells, the acetylation levels of these regions were already higher than those in control cells in the pre-senescent stage. Moreover, these acetylation levels in SIRT1-depleted cells were significantly higher than those in control cells during cellular senescence. These results suggest that SIRT1 repressed the expression of SASP factors through the deacetylation of histones in their promoter regions.

  16. Isolation and partial characterization of excretory/secretory antigens of Gastrothylax crumenifer.

    Science.gov (United States)

    Saifullah, Mohammad K; Ahmad, Gul; Abidi, Syed M A

    2011-08-25

    The present study was carried out to identify the excretory/secretory (E/S) antigens of the rumen infecting digenetic trematode Gastrothylax crumenifer that may be useful for the immunodiagnosis of rumen amphistomosis particularly during the pre-monsoon season during which this rumen parasite stops shedding eggs. The in vitro released E/S proteins were purified on a Sephadex G-200 column. The gel filtration profile revealed three distinct fractions F1-F3 where F1 and F3 appeared as sharp peaks while the F2 fraction was dispersed. The antibody titre against each of the purified E/S fractions was determined by ELISA using anti-whole E/S polyclonal antibodies raised in rabbit. Among the three fractions, the antibody titre against F1 was highest (1:12,800) whereas IgG titre was very low (1:50) for fraction F2 and F3 (1:100). Of the total polypeptides resolved on gradient SDS-PAGE, only a few antigenic polypeptides were detected in each fraction with hyperimmune anti-serum as revealed by Western Blot analysis. However, a 33 kDa antigen detected in each fraction appeared to be immunodominant which could be exploited for the diagnosis of the pouched amphistome. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. The cytological features of mammary analogue secretory carcinoma: a series of 6 molecularly confirmed cases.

    Science.gov (United States)

    Griffith, Christopher C; Stelow, Edward B; Saqi, Anjali; Khalbuss, Walid E; Schneider, Frank; Chiosea, Simion I; Seethala, Raja R

    2013-05-01

    Mammary analogue secretory carcinoma (MASC) of the salivary glands is a newly described tumor entity associated with the t(12;15)(p13;q25) ETV6-NTRK3 translocation. Early studies have shown this tumor to be a distinct entity with histologic, biologic, and clinical differences from acinic cell carcinoma and adenocarcinoma, not otherwise specified. Because this tumor was described only recently, it remains relatively unknown outside of head and neck specialty pathology centers. In the current study, 6 cases of fine-needle aspiration cytology from histologically and/or molecularly confirmed cases of MASC are presented. Using cytomorphology, MASC primarily raises the differential diagnosis of an oncocytic salivary gland tumor but there are some features that can suggest the specific diagnosis of MASC. The 6 cases presented in the current study all demonstrated at least focal cytoplasmic vacuolization and papillary formations on smears. MASC can be differentiated from acinic cell carcinoma by a lack of periodic acid-Schiff diastase-positive zymogen granules and S-100 protein positivity. The results of the current study the ability of ETV6 break-apart fluorescence in situ hybridization to detect gene rearrangement on cell block material. This is the first report of a case of MASC prospectively diagnosed on a cytology specimen. Copyright © 2012 American Cancer Society.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  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. 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.

  9. 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.

  10. Glucoamylase : green fluorescent protein fusions to monitor protein secretion in Aspergillus niger

    NARCIS (Netherlands)

    Gordon, C.L.; Khalaj, V.; Ram, A.F.J.; Archer, D.B.; Brookman, J.L.; Trinci, A.P.J.; Jeenes, D.J.; Doonan, J.H.; Wells, B.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Robson, G.D.

    2000-01-01

    A glucoamylase: :green fluorescent protein fusion (GLA: :sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA: :sGFP which employed slightly different

  11. Inhibition of the Secretory pathway by Foot-and-Mouth disease virus 2BC protein is reproduced by co-expression of 2B with 2C, and the site of inhibition is determined by the subcellular location of 2C

    DEFF Research Database (Denmark)

    Moffat, Katy; Knox, Caroline; Howell, Gareth

    2007-01-01

    immune responses in vivo. Foot-and-mouth disease virus (FMDV), another picornavirus, can cause persistent infection of ruminants, suggesting it too may inhibit immune responses. Endoplasmic reticulum (ER)-to-Golgi apparatus transport of proteins is blocked by the FMDV 2BC protein. The observation that 2...... blocked in FMDV-infected cells. The block could be reconstituted by coexpression of 2B and 2C, showing that processing of 2BC did not compromise the ability of FMDV to slow secretion. Under these conditions, 2C was located to the Golgi apparatus, and the block in transport also occurred in the Golgi...... apparatus. Interestingly, the block in transport could be redirected to the ER when 2B was coexpressed with a 2C protein fused to an ER retention element. Thus, for FMDV a block in secretion is dependent on both 2B and 2C, with the latter determining the site of the block....

  12. 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.

  13. 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...

  14. Mammary analogue secretory carcinoma of salivary glands is a lipid-rich tumour, and adipophilin can be valuable in its identification.

    Science.gov (United States)

    Mariano, Fernanda Viviane; dos Santos, Harim Tavares; Azañero, Wilson Delgado; da Cunha, Isabela Werneck; Coutinho-Camilo, Claudia Malheiros; de Almeida, Oslei Paes; Kowalski, Luiz Paulo; Altemani, Albina

    2013-10-01

    Mammary analogue secretory carcinoma (MASC) of salivary glands shows morphological similarities to milk-secreting mammary epithelial cells. The aim of this study was to analyse the immunohistochemical expression of adipophilin (a component of milk lipid globule membranes) and of proteins related to secretory mechanisms (STAT5a and mammaglobin) in MASC and other salivary tumours. Ten cases of MASC (all with ETV6 translocation) and 83 other salivary carcinomas were studied. In all MASC cases, adipophilin stained numerous large lipid droplets. These droplets were minute in other salivary carcinomas, except for sebaceous carcinoma. Overexpression of STAT5a was detected in all MASC cases, but only occasionally in other carcinomas. Mammaglobin expression occurred frequently in MASC (70% of cases), whereas, in other carcinomas, it was uncommon and limited. Only MASC showed cytoplasmic reactivity for p63, particularly in papillary-cystic areas. Positivity for S100, vimentin and high molecular weight keratin was observed in 100% of MASC cases. MASC is a lipid-rich tumour containing large lipid droplets covered by adipophilin. This finding can be included among its defining immunohistochemical features, and possibly represents lactation-like secretory differentiation. Strong expression of STAT5a and cytoplasmic p63 in MASC reinforces this hypothesis. © 2013 John Wiley & Sons Ltd.

  15. Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

    Science.gov (United States)

    Chen, Sun-Xia; Xu, Xiao-En; Wang, Xiao-Qing; Cui, Shu-Jian; Xu, Lei-Lei; Jiang, Ying-Hua; Zhang, Yang; Yan, Hai-Bo; Zhang, Qian; Qiao, Jie; Yang, Peng-Yuan; Liu, Feng

    2014-10-14

    Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel

  16. Epithelial disruptions, but not immune cell invasion, induced secretory dysfunction following innate immune activation in a novel model of acute salivary gland injury.

    Science.gov (United States)

    Shaalan, Abeer; Carpenter, Guy; Proctor, Gordon

    2017-11-21

    Salivary gland (SG) injurious agents are all translated into loss of salivation (xerostomia). An association has been established between activation of innate immunity and salivary gland injury and dysfunction. However, it remains unclear how the secretory epithelia respond by halting saliva production. C57BL/6 submandibular glands (SMGs) were acutely challenged using a single dose of the innate immune stimulant: polyinosinic-polycytidylic acid (poly (I:C)). Secretory capacity of the infected SMGs was substantiated by assessing the flow rate in response to pilocarpine stimulation. Depletion of the acute inflammatory cells was achieved by pre-treating mice with RB6-8C5 depletion antibody. Flow cytometry, histology and immunohistochemistry were conducted to verify the immune cell depletion. Epithelial expression of saliva-driving molecules: muscarinic 3 receptor (M3R), aquaporin 5 water channel (AQP5), Na-K- CL-Cotransporter 1 (NKCC1) and transmembrane member 16A (TMEM16A), were characterized using RT-qPCR and immunohistochemistry. Tight junction (TJ) protein; zonula occludens (ZO-1) and basement membrane (BM) protein; laminin were assessed by immunohistochemistry. Innate immune challenge prompted dysfunction in the exocrine salivary glands (SGs). Dysregulated gene and protein expression of molecules that drive saliva secretion was substantiated. Aberrant expression of TJ and BM proteins followed innate immune activation. Hyposalivation in the current model was independent of myeloperoxidase (MPO)-positive, acute inflammatory cells. In the present study, we developed a novel injury model of the SGs, featuring acute secretory dysfunction and immediate structural disruptions. Our results ruled out the injurious role of aggressively infiltrating inflammatory cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  17. Proteomic analysis of the secretory response of Aspergillus niger to D-maltose and D-xylose.

    Directory of Open Access Journals (Sweden)

    José Miguel P Ferreira de Oliveira

    Full Text Available Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on D-sorbitol, small amounts of D-maltose or D-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by D-maltose or D-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on D-maltose and β-xylosidase D on D-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra D-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of D-xylose or D-maltose. Furthermore, D-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15 and vesicular transport (e.g., the endosomal-cargo receptor Erv14. Millimolar amounts of the inducers D-maltose and D-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by D-maltose or D-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for D-xylose induction, D-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation.

  18. TISs-ST: a web server to evaluate polymorphic translation initiation sites and their reflections on the secretory targets

    Directory of Open Access Journals (Sweden)

    Menossi Marcelo

    2007-05-01

    Full Text Available Abstract Background The nucleotide sequence flanking the translation initiation codon (start codon context affects the translational efficiency of eukaryotic mRNAs, and may indicate the presence of an alternative translation initiation site (TIS to produce proteins with different properties. Multi-targeting may reflect the translational variability of these other protein forms. In this paper we present a web server that performs computations to investigate the usage of alternative translation initiation sites for the synthesis of new protein variants that might have different functions. Results An efficient web-based tool entitled TISs-ST (Translation Initiation Sites and Secretory Targets evaluates putative translation initiation sites and indicates the prediction of a signal peptide of the protein encoded from this site. The TISs-ST web server is freely available to both academic and commercial users and can be accessed at http://ipe.cbmeg.unicamp.br/pub/TISs-ST. Conclusion The program can be used to evaluate alternative translation initiation site consensus with user-specified sequences, based on their composition or on many position weight matrix models. TISs-ST provides analytical and visualization tools for evaluating the periodic frequency, the consensus pattern and the total information content of a sequence data set. A search option allows for the identification of signal peptides from predicted proteins using the PrediSi software.

  19. 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.

  20. Isolation of a sesquiterpene synthase expressing in specialized epithelial cells surrounding the secretory cavities in rough lemon (Citrus jambhiri).

    Science.gov (United States)

    Uji, Yuya; Ozawa, Rika; Shishido, Hodaka; Taniguchi, Shiduku; Takabayashi, Junji; Akimitsu, Kazuya; Gomi, Kenji

    2015-05-15

    Volatile terpenoids such as monoterpenes and sesquiterpenes play multiple roles in plant responses and are synthesized by terpene synthases (TPSs). We have previously isolated a partial TPS gene, RlemTPS4, that responds to microbial attack in rough lemon. In this study, we isolated a full length RlemTPS4 cDNA from rough lemon. RlemTPS4 localized in the cytosol. The recombinant RlemTPS4 protein was obtained using a prokaryotic expression system and GC-MS analysis of the terpenes produced by the RlemTPS4 enzymatic reaction determined that RlemTPS4 produces some sesquiterpenes such as δ-elemene. The RlemTPS4 gene was specifically expressed in specialized epithelial cells surrounding the oil secretory cavities in rough lemon leaf tissue. Copyright © 2015 Elsevier GmbH. All rights reserved.

  1. Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

    Science.gov (United States)

    Fletcher, Sarah J; Iqbal, Mudassar; Jabbari, Sara; Stekel, Dov; Rappoport, Joshua Z

    2014-01-01

    Tight junctions (TJs) link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK) epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes). By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

  2. Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

    Directory of Open Access Journals (Sweden)

    Sarah J Fletcher

    Full Text Available Tight junctions (TJs link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes. By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  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

    2015-01-01

    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

  9. 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

  10. Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures

    DEFF Research Database (Denmark)

    Tanner, V A; Ploug, Thorkil; Tao-Cheng, J H

    1996-01-01

    substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types...... membranes. Furthermore, whereas SV2 is localized on the membranes of the LDCVs, chromogranin A, an acidic protein in secretory granules, is clearly in the core of the LDCVs. This is the first demonstration of these two antigens in such dose (approximately 20 nm) yet distinct compartments within a single...

  11. Mammary analogue secretory carcinoma of salivary glands, containing the ETV6-NTRK3 fusion gene: a hitherto undescribed salivary gland tumor entity.

    Science.gov (United States)

    Skálová, Alena; Vanecek, Tomas; Sima, Radek; Laco, Jan; Weinreb, Ilan; Perez-Ordonez, Bayardo; Starek, Ivo; Geierova, Marie; Simpson, Roderrick H W; Passador-Santos, Fabricio; Ryska, Ales; Leivo, Ilmo; Kinkor, Zdenek; Michal, Michal

    2010-05-01

    We present a series of 16 salivary gland tumors with histomorphologic and immunohistochemical features reminiscent of secretory carcinoma of the breast. This is a hitherto undescribed and distinctive salivary gland neoplasm, with features resembling both salivary acinic cell carcinoma (AciCC) and low-grade cystadenocarcinoma, and displaying strong similarities to breast secretory carcinoma. Microscopically, the tumors have a lobulated growth pattern and are composed of microcystic and glandular spaces with abundant eosinophilic homogenous or bubbly secretory material positive for periodic acid-Schiff, mucicarmine, MUC1, MUC4, and mammaglobin. The neoplasms also show strong vimentin, S-100 protein, and STAT5a positivity. For this tumor, we propose a designation mammary analogue secretory carcinoma of salivary glands (MASC). The 16 patients comprised 9 men and 7 women, with a mean age of 46 years (range 21 to 75). Thirteen cases occurred in the parotid gland, and one each in the minor salivary glands of the buccal mucosa, upper lip, and palate. The mean size of the tumors was 2.1 cm (range 0.7 to 5.5 cm). The duration of symptoms was recorded in 11 cases and ranged from 2 months to 30 years. Clinical follow-up was available in 13 cases, and ranged from 3 months to 10 years. Four patients suffered local recurrences. Two patients died, 1 of them owing to multiple local recurrences with extension to the temporal bone, and another owing to metastatic dissemination to cervical lymph nodes, pleura, pericardium, and lungs. We have shown a t(12;15) (p13;q25) ETV6-NTRK3 translocation in all but one case of MASC suitable for analysis. One case was not analyzable and another was not available for testing. This translocation was not found in any conventional salivary AciCC (12 cases), nor in other tumor types including pleomorphic adenoma (1 case) and low-grade cribriform cystadenocarcinoma (1 case), whereas ETV6-NTRK3 gene rearrangements were proven in all 3 tested cases of

  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. 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.

  14. Immunolocalization and immunodetection of the excretory/secretory (ES antigens of Fasciola gigantica.

    Directory of Open Access Journals (Sweden)

    M A Hannan Khan

    Full Text Available The digenetic trematode Fasciola gigantica is a parasite of great agricultural and economic importance. Along with Fasciola hepatica, F. gigantica incurs huge economic losses to the agricultural sector. Because of unavailability of an effective and commercial vaccine, the earliest diagnosis of the disease is the only way to control the disease. The conventional coprological techniques are able to detect the disease only after the parasites get matured and starts releasing their eggs with the faeces of host, therefore prepatent infection remain undiagnosed. The alternative method is by serological tests that uses circulatory antigens. Despite high sensitivity, their reliability is quite low because of the common antigens shared between different helminth parasites. To overcome this, investigation was shifted to identify the copro-antigens which could be more sensitive and reliable. In the present study, we tried to identify some of the immunodominant proteins from the Excretory Secretory (ES product of F. gigantica which can be further characterized and used for early detection of infection and also as drug and vaccine candidates. The ES products of F. gigantica were collected and used for raising the polyclonal antibody in rabbit. The polypeptide profile was generated as well as immunogenic polypeptides were identified. The Source of ES antigen was immunolocalized using confocal microscopy and dot blot assay was performed to diagnose field infection. The polypeptide profile of ES products revealed a total of 24 polypeptides out of which 12 immunogenic polypeptides were identified by western blotting. Confocal micrographs showed the immunolocalization of antigens in the intestinal caecae, vitalline glands, gonads as well as in the tegument of the worm. The dot blot assay confirmed the utility of ES products for the detection of field infection. Subsequently, cross reactivity was found negative with Gigantocotyle explanatum; an amphitome parasite

  15. Mammary analogue secretory carcinoma (MASC) of salivary gland in four Mexican patients.

    Science.gov (United States)

    Serrano-Arévalo, Mónica L; 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.

  16. Astrocytes in the aging brain express characteristics of senescence-associated secretory phenotype.

    Science.gov (United States)

    Salminen, Antero; Ojala, Johanna; Kaarniranta, Kai; Haapasalo, Annakaisa; Hiltunen, Mikko; Soininen, Hilkka

    2011-07-01

    Cellular stress increases progressively with aging in mammalian tissues. Chronic stress triggers several signaling cascades that can induce a condition called cellular senescence. Recent studies have demonstrated that senescent cells express a senescence-associated secretory phenotype (SASP). Emerging evidence indicates that the number of cells expressing biomarkers of cellular senescence increases in tissues with aging, which implies that cellular senescence is an important player in organismal aging. In the brain, the aging process is associated with degenerative changes, e.g. synaptic loss and white matter atrophy, which lead to progressive cognitive impairment. There is substantial evidence for the presence of oxidative, proteotoxic and metabolic stresses in aging brain. A low-level, chronic inflammatory process is also present in brain during aging. Astrocytes demonstrate age-related changes that resemble those of the SASP: (i) increased level of intermediate glial fibrillary acidic protein and vimentin filaments, (ii) increased expression of several cytokines and (iii) increased accumulation of proteotoxic aggregates. In addition, in vitro stress evokes a typical senescent phenotype in cultured astrocytes and, moreover, isolated astrocytes from aged brain display the proinflammatory phenotype. All of these observations indicate that astrocytes are capable of triggering the SASP and the astrocytes in aging brain display typical characteristics of cellular senescence. Bearing in mind the many functions of astrocytes, it is evident that the age-related senescence of astrocytes enhances the decline in functional capacity of the brain. We will review the astroglial changes occurring during aging and emphasize that senescent astrocytes can have an important role in age-related neuroinflammation and neuronal degeneration. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  17. Dynamics of peptidergic secretory granule transport are regulated by neuronal stimulation

    Directory of Open Access Journals (Sweden)

    Cowan Ann E

    2010-03-01

    Full Text Available Abstract Background Peptidergic neurons store and secrete the contents of large dense core vesicles (LDCVs from axon terminals and from dendrites. Secretion of peptides requires a highly regulated exocytotic mechanism, plus coordinated synthesis and transport of LDCVs to their sites of release. Although these trafficking events are critical to function, little is known regarding the dynamic behavior of LDCVs and the mechanisms by which their transport is regulated. Sensory neurons also package opiate receptors in peptide-containing LDCVs, which is thought to be important in pain sensation. Since peptide granules cannot be refilled locally after their contents are secreted, it is particularly important to understand how neurons support regulated release of peptides. Results A vector encoding soluble peptidylglycine α-hydroxylating monooxygenase fused to green fluorescent protein was constructed to address these questions in cultured primary peptidergic neurons of the trigeminal ganglion using time lapse confocal microscopy. The time course of release differs with secretagogue; the secretory response to depolarization with K+ is rapid and terminates within 15 minutes, while phorbol ester stimulation of secretion is maintained over a longer period. The data demonstrate fundamental differences between LDCV dynamics in axons and growth cones under basal conditions. Conclusions Under basal conditions, LDCVs move faster away from the soma than toward the soma, but fewer LDCVs travel anterograde than retrograde. Stimulation decreased average anterograde velocity and increases granule pausing. Data from antibody uptake, quantification of enzyme secretion and appearance of pHluorin fluorescence demonstrate distributed release of peptides all along the axon, not just at terminals.

  18. Most Non-Parotid “Acinic Cell Carcinomas” Represent Mammary Analogue Secretory Carcinomas

    Science.gov (United States)

    Bishop, Justin A.; Yonescu, Raluca; Batista, Denise; Eisele, David W.; Westra, William H.

    2013-01-01

    Acinic cell carcinoma (ACC) is a low grade salivary gland malignancy characterized by serous acinar differentiation. Most ACCs arise in the parotid gland, but ACCs have been reported to originate in non-parotid salivary glands where where serous acini are less abundant. Given the recent discovery of mammary analogue secretory carcinoma (MASC) – a salivary malignancy that histologically mimics ACC – a retrospective re-evaluation of non-parotid ACCs is warranted. The surgical pathology archives of The Johns Hopkins Hospital were searched for all ACCs arising outside of the parotid gland. For each case, the histologic slides were reviewed; immunohistochemistry (mammaglobin, S100 protein) was performed; and confirmatory ETV6 break-apart FISH assay was completed. Demographic and clinical data was obtained from the medical records. Fourteen extra-parotid tumors diagnosed as ACC were identified. Eleven of 14 (79%) tumors harbored the ETV6 translocation (oral cavity = 9 of 11; submandibular gland = 2 of 2). The translocation positive tumors occurred in 7 women and 4 men ranging in age from 20–86 years (mean, 56), and usually presented as painless masses. Immunohistochemistry for mammaglobin and S100 was positive in all 11 translocation positive tumors, but negative in the 3 translocation negative tumors. Histologically, the translocation positive tumors exhibited uniform cells with vacuolated cytoplasm, microcystic/cystic and papillary architecture, and intraluminal secretions; but the presence of basophilic cytoplasmic granules was conspicuously absent. Basophilic cytoplasmic granules, indicative of true serous acinar differentiation, were present in the 3 translocation negative tumors. Of the translocation positive tumors, only one locally recurred, and none metastasized. Most alleged ACCs of non-parotid origin actually represent misclassified MASCs. The impact of diagnostic error is mitigated by the low grade nature of MASC that, like ACCs, do not appear to be

  19. Proteomic analysis of the excretory-secretory products from larval stages of Ascaris suum reveals high abundance of glycosyl hydrolases.

    Directory of Open Access Journals (Sweden)

    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.

  20. 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.

  1. [Histochemical properties of secretory granules and fine structure of terminal portion in the Japanese Macaque labial gland].

    Science.gov (United States)

    Tsutusumi, T; Kurabuchi, S; Aiyama, S

    1989-06-01

    The terminal portion of the Japanese macaque (Macaca fuscata) labial gland was examined ultrastructurally and histochemically. The results obtained are as follows. 1) The Japanese macaque, Macaca fuscata has the upper and the lower labial glands, which can be described as a compound tubulo-acinar gland. 2) The terminal portion of the labial gland appears to be consisted of the mucous acini and the demilunes when examined with the light microscope. 3) The secretory granules containing in the glandular cells of the mucous acini and the demilunes are negative with napthol yellow S, ninhydrin-Schiff and DMAB nitrite. 4) The secretory granules containing in the glandular cells of mucous acini stain intensely with PAS, alcian blue (pH1.0, 2.5, 3.5), colloidal iron and PA-methenamine silver, while those of demilunes are negative with alcian blue (pH1.0). The glandular cells of demilune with the PA-methenamine silver method shows weak positive granules and positive granules which are limited to the hallo of them. 5) In the mucous acini the mucous granules are ejected from glandular cells by the process of exocytosis. 6) The myoepithelial cell can be seen in the terminal portion. This cell surrounds the acini with long processes. These findings suggest that the glandular cells of the demilune have the granules containing mucopoly saccharides and a small quantity of protein in addition to the mucous granules, although the terminal portion of the Japanese macaque labial gland is nearly composed of mucous cells.

  2. Lysozyme Expression Can be Useful to Distinguish Mammary Analog Secretory Carcinoma from Acinic Cell Carcinoma of Salivary Glands.

    Science.gov (United States)

    Mariano, Fernanda Viviane; Gómez, Camila Andrea Concha; de Souza do Nascimento, Juliana; Dos Santos, Harim Tavares; Egal, Erika Said; Montalli, Victor Angelo Martins; Vargas, Pablo Agustin; de Almeida, Oslei Paes; Altemani, Albina

    2016-12-01

    Lysozyme is an enzymatic marker of acinar and intercalated duct cells of normal salivary glands. The aim of this study was to verify whether lysozyme expression could be useful to distinguish acinic cell carcinoma (ACC) from its main mimic, mammary analog secretory carcinoma (MASC). For comparison, DOG1 expression was analyzed as well. Seventeen cases of ACC, 15 MASC, and 125 other salivary tumors were studied. Lysozyme expression was found in tumor cells as well as in secreted material of MASC (86.6 % of cases) and in ductal cells of epithelial-myoepithelial carcinoma (EMC-53.8 %), pleomorphic adenoma (PA-29.1 %) and polymorphous low-grade adenocarcinoma (PLGA-23.8 %). However, in ACC, lysozyme was not expressed. Three patterns of DOG1 staining were seen: apical-luminal, cytoplasmic, and mixed cytoplasmic/membranous. The apical-luminal pattern was detected in ductal cells of ACC (58.8 % of cases), EMC (38.4 %), adenoid-cystic carcinoma (AdCC-35.3 %), PA (8.3 %), and PLGA (4.8 %). These tumors also showed mixed membranous/cytoplasmic staining for DOG1. MASC, mucoepidermoid, and salivary duct carcinomas exhibited only DOG1 cytoplasmic staining. In conclusion, lysozyme cannot be used as a marker of acinar differentiation in salivary tumors. However, lysozyme expression can be helpful to distinguish MASC from ACC due to its high frequency in the former and absence in ACC. It is likely that in MASC, lysozyme expression may reflect a lactational-like secretory differentiation since lysozyme belongs to breast milk proteins. Regarding DOG1 expression, the apical-luminal pattern is related to acinar and intercalated duct differentiation whereas the cytoplasmic staining does not seem to be associated with a specific cellular phenotype.

  3. 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.

  4. Rat peritubular cell secretory activity: modulation by vitamin A.

    Science.gov (United States)

    Galdieri, M; Provenzano, D

    1996-10-01

    Protein synthesis and secretion by rat peritubular cells cultured in the presence or in the absence of vitamin A were analyzed after labelling the cells with different amino acids. Vitamin A reduces the amount of proline incorporated into the newly-synthesized proteins secreted by the peritubular cells. By mono- and two-dimensional gel electrophoresis analysis, qualitative differences induced by retinol addition were detected in the proteins secreted by the proline-labelled cells. In particular we demonstrate that the amount of fibronectin secreted by the cells is reduced after retinol addition. Northern blot analysis indicates that fibronectin messenger RNA transcription is also reduced as a consequence of the vitamin addition.

  5. Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization.

    Science.gov (United States)

    Heiss, Silvia; Puxbaum, Verena; Gruber, Clemens; Altmann, Friedrich; Mattanovich, Diethard; Gasser, Brigitte

    2015-07-01

    Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.

  6. Modulation of the Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus cascavella Induced by Naringin

    Directory of Open Access Journals (Sweden)

    Marcos H. Toyama

    2011-01-01

    Full Text Available In this work we have characterized the action of the naringin, a flavonoid found in grapefruit and known for its various pharmacological effects, which include antioxidant blood lipid lowering and anticancer activity, on the structure and biochemical activities of a secretory phospholipase A (sPLA2 from Crotalus durissus cascavella, an important protein involved in the releasinge of arachidonic acid in phospholipid membranes. sPLA2 was incubated with naringin (mol:mol at 37 °C and a discrete reduction in the UV scanning signal and a modification of the circular dichroism spectra were observed after treatment with naringin, suggesting modifications of the secondary structure of the protein. This flavonoid was able to decrease enzymatic activity and some pharmacological effects, such as myonecrosis, platelet aggregation, and neurotoxic activity caused by sPLA2, however, the inflammatory effect was not affected by naringin. In addition, small angle X-ray scattering (SAXS data were collected for sPLA2 and naringin-treated sPLA2 to evaluate possible modifications of the protein structure. These structural investigations have shown that sPLA2 is an elongated dimer in solution and after treatment with naringin a conformational change in the dimeric configuration was observed. Our results suggest that structural modification may be correlated with the loss of enzymatic activity and alterations in pharmacological properties.

  7. [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.

  8. 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...

  9. 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...

  10. 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.

  11. 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.

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

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  20. Amelogenins as potential buffers during secretory-stage amelogenesis

    NARCIS (Netherlands)

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

    2015-01-01

    Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would

  1. Secretory expression of active clostripain in Escherichia coli.

    Science.gov (United States)

    Kim, Chang-Kyu; Lee, Sang-Yong; Kwon, Oh-Joong; Lee, Sang-Mok; Nah, Seung-Yeol; Jeong, Sang Min

    2007-09-15

    In this study, the clostripain gene was modified and its signal sequence was replaced with that of penicillin G acylase (PGA). The core clostripain protein fused to the PGA signal peptide was also prepared. With regard to the expression of the clostripain precursors, the majority of clostripain activity was observed in the culture media, thereby indicating that both the clostripain signal peptide and the PGA signal peptide were recognized in the E. coli secretion pathway, and the precursors successfully matured into the active form. Otherwise, the activity was rather low when the core protein was expressed, which indicates that the clostripain pro-peptide is important in the formation of the active enzyme in E. coli. Enzyme activity reached a value of 3200U/L in CGY media for high expression. The recombinant clostripain and porcine carboxypeptidase B were used in the conversion of a proinsulin fusion protein into insulin. The leader peptide (LP) and the proinsulin C-peptide appeared to have been removed simultaneously, and the final cleavage product evidenced an HPLC retention time identical to that of the insulin standard, thereby implying that the clostripain specifically cleaved the arginine residues in the LP and in the C-peptide. We have also demonstrated the possibility that the recombinant clostripain might prove useful in the production of insulin from the proinsulin fusion protein.

  2. 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.

  3. 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....

  4. 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.

  5. 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

  6. 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.

  7. 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

  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. 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.

  10. 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

  11. 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.

  12. 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 ...

  13. 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...

  14. 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

  15. 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

  16. 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.

  17. A Cooperia punctata gene family encoding 14 kDa excretory-secretory antigens conserved for trichostrongyloid nematodes.

    Science.gov (United States)

    Yatsuda, A P; De Vries, E; Vieira Bressan, M C; Eysker, M

    2001-12-01

    A polymorphic set of 14 kDa excretory-secretory (E-S) antigen-encoding cDNAs, with similarity to a previously characterized 15 kDa E-S antigen of Haemonchus contortus, was cloned from Cooperia punctata. Five cDNAs encoding predicted proteins of 70-80% identity were sequenced. Genomic analyses of individuals proved the existence of three 14 kDa E-S antigen-encoding genes, excluding that the differences reflected polymorphisms between individuals in a population. Southern blots indicated the presence of additional members of this gene family. Thus, despite the fact that heterologously expressed C. punctata 14 kDa E-S products are shown to be recognized by immune sera, potential pitfalls in the development of a recombinant vaccine are presented by this genetic diversity. Vaccine design could be further rationalized by knowledge of the function, and possible redundancy in function, of the E-S products which is presently lacking. The limitations encountered in assigning a function to the 14/15 kDa family of E-S proteins that is thus far unique to the trichostrongyloid nematodes are discussed.

  18. 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.

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

    OpenAIRE

    Balic, Anamaria; Mina, Mina

    2010-01-01

    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 studie...

  20. Functional analysis of an Aspergillus ficuum phytase gene in Saccharomyces cerevisiae and its root-specific, secretory expression in transgenic soybean plants.

    Science.gov (United States)

    Li, Guilan; Yang, Shaohui; Li, Minggang; Qiao, Yake; Wang, Jiehua

    2009-08-01

    Phytases release inorganic phosphates from phytate in soil. A gene encoding phytase (AfPhyA) was isolated from Aspergillus ficuum and its ability to degrade phytase and release phosphate was demonstrated in Saccharomyces cerevisiae. A promoter from the Arabidopsis Pky10 gene and the carrot extensin signal peptide were used to drive the root-specific and secretory expression of the AfPhyA gene in soybean plants. The phytase activity and inorganic phosphate levels in transgenic soybean root secretions were 4.7 U/mg protein and 439 microM, respectively, compared to 0.8 U/mg protein and 120 microM, respectively, in control soybeans. Our results demonstrated the potential usefulness of the root-specific promoter for the exudation of recombinant phytases and offered a new perspective on the mobilization of phytate in soil to inorganic phosphates for plant uptake.

  1. Transcriptome profiles of the protoscoleces of Echinococcus granulosus reveal that excretory-secretory products are essential to metabolic adaptation.

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  3. 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

  4. [Controls of the plant endomembrane-secretory pathway]. Final report

    Energy Technology Data Exchange (ETDEWEB)

    1991-12-31

    These studies are focused on elucidating the molecular structure of plant cell membranes with special reference to cell surface glycoproteins. The studies reported herein include use of monoclonal antibodies to characterize cell surface epitopes, construction of cDNA libraries of cell surface proteins, isolation of plant cell mutants by flow cytometry, detection of beta-glucouronidase marker enzyme systems in plants, expression go VSVG (the major envelope glycoprotein of Vesicular Stomatis Virus) in plant cells, and control of gene expression of cell membrane glycoproteins.(DT)

  5. (Controls of the plant endomembrane-secretory pathway)

    Energy Technology Data Exchange (ETDEWEB)

    1991-01-01

    These studies are focused on elucidating the molecular structure of plant cell membranes with special reference to cell surface glycoproteins. The studies reported herein include use of monoclonal antibodies to characterize cell surface epitopes, construction of cDNA libraries of cell surface proteins, isolation of plant cell mutants by flow cytometry, detection of beta-glucouronidase marker enzyme systems in plants, expression go VSVG (the major envelope glycoprotein of Vesicular Stomatis Virus) in plant cells, and control of gene expression of cell membrane glycoproteins.(DT)

  6. Signal recognition partcle mediated protein targeting in Escherichia coli

    NARCIS (Netherlands)

    Valent, Q.A.

    2001-01-01

    The signal recognition particle (SRP) is a conserved ribonucleoprotein complex that binds to targeting sequences in nascent secretory and membrane proteins. The SRP guides these proteins to the cytoplasmic membrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes via an

  7. Effect of chlorogenic acid (5-Caffeoylquinic Acid) isolated from Baccharis oxyodonta on the structure and pharmacological activities of secretory phospholipase A2 from Crotalus durissus terrificus.

    Science.gov (United States)

    Toyama, Daniela O; Ferreira, Marcelo J P; Romoff, Paulete; Fávero, Oriana A; Gaeta, Henrique H; Toyama, Marcos H

    2014-01-01

    The aim of this paper was to investigate the effect of chlorogenic acid (5-caffeoylquinic acid, 5CQA), isolated from Baccharis oxyodonta, on the structure and pharmacological effect of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus. All in vitro and in vivo experiments were conducted using a purified sPLA2 compared under the same experimental conditions with sPLA2 : 5CQA. 5CQA induced several discrete modifications in the secondary structure and the hydrophobic characteristics of native sPLA2 that induced slight changes in the α-helical content, increase in the random coil structure, and decrease of fluorescence of native sPLA2. Moreover, 5CQA significantly decreased the enzymatic activity and the oedema and myonecrosis induced by native sPLA2. As the catalytic activity of sPLA2 plays an important role in several of its biological and pharmacological properties, antibacterial activity was used to confirm the decrease in its enzymatic activity by 5CQA, which induced massive bacterial cell destruction. We found that 5CQA specifically abolished the enzymatic activity of sPLA2 and induced discrete protein unfolding that mainly involved the pharmacological site of sPLA2. These results showed the potential application of 5CQA in the snake poisoning treatment and modulation of the pathological effect of inflammation induced by secretory PLA2.

  8. Characterization of mammary analogue secretory carcinoma of the salivary gland: discrimination from its mimics by the presence of the ETV6-NTRK3 translocation and novel surrogate markers.

    Science.gov (United States)

    Urano, Makoto; Nagao, Toshitaka; Miyabe, Satoru; Ishibashi, Kenichiro; Higuchi, Kayoko; Kuroda, Makoto

    2015-01-01

    Mammary analogue secretory carcinoma (MASC) is a recently recognized salivary gland tumor harboring an ETV6-NTRK3 translocation similar to secretory carcinoma of the breast. Histologically, MASC mimics papillary-cystic, microcystic, and follicular-type acinic cell carcinoma (AciCC) and low-grade cribriform cystadenocarcinoma (LGCCC) of the salivary gland. Using histology, immunohistochemistry (IHC), and molecular genetic techniques, we reevaluated 18 cases originally diagnosed as AciCC between 1993 and 2012. The last of these methods was used to detect the ETV6-NTRK3 translocation. The results reconfirmed 6 cases as AciCC (3 men; average age, 63 years) and helped us reclassify 10 cases as MASC (6 men; mean age, 46 years) and 2 as LGCCC (2 women; mean age, 48 years). Using IHC, we identified the 3 histologic types according to the expression patterns of vimentin, high-molecular-weight cytokeratin, cytokeratin 19, S-100, mammaglobin, MUC1, GATA-binding protein 3, adipophilin, α-amylase, DOG-1, SOX-10, and p63. The number of tumors diagnosed as MASC indicates that AciCC includes bona fide MASC cases. Because differential diagnosis among zymogen granule-poor AciCC, MASC, and LGCCC tumors is challenging, we recommend using molecular genetic tests for ETV6-NTRK3 for accurate diagnosis. Furthermore, detailed analyses of hematoxylin and eosin-stained tissues and IHC studies using the markers described here should be incorporated into routine practices. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. 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

  10. 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

  11. In Vtro Effects of Triclabendazole (TCBZ on the Excretory-Secretory Products (ESP of Fasciola Spp Parasites

    Directory of Open Access Journals (Sweden)

    Taghi Golmohamdi

    2012-03-01

    Full Text Available Fascioliasis is an endemic disease in Iran and triclabendazole (TCBZ is using for treatment of domestic animals and infected people. Excretory-secretory products (ESP play an important role in the host biochemical defense by means of activities of detoxifying and antioxidant glutathione S-transferase (GST and superoxide dismutase (SOD enzymes respectively. Therefore, the aim of this comparative study was to evaluate fasciola protection against TCBZ drug by detection of enzymatic activities, GST and SOD, in TCBZ treated Fasciola hepatica / Fasciola gigantica and control ESP samples. F. gigantic and F. hepatica helminthes were collected and cultured within buffer media (TCBZ treated and untreated or control for 4 h at 37 °C. Three TCBZ treated and 1 control ESP samples for each species were collected, centrifuged and supernatants were stored at -20°C. ESP samples protein concentrations were measured by Bradford method. SOD and GST enzymes activities of ESP samples were estimated photometrically. To determine the statistically significant difference between ESP of treated and control samples, t-test was conducted. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE. Protein concentrations in treated F. hepatica and F. gigantica ESP samples were estimated 204.88, 428, 130.4 and 288.2, 488.2, 308.2 µg/ml respectively. Protein concentrations in control samples were estimated 488.18 and 124.8 ug/ml respectively. SOD enzyme specific activities level in treated F.hepatica and F. gigantica ESP samples were determined 0.14, 0.31, 3.96 and 11.11, 13.54, 19.95 U/mg/protein respectively. SOD activities level in control samples were detected 70.69 and 10.92 U/mg/protein. GST specific activities level in treated F.hepatica and F. gigantica ESP samples were calculated 25.3, 85.5, 37.3 and 1823, 1314.3, 1320.8 U/mg respectively. GST activities levels in control samples were detected 98.6 and 1083.9 U/mg/protein

  12. In vitro effects of triclabendazole (TCBZ) on the excretory-secretory products (ESP) of Fasciola spp parasites.

    Science.gov (United States)

    Farahnak, Ali; Golmohamdi, Taghi; Eshraghian, Mohamadreza

    2012-01-01

    Fascioliasis is an endemic disease in Iran and triclabendazole (TCBZ) is using for treatment of domestic animals and infected people. Excretory-secretory products (ESP) play an important role in the host biochemical defense by means of activities of detoxifying and antioxidant glutathione S-transferase (GST) and superoxide dismutase (SOD) enzymes respectively. Therefore, the aim of this comparative study was to evaluate fasciola protection against TCBZ drug by detection of enzymatic activities, GST and SOD, in TCBZ treated Fasciola hepatica / Fasciola gigantica and control ESP samples. F. gigantic and F. hepatica helminthes were collected and cultured within buffer media (TCBZ treated and untreated or control) for 4 h at 37 °C. Three TCBZ treated and 1 control ESP samples for each species were collected, centrifuged and supernatants were stored at -20°C. ESP samples protein concentrations were measured by Bradford method. SOD and GST enzymes activities of ESP samples were estimated photometrically. To determine the statistically significant difference between ESP of treated and control samples, t-test was conducted. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations in treated F. hepatica and F. gigantica ESP samples were estimated 204.88, 428, 130.4 and 288.2, 488.2, 308.2 µg/ml respectively. Protein concentrations in control samples were estimated 488.18 and 124.8 ug/ml respectively. SOD enzyme specific activities level in treated F.hepatica and F. gigantica ESP samples were determined 0.14, 0.31, 3.96 and 11.11, 13.54, 19.95 U/mg/protein respectively. SOD activities level in control samples were detected 70.69 and 10.92 U/mg/protein. GST specific activities level in treated F.hepatica and F. gigantica ESP samples were calculated 25.3, 85.5, 37.3 and 1823, 1314.3, 1320.8 U/mg respectively. GST activities levels in control samples were detected 98.6 and 1083.9 U/mg/protein respectively

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Genome analysis reveals interplay between 5'UTR introns and nuclear mRNA export for secretory and mitochondrial genes.

    Directory of Open Access Journals (Sweden)

    Can Cenik

    2011-04-01

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

  20. Effects of anti-secretory factor (ASF) on irritable bowel syndrome (IBS). A double-blind, randomized study.

    Science.gov (United States)

    Ekesbo, Rickard; Nilsson, Peter M; Sjölund, Kristina

    2008-01-01

    To evaluate the role of the endogenous protein anti-secretory factor (ASF) on the symptoms, especially loose stools, in irritable bowel ayndrome (IBS). A diet with specially processed cereals (SPC) known to induce ASF production was used in patients with IBS, in an eight-week randomized, placebo-controlled study. Eighty-two patients with IBS were randomized to a diet with either SPC or placebo. The overall clinical condition and the quality of life were measured by VAS and SF-36 questionnaire, respectively. The plasma levels of ASF were determined in 14 patients with dominating loose stools before and after diet. All patients significantly (pASF level and the improvement on the VAS. Both study groups improved significantly on the VAS but no additive effect was seen for the active treatment. In the sub-group with loose stools, the SPC diet induced ASF plasma levels in IBS patients and was correlated to significant symptom improvement in the individual patient.

  1. Uptake of dehydroascorbic acid and ascorbic acid to isolated nerve terminals and secretory granules from ox neurohypophyses

    DEFF Research Database (Denmark)

    Thorn, N A; Nielsen, F S; Jeppesen, C K

    1986-01-01

    When uptake of L-[14C]ascorbic acid ([14C]AA) to various organs in guinea-pigs was studied after intracardiac injection, the adenohypophysis, pars intermedia, and the neurohypophysis had an uptake per milligramme protein which was about half of the uptake to the adrenals. Adrenal uptake was 20...... labelled dehydroascorbic acid ([14C]DHA), the uptake was much slower than when the medium contained labelled ascorbic acid. The uptake of [14C]DHA showed a linear dependence on concentration, and was not influenced by addition of Mg2+ and ATP. Addition of Mg2+ + ATP, omission of Ca2+ and Mg2+ or exchange...... of Na+ in the medium with K+ had no effect on the uptake of ascorbic acid. When isolated secretory granules from ox neurohypophyses were incubated with a medium containing [14C]DHA, uptake was considerably faster than the uptake when they were incubated in a medium containing [14C]AA. The uptake...

  2. Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

  4. 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.

  5. Mammary Analogue Secretory Carcinoma Presenting as a Cervical Lymph Node Metastasis of Unknown Primary Site: A Case Report.

    Science.gov (United States)

    Suzuki, Kensuke; Yagi, Masao; Kanda, Akira; Kobayashi, Yoshiki; Konishi, Masaya; Miyasaka, Chika; Tashiro, Takashi; Iwai, Hiroshi

    2017-01-01

    Mammary analogue secretory carcinoma (MASC) is a pathological entity arising in the salivary glands first described by Skalova et al. [Am J Surg Pathol 2010;34: 599-608]. Here, we report the first case of MASC presenting as a cervical lymph node metastasis of unknown primary site together with a brief review of the literature. We present a 74-year-old male with a painless lump in his left neck. Based on the fine-needle aspiration cytological findings, a possible malignant tumor was suspected. No evidence of a primary lesion was observed using imaging modalities including positron emission tomography/computed tomography. The patient underwent an ipsilateral modified radical neck dissection. Immunohistochemical staining showed that the neoplastic cells were positive for S100 protein and GATA3. A rearrangement of the ETV6 gene was noted during fluorescence in situ hybridization, and the final histopathological diagnosis was MASC. We encountered a MASC presenting as a cervical lymph node metastasis of unknown primary site. No adjuvant therapy was administered, and no local recurrence or metastatic disease has been detected during a follow-up period of 9 months. This is the first case report of MASC presenting as a cervical lymph node metastasis of unknown primary site and suggests the new properties of MASC.

  6. Mammary Analogue Secretory Carcinoma Presenting as a Cervical Lymph Node Metastasis of Unknown Primary Site: A Case Report

    Directory of Open Access Journals (Sweden)

    Kensuke Suzuki

    2017-02-01

    Full Text Available Background: Mammary analogue secretory carcinoma (MASC is a pathological entity arising in the salivary glands first described by Skalova et al. [Am J Surg Pathol 2010;34: 599–608]. Here, we report the first case of MASC presenting as a cervical lymph node metastasis of unknown primary site together with a brief review of the literature. Case Report: We present a 74-year-old male with a painless lump in his left neck. Based on the fine-needle aspiration cytological findings, a possible malignant tumor was suspected. No evidence of a primary lesion was observed using imaging modalities including positron emission tomography/computed tomography. The patient underwent an ipsilateral modified radical neck dissection. Immunohistochemical staining showed that the neoplastic cells were positive for S100 protein and GATA3. A rearrangement of the ETV6 gene was noted during fluorescence in situ hybridization, and the final histopathological diagnosis was MASC. Conclusion: We encountered a MASC presenting as a cervical lymph node metastasis of unknown primary site. No adjuvant therapy was administered, and no local recurrence or metastatic disease has been detected during a follow-up period of 9 months. This is the first case report of MASC presenting as a cervical lymph node metastasis of unknown primary site and suggests the new properties of MASC.

  7. Therapy of pathology secretory disease of the breast

    Directory of Open Access Journals (Sweden)

    G. Kh. Khanafiev

    2016-01-01

    Full Text Available Pathogenesis of serous discharge from the nipples of the breast is complex and includes the following components: hormonal imbalance as a backdrop, stimulating increased secretion in the ducts of serous content, resorption of the liquid component of a secret, resulting in local pathological effects of protein resorption, hormone content, autoimmune reactions ductless with subsequent development of proliferative processes in them. Under adverse conditions, infection occurs from an external or internal source of microorganisms. The presence of fluid in the ducts, mammary system ductal communication with the external environment, is an “open gate” for content ductal infection of the mammary glands. This creates the prerequisites for the development of proliferative processes in the first phase of the inflammatory origin. Application of 194 women with serous secretions from the nipples of the breast combined combinations of enzymes, glucocorticoids, progestogens, gonadoliberins and other agonists, suppress the proliferation of drugs that helped to put an end to abnormal secretion and prevent changes in the walls of the ducts and periductal breast tissue at different stages of disease.

  8. Proteins of the lactococcin A secretion system : lcnD encodes two in-frame proteins

    NARCIS (Netherlands)

    Varcamonti, M; Nicastro, G; Venema, G; Kok, J

    2001-01-01

    Polyclonal antibodies were raised against LcnC and LcnD proteins of the Lactococcus lactis bacteriocin lactococcin A secretory system to examine their cellular location and interaction. Two major reacting bands were detected by Western immunoblot with the anti-LcnD antibody: one of 52 kDa (LcnD) and

  9. 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...

  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; 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

  11. 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.

  12. 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...

  13. 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

  14. 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.

  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. 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.

  17. 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.

  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. 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.

  1. Developmental and Diurnal Expression of the Synaptosomal-Associated Protein 25 (Snap25) in the Rat Pineal Gland

    DEFF Research Database (Denmark)

    Karlsen, Anna S; Rath, Martin Fredensborg; Rohde, Kristian

    2013-01-01

    Snap25 (synaptosomal-associated protein) is a 25 kDa protein, belonging to the SNARE-family (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) of proteins, essential for synaptic and secretory vesicle exocytosis. Snap25 has by immunohistochemistry been demonstrated in the rat...

  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. Moderate expression of SEC16 increases protein secretion by Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bao, Jichen; Huang, Mingtao; Petranovic, Dina

    2017-01-01

    The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous α-amylase, a model protein used for studying the protein secretory pathwa...... were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-α-glucosidase, indicating that this mechanism is of general relevance....

  4. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Withers, III, Sydnor T.; Dominguez, Miguel A.; DeLisa, Matthew P.; Haitjema, Charles H.

    2017-02-21

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  5. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

    2016-08-09

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  6. MscL of Bacillus subtilis prevents selective release of cytoplasmic proteins in a hypotonic environment

    NARCIS (Netherlands)

    Kouwen, Thijs R. H. M.; Antelmann, Haike; van der Ploeg, Rene; Denham, Emma L.; Hecker, Michael; van Dijl, Jan Maarten

    Bacillus subtilis serves as an excellent model to study protein secretion at a proteomic scale. Most of the extracellular proteins are exported from the cytoplasm via the secretory (Sec) pathway. Despite extensive studies, the secretion mechanisms of about 25% of the extracellular proteins are

  7. Protein Secretion and the Endoplasmic Reticulum

    OpenAIRE

    Benham, Adam M.

    2012-01-01

    In a complex multicellular organism, different cell types engage in specialist functions, and as a result, the secretory output of cells and tissues varies widely. Whereas some quiescent cell types secrete minor amounts of proteins, tissues like the pancreas, producing insulin and other hormones, and mature B cells, producing antibodies, place a great demand on their endoplasmic reticulum (ER). Our understanding of how protein secretion in general is controlled in the ER is now quite sophisti...

  8. The Unfolded Protein Response and Cell Fate Control.

    Science.gov (United States)

    Hetz, Claudio; Papa, Feroz R

    2017-10-25

    The secretory capacity of a cell is constantly challenged by physiological demands and pathological perturbations. To adjust and match the protein-folding capacity of the endoplasmic reticulum (ER) to changing secretory needs, cells employ a dynamic intracellular signaling pathway known as the unfolded protein response (UPR). Homeostatic activation of the UPR enforces adaptive programs that modulate and augment key aspects of the entire secretory pathway, whereas maladaptive UPR outputs trigger apoptosis. Here, we discuss recent advances into how the UPR integrates information about the intensity and duration of ER stress stimuli in order to control cell fate. These findings are timely and significant because they inform an evolving mechanistic understanding of a wide variety of human diseases, including diabetes mellitus, neurodegeneration, and cancer, thus opening up the potential for new therapeutic modalities to treat these diverse diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. A New Hitherto Unreported Histopathologic Manifestation of Mammary Analogue Secretory Carcinoma: "Masked MASC" Associated With Low-grade Mucinous Adenocarcinoma and Low-grade In Situ Carcinoma Components.

    Science.gov (United States)

    Petersson, Fredrik; Michal, Michael; Kazakov, Dmitry V; Grossmann, Petr; Michal, Michal

    2016-10-01

    We present a salivary gland tumor of the parotid gland in a 54-year-old woman, which contained a minor mammary analogue secretory carcinoma (MASC) component (20%) intermixed with a morphologically entirely different mucinous adenocarcinomatous component that comprised 80% of the tumor mass and a morphologically nondescript low-grade intraductal carcinoma (in situ) component. On fluorescence in situ hybridization, a break in the ETV6 gene was documented in the mucinous adenocarcinomatous, the conventional MASC, and the intraductal (in situ) components. RT-PCR failed to reveal an ETV6-NTRK3 fusion. The entire conventional MASC and only rare mucinous adenocarcinoma tumor cells were mammaglobin positive, whereas the low-grade intraductal carcinoma (in-situ) component was negative. S-100 protein stained only the MASC component.

  10. Mammary analog secretory carcinoma of the thyroid gland: A primary thyroid adenocarcinoma harboring ETV6–NTRK3 fusion

    Science.gov (United States)

    Dogan, Snjezana; Wang, Lu; Ptashkin, Ryan N; Dawson, Robert R; Shah, Jatin P; Sherman, Eric J; Tuttle, R Michael; Fagin, James A; Klimstra, David S; Katabi, Nora; Ghossein, Ronald A

    2016-01-01

    ETV6–NTRK3 fusion was identified in several cancers including the recently described mammary analog secretory carcinoma (MASC) of the salivary glands and a minority of papillary thyroid carcinomas. We describe three cases of primary MASC of the thyroid gland and provide a detailed clinical and pathological characterization of the tumor morphology, immunoprofile, and genetic background. Immunohistochemistry for PAX8, TTF-1, thyroglobulin, mammaglobin, GCDFP-15, S-100 protein, and p63 was used to define the tumor immunophenotype. Fluorescence in situ hybridization for ETV6 rearrangement was performed in three, and the next-generation sequencing assay MSK-IMPACT™ (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) was performed in two cases. Primary MASC of the thyroid occurred in two women and one man, age 47–72 years. All patients presented with high T stage, infiltrative, locally aggressive tumors with extrathyroidal extension. Two cases were associated with well-differentiated papillary thyroid carcinoma. Histologically, they appeared as low-grade tumors, resembling MASC of the salivary glands and labeled positive for mammaglobin, GCDFP-15, S-100 protein, p63, weakly positive for PAX8, and negative for TTF-1 and thyroglobulin. Fluorescence in situ hybridization revealed ETV6 rearrangement in all cases. In two tested cases MSK-IMPACT™ confirmed the presence of ETV6–NTRK3 gene fusion. Two patients had at least two local recurrences, one was alive with disease, and one was alive and free of disease after 14 and 17 years, respectively. The third patient was alive and free of disease after 2 years. MASC of the thyroid is histologically, immunophenotypically, and genetically similar to its salivary gland counterpart. Thyroid MASC can be associated with a well-differentiated papillary thyroid carcinoma component, supporting follicular cell origin. Clinically, these carcinomas may show frequent recurrences but are associated

  11. Type II PKAs are anchored to mature insulin secretory granules in INS-1 β-cells and required for cAMP-dependent potentiation of exocytosis.

    Science.gov (United States)

    Villalpando, Sabrina; Cazevieille, Chantal; Fernandez, Anne; Lamb, Ned J; Hani, El-Habib

    2016-06-01

    Specificity of the cAMP-dependent protein kinase (PKA) pathway relies on an extremely sophisticated compartmentalization mechanism of the kinase within a given cell, based on high-affinity binding of PKA tetramer pools to different A-Kinase Anchoring Proteins (AKAPs). We and others have previously shown that AKAPs-dependent PKA subcellular targeting is a requisite for optimal cAMP-dependent potentiation of insulin exocytosis. We thus hypothesized that a PKA pool may directly anchor to the secretory compartment to potentiate insulin exocytosis. Here, using immunofluorescence analyses combined to subcellular fractionations and purification of insulin secretory granules (ISGs), we identified discrete subpools of type II PKAs, RIIα and RIIβ PKAs, along with the catalytic subunit, physically associated with ISGs within pancreatic insulin-secreting β-cells. Ultrastructural analysis of native rodent β-cells confirmed in vivo the occurrence of PKA on dense-core ISGs. Isoform-selective disruption of binding of PKAs to AKAPs reinforced the requirement of type II PKA isoforms for cAMP potentiation of insulin exocytosis. This granular localization of PKA was of critical importance since siRNA-mediated depletion of either RIIα or RIIβ PKAs resulted in a significant reduction of cAMP-dependent potentiation of insulin release. The present work provides evidence for a previously unrecognized pool of type II PKAs physically anchored to the β-cell ISGs compartment and supports a non-redundant function for type II PKAs during cAMP potentiation of exocytosis. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  12. Cellular adaptation of the rat small intestine after proximal enterectomy: changes in microvillous enzymes and in the secretory component of immunoglobulins.

    Science.gov (United States)

    Buts, J P; De Keyser, N; Dive, C

    1987-07-01

    To investigate the adaptation of functions expressed by the villous and crypt cell of the intestinal mucosa after intestinal resection, a 50% proximal enterectomy or a single transection was performed in 16 growing rats weighing 175-200 g. Ten days following the enterectomy, we determined the mucosal mass parameters (weight, protein, and DNA content), the activity of microvillous enzymes (lactase, sucrase, and aminopeptidase) in villus cells, and the concentration of the secretory component of immunoglobulins in crypt cells isolated from the proximal intestinal remnant. Mucosal hyperplasia was attested by the finding that mucosal weight, protein, and DNA content per cm of intestinal length were significantly (p less than 0.01) higher (+29 to +48%) in the resected group than in transected controls. The specific activity of lactase, sucrase, and aminopeptidase were significantly (p less than 0.05) lower (-23 to -56%) in villous cells isolated from the intestinal remnant of resected rats compared to controls. Sucrase activity was depressed in each cell fraction of the entire villous-crypt unit resulting in a lower villous to crypt gradient of enzyme activity. Km for the enzyme determined in villous cells was similar in both groups but the Vmax was reduced proportionally to the enzyme activity in the resected group indicating less enzyme per cell. By contrast, the concentration of secretory component measured by an immunoradiometric assay in both villous and crypt cells was significantly (p less than 0.05) increased (+37 to 45%) following proximal enterectomy. Our data indicate that the response of the epithelial cell to intestinal resection varies according to the metabolic function and that the mechanism of adaptation at the cellular level is complex.

  13. 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 p