Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking

DEFF Research Database (Denmark)

Hansen, Gert Helge; Niels-Christiansen, L L; Thorsen, Evy

2000-01-01

Intestinal brush border enzymes, including aminopeptidase N and sucrase-isomaltase, are associated with "rafts" (membrane microdomains rich in cholesterol and sphingoglycolipids). To assess the functional role of rafts in the present work, we studied the effect of cholesterol depletion on apical......, the rates of the Golgi-associated complex glycosylation and association with rafts of newly synthesized aminopeptidase N were reduced, and less of the enzyme had reached the brush border membrane after 2 h of labeling. In contrast, the basolateral Na(+)/K(+)-ATPase was neither missorted nor raft......-associated. Our results implicate the Golgi complex/trans-Golgi network in raft formation and suggest a close relationship between this event and apical membrane trafficking....

(Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: Presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit

Energy Technology Data Exchange (ETDEWEB)

Marxer, A.; Stieger, B.; Quaroni, A.; Kashgarian, M.; Hauri, H.P. (Univ. of Basel (Switzerland))

1989-09-01

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

Immunization with the Malaria Diversity-Covering Blood-Stage Vaccine Candidate Plasmodium falciparum Apical Membrane Antigen 1 DiCo in Complex with Its Natural Ligand PfRon2 Does Not Improve the In Vitro Efficacy

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Holger Spiegel

2017-06-01

Full Text Available The blood-stage malaria vaccine candidate Plasmodium falciparum apical membrane antigen 1 (PfAMA1 can induce strong parasite growth-inhibitory antibody responses in animals but has not achieved the anticipated efficacy in clinical trials. Possible explanations in humans are the insufficient potency of the elicited antibody responses, as well as the high degree of sequence polymorphisms found in the field. Several strategies have been developed to improve the cross-strain coverage of PfAMA1-based vaccines, whereas innovative concepts to increase the potency of PfAMA1-specific IgG responses have received little attention even though this may be an essential requirement for protective efficacy. A previous study has demonstrated that immunization with a complex of PyAMA1 and PyRON2, a ligand with an essential functional role in erythrocyte invasion, leads to protection from lethal Plasmodium yoelli challenge in an animal model and suggested to extend this strategy toward improved strain coverage by using multiple PfAMA1 alleles in combination with PfRon2L. As an alternative approach along this line, we decided to use PfRon2L in combination with three PfAMA1 diversity covering variants (DiCo to investigate the potential of this complex to induce more potent parasite growth inhibitory immune response in combination with better cross-strain-specific efficacy. Within the limits of the study design, the ability of the PfAMA1 DiCo-Mix to induce cross-strain-specific antibodies was not affected in all immunization groups, but the DiCo–PfRon2L complexes did not improve the potency of PfAMA1-specific IgG responses.

Analysis of polymorphisms and selective pressures on ama1 gene ...

Indian Academy of Sciences (India)

The apical membrane antigen-1 (AMA-1) of Plasmodium spp. is a merozoite surface antigen that is essential for the recognition and invasion of erythrocytes. Polymorphisms occurring in this surface antigen will cause major obstacles in developing effective malaria vaccines based on AMA-1. The objective of this study was ...

Recycling endosomes in apical plasma membrane domain formation and epithelial cell polarity

NARCIS (Netherlands)

Golachowska, Magdalena R.; Hoekstra, Dick; van IJzendoorn, Sven C. D.

2010-01-01

Recycling endosomes have taken central stage in the intracellular sorting and polarized trafficking of apical and basolateral plasma membrane components. Molecular players in the underlying mechanisms are now emerging, including small GTPases, class V myosins and adaptor proteins. In particular,

Prostate Specific Membrane Antigen (PSMA) Targeted Bio-orthogonal Therapy for Metastatic Prostate Cancer

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-16-1-0595 TITLE: Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate Cancer...Sep 2016 - 14 Sep 2017 4. TITLE AND SUBTITLE Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate

Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation.

Science.gov (United States)

Castillon, Guillaume Alain; Michon, Laetitia; Watanabe, Reika

2013-06-01

Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin-Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.

MST4 kinase phosphorylates ACAP4 protein to orchestrate apical membrane remodeling during gastric acid secretion.

Science.gov (United States)

Yuan, Xiao; Yao, Phil Y; Jiang, Jiying; Zhang, Yin; Su, Zeqi; Yao, Wendy; Wang, Xueying; Gui, Ping; Mullen, McKay; Henry, Calmour; Ward, Tarsha; Wang, Wenwen; Brako, Larry; Tian, Ruijun; Zhao, Xuannv; Wang, Fengsong; Cao, Xinwang; Wang, Dongmei; Liu, Xing; Ding, Xia; Yao, Xuebiao

2017-09-29

Digestion in the stomach depends on acidification of the lumen. Histamine-elicited acid secretion is triggered by activation of the PKA cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. Our recent study revealed the functional role of PKA-MST4-ezrin signaling axis in histamine-elicited acid secretion. However, it remains uncharacterized how the PKA-MST4-ezrin signaling axis operates the insertion of H,K-ATPases into the apical plasma membranes of gastric parietal cells. Here we show that MST4 phosphorylates ACAP4, an ARF6 GTPase-activating protein, at Thr 545 Histamine stimulation activates MST4 and promotes MST4 interaction with ACAP4. ACAP4 physically interacts with MST4 and is a cognate substrate of MST4 during parietal cell activation. The phosphorylation site of ACAP4 by MST4 was mapped to Thr 545 by mass spectrometric analyses. Importantly, phosphorylation of Thr 545 is essential for acid secretion in parietal cells because either suppression of ACAP4 or overexpression of non-phosphorylatable ACAP4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, persistent overexpression of MST4 phosphorylation-deficient ACAP4 results in inhibition of gastric acid secretion and blockage of tubulovesicle fusion to the apical membranes. Significantly, phosphorylation of Thr 545 enables ACAP4 to interact with ezrin. Given the location of Thr 545 between the GTPase-activating protein domain and the first ankyrin repeat, we reason that MST4 phosphorylation elicits a conformational change that enables ezrin-ACAP4 interaction. Taken together, these results define a novel molecular mechanism linking the PKA-MST4-ACAP4 signaling cascade to polarized acid secretion in gastric parietal cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation

International Nuclear Information System (INIS)

Bartles, J.R.; Feracci, H.M.; Stieger, B.; Hubbard, A.L.

1987-01-01

We have used pulse-chase metabolic radiolabeling with L-[ 35 S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates

Apical membrane P2Y4 purinergic receptor controls K+ secretion by strial marginal cell epithelium

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Scofield Margaret A

2005-11-01

Full Text Available Abstract Background It was previously shown that K+ secretion by strial marginal cell epithelium is under the control of G-protein coupled receptors of the P2Y family in the apical membrane. Receptor activation by uracil nucleotides (P2Y2, P2Y4 or P2Y6 leads to a decrease in the electrogenic K+ secretion. The present study was conducted to determine the subtype of the functional purinergic receptor in gerbil stria vascularis, to test if receptor activation leads to elevation of intracellular [Ca2+] and to test if the response to these receptors undergoes desensitization. Results The transepithelial short circuit current (Isc represents electrogenic K+ secretion and was found to be decreased by uridine 5'-triphosphate (UTP, adenosine 5'-triphosphate (ATP and diadenosine tetraphosphate (Ap4A but not uridine 5'-diphosphate (UDP at the apical membrane of marginal cells of the gerbil stria vascularis. The potencies of these agonists were consistent with rodent P2Y4 and P2Y2 but not P2Y6 receptors. Activation caused a biphasic increase in intracellular [Ca2+] that could be partially blocked by 2-aminoethoxy-diphenyl borate (2-APB, an inhibitor of the IP3 receptor and store-operated channels. Suramin (100 μM did not inhibit the effect of UTP (1 μM. The ineffectiveness of suramin at the concentration used was consistent with P2Y4 but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in the stria vascularis. Sustained exposure to ATP or UTP for 15 min caused a depression of Isc that appeared to have two components but with apparently no chronic desensitization. Conclusion The results support the conclusion that regulation of K+ secretion across strial marginal cell epithelium occurs by P2Y4 receptors at the apical membrane. The apparent lack of desensitization of the response is consistent with two processes: a rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP2.

Prostate-specific membrane antigen and its truncated form PSM'

Czech Academy of Sciences Publication Activity Database

Mlčochová, Petra; Bařinka, Cyril; Tykvart, Jan; Šácha, Pavel; Konvalinka, Jan

2009-01-01

Roč. 69, č. 5 (2009), s. 471-479 ISSN 0270-4137 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : prostate specific membrane antigen * glutamate carboxypeptidase II * prostate cancer Subject RIV: CE - Biochemistry Impact factor: 3.081, year: 2009

New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells.

Science.gov (United States)

Almássy, János; Siguenza, Elias; Skaliczki, Marianna; Matesz, Klara; Sneyd, James; Yule, David I; Nánási, Péter P

2018-04-01

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl - efflux and the subsequent paracellular Na + transport. In this model, the Na + -K + pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl - transport via basolateral Na + -K + -2Cl - cotransport is generated by the Na + -K + pump. In addition, the continuous electrochemical gradient for Cl - flow during acinar cell stimulation is maintained by the basolateral K + efflux. However, using a combination of single-cell electrophysiology and Ca 2+ -imaging, we demonstrate that photolysis of Ca 2+ close to the apical membrane of parotid acinar cells triggered significant K + current, indicating that a substantial amount of K + is secreted into the lumen during stimulation. Nevertheless, the K + content of the primary saliva is relatively low, suggesting that K + might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na + -K + pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K + from and secretes Na + to the lumen, which can partially supplement the paracellular Na + pathway.

Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport-type familial nephritis.

Science.gov (United States)

Kashtan, C; Fish, A J; Kleppel, M; Yoshioka, K; Michael, A F

1986-10-01

We probed epidermal basement membranes (EBM) of acid-urea denatured skin from members of kindreds with Alport-type familial nephritis (FN) for the presence of antigens reactive with Goodpasture sera (GPS) and serum (FNS) from an Alport patient who developed anti-glomerular basement membrane (GBM) nephritis in a renal allograft. By immunoblotting, GPS reacted primarily with the 28,000 molecular weight (mol wt) monomer but also the 24,000 mol wt and 26,000 mol wt monomers of the noncollagenous globular domain (NC1) of type IV collagen from normal human GBM, while FNS identified only the 26,000-mol wt monomer. FNS reacted with EBM of 12 controls and nine unaffected male kindred members but not EBM of eight affected males. Five affected females exhibited interrupted reactivity of FNS with EBM. GPS showed variable reactivity with EBM and was not discriminating with respect to Alport-type FN. FNS did not stain renal basement members of five affected males. However, the EBM, tubular basement membrane, and Bowman's capsules of affected males contained antigens reactive with GPS. These immunochemical studies suggest that the FNS antigen is distinct from Goodpasture antigen(s). The expression of FNS antigen located on the NC1 domain of type IV collagen is altered in basement membranes of patients with Alport-type FN, and the distribution of this antigenic anomaly within kindreds suggests X-linked dominant transmission of a defective gene.

Quantification of an Epstein-Barr virus-associated membrane antigen component

International Nuclear Information System (INIS)

North, J.R.; Morgan, A.J.; Thompson, J.L.; Epstein, M.A.

1982-01-01

A method is described for the preparation of a 125 I-labelled membrane antigen (MA) component (gp340) from B95-8 cell membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Good yields of antigenic material were obtained when renaturation of the [ 125 I]gp340 was carried out by removal of SDS in the presence of urea and subsequent removal of the urea. The availability of purified, radiolabelled gp340 has provided the essential basis for the development of a radioimmunoassay which, for the first time, permits quantification of this antigen. The assay has been used to demonstrate that cell membrane MA is a better source of gp340 for large-scale work than is the Epstein-Barr virus envelope and to measure the increase in expression of gp340 following treatment of cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). (Auth.)

Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

Directory of Open Access Journals (Sweden)

Daniel L Glauser

Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.

Quantitative radioimmunoassay for membranous and soluble H-Y antigen typing

Energy Technology Data Exchange (ETDEWEB)

Casanova-Bettane, M.; Latron, F.; Jakob, H.; Fellous, M.

1981-01-01

Two sensitive and quantitative methods for membranous or soluble H-Y antigen typing using rat anti-H-Y immune sera and /sup 125/I labelled protein A were carried out. These techniques were used to study H-Y antigen expression in human cell lines, and to refine the hypothesis that ..beta../sub 2/m serves as an anchorage point for H-Y antigen.

Effects of radiation on the expression of antigens on the membranes of human adenocarcinoma cells

International Nuclear Information System (INIS)

Hareyama, Masato; Imai, Kozo; Oouchi, Atsushi; Shidou, Mitsuo; Takahashi, Hiroki; Koshiba, Hirohumi; Yachi, Akira; Morita, Kazuo

1992-01-01

We have investigated the effects of irradiation on the membranes of tumor cells. X-ray irradiation caused remarkable increases in the expression of tumor-associated antigens (YH206, CEA) and c-erbB-2 protein by flow cytometry, whereas IFN had no obvious effect on the expression of tumor-associated antigens and c-erbB-2 protein. On the other hand, the expression of MHC Class I and ICAM-1 antigen on the membrane by flow cytometry was enhanced by both irradiation and IFN. In addition, the irradiated cells, when analyzed using a CEA specific probe, showed remarkable increases in the CEA mRNA compared to IFN-treated cells. It is possible that enhancement of the expression of tumor-associated antigens and c-erbB-2 protein, together with the enhancement of that of MHC-Class I and ICAM-1, would help cytotoxic killer cells recognize the tumor cells. (author)

19-DEJ-1, a hemidesmosome-anchoring filament complex-associated monoclonal antibody. Definition of a new skin basement membrane antigenic defect in junctional and dystrophic epidermolysis bullosa

DEFF Research Database (Denmark)

Fine, J D; Horiguchi, Y; Couchman, J R

1989-01-01

A murine monoclonal antibody (19-DEJ-1) was recently produced that recognizes a unique antigenic epitope of human skin basement membrane localized to the midlamina lucida exclusively in those areas bordered by overlying hemidesmosomes. To determine whether the antigen defined by 19-DEJ-1 is norma...

Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

Directory of Open Access Journals (Sweden)

Deirdre R Ducken

Full Text Available Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to

Immunochemical identification of human trophoblast membrane antigens using monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Brown, P J; Molloy, C M; Johnson, P M [Liverpool Univ. (UK). Dept. of Immunology

1983-11-01

Human trophoblast membrane antigens recognised by monoclonal antibodies (H310, H315, H316 and H317) have been identified using combinations of radioimmunoprecipitation, SDS-PAGE, electroblotting, chromatographic and ELISA-type techniques. H317 is known to identify heat-stable placental-type alkaline phosphatase and accordingly was shown to react with a protein of subunit Msub(r) of 68000. H310 and H316 both recognise an antigen with a subunit Msub(r) of 34000 under reducing conditions. In non-reducing conditions, the H310/316 antigen gave oligomers of a component of Msub(r) 62000. It is unknown whether this 62000 dalton component is a dimer of the 34000 dalton protein with either itself or a second protein chain of presumed Msub(r) around 28000. H315 recognises an antigen with subunit Msub(r) of 36000; in non-reducing conditions this component readily associates to oligomeric structures. The epitope recognised by H315 may be sensitive to SDS. The two proteins recognised by H310/316 and H315 have been termed the p34 and p36 trophoblast membrane proteins, respectively.

Expression of the Gastrin-Releasing Peptide Receptor, the Prostate Stem Cell Antigen and the Prostate-Specific Membrane Antigen in Lymph Node and Bone Metastases of Prostate Cancer

NARCIS (Netherlands)

Ananias, Hildo J. K.; van den Heuvel, Marius C.; Helfrich, Wijnand; de Jong, Igle J.

2009-01-01

OBJECTIVE. Cell membrane antigens like the gastrin-releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate-specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we

Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes

DEFF Research Database (Denmark)

Hansen, Gert H; Pedersen, Jens; Niels-Christiansen, Lise-Lotte

2003-01-01

microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident...... lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized...... brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion...

Exposure to the Epstein–Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo

Directory of Open Access Journals (Sweden)

Yakov Lomakin

2017-07-01

Full Text Available Multiple sclerosis (MS is an autoimmune chronic inflammatory disease of the central nervous system (CNS. Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP-specific antibodies from MS patients cross-react with Epstein–Barr virus (EBV latent membrane protein 1 (LMP1. In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20–50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

Exposure to the Epstein–Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo

Science.gov (United States)

Lomakin, Yakov; Arapidi, Georgii Pavlovich; Chernov, Alexander; Ziganshin, Rustam; Tcyganov, Evgenii; Lyadova, Irina; Butenko, Ivan Olegovich; Osetrova, Maria; Ponomarenko, Natalia; Telegin, Georgy; Govorun, Vadim Markovich; Gabibov, Alexander; Belogurov, Alexey

2017-01-01

Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20–50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading. PMID:28729867

Exposure to the Epstein-Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo.

Science.gov (United States)

Lomakin, Yakov; Arapidi, Georgii Pavlovich; Chernov, Alexander; Ziganshin, Rustam; Tcyganov, Evgenii; Lyadova, Irina; Butenko, Ivan Olegovich; Osetrova, Maria; Ponomarenko, Natalia; Telegin, Georgy; Govorun, Vadim Markovich; Gabibov, Alexander; Belogurov, Alexey

2017-01-01

Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo . We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20-50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

Science.gov (United States)

Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

2014-10-13

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

Aminopeptidase N is directly sorted to the apical domain in MDCK cells

DEFF Research Database (Denmark)

Wessels, H P; Hansen, Gert Helge; Fuhrer, C

1990-01-01

In different epithelial cell types, integral membrane proteins appear to follow different sorting pathways to the apical surface. In hepatocytes, several apical proteins were shown to be transported there indirectly via the basolateral membrane, whereas in MDCK cells a direct sorting pathway from...

Characterization of plant plasma membrane antigens: [Annual] progress report

International Nuclear Information System (INIS)

Galbraith, D.W.; Afonso, C.L.; Meyer, D.; Harkins, K.R.

1987-01-01

Protoplast plasma membranes were used to raise antibodies in mice to cell surface antigens. Monoclonal antibodies were selected from those produced and used for indirect immunofluorescence microscopic analysis of N. tabacum cells. In parallel studies cDNA expression libraries were prepared. (DT)

Cancer Patient T Cells Genetically Targeted to Prostate-Specific Membrane Antigen Specifically Lyse Prostate Cancer Cells and Release Cytokines in Response to Prostate-Specific Membrane Antigen

Directory of Open Access Journals (Sweden)

Michael C. Gong

1999-06-01

Full Text Available The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells

International Nuclear Information System (INIS)

Sadeghi, H.; Da Silva, A.M.; Klein, C.

1988-01-01

The authors describe the biochemical features of the putative cell cohesion molecule antigen 117, indicating that it is anchored to the plasma membrane by a glycolipid tail. Antigen 117 can be radiolabeled with [ 3 H]myristate, [ 3 H]palmitate, and [ 14 C]ethanolamine. The fatty acid label is removed by periodate oxidation and nitrous acid deamination, indicating that the fatty acid is attached to the protein by a structure containing carbohydrate and an unsubstituted glucosamine. As cells develop aggregation competence, the antigen is released from the cell surface in a soluble form that can still be radiolabeled with [ 14 C]ethanolamine but not with [ 3 H]myristate of [ 3 H]-palmitate. The molecular weight of the released antigen is similar to that found in the plasma membrane, but it preferentially partitions in Triton X-114 as a hydrophilic, as opposed to a hydrophobic, protein. Plasma membranes contain the enzyme activity responsible for the release of the antigen in a soluble form

Macrophage polarization differs between apical granulomas, radicular cysts, and dentigerous cysts.

Science.gov (United States)

Weber, Manuel; Schlittenbauer, Tilo; Moebius, Patrick; Büttner-Herold, Maike; Ries, Jutta; Preidl, Raimund; Geppert, Carol-Immanuel; Neukam, Friedrich W; Wehrhan, Falk

2018-01-01

Apical periodontitis can appear clinically as apical granulomas or radicular cysts. There is evidence that immunologic factors are involved in the pathogenesis of both pathologies. In contrast to radicular cysts, the dentigerous cysts have a developmental origin. Macrophage polarization (M1 vs M2) is a main regulator of tissue homeostasis and differentiation. There are no studies comparing macrophage polarization in apical granulomas, radicular cysts, and dentigerous cysts. Forty-one apical granulomas, 23 radicular cysts, and 23 dentigerous cysts were analyzed in this study. A tissue microarray (TMA) of the 87 consecutive specimens was created, and CD68-, CD11c-, CD163-, and MRC1-positive macrophages were detected by immunohistochemical methods. TMAs were digitized, and the expression of macrophage markers was quantitatively assessed. Radicular cysts are characterized by M1 polarization of macrophages while apical granulomas show a significantly higher degree of M2 polarization. Dentigerous cysts have a significantly lower M1 polarization than both analyzed periapical lesions (apical granulomas and radicular cysts) and accordingly, a significantly higher M2 polarization than radicular cysts. Macrophage cell density in dentigerous cysts is significantly lower than in the periapical lesions. The development of apical periodontitis towards apical granulomas or radicular cysts might be directed by macrophage polarization. Radicular cyst formation is associated with an increased M1 polarization of infiltrating macrophages. In contrast to radicular cysts, dentigerous cysts are characterized by a low macrophage infiltration and a high degree of M2 polarization, possibly reflecting their developmental rather than inflammatory origin. As M1 polarization of macrophages is triggered by bacterial antigens, these results underline the need for sufficient bacterial clearance during endodontic treatment to prevent a possible M1 macrophage-derived stimulus for radicular cyst

Comparative analysis of monoclonal antibodies against prostate-specific membrane antigen (PSMA)

Czech Academy of Sciences Publication Activity Database

Tykvart, Jan; Navrátil, Václav; Sedlák, František; Corey, E.; Colombatti, M.; Fracasso, G.; Koukolík, F.; Bařinka, Cyril; Šácha, Pavel; Konvalinka, Jan

2014-01-01

Roč. 74, č. 16 (2014), s. 1674-1690 ISSN 0270-4137 R&D Projects: GA ČR GAP304/12/0847; GA MŠk LO1302; GA ČR GAP301/12/1513; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388963 ; RVO:86652036 Keywords : glutamate carboxypeptidase II * prostate -specific membrane antigen * folate hydrolase * NAALADase * Western blot * immunohistochemistry * ELISA * flow cytometry * surface plasmon resonance Subject RIV: CE - Biochemistry Impact factor: 3.565, year: 2014

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

International Nuclear Information System (INIS)

Dominguez, J.M.; Lanoix, J.; Paiement, J.

1991-01-01

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha- 32 P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha- 32 P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations

Surface co-expression of two different PfEMP1 antigens on single Plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1

DEFF Research Database (Denmark)

Joergensen, Louise; Bengtsson, Dominique C; Bengtsson, Anja

2010-01-01

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var...

Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

Science.gov (United States)

Zeelenberg, Ingrid S; Ostrowski, Matias; Krumeich, Sophie; Bobrie, Angélique; Jancic, Carolina; Boissonnas, Alexandre; Delcayre, Alain; Le Pecq, Jean-Bernard; Combadière, Béhazine; Amigorena, Sebastian; Théry, Clotilde

2008-02-15

Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

Science.gov (United States)

Lee, Seung S; Roche, Philip Jr; Giannopoulos, Paresa N; Mitmaker, Elliot J; Tamilia, Michael; Paliouras, Miltiadis; Trifiro, Mark A

2017-03-01

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells

Science.gov (United States)

Perez Bay, Andres E.; Schreiner, Ryan; Benedicto, Ignacio; Paz Marzolo, Maria; Banfelder, Jason; Weinstein, Alan M.; Rodriguez-Boulan, Enrique J.

2016-01-01

The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station. PMID:27180806

Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

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Stewart Nuttall

2013-01-01

Full Text Available The single variable new antigen receptor domain antibody fragments (VNARs derived from shark immunoglobulin new antigen receptor antibodies (IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1 from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

Calmodulin and CaMKII modulate ENaC activity by regulating the association of MARCKS and the cytoskeleton with the apical membrane.

Science.gov (United States)

Alli, Abdel A; Bao, Hui-Fang; Liu, Bing-Chen; Yu, Ling; Aldrugh, Summer; Montgomery, Darrice S; Ma, He-Ping; Eaton, Douglas C

2015-09-01

Phosphatidylinositol bisphosphate (PIP2) regulates epithelial sodium channel (ENaC) open probability. In turn, myristoylated alanine-rich C kinase substrate (MARCKS) protein or MARCKS-like protein 1 (MLP-1) at the plasma membrane regulates the delivery of PIP2 to ENaC. MARCKS and MLP-1 are regulated by changes in cytosolic calcium; increasing calcium promotes dissociation of MARCKS from the membrane, but the calcium-regulatory mechanisms are unclear. However, it is known that increased intracellular calcium can activate calmodulin and we show that inhibition of calmodulin with calmidazolium increases ENaC activity presumably by regulating MARCKS and MLP-1. Activated calmodulin can regulate MARCKS and MLP-1 in two ways. Calmodulin can bind to the effector domain of MARCKS or MLP-1, inactivating both proteins by causing their dissociation from the membrane. Mutations in MARCKS that prevent calmodulin association prevent dissociation of MARCKS from the membrane. Calmodulin also activates CaM kinase II (CaMKII). An inhibitor of CaMKII (KN93) increases ENaC activity, MARCKS association with ENaC, and promotes MARCKS movement to a membrane fraction. CaMKII phosphorylates filamin. Filamin is an essential component of the cytoskeleton and promotes association of ENaC, MARCKS, and MLP-1. Disruption of the cytoskeleton with cytochalasin E reduces ENaC activity. CaMKII phosphorylation of filamin disrupts the cytoskeleton and the association of MARCKS, MLP-1, and ENaC, thereby reducing ENaC open probability. Taken together, these findings suggest calmodulin and CaMKII modulate ENaC activity by destabilizing the association between the actin cytoskeleton, ENaC, and MARCKS, or MLP-1 at the apical membrane. Copyright © 2015 the American Physiological Society.

Stem Cell Antigen-1 in Skeletal Muscle Function

OpenAIRE

Bernstein, Harold S.; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J.; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

2013-01-01

Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1...

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

Science.gov (United States)

Ratner, Martha A; Decker, Sarah E; Aller, Stephen G; Weber, Gerhard; Forrest, John N

2006-03-01

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action. Copyright 2006 Wiley-Liss, Inc.

Actin filaments and microtubules are involved in different membrane traffic pathways that transport sphingolipids to the apical surface of polarized HepG2 cells

NARCIS (Netherlands)

Zegers, MMP; Zaal, KJM; van Ijzendoorn, SCD; Klappe, K; Hoekstra, D

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and

A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells.

Science.gov (United States)

Park, Sungjin; Szumlanski, Amy L; Gu, Fangwei; Guo, Feng; Nielsen, Erik

2011-07-17

In plants, cell shape is defined by the cell wall, and changes in cell shape and size are dictated by modification of existing cell walls and deposition of newly synthesized cell-wall material. In root hairs, expansion occurs by a process called tip growth, which is shared by root hairs, pollen tubes and fungal hyphae. We show that cellulose-like polysaccharides are present in root-hair tips, and de novo synthesis of these polysaccharides is required for tip growth. We also find that eYFP-CSLD3 proteins, but not CESA cellulose synthases, localize to a polarized plasma-membrane domain in root hairs. Using biochemical methods and genetic complementation of a csld3 mutant with a chimaeric CSLD3 protein containing a CESA6 catalytic domain, we provide evidence that CSLD3 represents a distinct (1→4)-β-glucan synthase activity in apical plasma membranes during tip growth in root-hair cells.

Differential subcellular membrane recruitment of Src may specify its downstream signalling

International Nuclear Information System (INIS)

Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah; D'Auria, Ludovic; Van Der Smissen, Patrick; Platek, Anna; Mettlen, Marcel; Caplanusi, Adrian; Hove, Marie-France van den; Tyteca, Donatienne; Courtoy, Pierre J.

2008-01-01

Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe (∼ 70%) cholesterol extraction with methyl-β-cyclodextrin (MβCD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MβCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined

Membrane domains and polarized trafficking of sphingolipids

NARCIS (Netherlands)

Maier, O; Slimane, TA; Hoekstra, D

The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid-cholesterol enriched membrane microdomains, so called rafts. In

Development of a Novel, Ultra-rapid Biosensor for the Qualitative Detection of Hepatitis B Virus-associated Antigens and Anti-HBV, Based on “Membrane-engineered” Fibroblast Cells with Virus-Specific Antibodies and Antigens

Directory of Open Access Journals (Sweden)

Antonios Perdikaris

2009-03-01

Full Text Available A novel miniature cell biosensor detection system for the detection of Hepatis B virus (HBV-associated antigens and anti-HBV is described. The biosensor is based on “membrane-engineered” Vero fibroblast cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of anti-HBV specific antibodies (anti-HBs, anti-HBe or antigens (HBsAg in the membranes of the Vero cells. The attachment of a homologous antigen to the electroinserted antibody (or, respectively, of the antibody to the electroinserted antigen triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the Bioelectric Recognition Assay (BERA. The sensor was used for screening 133 clinical blood serum samples according to a double-blind protocol. Considerably higher sensor responses were observed against HBV-positive samples, compared with responses against negative samples or samples positive for heterologous hepatitis viruses such as Hepatitis C (HCV virus. Detection of anti-HBs antibodies was made possible by using a biosensor based on immobilized Vero cells bearing the respective antigen (HBsAg. The observed response was rapid (45 sec and quite reproducible. Fluorescence microscopy observations showed that attachment of HBV particles to cells membrane-engineered with anti-HBs was associated with a decrease of [Ca2+]cyt. The perspectives for using the novel biosensor as a qualitative, rapid screening, high throughput assay for HBV antigens and anti-HBs in clinical samples is discussed.

Screening of random peptide library of hemagglutinin from pandemic 2009 A(H1N1 influenza virus reveals unexpected antigenically important regions.

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Wanghui Xu

Full Text Available The antigenic structure of the membrane protein hemagglutinin (HA from the 2009 A(H1N1 influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify "hot spots" or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins.

Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1

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Patarroyo Manuel E

2011-10-01

Full Text Available Abstract Background Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1 and examine its antigenicity in natural P. vivax infections. Methods The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions This study shows the identification and characterization of

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes

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Arnot David E

2008-06-01

Full Text Available Abstract Background The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development. Methods A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy Results Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1. Conclusion A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

International Nuclear Information System (INIS)

Kalra, Rajkumar S.; Wadhwa, Renu

2015-01-01

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

Energy Technology Data Exchange (ETDEWEB)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp [Cell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST Central 4), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

The establishment of polarized membrane traffic in Xenopus laevis embryos.

Science.gov (United States)

Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

1992-09-01

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin

Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens.

Science.gov (United States)

Ondigo, Bartholomew N; Park, Gregory S; Gose, Severin O; Ho, Benjamin M; Ochola, Lyticia A; Ayodo, George O; Ofulla, Ayub V; John, Chandy C

2012-12-21

Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in

Correlation between prostate brachytherapy-related urethral stricture and peri-apical urethral dosimetry: A matched case–control study

International Nuclear Information System (INIS)

Earley, James J.; Abdelbaky, Ather M.; Cunningham, Melanie J.; Chadwick, Eliot; Langley, Stephen E.M.; Laing, Robert W.

2012-01-01

Background and purpose: Radiation dose to the bulbomembranous urethra has been shown to correlate with urethral stricture formation. This retrospective case–control study was designed to explore the relationship between dose to the apical/peri-apical regions of the urethra and development of brachytherapy (BXT)-related urethral stricture. Materials and methods: Cases were patients who developed urethral stricture after treatment with BXT as monotherapy and who had urethral dosimetry post-implant. Each case was matched with a control that had not developed urethral stricture. Dosimetry was compared between cases and controls. Results: Twenty-three cases were pair matched with 23 controls. There were no significant differences between the two groups in terms of age, presenting Prostate Specific Antigen (PSA), International Prostate Symptom Score (IPSS) or Gleason score. The dose delivered to the peri-apical and apical urethra was significantly higher for cases when compared with controls (peri-apical urethra: mean V 150 1.1 Vs 0.8 cc [p = 0.02]; apical urethra: mean dose 200 Vs 174 Gy [p = 0.01]). The distance from the prostate apex to isodose lines was also found to be significant in predicting stricture formation. Conclusion: There was evidence to suggest that the development of BXT-related stricture was associated with radiation dose at the apical and peri-apical urethra. Attention to the dose delivered to those areas may minimise the risk of developing such morbidity.

Decreased Expression of Semaphorin3A/Neuropilin-1 Signaling Axis in Apical Periodontitis

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Ying Lin

2017-01-01

Full Text Available Apical periodontitis (AP is a chronic infection of endodontic origin accompanied with bone destruction around the apical region. Semaphorin3A (Sema3A and neuropilin-1 (Nrp1 are regarded as a pair of immune regulators in bone metabolism. In this study, we firstly investigated the expression pattern of Sema3A/Nrp1 in apical periodontitis and its correlation with bone destruction. Using rat animal model, we analysed the level of mandibular bone destruction and the expression of Sema3A/Nrp1 on days 0, 7, 14, 21, 28, and 35 after pulp exposure. In addition, clinical samples from apical periodontitis patients were obtained to analyse the expression of Sema3A/Nrp1. These results indicated that the bone destruction level expanded from days 7 to 35. The number of positive cells and level of mRNA expression of Sema3A/Nrp1 were significantly decreased from days 7 to 35, with a negative correlation with bone destruction. Moreover, expression of Sema3A/Nrp1 in the AP group was reduced compared to the control group of clinical samples. In conclusion, decreased expression of Sema3A/Nrp1 was observed in periapical lesions and is potentially involved in the bone resorption of the periapical area, suggesting that Sema3A/Nrp1 may contribute to the pathological development of apical periodontitis.

Asymmetric localization of Arabidopsis SYP124 syntaxin at the pollen tube apical and sub-apical zones is involved in tip growth

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Silva Pedro

2010-08-01

Full Text Available Abstract Background The continuous polarized vesicle secretion in pollen tubes is essential for tip growth but the location of endo- and exocytic sub-domains remains however controversial. In this report we aimed to show that Arabidopsis thaliana syntaxins are involved in this process and contribute to spatially define exocytosis and membrane recycling. Results Using GFP-fusion constructs, we imaged the distribution of pollen-specific (AtSYP124 and non-pollen syntaxins (AtSYP121 and AtSYP122 in transiently transformed Nicotiana tabacum pollen tubes. All three proteins associate with the plasma membrane and with apical vesicles indicating a conserved action mechanism for all SYPs. However, the GFP tagged SYP124 showed a specific distribution with a higher labelling at the plasma membrane flanks, 10-25 μm behind the apex. This distribution is affected by Ca2+ fluxes as revealed by treatment with Gd3+ (an inhibitor of extracellular Ca2+ influx and TMB-8 (an inhibitor of intracellular Ca2+ release. Both inhibitors decreased growth rate but the distribution of SYP124 at the plasma membrane was more strongly affected by Gd3+. Competition with a related dominant negative mutant affected the specific distribution of SYP124 but not tip growth. In contrast, co-expression of the phosphatidylinositol-4-monophosphate 5-kinase 4 (PIP5K4 or of the small GTPase Rab11 perturbed polarity and the normal distribution of GFP-SYP but did not inhibit the accumulation in vesicles or at the plasma membrane. Conclusions The results presented suggest that in normal growing pollen tubes, a net exocytic flow occurs in the flanks of the tube apex mediated by SYP124. The specific distribution of SYP124 at the plasma membrane is affected by changes in Ca2+ levels in agreement with the importance of this ion for exocytosis. Apical growth and the specific localization of SYP124 were affected by regulators of membrane secretion (Ca2+, PIP5K4 and Rab11 but competition with a

Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

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Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

2016-01-01

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

Aquaporin-2 membrane targeting

DEFF Research Database (Denmark)

Olesen, Emma T B; Fenton, Robert A

2017-01-01

The targeting of the water channel aquaporin-2 (AQP2) to the apical plasma membrane of kidney collecting duct principal cells is regulated mainly by the antidiuretic peptide hormone arginine vasopressin (AVP). This process is of crucial importance for the maintenance of body water homeostasis...... of aquaporin-2 (AQP2) to the apical plasma membrane of collecting duct (CD) principal cells (10, 20). This process is mainly regulated by the actions of AVP on the type 2 AVP receptor (V2R), although the V1a receptor may also play a minor role (26). The V2R is classified within the group of 7-transmembrane....... For example, 1) stimulation with the nonspecific AC activator forskolin increases AQP2 membrane accumulation in a mouse cortical collecting duct cell line [e.g., Norregaard et al. (16)]; 2) cAMP increases CD water permeability (15); 3) the cAMP-activated protein kinase A (PKA) can phosphorylate AQP2 on its...

Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.

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Hemavathy Harikrishnan

Full Text Available Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.

Plasma membrane associated, virus-specific polypeptides required for the formation of target antigen complexes recognized by virus-specific cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Domber, E.A.

1986-01-01

These studies were undertaken to define some of the poxvirus-specific target antigens which are synthesized in infected cells and recognized by vaccinia virus-specific CTLs (VV-CTLs). Since vaccinia virus infected, unmanipulated target cells express numerous virus-specific antigens on the plasma membrane, attempts were made to manipulate expression of the poxvirus genome after infection so that one or a few defined virus-specified antigens were expressed on the surface of infected cells. In vitro [ 51 Cr]-release assays determined that viral DNA synthesis and expression of late viral proteins were not necessary to form a target cell which was fully competent for lysis by VV-CTLs. Under the conditions employed in these experiments, 90-120 minutes of viral protein synthesis were necessary to produce a competent cell for lysis by VV-CTLs. In order to further inhibit the expression of early viral proteins in infected cells, partially UV-inactivated vaccinia virus was employed to infect target cells. It was determined that L-cells infected with virus preparations which had been UV-irradiated for 90 seconds were fully competent for lysis by VV-CTLs. Cells infected with 90 second UV-irr virus expressed 3 predominant, plasma membrane associated antigens of 36-37K, 27-28K, and 19-17K. These 3 viral antigens represent the predominant membrane-associated viral antigens available for interaction with class I, major histocompatibility antigens and hence are potential target antigens for VV-CTLs

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

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Isabel Correa

2018-03-01

Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

Apical cap

International Nuclear Information System (INIS)

McLoud, T.C.; Isler, R.J.; Novelline, R.A.; Putman, C.E.; Simeone, J.; Stark, P.

1981-01-01

Apical caps, either unilateral or bilateral, are a common feature of advancing age and are usually the result of subpleural scarring unassociated with other diseases. Pancoast (superior sulcus) tumors are a well recognized cause of unilateral asymmetric apical density. Other lesions arising in the lung, pleura, or extrapleural space may produce unilateral or bilateral apical caps. These include: (1) inflammatory: tuberculosis and extrapleural abscesses extending from the neck; (2) post radiation fibrosis after mantle therapy for Hodgkin disease or supraclavicular radiation in the treatment of breast carcinoma; (3) neoplasm: lymphoma extending from the neck or mediastinum, superior sulcus bronchogenic carcinoma, and metastases; (4) traumatic: extrapleural dissection of blood from a ruptured aorta, fractures of the ribs or spine, or hemorrhage due to subclavian line placement; (5) vascular: coarctation of the aorta with dilated collaterals over the apex, fistula between the subclavian artery and vein; and (6) miscellaneous: mediastinal lipomatosis with subcostal fat extending over the apices

Biosynthetic incorporation of [75Se]selenomethionine: a new method for labelling lymphocyte membrane antigens

International Nuclear Information System (INIS)

Dosseto, M.; Rohner, C.; Pierres, M.; Goridis, C.

1981-01-01

A novel approach for radiolabelling lymphocyte membrane antigens is described. This technique is based on the use of the γ-emitting amino acid analogue [ 75 Se]selenomethionine. Human HLA-A, B, C and DR heavy and light chains and mouse Ia antigens were efficiently labelled by this technique and were precipitated with monoclonal antibodies. Approximately the same radioactivity was incorporated into the HLA-A, B, C chains whether [ 75 Se]selenomethionine, [ 35 S]methionine or [ 3 H]leucine were used as precursors. Easily detectable as a γ-emitter, [ 75 Se]selenomethionine thus constitutes a useful biosynthetic label of lymphocyte surface antigens. The same method was used to label immunoglobulins produced by hybridomas and to determine the nature of the secreted light chains. (Auth.)

Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery.

Science.gov (United States)

Davoodi-Semiromi, Abdoreza; Schreiber, Melissa; Nalapalli, Samson; Verma, Dheeraj; Singh, Nameirakpam D; Banks, Robert K; Chakrabarti, Debopam; Daniell, Henry

2010-02-01

Cholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin-B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB-AMA1 and CTB-MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce, respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen-specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross-reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB-specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin-10(+) T cell but not Foxp3(+) regulatory T cells, suppression of interferon-gamma and absence of interleukin-17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast-derived vaccine antigens for long-term (>300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity.

Relationship between the Apical Preparation Diameter and the Apical Seal: An In Vitro Study

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Kaoutar Laslami

2018-01-01

Full Text Available Objectives. The aim of the study is to define the relationship between the apical preparation diameter and the apical sealing ability to highlight the importance of the preservation of the diameter and the original position of the apical foramen. Materials and Methods. 50 extracted maxillary incisors were randomly allocated into three groups of 15 teeth each (n = 15 according to the apical preparation size: Group 1: finishing file F1 corresponding to size 20 reached the working length (ProTaper Universal system Dentsply®; Group 2: prepared up to size 30 corresponding to finishing file F30; Group 3: prepared up to size 50 corresponding to finishing file F5. Five teeth were assigned to positive and negative control groups. After the filling of the root canals, the teeth were isolated and immersed in a dye solution, then cut longitudinally, photographed, and the dye penetration were calculated using a computer software. Results. Comparison of the three different apical preparation sizes showed no statistically significant differences regarding the apical microleakage. Conclusion. The most important value of the dye penetration was observed in the group with the largest apical diameter.

Tissue expression and enzymologic characterization of human prostate specific membrane antigen and its rat and pig orthologs

Czech Academy of Sciences Publication Activity Database

Rovenská, Miroslava; Hlouchová, Klára; Šácha, Pavel; Mlčochová, Petra; Horák, Vratislav; Zámečník, J.; Bařinka, C.; Konvalinka, Jan

2008-01-01

Roč. 68, č. 2 (2008), s. 171-182 ISSN 0270-4137 R&D Projects: GA MŠk 1M0508; GA ČR GA524/04/0102 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50450515 Keywords : prostate specific membrane antigen * glutamate carboxypeptidase II * animal orthologs * prostate cancer * animal model Subject RIV: CE - Biochemistry Impact factor: 3.069, year: 2008

Antigen sequence typing of outer membrane protein (fetA gene of Neisseria meningitidis serogroup A from Delhi & adjoining areas

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S Dwivedi

2014-01-01

Full Text Available Background & objectives: Meningitis caused by Neisseria meningitidis is a fatal disease. Meningococcal meningitis is an endemic disease in Delhi and irregular pattern of outbreaks has been reported in India. All these outbreaks were associated with serogroup A. Detailed molecular characterization of N. meningitidis is required for the management of this fatal disease. In this study, we characterized antigenic diversity of surface exposed outer membrane protein (OMP FetA antigen of N. meningitidis serogroup A isolates obtained from cases of invasive meningococcal meningitis in Delhi, India. Methods: Eight isolates of N. meningitidis were collected from cerebrospinal fluid during October 2008 to May 2011 from occasional cases of meningococcal meningitis. Seven isolates were from outbreaks of meningococcal meningitis in 2005-2006 in Delhi and its adjoining areas. These were subjected to molecular typing of fetA gene, an outer membrane protein gene. Results: All 15 N. meningitides isolates studied were serogroup A. This surface exposed porin is putatively under immune pressure. Hence as a part of molecular characterization, genotyping was carried out to find out the diversity in outer membrane protein (FetA gene among the circulating isolates of N. meningitidis. All 15 isolates proved to be of the same existing allele type of FetA variable region (VR when matched with global database. The allele found was F3-1 for all the isolates. Interpretation & conclusions: There was no diversity reported in the outer membrane protein FetA in the present study and hence this protein appeared to be a stable molecule. More studies on molecular characterization of FetA antigen are required from different serogroups circulating in different parts of the world.

A transferrin-like GPI-linked iron-binding protein in detergent-insoluble noncaveolar microdomains at the apical surface of fetal intestinal epithelial cells

DEFF Research Database (Denmark)

Danielsen, E M; van Deurs, B

1995-01-01

of ultracryosections of mucosal tissue, the protein was localized to the apical surface of the enterocytes, whereas it was absent from the basolateral plasma membrane. Interestingly, it was mainly found in patches of flat or invaginated apical membrane domains rather than at the surface of microvilli. Caveolae were...

TRPP2 and TRPV4 form an EGF-activated calcium permeable channel at the apical membrane of renal collecting duct cells.

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Zhi-Ren Zhang

Full Text Available Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+ or in which cilia are absent (cilia (-. This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (- compared to cilia (+ cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF stimulated TRPP2\\TRPV4 channel through the EGF receptor (EGFR tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (- but not cilia (+ cells. In addition EGF increased cell proliferation in cilia (- cell that was dependent upon TRPP2\\TRPV4 channel mediated increase in intracellular calcium.We conclude that in the absence of cilia, an EGF activated TRPP2\\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s

DEFF Research Database (Denmark)

Eriksson, H; Nissen, Mogens Holst

1990-01-01

weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the alpha 2-and alpha 3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC...... class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the alpha 1-and alpha 2-domains which...

Neuroelectric Tuning of Cortical Oscillations by Apical Dendrites in Loop Circuits

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David LaBerge

2017-06-01

Full Text Available Bundles of relatively long apical dendrites dominate the neurons that make up the thickness of the cerebral cortex. It is proposed that a major function of the apical dendrite is to produce sustained oscillations at a specific frequency that can serve as a common timing unit for the processing of information in circuits connected to that apical dendrite. Many layer 5 and 6 pyramidal neurons are connected to thalamic neurons in loop circuits. A model of the apical dendrites of these pyramidal neurons has been used to simulate the electric activity of the apical dendrite. The results of that simulation demonstrated that subthreshold electric pulses in these apical dendrites can be tuned to specific frequencies and also can be fine-tuned to narrow bandwidths of less than one Hertz (1 Hz. Synchronous pulse outputs from the circuit loops containing apical dendrites can tune subthreshold membrane oscillations of neurons they contact. When the pulse outputs are finely tuned, they function as a local “clock,” which enables the contacted neurons to synchronously communicate with each other. Thus, a shared tuning frequency can select neurons for membership in a circuit. Unlike layer 6 apical dendrites, layer 5 apical dendrites can produce burst firing in many of their neurons, which increases the amplitude of signals in the neurons they contact. This difference in amplitude of signals serves as basis of selecting a sub-circuit for specialized processing (e.g., sustained attention within the typically larger layer 6-based circuit. After examining the sustaining of oscillations in loop circuits and the processing of spikes in network circuits, we propose that cortical functioning can be globally viewed as two systems: a loop system and a network system. The loop system oscillations influence the network system’s timing and amplitude of pulse signals, both of which can select circuits that are momentarily dominant in cortical activity.

Systemic antibiotics for symptomatic apical periodontitis and acute apical abscess in adults.

Science.gov (United States)

Cope, Anwen; Francis, Nick; Wood, Fiona; Mann, Mala K; Chestnutt, Ivor G

2014-06-26

Dental pain can have a considerable detrimental effect on an individual's quality of life. Symptomatic apical periodontitis and acute apical abscess are common causes of dental pain and arise from an inflamed or necrotic dental pulp, or infection of the pulpless root canal system. Clinical guidelines recommend that the first-line treatment for teeth with symptomatic apical periodontitis or an acute apical abscess should be removal of the source of inflammation or infection by local, operative measures, and that systemic antibiotics are currently only recommended for situations where there is evidence of spreading infection (cellulitis, lymph node involvement, diffuse swelling) or systemic involvement (fever, malaise). Despite this, there is evidence that dentists continue to prescribe antibiotics for these conditions. There is concern that this could contribute to the development of antibiotic-resistant bacterial colonies both within the individual and within the community as a whole. To evaluate the effects of systemic antibiotics provided with or without surgical intervention (such as extraction, incision and drainage of a swelling or endodontic treatment), with or without analgesics, for symptomatic apical periodontitis or acute apical abscess in adults. We searched the following electronic databases: Cochrane Oral Health Group's Trials Register (to 1 October 2013); Cochrane Central Register of Controlled Trials (The Cochrane Library 2013, Issue 9); MEDLINE via OVID (1946 to 1 October 2013); EMBASE via OVID (1980 to 1 October 2013) and CINAHL via EBSCO (1980 to 1 October 2013). We searched the World Health Organization (WHO) International Trials Registry Platform and the US National Institutes of Health Trials Registry (ClinicalTrials.gov) on 1 October 2013 to identify ongoing trials. We searched for grey literature using OpenGrey (to 1 October 2013) and ZETOC Conference Proceedings (1993 to 1 October 2013). We placed no restrictions on the language or date of

Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

International Nuclear Information System (INIS)

Wu, J.S.R.; Lever, J.E.

1987-01-01

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na + /glucose symporter by a Na + -dependent mechanism but is not itself transported across the membrane. The authors have isolated a panel of monoclonal antibodies that influence high-affinity, Na + -dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK 1 , a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG/sub 2b/, reproducibly stimulated Na + -dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na + -dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na + /glucose symporter

Immunoprotection of mice against Schistosomiasis mansoni using solubilized membrane antigens.

Directory of Open Access Journals (Sweden)

Guidenn Sulbarán

Full Text Available BACKGROUND: Schistosomiasis continues to be one of the most prevalent parasitic diseases in the world. Despite the existence of a highly effective antischistosome drug, the disease is spreading into new areas, and national control programs do not arrive to complete their tasks particularly in low endemic areas. The availability of a vaccine could represent an additional component to chemotherapy. Experimental vaccination studies are however necessary to identify parasite molecules that would serve as vaccine candidates. In the present work, C57BL/6 female mice were subcutaneously immunized with an n-butanol extract of the adult worm particulate membranous fraction (AWBE and its protective effect against a S. mansoni challenge infection was evaluated. METHODOLOGY AND FINDINGS: Water-saturated n-butanol release into the aqueous phase a set of membrane-associated (glycoproteins that are variably recognized by antibodies in schistosome-infected patients; among the previously identified AWBE antigens there is Alkaline Phosphatase (SmAP which has been associated with resistance to the infection in mice. As compared to control, a significantly lower number of perfuse parasites was obtained in the immunized/challenged mouse group (P<0.05, t test; and consequently, a lower number of eggs and granulomas (with reduced sizes, overall decreasing pathology. Immunized mice produced high levels of sera anti-AWBE IgG recognizing antigens of ∼190-, 130-, 98-, 47-, 28-23, 14-, and 9-kDa. The ∼130-kDa band (the AP dimer exhibited in situ SmAP activity after addition of AP substrate and the activity was not apparently inhibited by host antibodies. A preliminary proteomic analysis of the 25-, 27-, and 28-kDa bands in the immunodominant 28-23 kDa region suggested that they are composed of actin. CONCLUSIONS: Immunization with AWBE induced the production of specific antibodies to various adult worm membrane molecules (including AP and a partial (43% protection

A Comparative Study of Apical Healing of Open Apices Using MTA and Ca(OH2 Apical Plugs in Cats

Directory of Open Access Journals (Sweden)

M. H. Zarrabi

2005-06-01

Full Text Available Statement of problem: Endodontic treatment of necrotic teeth with open apices is a challenge. After ruling out surgery as a treatment scheme and introduction of the multivisit apexification which in turn had its disadvantages, apical plug seems to be a suitable substitute treatment plan for such cases. Apical plug makes the treatment through formation of a barrier against the obturating material in a single visit.Purpose: The purpose of this study was to compare histologically the periapical healing using MTA and calcium hydroxide apical plugs after intervals of 4 and 12 weeks in cats.Materials and Methods: In this clinical trial study 64 canines of 16 healthy and mature cats were divided into 3 groups after a periapical lesion formation by over instrumentation in the apical area with files up to no.120. The first group included 24 teeth on which MTA apical plug was applied. The second group included 24 teeth on which Ca (OH 2 apical plug was applied. In both groups the canals were filled with gutta percha and sealer. The third group included 16 control teeth whose canals were left empty after instrumentation and debridement. The access cavities of all teeth were sealed with varnish and amalgam and the vital perfusion of cats was performed in 4 and 12 week intervals. Statistical analysis was established by χ2 and independence test.Results: After 4 weeks, periapical healing in the first group was 90%, in the second group 80% and in the third group, it was only 12.5 %. After 12 weeks, periapical healing occurred in 100% of the MTA group, while it was 57.1% in the second and 40%in the third group .Generally, in the study of histological parameters of healing, no statistical significant difference was observed between the 2 experimental groups,although the MTA group results were much better than the Ca (OH 2 group especially at 12 weeks.Conclusion: The use of MTA apical plug is more effective than Ca (OH 2 in treatment of necrotic teeth with open

Development of a PET Prostate-Specific Membrane Antigen Imaging Agent: Preclinical Translation for Future Clinical Application

Science.gov (United States)

2017-10-01

are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by...phase 0) application to the FDA by the end of the funding period. The small molecule imaging agents under study home to prostate specific membrane...funding period. The small molecule imaging agents under study home to prostate specific membrane antigen (PSMA) that is prevalent on a majority of

Lipid rafts in epithelial brush borders: atypical membrane microdomains with specialized functions

DEFF Research Database (Denmark)

Danielsen, E Michael; Hansen, Gert H

2003-01-01

of the apical surface sterically accessible for membrane fusion/budding events. Many of these invaginations appear as pleiomorphic, deep apical tubules that extend up to 0.5-1 microm into the underlying terminal web region. Their sensitivity to methyl-beta-cyclodextrin suggests them to contain cholesterol...

Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

Science.gov (United States)

Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

2013-11-01

Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

Science.gov (United States)

Hönig, Ellena; Ringer, Karina; Dewes, Jenny; von Mach, Tobias; Kamm, Natalia; Kreitzer, Geri; Jacob, Ralf

2018-05-10

Epithelial cells require a precise intracellular transport and sorting machinery in order to establish and maintain their polarized architecture. This machinery includes beta-galactoside binding galectins for glycoprotein targeting to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analyzed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, galectin-3 knockout results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells. © 2018. Published by The Company of Biologists Ltd.

Role of apical oxygen in 2-1-4 electron-doped superconductors

Energy Technology Data Exchange (ETDEWEB)

Richard, P.; Riou, G.; Jandl, S.; Poirier, M.; Fournier, P.; Nekvasil, V.; Divis, M

2004-08-01

We report a crystal-field infrared transmission and Raman study of oxygenated and reduced Nd{sub 2-x}Ce{sub x}CuO{sub 4} single crystals. Some Nd{sup 3+} crystal-field absorption bands corresponding to rare-earth ions in non-regular sites are attributed to Nd{sup 3+} ions in the vicinity of apical oxygens. This is correlated with a study of the A{sup *} ({approx}580 cm{sup -1}) Raman local mode and with the transport properties of undoped materials. We show that the apical oxygen is not removed by the reduction.

A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules

Czech Academy of Sciences Publication Activity Database

Zhang, A.X.; Murelli, R.P.; Bařinka, Cyril; Michel, J.; Cocleaza, A.; Jorgensen, W.L.; Lubkowski, J.; Spiegel, D.A.

2010-01-01

Roč. 132, č. 36 (2010), s. 12711-12716 ISSN 0002-7863 Institutional research plan: CEZ:AV0Z50520701 Keywords : Prostate -specific membrane antigen * antibody recruiting molecules * Structure-activity relationship Subject RIV: CE - Biochemistry Impact factor: 9.019, year: 2010

Characterization of Prostate-Specific Membrane Antigen (PSMA) for Use in Therapeutic and Diagnostic Strategies Against Prostate Cancer

National Research Council Canada - National Science Library

O'Keefe, Denise

2002-01-01

Prostate-Specific Membrane Antigen (PSMA) appears to be an ideal prostate cancer marker and potential therapeutic target, however there have been reports of PSMA expression in non-prostatic tissues, including brain, kidney and liver...

The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit.

Science.gov (United States)

Lobato-Álvarez, Jorge A; Roldán, María L; López-Murillo, Teresa Del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

2016-01-01

Na + , K + -ATPase, or the Na + pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β 1 subunit of Na + , K + -ATPase plays an important role in this mechanism because homotypic β 1 -β 1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na + pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β 2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na + , K + -ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na + pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β 2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β 2 subunit. qPCR results showed a time-dependent increase in the level of β 2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β 2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α 2 subunit in that domain. Our results demonstrate that the apical polarization of Na + , K + -ATPase in RPE cells depends on the expression of the β 2 subunit.

Apical accumulation of the Sevenless receptor tyrosine kinase during Drosophila eye development is promoted by the small GTPase Rap1.

Science.gov (United States)

Baril, Caroline; Lefrançois, Martin; Sahmi, Malha; Knævelsrud, Helene; Therrien, Marc

2014-08-01

The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other co-required signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, we identified the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. Here, we show that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell-cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity. Copyright © 2014 by the Genetics Society of America.

Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport-type familial nephritis.

OpenAIRE

Kashtan, C; Fish, A J; Kleppel, M; Yoshioka, K; Michael, A F

1986-01-01

We probed epidermal basement membranes (EBM) of acid-urea denatured skin from members of kindreds with Alport-type familial nephritis (FN) for the presence of antigens reactive with Goodpasture sera (GPS) and serum (FNS) from an Alport patient who developed anti-glomerular basement membrane (GBM) nephritis in a renal allograft. By immunoblotting, GPS reacted primarily with the 28,000 molecular weight (mol wt) monomer but also the 24,000 mol wt and 26,000 mol wt monomers of the noncollagenous ...

A solid phase radio immunoassay on hydrophobic membrane filters: detection of antibodies to gonocal surface antigens

International Nuclear Information System (INIS)

Lambden, P.R.; Watt, P.J.

1978-01-01

A solid phase radioimmunoassay (SPRIA) has been developed for detection of IgG antibodies to gonococcal outer membrane components. Gonococcal antigens was immobilised on a solid support by covalent coupling to CNBr-activated Sepharose in the presence of the detergent Triton X-100. Binding of specific antibody to the Sepharose-antigen complex was detected using radiolabelled Protein A as the antiglobulin. Protein A was labelled by radioacetylation with tritiated acetic anhydride, yielding a product of high specific activity and high stability. No detectable loss of activity was observed over a ten month period. The entire assay was performed on Mitex teflon hydrophobic membrane filters which held the Sepharose beads and aqueous supernatant as a discrete drop of liquid. The supernatants and incubation were easily and rapidly removed from the beads by suction on a specially-designed manifold system. This procedure removed the need for repeated and time-consuming centrifugations. Titres were obtained graphically from double log plots of cpm bound versus antiserum dilution by extrapolation of the straight line to a point corresponding to twice the control level of radioactivity binding. The assay proved to be a very reliable and simple procedure for the detection of IgG antibodies to gonococcal surface antigens. (Auth.)

Characterization of an apically derived epithelial membrane glycoprotein from bovine milk, which is expressed in capillary endothelia in diverse tissues.

Science.gov (United States)

Greenwalt, D E; Mather, I H

1985-02-01

A glycoprotein (PAS IV) of apparent Mr 76,000 was purified from bovine milk-fat-globule membrane and partially characterized. PAS IV contained mannose, galactose, and sialic acid as principal sugars (approximately 5.3% total carbohydrate [wt/wt]) and existed in milk in at least four isoelectric variants. The glycoprotein appeared to be an integral membrane protein by several criteria. PAS IV was recovered in the detergent phase of Triton X-114 extracts of milk-fat-globule membrane at room temperature. When bound to membrane, PAS IV was resistant to digestion by a number of proteinases, although after solubilization with non-ionic detergents, the protein was readily degraded. Amino acid analysis of the purified protein revealed a high percentage of amino acids with nonpolar residues. The location of PAS IV was determined in bovine tissues by using immunofluorescence techniques. In mammary tissue, PAS IV was located on both the apical surfaces of secretory epithelial cells and endothelial cells of capillaries. This glycoprotein was also detected in endothelial cells of heart, liver, spleen, pancreas, salivary gland, and small intestine. In addition to mammary epithelial cells, PAS IV was also located in certain other epithelial cells, most notably the bronchiolar epithelial cells of lung. The potential usefulness of this protein as a specific marker of capillary endothelial cells in certain tissues is discussed.

Effect of the angle of apical resection on apical leakage, measured with a computerized fluid filtration device.

Science.gov (United States)

Garip, Hasan; Garip, Yıldız; Oruçoğlu, Hasan; Hatipoğlu, Seda

2011-03-01

We determined the effect of the angle of apical resection on apical leakage using a computerized fluid filtration meter with a laser system and a digital air pressure regulator in 46 extracted single-rooted human teeth. Orthograde endodontic treatment was performed. The root canals were prepared up to a size 50 K-type file with 17% EDTA solution (Roth International, Chicago, IL) and 5% NaOCl solution as the irrigant. Gates Glidden burs (Maillefer Instruments, Ballaigues, Switzerland) were used to flare the coronal two thirds of the canal. All canals were dried with paper points and then obturated using cold lateral condensation (except for the positive controls) of gutta-percha points and AH plus (Dentsply DeTrey, Konstanz, Germany). All 40 roots were sectioned 3 mm from the apex. Forty teeth were assigned randomly into 1 of 4 experimental groups of 10 teeth each: in group 1, the teeth were resected apically (90° angle) and the cavities were obturated with mineral trioxide aggregate (MTA); in group 2, after apical resection (90° angle), a root-end cavity was prepared using ultrasonic diamond retrotips and the cavities were obturated with MTA; in group 3, the teeth were resected apically (∼45° angle) and the cavities were obturated with MTA; and in group 4, after apical resection (∼45° angle), a root-end cavity was prepared using ultrasonic diamond retrotips and the cavities were obturated with MTA. An additional 6 teeth were used as controls (3 each, negative and positive controls). Apical leakage was measured using a computerized fluid filtration meter with a laser system. The mean apical microleakage was 2.0 ± 0.4 × 10(-4), 1.6 ± 0.6 × 10(-4), 1.6 ± 0.9 × 10(-4), and 1.8 ± 0.7 × 10(-4) μL/cmH(2)O/min(-1) at 1.2 atm, in groups 1 to 4, respectively. Although the mean apical microleakage was greater in group 1, the differences among the 4 groups were not statistically significant (P > .05). The results of these in vitro studies showed that when an

Radio-iodination of plasma membranes of toad bladder epithelium

Energy Technology Data Exchange (ETDEWEB)

Rodriguez, H J; Edelman, I S [California Univ., San Francisco (USA). Cardiovascular Research Inst.; California Univ., San Francisco (USA). Dept. of Medicine; California Univ., San Francisco (USA). Dept. of Biochemistry and Biophysics)

1979-01-01

The present report describes high yield enzymatic radio-iodination of the apical and basal-lateral plasma membranes of toad bladder epithelium with /sup 125/I-Na, by a procedure that does not breach the functional integrity of the epithelium, as assessed by the basal and vasopressin-sensitive short-circuit current (SCC). Iodination of basal-lateral plasma membranes, at a yield comparable to that obtained with apical labelling, was attained after about 30 min of exposure of the intact bladder to the labelling solutions. Approximately 25% of the basal-lateral labeling was lost when the epithelial cells were harvested after collagenase treatment, implying that some iodination of the basement membrane had taken place. Less than 10% of iodination of the apical or basal-lateral surfaces was accounted for by lipid-labeling. Analysis of the labeled apical and basal-lateral species by enzymatic digestion and thin layer chromatography disclosed that virtually all the radioactivity was present as mono-iodotyrosine (MIT). (orig./AJ).

Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

Directory of Open Access Journals (Sweden)

Ana Teresa B.F. Antonangelo

2012-09-01

Full Text Available Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC. The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

Detection of zinc translocation into apical dendrite of CA1 pyramidal neuron after electrical stimulation.

Science.gov (United States)

Suh, Sang Won

2009-02-15

Translocation of the endogenous cation zinc from presynaptic terminals to postsynaptic neurons after brain insult has been implicated as a potential neurotoxic event. Several studies have previously demonstrated that a brief electrical stimulation is sufficient to induce the translocation of zinc from presynaptic vesicles into the cytoplasm (soma) of postsynaptic neurons. In the present work I have extended those findings in three ways: (i) providing evidence that zinc translocation occurs into apical dendrites, (ii) presenting data that there is an apparent translocation into apical dendrites when only a zinc-containing synaptic input is stimulated, and (iii) presenting data that there is no zinc translocation into apical dendrite of ZnT3 KO mice following electrical stimulation. Hippocampal slices were preloaded with the "trappable" zinc fluorescent probe, Newport Green. After washout, a single apical dendrite in the stratum radiatum of hippocampal CA1 area was selected and focused on. Burst stimulation (100Hz, 500microA, 0.2ms, monopolar) was delivered to either the adjacent Schaffer-collateral inputs (zinc-containing) or to the adjacent temporo-ammonic inputs (zinc-free) to the CA1 dendrites. Stimulation of the Schaffer collaterals increased the dendritic fluorescence, which was blocked by TTX, low-Ca medium, or the extracellular zinc chelator, CaEDTA. Stimulation of the temporo-ammonic pathway caused no significant rise in the fluorescence. Genetic depletion of vesicular zinc by ZnT3 KO showed no stimulation-induced apical dendrite zinc rise. The present study provides evidence that synaptically released zinc translocates into postsynaptic neurons through the apical dendrites of CA1 pyramidal neurons during physiological synaptic activity.

Discriminatory Role of Detergent-Resistant Membranes in the Dimerization and Endocytosis of Prostate-Specific Membrane Antigen

Science.gov (United States)

Schmidt, Sonja; Gericke, Birthe; Fracasso, Giulio; Ramarli, Dunia; Colombatti, Marco; Naim, Hassan Y.

2013-01-01

Prostate-specific membrane antigen (PSMA) is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in the endosomes. PSMA associates with different types of detergent-resistant membranes (DRMs) along the secretory pathway. Its mature form is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. To understand the mechanism of PSMA internalization we investigated its association during internalization with DRMs. For this purpose, internalization was induced by antibody cross-linking. We demonstrate at the biochemical and cell biological levels that: [i] exclusively homodimers of PSMA are associated with Lubrol WX-DRMs, [ii] antibody-induced cross-linking of PSMA molecules results in a time-dependent partitioning into another DRMs type, namely Triton X-100-DRMs, and [iii] concomitant with its association with Triton-X-100-DRMs internalization of PSMA occurs along tubulin filaments. In a previous work (Colombatti et al. (2009) PLoS One 4: e4608) we demonstrated that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during antibody cross-linking. As downstream effects of this activation we observed a strong induction of NF-kB associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically. These observations together with findings reported here hypothesize a fundamental role of DRMs during activation of PSMA as platforms for trafficking, endocytosis and signalling. Understanding these mechanisms constitutes an essential prerequisite for utilization of PSMA as

Discriminatory Role of Detergent-Resistant Membranes in the Dimerization and Endocytosis of Prostate-Specific Membrane Antigen.

Directory of Open Access Journals (Sweden)

Sonja Schmidt

Full Text Available Prostate-specific membrane antigen (PSMA is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in the endosomes. PSMA associates with different types of detergent-resistant membranes (DRMs along the secretory pathway. Its mature form is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. To understand the mechanism of PSMA internalization we investigated its association during internalization with DRMs. For this purpose, internalization was induced by antibody cross-linking. We demonstrate at the biochemical and cell biological levels that: [i] exclusively homodimers of PSMA are associated with Lubrol WX-DRMs, [ii] antibody-induced cross-linking of PSMA molecules results in a time-dependent partitioning into another DRMs type, namely Triton X-100-DRMs, and [iii] concomitant with its association with Triton-X-100-DRMs internalization of PSMA occurs along tubulin filaments. In a previous work (Colombatti et al. (2009 PLoS One 4: e4608 we demonstrated that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during antibody cross-linking. As downstream effects of this activation we observed a strong induction of NF-kB associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically. These observations together with findings reported here hypothesize a fundamental role of DRMs during activation of PSMA as platforms for trafficking, endocytosis and signalling. Understanding these mechanisms constitutes an essential prerequisite for

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

Science.gov (United States)

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Tolerance of brightness and contrast adjustments on chronic apical abscess and apical granuloma interpretation

Science.gov (United States)

Purnamasari, L.; Iskandar, H. H. B.; Makes, B. N.

2017-08-01

In digitized radiography techniques, adjusting the image enhancement can improve the subjective image quality by optimizing the brightness and contrast for diagnostic needs. To determine the value range of image enhancement (brightness and contrast) on chronic apical abscess and apical granuloma interpretation. 30 periapical radiographs that diagnosed chronic apical abscess and 30 that diagnosed apical granuloma were adjusted by changing brightness and contrast values. The value range of brightness and contrast adjustment that can be tolerated in radiographic interpretations of chronic apical abscess and apical granuloma spans from -10 to +10. Brightness and contrast adjustments on digital radiographs do not affect the radiographic interpretation of chronic apical abscess and apical granuloma if conducted within the value range.

Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA)

OpenAIRE

Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A.; Saul, Allan; Gerke, Christiane

2014-01-01

Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Int...

Definitions of apical vaginal support loss: a systematic review.

Science.gov (United States)

Meister, Melanie R L; Sutcliffe, Siobhan; Lowder, Jerry L

2017-03-01

We sought to identify and summarize definitions of apical support loss utilized for inclusion, success, and failure in surgical trials for treatment of apical vaginal prolapse. Pelvic organ prolapse is a common condition affecting more than 3 million women in the US, and the prevalence is increasing. Prolapse may occur in the anterior compartment, posterior compartment or at the apex. Apical support is considered paramount to overall female pelvic organ support, yet apical support loss is often underrecognized and there are no guidelines for when an apical support procedure should be performed or incorporated into a procedure designed to address prolapse. A systematic literature search was performed in 8 search engines: PubMed 1946-, Embase 1947-, Cochrane Database of Systematic Reviews, Cochrane Database of Abstracts of Review Effects, Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, Proquest Dissertations and Theses, and FirstSearch Proceedings, using key words for apical pelvic organ prolapse and apical suspension procedures through April 2016. Searches were limited to human beings using human filters and articles published in English. Study authors (M.R.L.M., J.L.L.) independently reviewed publications for inclusion based on predefined variables. Articles were eligible for inclusion if they satisfied any of the following criteria: (1) apical support loss was an inclusion criterion in the original study, (2) apical support loss was a surgical indication, or (3) an apical support procedure was performed as part of the primary surgery. A total of 4469 publications were identified. After review, 35 articles were included in the analysis. Prolapse-related inclusion criteria were: (1) apical prolapse (n = 20, 57.1%); (2) overall prolapse (n = 8, 22.8%); or (3) both (n = 6, 17.1%). Definitions of apical prolapse (relative to the hymen) included: (1) apical prolapse >-1 cm (n = 13, 50.0%); (2) apical prolapse >+1 cm (n = 7, 26.9%); (3) apical

Advances in prostate-specific membrane antigen PET of prostate cancer.

Science.gov (United States)

Bouchelouche, Kirsten; Choyke, Peter L

2018-05-01

In recent years, a large number of reports have been published on prostate-specific membrane antigen (PSMA)/PET in prostate cancer (PCa). This review highlights advances in PSMA PET in PCa during the past year. PSMA PET/computed tomography (CT) is useful in detection of biochemical recurrence, especially at low prostate-specific antigen (PSA) values. The detection rate of PSMA PET is influenced by PSA level. For primary PCa, PSMA PET/CT shows promise for tumour localization in the prostate, especially in combination with multiparametric MRI (mpMRI). For primary staging, PSMA PET/CT can be used in intermediate and high-risk PCa. Intraoperative PSMA radioligand guidance seems promising for detection of malignant lymph nodes. While the use of PSMA PET/MRI in primary localized disease is limited to high and intermediate-risk patients and localized staging, in the recurrence setting, PET/MRI can be particularly helpful when the lesions are subtle. PSMA PET/CT is superior to choline PET/CT and other conventional imaging modalities. Molecular imaging with PSMA PET continues to pave the way for personalized medicine in PCa.However, large prospective clinical studies are still needed to fully evaluate the role of PSMA PET/CT and PET/MRI in the clinical workflow of PCa.

Effect of colchicine on rat small intestinal absorptive cells. II. Distribution of label after incorporation of [3H]fucose into plasma membrane glycoproteins

International Nuclear Information System (INIS)

Ellinger, A.; Pavelka, M.; Gangl, A.

1983-01-01

By means of radioautography the influence was tested of various periods (5, 15, 30, 40 min, 2 hr) of pretreatment with colchicine, administered intraperitoneally to rats at a dosage of 0.5 mg/100 g of body weight, on the intracellular pathway of [ 3 H]fucose in absorptive cells of the small intestine. Administration of colchicine for 30 min and longer time intervals causes delay in the insertion of [ 3 H]fucose into the oligosaccharide chains of glycoconjugates in the Golgi apparatus, and results in redistribution of the label apparent over the different portions of the plasma membrane. In controls, at 2 and 4 hr after administration of [ 3 H]fucose the apical plasma membrane is strongly labeled. Colchicine causes equalization of the reaction of apical and basolateral regions of the plasma membrane: the number of silver grains attributable to the apical plasma membrane is reduced; following treatment with colchicine, apical portions of the plasma membrane comprise 31.6 +/- 1.8% of the silver grains, 38.6 +/- 3.8% are attributable to basolateral membrane regions. The colchicine-induced equalization of the density of label of apical and basolateral regions of the plasma membrane, in addition to the occurrence of basolateral microvillus borders, suggests microtubules to be important in the maintenance of the polar organization of small intestinal absorptive cells

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

Science.gov (United States)

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

2016-07-01

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. Copyright © 2016 Elsevier B.V. All rights reserved.

House Dust Mite Der p 1 Effects on Sinonasal Epithelial Tight Junctions

Science.gov (United States)

Henriquez, Oswaldo A.; Beste, Kyle Den; Hoddeson, Elizabeth K.; Parkos, Charles A.; Nusrat, Asma; Wise, Sarah K.

2013-01-01

Background Epithelial permeability is highly dependent upon the integrity of tight junctions, cell-cell adhesion complexes located at the apical aspect of the lateral membrane of polarized epithelial cells. We hypothesize that sinonasal epithelial exposure to Der p 1 house dust mite antigen decreases expression of tight junction proteins (TJPs), representing a potential mechanism for increased permeability and presentation of antigens across the sinonasal epithelial layer. Methods Confluent cultured primary human sinonasal epithelial cells were exposed to recombinant Der p 1 antigen versus control, and transepithelial resistance measurements were performed over 24 hours. Antibody staining for a panel of tight junction proteins was examined with immunofluorescence/confocal microscopy and Western blotting. Tissue for these experiments was obtained from 4 patients total. Results Der p 1 exposed sinonasal cells showed a marked decrease in transepithelial resistance when compared to control cells. In addition, results of Western immunoblot and immunofluorescent labeling demonstrated decreased expression of TJPs claudin-1 and junction adhesion molecule-A (JAM-A) in Der p 1 exposed cultured sinonasal cells versus controls. Conclusion Der p 1 antigen exposure decreases sinonasal epithelium TJP expression, most notably seen in JAM-A and claudin-1 in these preliminary experiments. This decreased TJP expression likely contributes to increased epithelial permeability and represents a potential mechanism for transepithelial antigen exposure in allergic rhinitis. PMID:23592402

Identification of Eimeria acervulina conoid antigen using chicken monoclonal antibody.

Science.gov (United States)

Matsubayashi, Makoto; Minoura, Chisa; Kimura, Shintaro; Tani, Hiroyuki; Furuya, Masaru; Lillehoj, Hyun S; Matsuda, Haruo; Takenaka, Shigeo; Hatta, Takeshi; Tsuji, Naotoshi; Sasai, Kazumi

2016-11-01

In the poultry industry, Eimeria spp. is one of the important pathogens which cause significant economic losses. We have previously generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina and with cross-reactive among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium spp. Furthermore, the protein of Cryptosporidium parvum recognized by the 6D-12-G10 has been identified as elongation factor-1α (EF-1α). In the present study, to identify the target molecule of E. acervulina by the mAb, we performed two-dimensional Western blotting analysis. Finally, we found two positive molecules which are identified as EF-1α and a related protein. Our previous finding using C. parvum and the results in this study suggest that EF-1α could be associated with the invasion facilitated by the cytoskeleton at the apical region of zoites.

Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

Science.gov (United States)

Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

2004-10-01

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized form MDCK cells and mainly basolaterally from Caco-2 cells

DEFF Research Database (Denmark)

Vogel, L K; Suske, G; Beato, M

1993-01-01

A complete cDNA encoding rabbit uteroglobin was constructed and expressed in MDCK and Caco-2 cells. The MDCK cells secrete uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco-2 cells secrete uteroglobin mainly to the basolateral side. Both MDCK...... and Caco-2 cells thus secrete uteroglobin in a non-sorted manner. It has, however, previously been shown that uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563-1573]. This suggests that either...... the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco-2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type....

Apical root resorption in orthodontically treated adults.

Science.gov (United States)

Baumrind, S; Korn, E L; Boyd, R L

1996-09-01

This study analyzed the relationship in orthodontically treated adults between upper central incisor displacement measured on lateral cephalograms and apical root resorption measured on anterior periapical x-ray films. A multiple linear regression examined incisor displacements in four directions (retraction, advancement, intrusion, and extrusion) as independent variables, attempting to account for observed differences in the dependent variable, resorption. Mean apical resorption was 1.36 mm (sd +/- 1.46, n = 73). Mean horizontal displacement of the apex was -0.83 mm (sd +/- 1.74, n = 67); mean vertical displacement was 0.19 mm (sd +/- 1.48, n = 67). The regression coefficients for the intercept and for retraction were highly significant; those for extrusion, intrusion, and advancement were not. At the 95% confidence level, an average of 0.99 mm (se = +/- 0.34) of resorption was implied in the absence of root displacement and an average of 0.49 mm (se = +/- 0.14) of resorption was implied per millimeter of retraction. R2 for all four directional displacement variables (DDVs) taken together was only 0.20, which implied that only a relatively small portion of the observed apical resorption could be accounted for by tooth displacement alone. In a secondary set of univariate analyses, the associations between apical resorption and each of 14 additional treatment-related variables were examined. Only Gender, Elapsed Time, and Total Apical Displacement displayed statistically significant associations with apical resorption. Additional multiple regressions were then performed in which the data for each of these three statistically significant variables were considered separately, with the data for the four directional displacement variables. The addition of information on Elapsed Time or Total Apical Displacement did not explain a significant additional portion of the variability in apical resorption. On the other hand, the addition of information on Gender to the

Presentation of lipid antigens to T cells.

Science.gov (United States)

Mori, Lucia; De Libero, Gennaro

2008-04-15

T cells specific for lipid antigens participate in regulation of the immune response during infections, tumor immunosurveillance, allergy and autoimmune diseases. T cells recognize lipid antigens as complexes formed with CD1 antigen-presenting molecules, thus resembling recognition of MHC-peptide complexes. The biophysical properties of lipids impose unique mechanisms for their delivery, internalization into antigen-presenting cells, membrane trafficking, processing, and loading of CD1 molecules. Each of these steps is controlled at molecular and celular levels and determines lipid immunogenicity. Lipid antigens may derive from microbes and from the cellular metabolism, thus allowing the immune system to survey a large repertoire of immunogenic molecules. Recognition of lipid antigens facilitates the detection of infectious agents and the initiation of responses involved in immunoregulation and autoimmunity. This review focuses on the presentation mechanisms and specific recognition of self and bacterial lipid antigens and discusses the important open issues.

Evaluation of three instrumentation techniques at the precision of apical stop and apical sealing of obturation

Directory of Open Access Journals (Sweden)

Özgür Genç

2011-08-01

Full Text Available OBJECTIVE: The aim of this study was to investigate the ability of two NiTi rotary apical preparation techniques used with an electronic apex locator-integrated endodontic motor and a manual technique to create an apical stop at a predetermined level (0.5 mm short of the apical foramen in teeth with disrupted apical constriction, and to evaluate microleakage following obturation in such prepared teeth. MATERIAL AND METHODS: 85 intact human mandibular permanent incisors with single root canal were accessed and the apical constriction was disrupted using a #25 K-file. The teeth were embedded in alginate and instrumented to #40 using rotary Lightspeed or S-Apex techniques or stainless-steel K-files. Distance between the apical foramen and the created apical stop was measured to an accuracy of 0.01 mm. In another set of instrumented teeth, root canals were obturated using gutta-percha and sealer, and leakage was tested at 1 week and 3 months using a fluid filtration device. RESULTS: All techniques performed slightly short of the predetermined level. Closest preparation to the predetermined level was with the manual technique and the farthest was with S-Apex. A significant difference was found between the performances of these two techniques (p<0.05. Lightspeed ranked in between. Leakage was similar for all techniques at either period. However, all groups leaked significantly more at 3 months compared to 1 week (p<0.05. CONCLUSIONS: Despite statistically significant differences found among the techniques, deviations from the predetermined level were small and clinically acceptable for all techniques. Leakage following obturation was comparable in all groups.

House dust mite allergen Der p 1 effects on sinonasal epithelial tight junctions.

Science.gov (United States)

Henriquez, Oswaldo A; Den Beste, Kyle; Hoddeson, Elizabeth K; Parkos, Charles A; Nusrat, Asma; Wise, Sarah K

2013-08-01

Epithelial permeability is highly dependent upon the integrity of tight junctions, which are cell-cell adhesion complexes located at the apical aspect of the lateral membrane of polarized epithelial cells. We hypothesize that sinonasal epithelial exposure to Der p 1 house dust mite antigen decreases expression of tight junction proteins (TJPs), representing a potential mechanism for increased permeability and presentation of antigens across the sinonasal epithelial layer. Confluent cultured primary human sinonasal epithelial cells were exposed to recombinant Der p 1 antigen vs control, and transepithelial resistance measurements were performed over 24 hours. Antibody staining for a panel of TJPs was examined with immunofluorescence/confocal microscopy and Western blotting. Tissue for these experiments was obtained from 4 patients total. Der p 1 exposed sinonasal cells showed a marked decrease in transepithelial resistance when compared to control cells. In addition, results of Western immunoblot and immunofluorescent labeling demonstrated decreased expression of TJPs claudin-1 and junction adhesion molecule-A (JAM-A) in Der p 1-exposed cultured sinonasal cells vs controls. Der p 1 antigen exposure decreases sinonasal epithelium TJP expression, most notably seen in JAM-A and claudin-1 in these preliminary experiments. This decreased TJP expression likely contributes to increased epithelial permeability and represents a potential mechanism for transepithelial antigen exposure in allergic rhinitis. © 2013 ARS-AAOA, LLC.

N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells.

Science.gov (United States)

Imjeti, Naga Salaija; Lebreton, Stéphanie; Paladino, Simona; de la Fuente, Erwin; Gonzalez, Alfonso; Zurzolo, Chiara

2011-12-01

Sorting of glycosylphosphatidyl-inositol--anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin-Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.

Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA-1.

Science.gov (United States)

Knight, J M; Zingales, B; Bottazzi, M E; Hotez, P; Zhan, B

2014-12-01

Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere. The toxicities and limited efficacies of current antitrypanosomal drugs have prompted a search for alternative technologies such as a therapeutic vaccine comprised of T. cruzi antigens, including a recombinant antigen encoding the N-terminal 65 kDa portion of Trypomastigote surface antigen-1 (TSA-1). With at least six known genetically distinct T. cruzi lineages, variability between the different lineages poses a unique challenge for the development of broadly effective therapeutic vaccine. The variability across the major lineages in the current vaccine candidate antigen TSA-1 has not previously been addressed. To assess the variation in TSA-1, we cloned and sequenced TSA-1 from several different T. cruzi strains representing three of the most clinically relevant lineages. Analysis of the different alleles showed limited variation in TSA-1 across the different strains and fit with the current theory for the evolution of the different lineages. Additionally, minimal variation in known antigenic epitopes for the HLA-A 02 allele suggests that interlineage variation in TSA-1 would not impair the range and efficacy of a vaccine containing TSA-1. © 2014 John Wiley & Sons Ltd.

Apical-to-basolateral transepithelial transport of cow's milk caseins by intestinal Caco-2 cell monolayers: MS-based quantitation of cellularly degraded α- and β-casein fragments.

Science.gov (United States)

Sakurai, Nao; Nishio, Shunsuke; Akiyama, Yuka; Miyata, Shinji; Oshima, Kenzi; Nadano, Daita; Matsuda, Tsukasa

2018-02-27

Casein is the major milk protein to nourish infants but, in certain population, it causes cow's milk allergy, indicating the uptake of antigenic casein and their peptides through the intestinal epithelium. Using human intestinal Caco-2 cell monolayers, the apical-to-basal transepithelial transport of casein was investigated. Confocal microscopy using component-specific antibodies showed that αs1-casein antigens became detectable as punctate signals at the apical-side cytoplasm and reached to the cytoplasm at a tight-junction level within a few hours. Such intracellular casein signals were more remarkable than those of the other antigens, β-lactoglobulin and ovalbumin, colocalized in part with an early endosome marker protein, EEA1, and decreased in the presence of cytochalasin D or sodium azide and also at lowered temperature at 4 °C. LC-MS analysis of the protein fraction in the basal-side medium identified the αs1-casein fragment including the N-terminal region and the αs2-casein fragment containing the central part of polypeptide at 100∼1000 fmol per well levels. Moreover, β-casein C-terminal overlapping peptides were identified in the peptide fraction below 10 kDa of the basal medium. These results suggest that caseins are partially degraded by cellular proteases and/or peptidases and immunologically active casein fragments are transported to basal side of the cell monolayers.

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes

DEFF Research Database (Denmark)

Bengtsson, Dominique; Sowa, Kordai M; Salanti, Ali

2008-01-01

BACKGROUND: The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used...... fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens....

Seroprevalence of Antibodies against Plasmodium falciparum Sporozoite Antigens as Predictive Disease Transmission Markers in an Area of Ghana with Seasonal Malaria Transmission.

Directory of Open Access Journals (Sweden)

Kwadwo A Kusi

Full Text Available As an increasing number of malaria-endemic countries approach the disease elimination phase, sustenance of control efforts and effective monitoring are necessary to ensure success. Mathematical models that estimate anti-parasite antibody seroconversion rates are gaining relevance as more sensitive transmission intensity estimation tools. Models however estimate yearly seroconversion and seroreversion rates and usually predict long term changes in transmission, occurring years before the time of sampling. Another challenge is the identification of appropriate antigen targets since specific antibody levels must directly reflect changes in transmission patterns. We therefore investigated the potential of antibodies to sporozoite and blood stage antigens for detecting short term differences in malaria transmission in two communities in Northern Ghana with marked, seasonal transmission.Cross-sectional surveys were conducted during the rainy and dry seasons in two communities, one in close proximity to an irrigation dam and the other at least 20 Km away from the dam. Antibodies against the sporozoite-specific antigens circumsporozoite protein (CSP and Cell traversal for ookinetes and sporozoites (CelTOS and the classical blood stage antigen apical membrane antigen 1 (AMA1 were measured by indirect ELISA. Antibody levels and seroprevalence were compared between surveys and between study communities. Antibody seroprevalence data were fitted to a modified reversible catalytic model to estimate the seroconversion and seroreversion rates.Changes in sporozoite-specific antibody levels and seroprevalence directly reflected differences in parasite prevalence between the rainy and dry seasons and hence the extent of malaria transmission. Seroconversion rate estimates from modelled seroprevalence data did not however support the above observation.The data confirms the potential utility of sporozoite-specific antigens as useful markers for monitoring short term

Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

International Nuclear Information System (INIS)

Rajendran, V.M.; Kashgarian, M.; Binder, H.J.

1989-01-01

Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats

EBIO, an agent causing maintained epithelial chloride secretion by co-ordinate actions at both apical and basolateral membranes.

Science.gov (United States)

MacVinish, L J; Keogh, J; Cuthbert, A W

2001-01-01

The effect of 1-ethyl-2-benzimidazolone (EBIO) on electrogenic chloride secretion in murine colonic and nasal epithelium was investigated by the short-circuit technique. In the colon, EBIO produces a sustained current increase in the presence of amiloride, which is sensitive to furosemide. In nasal epithelium EBIO causes only a small, transient current increase. Sustained increases in current were obtained in response to forskolin in both epithelia. To examine the mechanisms by which EBIO increases chloride secretion, the effects on intracellular mediators were measured in colonic crypts. There was no effect on [Ca(2+)]i but cAMP content was increased, more so in the presence of IBMX, indicating a direct effect on adenylate cyclase. In colonic epithelia in which the apical surface was permeabilized by nystatin, and the tissue subjected to an apical to basolateral K(+) gradient, EBIO caused a current increase that was entirely sensitive to charybdotoxin (ChTX). In similarly permeabilized colons Br-cAMP caused a current increase that was entirely sensitive to 293B. Thus EBIO increases chloride secretion in the colon by coordinated actions at both the apical and basolateral faces of the cells. These include direct and indirect actions on Ca(2+)-sensitive and cAMP-sensitive K(+) channels respectively, and indirect actions on the basolateral cotransporter and apical CFTR chloride channels via cAMP. In CF colonic epithelia EBIO did not evoke chloride secretion. It is not clear why the nasal epithelium responds poorly to EBIO whereas it gives a sustained response to the related compound chlorzoxazone.

Hierarchical, domain type-specific acquisition of antibodies to Plasmodium falciparum erythrocyte membrane protein 1 in Tanzanian children

DEFF Research Database (Denmark)

Cham, Gerald K K; Turner, Louise; Kurtis, Jonathan D

2010-01-01

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of malaria-infected erythrocytes. PfEMP1 attaches to the vascular lining and allows infected erythrocytes to avoid filtration through the spleen. Each parasite genome encodes about 60 diffe...... and play a major role in limiting parasite multiplication in the blood.......Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of malaria-infected erythrocytes. PfEMP1 attaches to the vascular lining and allows infected erythrocytes to avoid filtration through the spleen. Each parasite genome encodes about 60...... different PfEMP1 variants, each PfEMP1 comprises several domains in its extracellular region, and the PfEMP1 repertoire in different parasites contains domain types that are serologically cross-reactive. In this longitudinal study, we followed 672 children living in an area of high malaria transmission...

Bony change of apical lesion healing process using fractal analysis

Energy Technology Data Exchange (ETDEWEB)

Lee, Ji Min; Park, Hyok; Jeong, Ho Gul; Kim, Kee Deog; Park, Chang Seo [Yonsei University College of Medicine, Seoul (Korea, Republic of)

2005-06-15

To investigate the change of bone healing process after endodontic treatment of the tooth with an apical lesion by fractal analysis. Radiographic images of 35 teeth from 33 patients taken on first diagnosis, 6 months, and 1 year after endodontic treatment were selected. Radiographic images were taken by JUPITER computerized Dental X-ray System. Fractal dimensions were calculated three times at each area by Scion Image PC program. Rectangular region of interest (30 x 30) were selected at apical lesion and normal apex of each image. The fractal dimension at apical lesion of first diagnosis (L{sub 0}) is 0.940 {+-} 0.361 and that of normal area (N{sub 0}) is 1.186 {+-} 0.727 (p<0.05). Fractal dimension at apical lesion of 6 months after endodontic treatment (L{sub 1}) is 1.076 {+-} 0.069 and that of normal area (N{sub 1}) is 1.192 {+-} 0.055 (p<0.05). Fractal dimension at apical lesion of 1 year after endodontic treatment (L{sub 2}) is 1.163 {+-} 0.074 and that of normal area (N{sub 2}) is 1.225 {+-} 0.079 (p<0.05). After endodontic treatment, the fractal dimensions at each apical lesions depending on time showed statistically significant difference. And there are statistically significant different between normal area and apical lesion on first diagnosis, 6 months after, 1 year after. But the differences were grow smaller as time flows. The evaluation of the prognosis after the endodontic treatment of the apical lesion was estimated by bone regeneration in apical region. Fractal analysis was attempted to overcome the limit of subjective reading, and as a result the change of the bone during the healing process was able to be detected objectively and quantitatively.

Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis.

Science.gov (United States)

Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A; Hall, David H; Fleming, John T; Göbel, Verena

2011-09-18

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity through sphingolipid synthesis, and reveal ceramide glucosyltransferases (CGTs) as end-point biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids, CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and indicate that they sort new components to the expanding apical membrane.

Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens.

Directory of Open Access Journals (Sweden)

Edith S Málaga-Machaca

2017-11-01

Full Text Available Detection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease.Polyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA.Polyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status.

Role of apical oxygen in 2-1-4 electron-doped superconductors

Czech Academy of Sciences Publication Activity Database

Richard, P.; Riou, G.; Jandl, S.; Poirier, M.; Furnier, P.; Nekvasil, Vladimír; Diviš, M.

408-410, - (2004), s. 830-831 ISSN 0921-4534 R&D Projects: GA ČR GA202/03/0552 Institutional research plan: CEZ:AV0Z1010914 Keywords : Nd 2-x Ce x CuO 4 * crystal-field * apical oxygen * HTSC Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 1.072, year: 2004

Humoral immune responses to a single allele PfAMA1 vaccine in healthy malaria-naïve adults.

Directory of Open Access Journals (Sweden)

Edmond J Remarque

Full Text Available Plasmodium falciparum: apical membrane antigen 1 (AMA1 is a candidate malaria vaccine antigen expressed on merozoites and sporozoites. The polymorphic nature of AMA1 may compromise vaccine induced protection. The humoral response induced by two dosages (10 and 50 µg of a single allele AMA1 antigen (FVO formulated with Alhydrogel, Montanide ISA 720 or AS02 was investigated in 47 malaria-naïve adult volunteers. Volunteers were vaccinated 3 times at 4 weekly intervals and serum samples obtained four weeks after the third immunization were analysed for (i Antibody responses to various allelic variants, (ii Domain specificity, (iii Avidity, (iv IgG subclass levels, by ELISA and (v functionality of antibody responses by Growth Inhibition Assay (GIA. About half of the antibodies induced by vaccination cross reacted with heterologous AMA1 alleles. The choice of adjuvant determined the magnitude of the antibody response, but had only a marginal influence on specificity, avidity, domain recognition or subclass responses. The highest antibody responses were observed for AMA1 formulated with AS02. The Growth Inhibition Assay activity of the antibodies was proportional to the amount of antigen specific IgG and the functional capacity of the antibodies was similar for heterologous AMA1-expressing laboratory strains.ClinicalTrials.gov NCT00730782.

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy

DEFF Research Database (Denmark)

Denmeade, Samuel R; Mhaka, Annastasiah M; Rosen, D Marc

2012-01-01

adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment...... of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host...

Does Fetal antigen 1 (FA1) identify cells with regenerative, endocrine and neuroendocrine potentials?

DEFF Research Database (Denmark)

Jensen, Charlotte Floridon; Jensen, Charlotte Harken; Thorsen, Poul

2000-01-01

Fetal antigen 1 (FA1) is a circulating EGF multidomain glycoprotein. FA1 and its membrane-associated precursor is defined by the mRNAs referred to as delta-like (dlk), preadipocyte factor 1 (pref-1) or zona glomerulosa-specific factor (ZOG). Using a polyclonal antibody recognising both forms......, the localisation of FA1/dlk was analysed in embryonic and fetal tissues between week 5 to 25 of gestation and related to germinal origin and development. FA1 was observed in endodermally derived hepatocytes, glandular cells of the pancreas anlage, and in respiratory epithelial cells. FA1 was also present...... in mesodermally derived cells of the renal proximal tubules, adrenal cortex, Leydig and Hilus cells of the testes and ovaries, fetal chondroblasts, and skeletal myotubes. Ectodermally derived neuro- and adenohypophysial cells, cells in the floor of the 3rd ventricle and plexus choroideus were also FA1 positive...

Solid-phase radioimmunoassay for Epstein-Barr virus-associated membrane antigen prepared from B95-8 cell culture supernatants

International Nuclear Information System (INIS)

Doelken, G.; Klein, G.

1977-01-01

Epstein-Barr virus (EBV)-associated membrane antigen (MA) was concentrated from B95-8 cell culture media by precipitation with polyethylene glycol followed by chromatography on Bio-Gel A-50m. In a RAJI cell-binding assay, MA-positive material could only be found in the void volume of the column. After ultracentrifugation all antigenic activity appeared in the pellet, which suggested that MA was present in aggregates, presumably fragments of cellular membranes and/or virus envelopes. The MA-containing preparation was photopolymerized in polyacrylamide gel. The homogenized gel was used in a solid-phase radioimmunoassay with 125 I-labeled IgG from an anti-MA positive reference serum and an anti-MA negative control serum. The specificity of the reaction was confirmed in blocking tests with anti-EBV positive and negative sera. A good correlation was found between the results obtained in the radioimmunoassay and the results obtained in direct immunofluorescence tests for the detection of MA. The existence of at least two subspecificities of the MA complex could be confirmed by this radioimmunoassay

Participation of IAA in transduction of gravistimulus in apical cells of moss protonema

Science.gov (United States)

Oksyniuk, U. A.; Khorkavtsiv, O. Y.; Lesniak, Y. I.

carried out experiments it can be suggested that high concentrations of IAA and 1-NAA result in surplus of IAA cells led, probably, to a destruction of the apical-basal gradient in cells. Our results testify that NPA inhibits the gravitropism stronger than the growth of protonema. The peculiarity of moss protonema is that the growth orientation change is a result of a transference of growth zone in the apical cell dome caused by amyloplasts sedimentation inducing lateral asymmetry of Ca2+ and apical-basal IAA flow what in its turn manifests itself in distribution of IAA and/or Ca2+ channels in apical cell dome plasma membrane ( Schwuchow et al., 2001). The transport of IAA in apical cells, probably, functionally polarizes it and just that polarizing function is dominant in cells with tip growth.

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

Science.gov (United States)

Ashraf, S Q; Umana, P; Mössner, E; Ntouroupi, T; Brünker, P; Schmidt, C; Wilding, J L; Mortensen, N J; Bodmer, W F

2009-11-17

The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles.

Science.gov (United States)

Itoh, Kazuyoshi; Akimoto, Yoshihiro; Kondo, Shu; Ichimiya, Tomomi; Aoki, Kazuhiro; Tiemeyer, Michael; Nishihara, Shoko

2018-04-15

T antigen (Galβ1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization

Apical instrumentation in endodontic therapy

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Kurniasri Darliana

2007-07-01

Full Text Available Cleaning and shaping of the root canal as the foundation for successful endodontic therapy. Cleaning of the root canal as the removal of all the contents of the root canal systems before and during shaping. Mechanical cleaning as the most important part of the root canal therapy. Instrumentation of the apical region has long been considered to be an essential component in the cleaning and shaping process. The apical area as the critical zone for instrumentation. The apical portion of the root canal system can retain microorganisms that could potentially cause periradicular inflammation. The nickel-titanium rotary instrumentation system to facilitate the cleaning and shaping process. Larger instrumentation sizes not only allow proper irrigation but also significantly decrease remaining bacteria in the canal system. How the larger apical sizes preparation must be achieved to clinical success. This paper will describe the major factors impacting the selection of final apical size, the factors are the anatomy of the apical constriction, root canal diameter, apical instrumentation, and bacteria in dentin tubuli.

Advanced generation anti-prostate specific membrane antigen designer T cells for prostate cancer immunotherapy.

Science.gov (United States)

Ma, Qiangzhong; Gomes, Erica M; Lo, Agnes Shuk-Yee; Junghans, Richard P

2014-02-01

Adoptive immunotherapy by infusion of designer T cells (dTc) engineered with chimeric antigen receptors (CARs) for tumoricidal activity represents a potentially highly specific modality for the treatment of cancer. In this study, 2nd generation (gen) anti-prostate specific membrane antigen (PSMA) dTc were developed for improving the efficacy of previously developed 1st gen dTc for prostate cancer immunotherapy. The 1st gen dTc are modified with chimeric immunoglobulin-T cell receptor (IgTCR) while the 2nd gen dTc are engineered with an immunoglobulin-CD28-T cell receptor (IgCD28TCR), which incorporates a CD28 costimulatory signal for optimal T cell activation. A 2nd gen anti-PSMA IgCD28TCR CAR was constructed by inserting the CD28 signal domain into the 1st gen CAR. 1st and 2nd gen anti-PSMA dTc were created by transducing human T cells with anti-PSMA CARs and their antitumor efficacy was compared for specific activation on PSMA-expressing tumor contact, cytotoxicity against PSMA-expressing tumor cells in vitro, and suppression of tumor growth in an animal model. The 2nd gen dTc can be optimally activated to secrete larger amounts of cytokines such as IL2 and IFNγ than 1st gen and to proliferate more vigorously on PSMA-expressing tumor contact. More importantly, the 2nd gen dTc preserve the PSMA-specific cytotoxicity in vitro and suppress tumor growth in animal models with significant higher potency. Our results demonstrate that 2nd gen anti-PSMA designer T cells exhibit superior antitumor functions versus 1st gen, providing a rationale for advancing this improved agent toward clinical application in prostate cancer immunotherapy. © 2013 Wiley Periodicals, Inc.

Bony change of apical lesion healing process using fractal analysis

International Nuclear Information System (INIS)

Lee, Ji Min; Park, Hyok; Jeong, Ho Gul; Kim, Kee Deog; Park, Chang Seo

2005-01-01

To investigate the change of bone healing process after endodontic treatment of the tooth with an apical lesion by fractal analysis. Radiographic images of 35 teeth from 33 patients taken on first diagnosis, 6 months, and 1 year after endodontic treatment were selected. Radiographic images were taken by JUPITER computerized Dental X-ray System. Fractal dimensions were calculated three times at each area by Scion Image PC program. Rectangular region of interest (30 x 30) were selected at apical lesion and normal apex of each image. The fractal dimension at apical lesion of first diagnosis (L 0 ) is 0.940 ± 0.361 and that of normal area (N 0 ) is 1.186 ± 0.727 (p 1 ) is 1.076 ± 0.069 and that of normal area (N 1 ) is 1.192 ± 0.055 (p 2 ) is 1.163 ± 0.074 and that of normal area (N 2 ) is 1.225 ± 0.079 (p<0.05). After endodontic treatment, the fractal dimensions at each apical lesions depending on time showed statistically significant difference. And there are statistically significant different between normal area and apical lesion on first diagnosis, 6 months after, 1 year after. But the differences were grow smaller as time flows. The evaluation of the prognosis after the endodontic treatment of the apical lesion was estimated by bone regeneration in apical region. Fractal analysis was attempted to overcome the limit of subjective reading, and as a result the change of the bone during the healing process was able to be detected objectively and quantitatively.

Prostate specific membrane antigen- a target for imaging and therapy with radionuclides

DEFF Research Database (Denmark)

Bouchelouche, Kirsten; Choyke, Peter L; Capala, Jacek

2010-01-01

Prostate cancer continues to represent a major health problem, and yet there is no effective treatment available for advanced metastatic disease. Thus, there is an urgent need for the development of more effective treatment modalities that could improve the outcome. Because prostate specific...... membrane antigen (PSMA), a transmembrane protein, is expressed by virtually all prostate cancers, and its expression is further increased in poorly differentiated, metastatic, and hormone-refractory carcinomas, it is a very attractive target. Molecules targeting PSMA can be labelled with radionuclides...... to become both diagnostic and/or therapeutic agents. The use of PSMA binding agents, labelled with diagnostic and therapeutic radio-isotopes, opens up the potential for a new era of personalized management of metastatic prostate cancer....

Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

International Nuclear Information System (INIS)

Bonekamp, Nina A.; Vormund, Kerstin; Jacob, Ralf; Schrader, Michael

2010-01-01

The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.

The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA Which Displays Light-Dependent Gene and Protein Expressions

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Yuen K. Ip

2017-10-01

Full Text Available Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification in light. Light-enhanced calcification requires an increase in the supply of Ca2+ to, and simultaneously an augmented removal of H+ from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca2+-ATPase (PMCA from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa. The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca2+ in exchange for H+. More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA/PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam.

Floating retained root lesion mimicking apical periodontitis.

Science.gov (United States)

Chung, Ming-Pang; Chen, Chih-Ping; Shieh, Yi-Shing

2009-10-01

A case of a retained root tip simulating apical periodontitis on radiographic examination is described. The retained root tip, originating from the left lower first molar, floated under the left lower second premolar apical region mimicking apical periodontitis. It appeared as an ill-defined periapical radiolucency containing a smaller radiodense mass on radiograph. The differential diagnosis included focal sclerosing osteomyelitis (condensing osteitis) and ossifying fibroma. Upon exicisional biopsy, a retained root associated with granulation tissue was found. After 1-year follow-up, the patient was asymptomatic and the periradicular lesion was healing. Meanwhile, the associated tooth showed a normal response to stimulation testing.

Quantitative assessment of apical debris extrusion and intracanal debris in the apical third, using hand instrumentation and three rotary instrumentation systems.

Science.gov (United States)

H K, Sowmya; T S, Subhash; Goel, Beena Rani; T N, Nandini; Bhandi, Shilpa H

2014-02-01

Decreased apical extrusion of debris and apical one third debris have strong implications for decreased incidence of postoperative inflammation and pain. Thus, the aim of this study was to assess quantitatively the apical extrusion of debris and intracanal debris in the apical third during root canal instrumentation using hand and three different types of rotary instruments. Sixty freshly extracted single rooted human teeth were randomly divided into four groups. Canal preparation was done using step-back with hand instrumentation, crown-down technique with respect to ProTaper and K3, and hybrid technique with LightSpeed LSX. Irrigation was done with NaOCl, EDTA, and normal saline and for final irrigation, EndoVac system was used. The apically extruded debris was collected on the pre-weighed Millipore plastic filter disk and weighed using microbalance. The teeth were submitted to the histological processing. Sections from the apical third were analyzed by a trinocular research microscope that was coupled to a computer where the images were captured and analyzed using image proplus V4.1.0.0 software. The mean weight of extruded debris for each group and intracanal debris in the root canal was statistically analyzed by a Kruskal-Wallis one-way analysis of variance and Mann-Whitney U test. The result showed that, hand instrumentation using K files showed the highest amount of debris extrusion apically when compared to ProTaper, K3 and LightSpeed LSX. The result also showed that there was no statistically significant difference between the groups in relation to presence of intracanal debris in the apical one third. Based on the results, all instrumentation techniques produced debris extrusion. The engine driven Ni-Ti systems extruded significantly less apical debris than hand instrumentation. There was no statistically significant difference between the groups in relation to presence of intracanal debris in the apical one third.

Increased membrane cholesterol in lymphocytes diverts T-cells toward an inflammatory response.

Directory of Open Access Journals (Sweden)

Jacqueline Surls

Full Text Available Cell signaling for T-cell growth, differentiation, and apoptosis is initiated in the cholesterol-rich microdomains of the plasma membrane known as lipid rafts. Herein, we investigated whether enrichment of membrane cholesterol in lipid rafts affects antigen-specific CD4 T-helper cell functions. Enrichment of membrane cholesterol by 40-50% following squalene administration in mice was paralleled by an increased number of resting CD4 T helper cells in periphery. We also observed sensitization of the Th1 differentiation machinery through co-localization of IL-2Rα, IL-4Rα, and IL-12Rβ2 subunits with GM1 positive lipid rafts, and increased STAT-4 and STAT-5 phosphorylation following membrane cholesterol enrichment. Antigen stimulation or CD3/CD28 polyclonal stimulation of membrane cholesterol-enriched, resting CD4 T-cells followed a path of Th1 differentiation, which was more vigorous in the presence of increased IL-12 secretion by APCs enriched in membrane cholesterol. Enrichment of membrane cholesterol in antigen-specific, autoimmune Th1 cells fostered their organ-specific reactivity, as confirmed in an autoimmune mouse model for diabetes. However, membrane cholesterol enrichment in CD4(+Foxp3(+ T-reg cells did not alter their suppressogenic function. These findings revealed a differential regulatory effect of membrane cholesterol on the function of CD4 T-cell subsets. This first suggests that membrane cholesterol could be a new therapeutic target to modulate the immune functions, and second that increased membrane cholesterol in various physiopathological conditions may bias the immune system toward an inflammatory Th1 type response.

18F-DCFBC Prostate-Specific Membrane Antigen-Targeted PET/CT Imaging in Localized Prostate Cancer: Correlation With Multiparametric MRI and Histopathology.

Science.gov (United States)

Turkbey, Baris; Mena, Esther; Lindenberg, Liza; Adler, Stephen; Bednarova, Sandra; Berman, Rose; Ton, Anita T; McKinney, Yolanda; Eclarinal, Philip; Hill, Craig; Afari, George; Bhattacharyya, Sibaprasad; Mease, Ronnie C; Merino, Maria J; Jacobs, Paula M; Wood, Bradford J; Pinto, Peter A; Pomper, Martin G; Choyke, Peter L

2017-10-01

To assess the ability of (N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-F-fluorobenzyl-L-cysteine) (F-DCFBC), a prostate-specific membrane antigen-targeted PET agent, to detect localized prostate cancer lesions in correlation with multiparametric MRI (mpMRI) and histopathology. This Health Insurance Portability and Accountability Act of 1996-compliant, prospective, institutional review board-approved study included 13 evaluable patients with localized prostate cancer (median age, 62.8 years [range, 51-74 years]; median prostate-specific antigen, 37.5 ng/dL [range, 3.26-216 ng/dL]). Patients underwent mpMRI and F-DCFBC PET/CT within a 3 months' window. Lesions seen on mpMRI were biopsied under transrectal ultrasound/MRI fusion-guided biopsy, or a radical prostatectomy was performed. F-DCFBC PET/CT and mpMRI were evaluated blinded and separately for tumor detection on a lesion basis. For PET image analysis, MRI and F-DCFBC PET images were fused by using software registration; imaging findings were correlated with histology, and uptake of F-DCFBC in tumors was compared with uptake in benign prostatic hyperplasia nodules and normal peripheral zone tissue using the 80% threshold SUVmax. A total of 25 tumor foci (mean size, 1.8 cm; median size, 1.5 cm; range, 0.6-4.7 cm) were histopathologically identified in 13 patients. Sensitivity rates of F-DCFBC PET/CT and mpMRI were 36% and 96%, respectively, for all tumors. For index lesions, the largest tumor with highest Gleason score, sensitivity rates of F-DCFBC PET/CT and mpMRI were 61.5% and 92%, respectively. The average SUVmax for primary prostate cancer was higher (5.8 ± 4.4) than that of benign prostatic hyperplasia nodules (2.1 ± 0.3) or that of normal prostate tissue (2.1 ± 0.4) at 1 hour postinjection (P = 0.0033). The majority of index prostate cancers are detected with F-DCFBC PET/CT, and this may be a prognostic indicator based on uptake and staging. However, for detecting prostate cancer with high sensitivity, it

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy.

Science.gov (United States)

Denmeade, Samuel R; Mhaka, Annastasiah M; Rosen, D Marc; Brennen, W Nathaniel; Dalrymple, Susan; Dach, Ingrid; Olesen, Claus; Gurel, Bora; Demarzo, Angelo M; Wilding, George; Carducci, Michael A; Dionne, Craig A; Møller, Jesper V; Nissen, Poul; Christensen, S Brøgger; Isaacs, John T

2012-06-27

Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.

Association of human herpesvirus 6 subtypes with symptomatic apical periodontitis.

Science.gov (United States)

Hernádi, Katinka; Csoma, Eszter; Adám, Balázs; Szalmás, Anita; Gyöngyösi, Eszter; Veress, György; Ildikó-Márton; Kónya, József

2011-09-01

The occurrence of human herpesvirus (HHV) 6 subtypes A and B in apical periodontitis was determined. The relationship of HHV-6 subtypes to other disease associated herpesviruses, i.e., Epstein-Barr virus (EBV) and human cytomegalovirus, was also investigated. Forty apical periodontitis samples (17 symptomatic and 23 asymptomatic) and 40 healthy pulp control samples were collected. Nested polymerase chain reaction was used to detect HHV-6 DNA. HHV-6 DNA was observed in significantly higher frequencies in apical periodontitis samples than in control samples (20% vs. 2.5%; P = .03). Further classification of apical lesions revealed that subtype B of HHV-6 was significantly associated with large-sized and symptomatic lesions (P apical lesions (77%) harbored ≥1 of the tested herpesviruses: EBV was the most frequent herpesvirus (72.5%) in apical periodontitis, followed by HHV-6 (20%). Our findings suggest that EBV and HHV-6B infections can be associated with symptomatic apical periodontitis. Copyright © 2011 Mosby, Inc. All rights reserved.

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

Science.gov (United States)

Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

2012-01-01

Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

Directory of Open Access Journals (Sweden)

Ewoud Bernardus Compeer

2012-03-01

Full Text Available The cross-presentation of endocytosed antigen as peptide/class I MHC complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells (APC capable of antigen cross-presentation, description of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC, there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlight DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, recycling and maturation including the sorting of membrane proteins, dynamic remodeling of endosomal structures and cell-surface directed endosomal trafficking. We will conclude with description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

Lipocalins Are Required for Apical Extracellular Matrix Organization and Remodeling in Caenorhabditis elegans.

Science.gov (United States)

Forman-Rubinsky, Rachel; Cohen, Jennifer D; Sundaram, Meera V

2017-10-01

A lipid and glycoprotein-rich apical extracellular matrix (aECM) or glycocalyx lines exposed membranes in the body, and is particularly important to protect narrow tube integrity. Lipocalins ("fat cups") are small, secreted, cup-shaped proteins that bind and transport lipophilic cargo and are often found in luminal or aECM compartments such as mammalian plasma, urine, or tear film. Although some lipocalins can bind known aECM lipids and/or matrix metalloproteinases, it is not known if and how lipocalins affect aECM structure due to challenges in visualizing the aECM in most systems. Here we show that two Caenorhabditis elegans lipocalins, LPR-1 and LPR-3, have distinct functions in the precuticular glycocalyx of developing external epithelia. LPR-1 moves freely through luminal compartments, while LPR-3 stably localizes to a central layer of the membrane-anchored glycocalyx, adjacent to the transient zona pellucida domain protein LET-653 Like LET-653 and other C. elegans glycocalyx components, these lipocalins are required to maintain the patency of the narrow excretory duct tube, and also affect multiple aspects of later cuticle organization. lpr-1 mutants cannot maintain a continuous excretory duct apical domain and have misshapen cuticle ridges (alae) and abnormal patterns of cuticular surface lipid staining. lpr-3 mutants cannot maintain a passable excretory duct lumen, properly degrade the eggshell, or shed old cuticle during molting, and they lack cuticle barrier function. Based on these phenotypes, we infer that both LPR-1 and LPR-3 are required to build a properly organized aECM, while LPR-3 additionally is needed for aECM clearance and remodeling. The C. elegans glycocalyx provides a powerful system, amenable to both genetic analysis and live imaging, for investigating how lipocalins and lipids affect aECM structure. Copyright © 2017 by the Genetics Society of America.

Antibodies to AB blood group antigens mimic anti-salivary duct autoantibodies in patients with limited sicca symptoms.

Science.gov (United States)

Goldblatt, F; Beroukas, D; Gillis, D; Cavill, D; Bradwell, A; Rischmueller, M; Gordon, T P

2000-10-01

We evaluated the clinical relevance and pathogenic significance of anti-salivary duct autoantibodies (ASDA) in Sjögren's syndrome (SS) and rheumatoid arthritis (RA) by examining (1) their frequency in healthy controls, patients with sicca symptoms, and patients with various autoimmune and infective disorders; (2) their localization by confocal microscopy; and (3) their tissue distribution and cross reactivity with blood group antigens. Indirect immunofluorescence (IF) was performed on commercial cryostat sections of monkey parotid salivary gland. Sections were examined by fluorescence and confocal laser scanning microscopy. Sera giving positive staining on the ducts were tested by IF on a range of monkey tissues and salivary glands from several mammalian species. Blocking experiments were performed with human erythrocytes of different ABO blood groups and AB antigens. We identified 2 distinct ductal staining patterns. The first resembled ASDA described in earlier studies and showed patchy bright staining of the apical (luminal) surfaces of the ducts and staining of apical cytoplasmic vesicles. The other was only observed with anti-mitochondrial antibody positive sera and stained the mitochondrial-rich ductal epithelium in a distinctive punctate pattern. Antibodies staining the apical surface of ducts were detected rarely in patients with antiRo/La autoantibody-positive primary SS (1/76) and RA (1/36) and were found in only 1115 with RA and secondary SS. ASDA were detected in sera from 13/51 (25.5%) of patients referred to our clinic with limited sicca symptoms who were anti-Ro/La antibody-negative and had no typical clinical or laboratory features of classical primary SS. The apical ductal staining pattern was not observed with sera from 63 healthy controls without sicca symptoms or in patients with autoimmune and infective disorders. Twelve of the 13 patients whose sera gave ASDA-like staining were blood group O and one group A. Ductal staining was abolished in

Apical Revascularization after Delayed Tooth Replantation: An Unusual Case

Directory of Open Access Journals (Sweden)

Marília Pacífico Lucisano

2016-01-01

Full Text Available The aim of this paper is to present the clinical and radiological outcome of the treatment involving a delayed tooth replantation after an avulsed immature permanent incisor, with a follow-up of 1 year and 6 months. An 8-year-old boy was referred after dental trauma that occurred on the previous day. The permanent maxillary right central incisor (tooth 11 had been avulsed. The tooth was hand-held during endodontic therapy and an intracanal medication application with calcium hydroxide-based paste was performed. An apical plug with mineral trioxide aggregate (MTA was introduced into the apical portion of the canal. When the avulsed tooth was replanted with digital pressure, a blood clot had formed within the socket, which moved the MTA apical plug about 2 mm inside of the root canal. These procedures developed apical revascularization, which promoted a successful endodontic outcome, evidenced by apical closure, slight increase in root length, and absence of signs of external root resorption, during a follow-up of 1 year and 6 months.

Molecular mimics of the tumour antigen MUC1.

Directory of Open Access Journals (Sweden)

Tharappel C James

Full Text Available A key requirement for the development of cancer immunotherapy is the identification of tumour-associated antigens that are differentially or exclusively expressed on the tumour and recognized by the host immune system. However, immune responses to such antigens are often muted or lacking due to the antigens being recognized as "self", and further complicated by the tumour environment and regulation of immune cells within. In an effort to circumvent the lack of immune responses to tumour antigens, we have devised a strategy to develop potential synthetic immunogens. The strategy, termed mirror image phage display, is based on the concept of molecular mimicry as demonstrated by the idiotype/anti-idiotype paradigm in the immune system. Here as 'proof of principle' we have selected molecular mimics of the well-characterised tumour associated antigen, the human mucin1 protein (MUC1 from two different peptide phage display libraries. The putative mimics were compared in structure and function to that of the native antigen. Our results demonstrate that several of the mimic peptides display T-cell stimulation activity in vitro when presented by matured dendritic cells. The mimic peptides and the native MUC1 antigenic epitopes can cross-stimulate T-cells. The data also indicate that sequence homology and/or chemical properties to the original epitope are not the sole determining factors for the observed immunostimulatory activity of the mimic peptides.

Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

Directory of Open Access Journals (Sweden)

Scott A Walper

Full Text Available There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9 M to 7.0×10(-10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions.

Science.gov (United States)

Gildea, John J; Xu, Peng; Carlson, Julia M; Gaglione, Robert T; Bigler Wang, Dora; Kemp, Brandon A; Reyes, Camellia M; McGrath, Helen E; Carey, Robert M; Jose, Pedro A; Felder, Robin A

2015-12-01

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake. Copyright © 2015 the American Physiological Society.

Incidence of apical root cracks and apical dentinal detachments after canal preparation with hand and rotary files at different instrumentation lengths.

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Liu, Rui; Kaiwar, Anjali; Shemesh, Hagay; Wesselink, Paul R; Hou, Benxiang; Wu, Min-Kai

2013-01-01

The aim of this study was to compare the incidence of apical root cracks and dentinal detachments after canal preparation with hand and rotary files at different instrumentation lengths. Two hundred forty mandibular incisors were mounted in resin blocks with simulated periodontal ligaments, and the apex was exposed. The root canals were instrumented with rotary and hand files, namely K3, ProTaper, and nickel-titanium Flex K files to the major apical foramen (AF), short AF, or beyond AF. Digital images of the apical surface of every tooth were taken during the apical enlargement at each file change. Development of dentinal defects was determined by comparing these images with the baseline image. Multinomial logistic regression test was performed to identify influencing factors. Apical crack developed in 1 of 80 teeth (1.3%) with hand files and 31 of 160 teeth (19.4%) with rotary files. Apical dentinal detachment developed in 2 of 80 teeth (2.5%) with hand files and 35 of 160 teeth (21.9%) with rotary files. Instrumentation with rotary files terminated 2 mm short of AF and did not cause any cracks. Significantly less cracks and detachments occurred when instrumentation with rotary files was terminated short of AF, as compared with that terminated at or beyond AF (P hand instruments; instrumentation short of AF reduced the risk of dentinal defects. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Revascularization and periapical repair after endodontic treatment using apical negative pressure irrigation versus conventional irrigation plus triantibiotic intracanal dressing in dogs' teeth with apical periodontitis.

Science.gov (United States)

da Silva, Lea Assed Bezerra; Nelson-Filho, Paulo; da Silva, Raquel Assed Bezerra; Flores, Daniel Silva Herzog; Heilborn, Carlos; Johnson, James D; Cohenca, Nestor

2010-05-01

The objective of this study was to evaluate in vivo the revascularization and the apical and periapical repair after endodontic treatment using 2 techniques for root canal disinfection (apical negative pressure irrigation versus apical positive pressure irrigation plus triantibiotic intracanal dressing) in immature dogs' teeth with apical periodontitis. Two test groups of canals with experimentally induced apical periodontitis were evaluated according to the disinfection technique: Group 1, apical negative pressure irrigation (EndoVac system), and Group 2, apical positive pressure irrigation (conventional irrigation) plus triantibiotic intracanal dressing. In Group 3 (positive control), periapical lesions were induced, but no endodontic treatment was done. Group 4 (negative control) was composed of sound teeth. The animals were killed after 90 days and the maxillas and mandibles were subjected to histological processing. The sections were stained with hematoxylin and eosin and Mallory Trichrome and examined under light microscopy. A description of the apical and periapical features was done and scores were attributed to the following histopathological parameters: newly formed mineralized apical tissue, periapical inflammatory infiltrate, apical periodontal ligament thickness, dentin resorption, and bone tissue resorption. Intergroup comparisons were done by the Kruskal-Wallis and Dunn's tests (alpha = 0.05). Although statistically significant difference was found only for the inflammatory infiltrate (P irrigation with the EndoVac system can be considered as a promising disinfection protocol in immature teeth with apical periodontitis, suggesting that the use of intracanal antibiotics might not be necessary. Copyright (c) 2010 Mosby, Inc. All rights reserved.

ChAd63-MVA-vectored blood-stage malaria vaccines targeting MSP1 and AMA1: assessment of efficacy against mosquito bite challenge in humans.

Science.gov (United States)

Sheehy, Susanne H; Duncan, Christopher J A; Elias, Sean C; Choudhary, Prateek; Biswas, Sumi; Halstead, Fenella D; Collins, Katharine A; Edwards, Nick J; Douglas, Alexander D; Anagnostou, Nicholas A; Ewer, Katie J; Havelock, Tom; Mahungu, Tabitha; Bliss, Carly M; Miura, Kazutoyo; Poulton, Ian D; Lillie, Patrick J; Antrobus, Richard D; Berrie, Eleanor; Moyle, Sarah; Gantlett, Katherine; Colloca, Stefano; Cortese, Riccardo; Long, Carole A; Sinden, Robert E; Gilbert, Sarah C; Lawrie, Alison M; Doherty, Tom; Faust, Saul N; Nicosia, Alfredo; Hill, Adrian V S; Draper, Simon J

2012-12-01

The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1+AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also show that induction of strong cellular immunity against MSP1 and AMA1 is safe, but does not impact on parasite growth rates in the blood. In a subset of vaccinated volunteers, a delay in time to diagnosis was observed and sterilizing protection was observed in one volunteer coimmunized with MSP1+AMA1-results consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccines and suggest that the focus should remain on high-titer antibody induction against susceptible antigen targets.

Effect of apical clearing technique on the treatment outcome of teeth with asymptomatic apical periodontitis: A randomized clinical trial

OpenAIRE

Priya Mittal; Ajay Logani; Naseem Shah; R M Pandey

2016-01-01

Aim: This study aims to compare the periapical healing of teeth with asymptomatic apical periodontitis treated either by conventional apical preparation (CAP) or apical clearing technique (ACT). Materials and Methods: T wenty subjects with bilateral nonvital similar teeth exhibiting comparable periapical index (PAI) score were enrolled and randomly allocated. Group I (CAP, n = 20): Apical preparation three sizes greater (master apical file [MAF]) than the first binding file at the establis...

Protective effect of intranasal immunization with Neospora caninum membrane antigens against murine neosporosis established through the gastrointestinal tract

Science.gov (United States)

Ferreirinha, Pedro; Dias, Joana; Correia, Alexandra; Pérez-Cabezas, Begoña; Santos, Carlos; Teixeira, Luzia; Ribeiro, Adília; Rocha, António; Vilanova, Manuel

2014-01-01

Neospora caninum is an Apicomplexa parasite that in the last two decades was acknowledged as the main pathogenic agent responsible for economic losses in the cattle industry. In the present study, the effectiveness of intranasal immunization with N. caninum membrane antigens plus CpG adjuvant was assessed in a murine model of intragastrically established neosporosis. Immunized mice presented a lower parasitic burden in the brain on infection with 5 × 107 tachyzoites, showing that significant protection was achieved by this immunization strategy. Intestinal IgA antibodies raised by immunization markedly agglutinated live N. caninum tachyzoites whereas previous opsonization with IgG antibodies purified from immunized mice sera reduced parasite survival within macrophage cells. Although an IgG1 : IgG2a ratio < 1 was detected in the immunized mice before and after infection, indicative of a predominant T helper type 1 immune response, no increased production of interferon-γ was detected in the spleen or mesenteric lymph nodes of the immunized mice. Altogether, these results show that mucosal immunization with N. caninum membrane proteins plus CpG adjuvant protect against intragastrically established neosporosis and indicate that parasite-specific mucosal and circulating antibodies have a protective role against this parasitic infection. PMID:24128071

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

Apical P2XR contribute to [Ca2+]i signaling and Isc in mouse renal MCD.

Science.gov (United States)

Li, Liuzhe; Lynch, I Jeanette; Zheng, Wencui; Cash, Melanie N; Teng, Xueling; Wingo, Charles S; Verlander, Jill W; Xia, Shen-Ling

2007-08-03

We examined P2X receptor expression and distribution in the mouse collecting duct (CD) and their functional role in Ca(2+) signaling. Both P2X(1) and P2X(4) were detected by RT-PCR and Western blot. Immunohistochemistry demonstrated apical P2X(1) and P2X(4) immunoreactivity in principal cells in the outer medullary CD (OMCD) and inner medullary CD (IMCD). Luminal ATP induced an increase in Ca(2+) signaling in native medullary CD (MCD) as measured by fluorescence imaging. ATP also induced an increase in Ca(2+) signaling in MCD cells grown in primary culture but not in the presence of P2XR antagonist PPNDS. Short circuit current (I(sc)) measurement with mouse IMCD cells showed that P2XR agonist BzATP induced a larger I(sc) than did P2YR agonist UTP in the apical membrane. Our data reveal for the first time that P2X(1) and P2X(4) are cell-specific with prominent immunoreactivity in the apical area of MCD cells. The finding that P2XR blockade inhibits ATP-induced Ca(2+) signaling suggests that activation of P2XR is a key step in Ca(2+)-dependent purinergic signaling. The result that activation of P2XR produces large I(sc) indicates the necessity of P2XR in renal CD ion transport.

External apical root resorption diagnosis by using FII human dentine fraction and salivary IGg.

Science.gov (United States)

Da-Costa, Tânia Maris Pedrini Soares; Hidalgo, Mirian Marubayashi; Consolaro, Alberto; Lima, Carlos Eduardo de Oliveira; Tanaka, Evelise Ono; Itano, Eiko Nakagawa

2018-06-01

External apical root resorption as a consequence of orthodontic treatment is an inflammatory pathological process that results in permanent loss of tooth structure from the root apex. This study aimed to investigate the diagnostic potential of human dentine fractions and salivary IgG in external apical root resorption. Saliva samples were collected from 10 patients before (T0) and after 3 (T3), 6 (T6) and 12 (T12) months of orthodontic treatment. The total dentinal extract, obtained from human third molars, was fractioned by gel filtration chromatography in three fractions denominated FI, FII and FIII. The root resorption analysis of the upper central incisors was performed by digital image subtraction method. Reactivity of salivary IgG to antigenic fractions of dentine was determined by enzyme-linked immunosorbent assay (Elisa). Regardless of treatment, FI dentin fraction with high MM (root resorptions were detected. Our results suggest that FII human dentine fraction and salivary IgG have potential to be used in diagnosis and monitoring of external apical root resorption. The development of a practical and accessible biochemical test using saliva and FII dentine fraction may help in the prevention of severe root resorption. Copyright © 2018. Published by Elsevier Masson SAS.

Prostate-Specific Membrane Antigen Positron Emission Tomography-Computed Tomography for Prostate Cancer: Distribution of Disease and Implications for Radiation Therapy Planning.

Science.gov (United States)

Gupta, Sandeep K; Watson, Tahne; Denham, Jim; Shakespeare, Thomas P; Rutherford, Natalie; McLeod, Nicholas; Picton, Kevin; Ainsworth, Paul; Bonaventura, Tony; Martin, Jarad M

2017-11-01

To explore the prostate-specific membrane antigen (PSMA)-avid distribution of prostate cancer (PC) on positron emission tomography (PET), both at the time of initial diagnosis and at the time of relapse after definitive local treatment. A total of 179 PSMA PET scans in patients with nil or ≤3 lesions on conventional imaging were retrospectively categorized into 3 subgroups: group A, high-risk PC with no prior definitive therapy (n=34); group B, prior prostatectomy (n=75); and group C, prior radiation therapy (n=70). The numbers and locations of the PSMA-avid lesions were mapped. The PSMA-positive lesions were identified subjectively by a nuclear medicine physician on the basis of clinical experience and taking into account the recent literature and artefacts. A total of 893 PSMA-avid lesions were identified; at least 1 lesion was detected in 80% of all scans. A high detection rate was present even at very low serum PSA levels (eg, at PSA ≤0.20 ng/mL in group B, the detection rate was 46%). Thirty-eight percent of studies revealed extrapelvic disease (41%, 31%, and 46% in groups A, B, and C, respectively). Almost one-third of all studies showed only oligometastases (24%, 36%, and 31% in groups A, B, and C, respectively). A large proportion of these (40%) were a solitary lesion. Prostate-specific membrane antigen PET demonstrated a large number of otherwise unknown metastatic lesions. Therefore we recommend PSMA PET for more accurate assessment of disease burden in initial staging of high-risk PC, as well as for restaging in patients with prostate-specific antigen relapse after primary therapies. Furthermore, a high proportion of oligometastases on PSMA PET provides a prime opportunity to investigate the role of targeted local therapies for oligometastatic PCs. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Domestication-driven Gossypium profilin 1 (GhPRF1) gene transduces early flowering phenotype in tobacco by spatial alteration of apical/floral-meristem related gene expression.

Science.gov (United States)

Pandey, Dhananjay K; Chaudhary, Bhupendra

2016-05-13

Plant profilin genes encode core cell-wall structural proteins and are evidenced for their up-regulation under cotton domestication. Notwithstanding striking discoveries in the genetics of cell-wall organization in plants, little is explicit about the manner in which profilin-mediated molecular interplay and corresponding networks are altered, especially during cellular signalling of apical meristem determinacy and flower development. Here we show that the ectopic expression of GhPRF1 gene in tobacco resulted in the hyperactivation of apical meristem and early flowering phenotype with increased flower number in comparison to the control plants. Spatial expression alteration in CLV1, a key meristem-determinacy gene, is induced by the GhPRF1 overexpression in a WUS-dependent manner and mediates cell signalling to promote flowering. But no such expression alterations are recorded in the GhPRF1-RNAi lines. The GhPRF1 transduces key positive flowering regulator AP1 gene via coordinated expression of FT4, SOC1, FLC1 and FT1 genes involved in the apical-to-floral meristem signalling cascade which is consistent with our in silico profilin interaction data. Remarkably, these positive and negative flowering regulators are spatially controlled by the Actin-Related Protein (ARP) genes, specifically ARP4 and ARP6 in proximate association with profilins. This study provides a novel and systematic link between GhPRF1 gene expression and the flower primordium initiation via up-regulation of the ARP genes, and an insight into the functional characterization of GhPRF1 gene acting upstream to the flowering mechanism. Also, the transgenic plants expressing GhPRF1 gene show an increase in the plant height, internode length, leaf size and plant vigor. Overexpression of GhPRF1 gene induced early and increased flowering in tobacco with enhanced plant vigor. During apical meristem determinacy and flower development, the GhPRF1 gene directly influences key flowering regulators through ARP

Synergistic co-targeting of prostate-specific membrane antigen and androgen receptor in prostate cancer.

Science.gov (United States)

Murga, Jose D; Moorji, Sameer M; Han, Amy Q; Magargal, Wells W; DiPippo, Vincent A; Olson, William C

2015-02-15

Antibody-drug conjugates (ADCs) are an emerging class of cancer therapies that have demonstrated favorable activity both as single agents and as components of combination regimens. Phase 2 testing of an ADC targeting prostate-specific membrane antigen (PSMA) in advanced prostate cancer has shown antitumor activity. The present study examined PSMA ADC used in combination with potent antiandrogens (enzalutamide and abiraterone) and other compounds. Antiproliferative activity and expression of PSMA, prostate-specific antigen and androgen receptor were evaluated in the prostate cancer cell lines LNCaP and C4-2. Cells were tested for susceptibility to antiandrogens or other inhibitors, used alone and in combination with PSMA ADC. Potential drug synergy or antagonism was evaluated using the Bliss independence method. Enzalutamide and abiraterone demonstrated robust, statistically significant synergy when combined with PSMA ADC. Largely additive activity was observed between the antiandrogens and the individual components of the ADC (free drug and unmodified antibody). Rapamycin also synergized with PSMA ADC in certain settings. Synergy was linked in part to upregulation of PSMA expression. In androgen-dependent LNCaP cells, enzalutamide and abiraterone each inhibited proliferation, upregulated PSMA expression, and synergized with PSMA ADC. In androgen-independent C4-2 cells, enzalutamide and abiraterone showed no measurable antiproliferative activity on their own but increased PSMA expression and synergized with PSMA ADC nonetheless. PSMA expression increased progressively over 3 weeks with enzalutamide and returned to baseline levels 1 week after enzalutamide removal. The findings support exploration of clinical treatment regimens that combine potent antiandrogens and PSMA-targeted therapies for prostate cancer. © 2014 Wiley Periodicals, Inc.

Detection and partial characterization of a midlamina lucida-hemidesmosome-associated antigen (19-DEJ-1) present within human skin

DEFF Research Database (Denmark)

Fine, J D; Horiguchi, Y; Jester, J

1989-01-01

, esophagus, cervix, and cornea, and BMs surrounding smooth muscle in medium-sized vessels, placenta, uterus, and esophagus. When 16 human fetal skins (aged 54-142 gestational days) were examined, the antigen was first detected at 81 days. Using immunoperoxidase and immunogold staining techniques, indirect......A murine anti-human monoclonal antibody (19-DEJ-1) has been produced that binds to basement membranes (BMs) of the dermoepidermal junction and arrector pili muscles but not to either dermal glandular or vascular BMs. 19-DEJ-1 also recognizes BMs underneath epithelia of buccal mucosa, tongue......-specific proteoglycan that is present within BMs along the epithelial-connective tissue interface and around smooth muscle in skin and other selected organs. Its unique ultrastructural localization suggests the possibility that 19-DEJ-1 may recognize an antigenic epitope of either anchoring filaments or alternatively...

Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage.

Science.gov (United States)

Zaika, Oleg; Palygin, Oleg; Tomilin, Viktor; Mamenko, Mykola; Staruschenko, Alexander; Pochynyuk, Oleh

2016-02-15

Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K(+)-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 μM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 μM ouabain (Na(+)-K(+)-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM), virtually abolished whole cell K(+)-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 μM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na(+) reabsorption in the CCD. Copyright © 2016 the American Physiological Society.

Stem cell antigen-1 in skeletal muscle function.

Science.gov (United States)

Bernstein, Harold S; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

2013-08-15

Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1 in normal, post-natal muscle has not been thoroughly investigated. We systematically compared Sca-1-/- (KO) and Sca-1+/+ (WT) mice and hindlimb muscles to elucidate the tissue, contractile, and functional effects of Sca-1 in young and aging animals. Comparison of muscle volume, fibrosis, myofiber cross-sectional area, and Pax7+ myoblast number showed little differences between ages or genotypes. Exercise protocols, however, demonstrated decreased stamina in KO versus WT mice, with young KO mice achieving results similar to aging WT animals. In addition, KO mice did not improve with practice, while WT animals demonstrated conditioning over time. Surprisingly, myomechanical analysis of isolated muscles showed that KO young muscle generated more force and experienced less fatigue. However, KO muscle also demonstrated incomplete relaxation with fatigue. These findings suggest that Sca-1 is necessary for muscle conditioning with exercise, and that deficient conditioning in Sca-1 KO animals becomes more pronounced with age.

Global Analysis of Palmitoylated Proteins in Toxoplasma gondii.

Science.gov (United States)

Foe, Ian T; Child, Matthew A; Majmudar, Jaimeen D; Krishnamurthy, Shruthi; van der Linden, Wouter A; Ward, Gary E; Martin, Brent R; Bogyo, Matthew

2015-10-14

Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.

Dengue NS1 Antigen - for Early Detection of Dengue Virus Infection

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Amol Hartalkar

2015-08-01

Full Text Available Objectives: To evaluate the efficacy of NS1 antigen assay for early diagnosis of dengue virus infection in a tertiary care hospital. Methods: This cross sectional study was carried out in department of Medicine from August to December 2013. Total 100 patients with dengue fever were included. Complete blood count, alanine aminotransferase (ALT, aspartate aminotransferase (AST, Dengue NS1 antigen and IgM and IgG antibodies of dengue virus were done in all cases. Results: Of the 100 sera tested, 75% were positive for dengue virus infection based on dengue NS1 antigen, IgM antibody and IgG antibody. Dengue NS1 antigen and IgM, IgG antibody were able to detect dengue virus infection between day 1 to day 8 in 92% of samples, 86.7% of samples and 82.6% of samples respectively. Sixty nine percent (69% were found positive for dengue NS1 antigen, 65% were IgM positive and 62% were IgG positive. Based on the dengue NS1 antigen and IgM antibody combination, 74% were positive for dengue virus infections. Sensitivity of Dengue NS1 antigen was 92.3% and specificity of 74.28% in comparison to IgM antibody. Detection rate increased to 75%, based on the antigen and IgG antibody combination. Sensitivity of dengue NS1 antigen was 90.3% and specificity of 65.8% in comparison to IgG antibody. Conclusion: Dengue NS1 antigen is a useful, sensitive and specific test for early diagnosis of dengue virus infection and it improves diagnostic efficiency in combination with antibody test. Key words: Dengue fever, NS1 antigen. Introduction: Dengue fever (DF is the most common arboviral illness in humans. Each year, an estimated 50-100 million cases of dengue fever and 500,000 cases of dengue hemorrhagic fever occur worldwide, with 30000 deaths (mainly in children. Globally 2.5-3 billion people in approximately 112 tropical and subtropical countries are at risk of dengue.of samples respectively. Sixty nine percent (69% were found positive for dengue NS1 antigen, 65% were Ig

Mutations in ap1b1 cause mistargeting of the Na(+/K(+-ATPase pump in sensory hair cells.

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Rachel Clemens Grisham

Full Text Available The hair cells of the inner ear are polarized epithelial cells with a specialized structure at the apical surface, the mechanosensitive hair bundle. Mechanotransduction occurs within the hair bundle, whereas synaptic transmission takes place at the basolateral membrane. The molecular basis of the development and maintenance of the apical and basal compartments in sensory hair cells is poorly understood. Here we describe auditory/vestibular mutants isolated from forward genetic screens in zebrafish with lesions in the adaptor protein 1 beta subunit 1 (ap1b1 gene. Ap1b1 is a subunit of the adaptor complex AP-1, which has been implicated in the targeting of basolateral membrane proteins. In ap1b1 mutants we observed that although the overall development of the inner ear and lateral-line organ appeared normal, the sensory epithelium showed progressive signs of degeneration. Mechanically-evoked calcium transients were reduced in mutant hair cells, indicating that mechanotransduction was also compromised. To gain insight into the cellular and molecular defects in ap1b1 mutants, we examined the localization of basolateral membrane proteins in hair cells. We observed that the Na(+/K(+-ATPase pump (NKA was less abundant in the basolateral membrane and was mislocalized to apical bundles in ap1b1 mutant hair cells. Accordingly, intracellular Na(+ levels were increased in ap1b1 mutant hair cells. Our results suggest that Ap1b1 is essential for maintaining integrity and ion homeostasis in hair cells.

Dog Erythrocyte Antigen 1 (DEA 1): Mode of Inheritance and Initial Characterization

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Polak, Klaudia; Acierno, Michelle; Raj, Karthik; Mizukami, Keijiro; Siegel, Don L.; Giger, Urs

2015-01-01

Background The Dog Erythrocyte Antigen (DEA) 1 blood group system remains poorly defined. Objectives The purpose of the study was to determine the DEA 1 mode of inheritance and to characterize the DEA 1 antigen and alloantibodies. Animals Canine research colony families, clinic canine patients, and DEA 1.2+ blood bank dogs were studied. Methods Canine blood was typed by flow cytometry and immunochromatographic strips using anti-DEA 1 monoclonal antibodies. Gel column experiments with polyclonal and immunoblotting with monoclonal anti-DEA 1 antibodies were performed to analyze select samples. Cross-reactivity of human typing reagents against canine RBCs and one monoclonal anti-DEA 1 antibody against human RBC panels was assessed. Results Typing of 12 families comprising 144 dogs indicated an autosomal dominant inheritance with ≥4 alleles: DEA 1− (0) and DEA 1+ weak (1+), intermediate (2+) and strong (3+ and 4+). Samples from 6 dogs previously typed as DEA 1.2+ were typed as DEA 1+ or DEA 1− using monoclonal antibodies. Human typing reagents produced varied reactions in tube agglutination experiments against DEA 1+ and DEA 1− RBCs. Polypeptide bands were not detected on immunoblots using a monoclonal anti-DEA 1 antibody, therefore the anti-DEA 1 antibody may be specific for conformational epitopes lost during denaturation. Conclusions The autosomal dominant inheritance of DEA 1 with ≥4 alleles indicates a complex blood group system; the antigenicity of each DEA 1+ type will need to be determined. The biochemical nature of the DEA 1 antigen(s) appears different from human blood group systems tested. PMID:26291052

Spontaneous release of soluble HL-A antigens from platelets during conservation.

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Dautigny, A; Bernier, I; Colombani, J; Jollès, P

1975-01-01

Experiments with the aim of studying the solubilisation of HL-A antigens from blood platelets by methods which do not involve any biologically active processes (moderate, discontinuous agitation of a low concentration of platelets suspended in a saline medium, in the presence of an antiseptic; supernatants collected at frequent intervals) have shown that platelets release membrane proteins, including HL-A antigens, spontaneously. Optimal conditions for the treatment of membrane proteins have been perfected. The great stability of HL-A antigens under these conditions permits prolonged treatment. The products extracted are soluble and extremely complex. The molecular weight of the HL-A antigens is between 40,000 and 70,000.

Domains of increased thickness in microvillar membranes of the small intestinal enterocyte

DEFF Research Database (Denmark)

Kunding, Andreas H; Christensen, Sune M; Danielsen, E Michael

2010-01-01

The apical surface of the enterocyte is sculpted into a dense array of cylindrical microvillar protrusions by supporting actin filaments. Membrane microdomains (rafts) enriched in cholesterol and glycosphingolipids comprise roughly 50% of the microvillar membrane and play a vital role in orchestr......The apical surface of the enterocyte is sculpted into a dense array of cylindrical microvillar protrusions by supporting actin filaments. Membrane microdomains (rafts) enriched in cholesterol and glycosphingolipids comprise roughly 50% of the microvillar membrane and play a vital role...... in orchestrating absorptive/digestive action of dietary nutrients at this important cellular interface. Increased membrane thickness is believed to be a morphological characteristic of rafts. Thus, we investigated whether the high contents of lipid rafts in the microvillar membrane is reflected in local variations...... was clearly monophasic. The encountered domains of increased thickness (DITs) occupied 48% of the microvillar membrane and from the data we estimated the area of a single DIT to have a lower limit of 600 nm(2). In other experiments we mapped the organization of biochemically defined lipid rafts by immunogold...

Selective elution of HLA antigens and beta 2-microglobulin from human platelets by chloroquine diphosphate

International Nuclear Information System (INIS)

Kao, K.J.

1988-01-01

To determine whether chloroquine can specifically elute HLA antigens and beta 2-microglobulin (beta 2-M) from the platelet surface, quantitative immunofluorescence flow cytometry and monoclonal antibodies were used to show that HLA antigens and beta 2-M were proportionally eluted from the platelet surface without affecting the membrane glycoproteins IIb and IIIa. Second, an autoradiogram of electrophoresed I-125-labeled platelets showed that only beta 2-M but not other I-125-labeled membrane proteins could be eluted. Although HLA antigens were poorly labeled by I-125 and could not be detected on the autoradiogram, the eluted HLA antigens could be detected by anti-HLA monoclonal antibody and immunoblotting techniques. No loss of plasma membrane integrity was observed by transmission electron microscopy after chloroquine treatment of platelets. The results indicate that chloroquine selectively elutes HLA antigens and their noncovalently associated beta 2-M without affecting other integral platelet membrane proteins

Five-year longitudinal assessment of the prognosis of apical microsurgery.

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von Arx, Thomas; Jensen, Simon S; Hänni, Stefan; Friedman, Shimon

2012-05-01

Apical surgery is an important treatment option for teeth with post-treatment apical periodontitis. Knowledge of the long-term prognosis is necessary when weighing apical surgery against alternative treatments. This study assessed the 5-year outcome of apical surgery and its predictors in a cohort for which the 1-year outcome was previously reported. Apical microsurgery procedures were uniformly performed using SuperEBA (Staident International, Staines, UK) or mineral trioxide aggregate (MTA) (ProRoot MTA; Dentsply Tulsa Dental Specialties, Tulsa, OK) root-end fillings or alternatively Retroplast capping (Retroplast Trading, Rorvig, Denmark). Subjects examined at 1 year (n = 191) were invited for the 5-year clinical and radiographic examination. Based on blinded, independent assessment by 3 calibrated examiners, the dichotomous outcome (healed or nonhealed) was determined and associated with patient-, tooth-, and treatment-related variables using logistic regression. At the 5-year follow-up, 9 of 191 teeth were unavailable, 12 of 191 teeth were extracted, and 170 of 191 teeth were examined (87.6% recall rate). A total of 129 of 170 teeth were healed (75.9%) compared with 83.8% at 1 year, and 85.3% were asymptomatic. Two significant outcome predictors were identified: the mesial-distal bone level at ≤ 3 mm versus >3 mm from the cementoenamel junction (78.2% vs 52.9% healed, respectively; odds ratio = 5.10; confidence interval, 1.67-16.21; P apical microsurgery was 8% poorer than assessed at 1 year. It also suggested that the prognosis was significantly impacted by the interproximal bone levels at the treated tooth and by the type of root-end filling material used. Copyright © 2012 American Association of Endodontists. All rights reserved.

Histology of periapical lesions obtained during apical surgery.

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Schulz, Malte; von Arx, Thomas; Altermatt, Hans Jörg; Bosshardt, Dieter

2009-05-01

The aim of this was to evaluate the histology of periapical lesions in teeth treated with periapical surgery. After root-end resection, the root tip was removed together with the periapical pathological tissue. Histologic sectioning was performed on calcified specimens embedded in methylmethacrylate (MMA) and on demineralized specimens embedded in LR White (Fluka, Buchs, Switzerland). The samples were evaluated with light and transmission electron microscopy (TEM). The histologic findings were classified into periapical abscesses, granulomas, or cystic lesions (true or pocket cysts). The final material comprised 70% granulomas, 23% cysts and 5% abscesses, 1% scar tissues, and 1% keratocysts. Six of 125 samples could not be used. The cystic lesions could not be subdivided into pocket or true cysts. All cysts had an epithelium-lined cavity, two of them with cilia-lined epithelium. These results show the high incidence of periapical granulomas among periapical lesions obtained during apical surgery. Periapical abscesses were a rare occasion. The histologic findings from samples obtained during apical surgery may differ from findings obtained by teeth extractions. A determination between pocket and true apical cysts is hardly possible when collecting samples by apical surgery.

MMP-7 and TIMP-1, new targets in predicting poor wound healing in apical periodontitis.

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Letra, Ariadne; Ghaneh, Ghazaleh; Zhao, Min; Ray, Herbert; Francisconi, Carolina Favaro; Garlet, Gustavo Pompermaier; Silva, Renato Menezes

2013-09-01

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions. Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy. Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus. MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Antigenic Variation of TprK Facilitates Development of Secondary Syphilis

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Reid, Tara B.; Molini, Barbara J.; Fernandez, Mark C.; Lukehart, Sheila A.

2014-01-01

Although primary syphilis lesions heal spontaneously, the infection is chronic, with subsequent clinical stages. Healing of the primary chancre occurs as antibodies against outer membrane antigens facilitate opsonophagocytosis of the bacteria by activated macrophages. TprK is an outer membrane protein that undergoes antigenic variation at 7 variable regions, and variants are selected by immune pressure. We hypothesized that individual TprK variants escape immune clearance and seed new dissemi...

RNG1 is a Late Marker of the Apical Polar Ring in Toxoplasma gondii

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Tran, Johnson Q.; de Leon, Jessica C.; Li, Catherine; Huynh, My-Hang; Beatty, Wandy; Morrissette, Naomi S.

2010-01-01

The asexually proliferating stages of apicomplexan parasites cause acute symptoms of diseases such as malaria, cryptosporidiosis and toxoplasmosis. These stages are characterized by the presence of two independent microtubule organizing centers (MTOCs). Centrioles are found at the poles of the intranuclear spindle. The apical polar ring (APR), a MTOC unique to apicomplexans, organizes subpellicular microtubules which impose cell shape and apical polarity on these protozoa. Here we describe the characteristics of a novel protein that localizes to the APR of Toxoplasma gondii which we have named ring-1 (RNG1). There are related RNG1 proteins in Neospora caninum and Sarcocystis neurona but no obvious homologs in Plasmodium spp., Cryptosporidium spp. or Babesia spp. RNG1 is a small, low-complexity, detergent-insoluble protein that assembles at the APR very late in the process of daughter parasite replication. We were unable to knock-out the RNG1 gene, suggesting that its gene product is essential. Tagged RNG1 lines have also allowed us to visualize the APR during growth of Toxoplasma in the microtubule-disrupting drug oryzalin. Oryzalin inhibits nuclear division and cytokinesis although Toxoplasma growth continues, and similar to earlier observations of unchecked centriole duplication in oryzalin-treated parasites, the APR continues to duplicate during aberrant parasite growth. PMID:20658557

Conjugating recombinant proteins to Pseudomonas aeruginosa ExoProtein A: a strategy for enhancing immunogenicity of malaria vaccine candidates

OpenAIRE

Qian, Feng; Wu, Yimin; Muratova, Olga; Zhou, Hong; Dobrescu, Gelu; Duggan, Peter; Lynn, Lambert; Song, Guanhong; Zhang, Yanling; Reiter, Karine; MacDonald, Nicholas; Narum, David L.; Long, Carole A.; Miller, Louis H.; Saul, Allan

2007-01-01

Conjugation of polysaccharides to carrier proteins has been a successful approach for producing safe and effective vaccines. In an attempt to increase the immunogenicity of two malarial vaccine candidate proteins of Plasmodium falciparum, apical membrane antigen 1 (AMA1) for blood stage vaccines and surface protein 25 (Pfs25) for mosquito stage vaccines, each was chemically conjugated to the mutant, nontoxic Pseudomonas aeruginosa ExoProtein A (rEPA). AMA1 is a large (66 kD) relatively good i...

Phase variable O antigen biosynthetic genes control expression of the major protective antigen and bacteriophage receptor in Vibrio cholerae O1.

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Kimberley D Seed

2012-09-01

Full Text Available The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage.

Apical Hypertrophic Cardiomyopathy in Association with PulmonaryArtery Hypertension

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Mehdi Peighambari

2012-09-01

Full Text Available Apical Hypertrophic Cardiomyopathy is an uncommon condition constituting 1% -2% of the cases with Hypertrophic Cardiomyopathy (HCM diagnosis. We interestingly report two patients with apical hypertrophic cardiomyopathy in association with significant pulmonary artery hypertension without any other underlying reason for pulmonary hypertension. The patients were assessed by echocardiography, cardiac catheterization and pulmonary function parameters study.

Membrane lipidome of an epithelial cell line

DEFF Research Database (Denmark)

Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

2011-01-01

Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to generate an apical membrane domain that serves as a protective barrier for the epithelial sheet....

Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

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Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

2015-01-01

The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.

Radiation and chemical effects on viral transformation and tumor antigen expression. Annual progress report, August 1, 1978--May 1, 1979

International Nuclear Information System (INIS)

Coggin, J.H. Jr.

1979-01-01

Studies aimed at the biological, biochemical, and immunologic characterization of fetal antigens (EA) in hamsters and mice and locating and determining the distribution of fetal antigens in tumor tissues and in developing fetuses have been underway for several months. Progress has been made in isolating embryonic or fetal antigens from fetuses and from tumor cells. We have developed and reported a reliable lymphocyte transformation assay (LTA) which meets our needs in routinely assaying cell free tumor associated antigen (TAA) preparations from fetal and tumor cells. The assay correlated with transplantation resistance assays and has appropriate specificity. We have also developed the staph-A protein binding assay utilizing anti-serum derived against embryonic antigens present on SV40 tumor cells. In other studies, we have reported increases and perturbations in thymocytes during viral and chemical oncogenesis in hamsters, have developed a simple technique for preserving functional lymphocytes sensitized against TAA by freezing for use in our model system work, have reported the cross-reactivity of tranplantation resistance antigen on a spectrum of chemically induced tumors previously believed to only contain individually specific TSTAs and have recently reported the cross-reactivity of papovavirus induced transplantation resistance antigen in sarcoma cells induced by different viruses. We have concluded our studies of glycosyltransferases in the membranes of developing fetuses and noted no differences in their levels with advancing days of gestation using whold embryo cell populations

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

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Xiaodong Weng

2011-03-01

Full Text Available Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12 in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA. Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4% and CD86 (80.13 ± 2.81%] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL and IL-12 (249.57 ± 12.51 pg/mL production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05 than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018, indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

Science.gov (United States)

Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites

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Thompson Joanne

2007-05-01

Full Text Available Abstract Background Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites. Methods After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels. Results Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in suface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3. Conclusion Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

Science.gov (United States)

Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

2018-02-01

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

International Nuclear Information System (INIS)

Xu, Juan; Wang, Shixia; Gan, Weihua; Zhang, Wenhong; Ju, Liwen; Huang, Zuhu; Lu, Shan

2012-01-01

Highlights: ► EV71 is a major emerging infectious disease in many Asian countries. ► Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. ► Developing subunit based EV71 vaccines is significant and novel antigen design is needed. ► DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. ► Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

[New Radiopharmaceuticals Based on Prostate-Specific Inhibitors of Membrane Antigen for Diagnostics and Therapy of Metastatic Prostate Cancer].

Science.gov (United States)

Vlasova, O P; German, K E; Krilov, V V; Petriev, V M; Epstein, N B

2015-01-01

About 10.7% cases of prostate cancer were registered in Russia in 2011 (40,000 patients). More than half of cancer cases were revealed in advanced (III-IV) stages when metastases inevitably developed quickly. Clinical problem of early diagnostics and treatment of metastatic prostate cancer is still not solved. Anatomical imaging techniques have low sensitivity and specificity for the detection of this disease. Metabolic visualization methods which use prostate specific antigen (PSA) as a marker are also ineffective. This article describes prostate-specific membrane antigens (PSMA) that are proposed as a marker for diagnostics and therapy of prostate cancer. The most promising PSMA-based radiopharmaceutical agent for diagnostics has been developed and clinically tested in the European countries. These pharmaceuticals are based on small peptide molecules modified with urea, and have the highest affinity to PSMA. Favorable phannacokinetics, rapid accumulation in the tumor and rapid excretion from the body are beneficial features of these pharmaceuticals.

Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles

DEFF Research Database (Denmark)

Hummel, R; Nørgaard, P; Andreasen, P H

1992-01-01

The F-antigen is a prominent liver protein which has been extensively used in studies on natural and induced immunological tolerance. However, its intracellular localization and biological function have remained elusive. It has generally been assumed that the F-antigen is confined phylogenetically...... of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking. Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded. The abundance...

Conservation of myeloid surface antigens on primate granulocytes.

Science.gov (United States)

Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

1983-02-01

Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

A 16-kilodalton lipoprotein of the outer membrane of Serpulina (Treponema) hyodysenteriae.

Science.gov (United States)

Thomas, W; Sellwood, R; Lysons, R J

1992-08-01

Serpulina (Treponema) hyodysenteriae P18A and VS1 were extracted by using the detergent Triton X-114 and separated into detergent and aqueous phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis confirmed that a membrane-associated 16-kDa antigen was hydrophobic, since it was found in the detergent phase. A 45-kDa antigen partitioned into the aqueous phase, suggesting that it was hydrophilic and may be of periplasmic origin. When spirochetes were grown in the presence of [3H]palmitic acid, a predominant 16-kDa antigen was labeled; from the results of immunoprecipitation experiments, this antigen appeared to be the same as that recognized by both polyclonal and monoclonal antisera to a previously described 16-kDa antigen. This antigen was proteinase K sensitive and was not a component of the lipopolysaccharide, which, although [3H]palmitate labeled, was resistant to proteinase K digestion. The most probable explanation is that the 16-kDa antigen is a membrane-associated, surface-exposed, immunodominant lipoprotein.

Dental Apical Papilla as Therapy for Spinal Cord Injury.

Science.gov (United States)

De Berdt, P; Vanacker, J; Ucakar, B; Elens, L; Diogenes, A; Leprince, J G; Deumens, R; des Rieux, A

2015-11-01

Stem cells of the apical papilla (SCAP) represent great promise regarding treatment of neural tissue damage, such as spinal cord injury (SCI). They derive from the neural crest, express numerous neurogenic markers, and mediate neurite outgrowth and axonal targeting. The goal of the present work was to investigate for the first time their potential to promote motor recovery after SCI in a rat hemisection model when delivered in their original stem cell niche-that is, by transplantation of the human apical papilla tissue itself into the lesion. Control groups consisted of animals subjected to laminectomy only (shams) and to lesion either untreated or injected with a fibrin hydrogel with or without human SCAP. Basso-Beattie-Bresnahan locomotor scores at 1 and 3 d postsurgery confirmed early functional decline in all SCI groups. This significant impairment was reversed, as seen in CatWalk analyses, after transplantation of apical papilla into the injured spinal cord wound, whereas the other groups demonstrated persistent functional impairment. Moreover, tactile allodynia did not develop as an unwanted side effect in any of the groups, even though the SCAP hydrogel group showed higher expression of the microglial marker Iba-1, which has been frequently associated with allodynia. Notably, the apical papilla transplant group presented with reduced Iba-1 expression level. Masson trichrome and human mitochondria staining showed the preservation of the apical papilla integrity and the presence of numerous human cells, while human cells could no longer be detected in the SCAP hydrogel group at the 6-wk postsurgery time point. Altogether, our data suggest that the transplantation of a human apical papilla at the lesion site improves gait in spinally injured rats and reduces glial reactivity. It also underlines the potential interest for the application of delivering SCAP in their original niche, as compared with use of a fibrin hydrogel. © International & American

Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

Energy Technology Data Exchange (ETDEWEB)

Xu, Juan [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Microbiology and Immunology, Nanjing Medical University (China); Wang, Shixia [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States); Gan, Weihua [Department of Pediatrics, The Second Affiliated Hospital, Nanjing Medical University (China); Zhang, Wenhong [Department of Infectious Diseases, Huashan Hospital, Fudan University (China); Ju, Liwen [School of Public Health, Fudan University (China); Huang, Zuhu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Lu, Shan, E-mail: shan.lu@umassmed.edu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States)

2012-04-20

Highlights: Black-Right-Pointing-Pointer EV71 is a major emerging infectious disease in many Asian countries. Black-Right-Pointing-Pointer Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. Black-Right-Pointing-Pointer Developing subunit based EV71 vaccines is significant and novel antigen design is needed. Black-Right-Pointing-Pointer DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. Black-Right-Pointing-Pointer Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

Prostate-Specific Membrane Antigen Targeted Gold Nanoparticles for Theranostics of Prostate Cancer.

Science.gov (United States)

Mangadlao, Joey Dacula; Wang, Xinning; McCleese, Christopher; Escamilla, Maria; Ramamurthy, Gopalakrishnan; Wang, Ziying; Govande, Mukul; Basilion, James P; Burda, Clemens

2018-04-24

Prostate cancer is one of the most common cancers and among the leading causes of cancer deaths in the United States. Men diagnosed with the disease typically undergo radical prostatectomy, which often results in incontinence and impotence. Recurrence of the disease is often experienced by most patients with incomplete prostatectomy during surgery. Hence, the development of a technique that will enable surgeons to achieve a more precise prostatectomy remains an open challenge. In this contribution, we report a theranostic agent (AuNP-5kPEG-PSMA-1-Pc4) based on prostate-specific membrane antigen (PSMA-1)-targeted gold nanoparticles (AuNPs) loaded with a fluorescent photodynamic therapy (PDT) drug, Pc4. The fabricated nanoparticles are well-characterized by spectroscopic and imaging techniques and are found to be stable over a wide range of solvents, buffers, and media. In vitro cellular uptake experiments demonstrated significantly higher nanoparticle uptake in PSMA-positive PC3pip cells than in PSMA-negative PC3flu cells. Further, more complete cell killing was observed in Pc3pip than in PC3flu cells upon exposure to light at different doses, demonstrating active targeting followed by Pc4 delivery. Likewise, in vivo studies showed remission on PSMA-expressing tumors 14 days post-PDT. Atomic absorption spectroscopy revealed that targeted AuNPs accumulate 4-fold higher in PC3pip than in PC3flu tumors. The nanoparticle system described herein is envisioned to provide surgical guidance for prostate tumor resection and therapeutic intervention when surgery is insufficient.

Evaluation of Bacteriological Profile in the Apical Root Segment of the Patients with Primary Apical Periodontitis.

Science.gov (United States)

Tatikonda, Aravind; Sudheep, N; Biswas, Krishna P; Gowtham, K; Pujari, Sudarshan; Singh, Padam

2017-01-01

Apical periodontitis usually results from bacterial accumulation and contamination occurring in the root-canal system, and extending beyond the apical foramen to involve the periapical tissues. Literature has a paucity of the studies that stress on the division and analysis of the pulp canal segments. The reason for this disparity might be the technique used for collecting the samples from the pulp canals. Hence, we carried out the present study to evaluate the microbial flora in the apical part of the roots with necrotic pulp canals. The present study included the assessment of 40 freshly extracted teeth that had necrotized pulpal tissue along with the presence of periapical periodontal lesions. Removal of the soft tissue lesions attached to the root portion of the teeth along with apical periodontal lesions was done with the help of scalpel blade, after rinsing them with a sterile solution of saline. Thorough cleaning of the root surfaces was done with hydrogen peroxide followed by rapid disinfection with the help of sodium hypochlorite at varying concentrations. Sectioning of the root portion of all the specimens with the help of a disk was done perpendicular to the long axis of the teeth at a distance of roughly 5 to 6 mm from the teeth's apicalmost point. Cryotubes were used for transferring the specimens of apical portions containing 1 mL of buffer and were subjected to immediate frozen processing at a temperature of -20°C. A 10 K-type file was used for the initial collection of the samples followed by subsequent incubation of the files and paper pints in the incubation cabinet. Subsequent deoxyribonucleic acid (DNA) extraction from the samples was done following the procedure described by Siqueira et al. Paster et al's modification of the reverse-capture checkerboard assay was used in the present study. Semiquantitative data were used for overcoming the difficulties arising due to obtaining the counts of the polymerase chain reaction (PCR)-based analysis of

Modulation of apical constriction by Wnt signaling is required for lung epithelial shape transition.

Science.gov (United States)

Fumoto, Katsumi; Takigawa-Imamura, Hisako; Sumiyama, Kenta; Kaneiwa, Tomoyuki; Kikuchi, Akira

2017-01-01

In lung development, the apically constricted columnar epithelium forms numerous buds during the pseudoglandular stage. Subsequently, these epithelial cells change shape into the flat or cuboidal pneumocytes that form the air sacs during the canalicular and saccular (canalicular-saccular) stages, yet the impact of cell shape on tissue morphogenesis remains unclear. Here, we show that the expression of Wnt components is decreased in the canalicular-saccular stages, and that genetically constitutive activation of Wnt signaling impairs air sac formation by inducing apical constriction in the epithelium as seen in the pseudoglandular stage. Organ culture models also demonstrate that Wnt signaling induces apical constriction through apical actomyosin cytoskeletal organization. Mathematical modeling reveals that apical constriction induces bud formation and that loss of apical constriction is required for the formation of an air sac-like structure. We identify MAP/microtubule affinity-regulating kinase 1 (Mark1) as a downstream molecule of Wnt signaling and show that it is required for apical cytoskeletal organization and bud formation. These results suggest that Wnt signaling is required for bud formation by inducing apical constriction during the pseudoglandular stage, whereas loss of Wnt signaling is necessary for air sac formation in the canalicular-saccular stages. © 2017. Published by The Company of Biologists Ltd.

Effect of apical clearing technique on the treatment outcome of teeth with asymptomatic apical periodontitis: A randomized clinical trial.

Science.gov (United States)

Mittal, Priya; Logani, Ajay; Shah, Naseem; Pandey, R M

2016-01-01

This study aims to compare the periapical healing of teeth with asymptomatic apical periodontitis treated either by conventional apical preparation (CAP) or apical clearing technique (ACT). Twenty subjects with bilateral nonvital similar teeth exhibiting comparable periapical index (PAI) score were enrolled and randomly allocated. Group I (CAP, n = 20): Apical preparation three sizes greater (master apical file [MAF]) than the first binding file at the established working length. Group II (ACT, n = 20): Apical preparation three sizes greater than the MAF that was followed by dry reaming. Root canal therapy was accomplished in single-visit for all the teeth. They were pursued radiographically at 3, 6, 9 and 12 months. Pre- and post-treatment PAI scores were compared. To ascertain the proportion of healed teeth between the two groups, McNemar Chi-square test was applied. At 3, 6, and 9 months' time interval the proportion of healed teeth for Group II (ACT) was greater in comparison to Group I (CAP) (P < 0.05). However, at 12 months follow-up period this difference was not significant (P = 0.08). ACT enhanced the healing kinetics. However, the long-term (12 months) radiographic outcome was similar for either technique.

Localization of the terminal steps of O-antigen synthesis in Salmonella typhimurium

International Nuclear Information System (INIS)

McGrath, B.C.; Osborn, M.J.

1991-01-01

Previous immunoelectron microscopic studies have shown that both the final intermediate in O-antigen synthesis, undecaprenol-linked O polymer, and newly synthesized O-antigenic lipopolysaccharide are localized to the periplasmic face of the inner membrane. In vivo pulse-chase experiments now provide further evidence that attachment of O antigen to core lipopolysaccharide, as well as polymerization of O-specific polysaccharide chains, takes place at the periplasmic face of the membrane. Mutants doubly conditional in lipopolysaccharide synthesis [kdsA(Ts) pmi] were constructed in which synthesis of core lipopolysaccharide and O antigen are temperature sensitive and mannose dependent, respectively. Periplasmic orientation of O antigen:core lipopolysaccharide ligase was established by experiments showing rapid chase of undecaprenol-linked O polymer, previously accumulated at 42 degrees C in the absence of core synthesis, into lipopolysaccharide following resumption of core formation at 30 degrees C. In addition, chase of the monomeric O-specific tetrasaccharide unit into lipopolysaccharide was found in similar experiments in an O-polymerase-negative [rfc kdsA(Ts) pmi] mutant, suggesting that polymerization of O chains also occurs at the external face of the inner membrane

Age related changes in erythrocyte A and B antigen strength

Energy Technology Data Exchange (ETDEWEB)

Hollingsworth, J W; Hamilton, H B; Ishii, Goro

1961-11-01

The strength of A and B antigens of the erythrocyte, as indicated by agglutinability with dilutions of specific antibody, has been investigated in a group of subjects in Hiroshima. Antigen strength was found to rise to maximal levels at age 25 to 29, and decline with advancing years. Degree of irradiation from the Hiroshima atomic bomb in 1945 did not appear in the limited sample to affect this age-dependent structural property of erythrocytes. Antigen strength of females was somewhat less than that of males for those individuals from 20 to 40 years of age. When compared with group A or B subjects, individuals of group AB demonstrated full strength of both A and B antigens. Since Rh antigenicity also has been reported to change with age, it seems probable that multiple changes in the erythrocyte membrane occur with age. Further investigation into the nature of these changes may be fruitful to an understanding of aging processes at the cellular level. 13 references, 1 figure, 6 tables.

Proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing.

Science.gov (United States)

Lin, Louis M; Huang, George T-J; Rosenberg, Paul A

2007-08-01

There is continuing controversy regarding the potential for inflammatory apical cysts to heal after nonsurgical endodontic therapy. Molecular cell biology may provide answers to a series of related questions. How are the epithelial cell rests of Malassez stimulated to proliferate? How are the apical cysts formed? How does the lining epithelium of apical cysts regress after endodontic therapy? Epithelial cell rests are induced to divide and proliferate by inflammatory mediators, proinflammatory cytokines, and growth factors released from host cells during periradicular inflammation. Quiescent epithelial cell rests can behave like restricted-potential stem cells if stimulated to proliferate. Formation of apical cysts is most likely caused by the merging of proliferating epithelial strands from all directions to form a three-dimensional ball mass. After endodontic therapy, epithelial cells in epithelial strands of periapical granulomas and the lining epithelium of apical cysts may stop proliferating because of a reduction in inflammatory mediators, proinflammatory cytokines, and growth factors. Epithelial cells will also regress because of activation of apoptosis or programmed cell death through deprivation of survival factors or by receiving death signals during periapical wound healing.

Protective immunity induced in mice by F8.1 and F8.2 antigens purified from Schistosoma mansoni eggs

Directory of Open Access Journals (Sweden)

Claudia Campra Ferreira

1998-01-01

Full Text Available Schistosoma mansoni soluble egg antigens (SEA were fractionated by isoelectric focusing, resulting in 20 components, characterized by pH, absorbance and protein concentration. The higher absorbance fractions were submitted to electrophoresis, and fraction 8 (F8 presented a specific pattern of bands on its isoelectric point. Protein 3 was observed only on F8, and so, it was utilized to rabbit immunization, in order to evaluate its capacity of inducing protective immunity. IgG antibodies from rabbit anti-F8 serum were coupled to Sepharose, and used to obtain the specific antigen by affinity chromatography. This antigen, submitted to electrophoresis, presented two proteic bands (F8.1 and F8.2, which were transferred to nitrocellulose membrane (PVDF and sequenciated. The homology of F8.2 to known proteins was determined using the Basic Local Alignment Search Tool program (BLASTp. Significant homologies were obtained for the rabbit cytosolic Ca2+ uptake inhibitor, and for the bird a1-proteinase inhibitor. Immunization of mice with F8.1 and F8.2, in the presence of Corynebacterium parvum and Al(OH3 as adjuvant, induced a significant protection degree against challenge infection, as observed by the decrease on worm burden recovered from portal system.

MAL Is a Regulator of the Recruitment of Myelin Protein PLP to Membrane Microdomains

NARCIS (Netherlands)

Bijlard, Marjolein; de Jonge, Jenny C.; Klunder, Bert; Nomden, Anita; Hoekstra, Dick; Baron, Wia

2016-01-01

In oligodendrocytes (OLGs), an indirect, transcytotic pathway is mediating transport of de novo synthesized PLP, a major myelin specific protein, from the apical-like plasma membrane to the specialized basolateral-like myelin membrane to prevent its premature compaction. MAL is a well-known

Immediate Implant Placement in Sockets with Asymptomatic Apical Periodontitis.

Science.gov (United States)

Crespi, Roberto; Capparé, Paolo; Crespi, Giovanni; Lo Giudice, Giuseppe; Gastaldi, Giorgio; Gherlone, Enrico

2017-02-01

The purpose of the present study was to evaluate if the presence of granulation tissue in asymptomatic apical periodontitis compromised immediate implant placement. Patients requiring extraction of one tooth (maxillary and mandibular incisive, canine or premolar) with asymptomatic apical periodontitis, were recruited for this prospective study. They were randomly scheduled into two groups: in first group (A) including 30 teeth, reactive soft tissue was debrided before implant placement, and in second group (B) including 30 teeth, reactive soft tissue was left in the apical lesion. Implants were positioned immediately after tooth extraction, and were loaded after 3 months in both groups. Cone beam computed tomography was performed before tooth extraction and at 1-year follow-up to evaluate the radiolucency around the root apex and the implant, bucco-lingual bone levels were also checked. Sixty patients were included in this study. Sixty implants were placed immediately after tooth extraction and, at 1-year follow-up, a survival rate of 100% was reported. After one year both groups showed absence of radiolucent zone at the apical region of implants. All fresh sockets presented a buccal-palatal bone reduction in both groups after one year, even if not statistically significant differences were found between baseline bone levels and within groups. Within the limitations of the present study, the immediate placement of implants into the extraction sockets with asymptomatic apical periodontitis, in presence of primary stability, did not lead to an increased rate of complications and rendered an equally favorable type of tissue integration. © 2016 Wiley Periodicals, Inc.

N-linked glycans do not affect plasma membrane localization of multidrug resistance protein 4 (MRP4) but selectively alter its prostaglandin E2 transport activity.

Science.gov (United States)

Miah, M Fahad; Conseil, Gwenaëlle; Cole, Susan P C

2016-01-22

Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Dexamethasone concentration gradients along scala tympani after application to the round window membrane.

Science.gov (United States)

Plontke, Stefan K; Biegner, Thorsten; Kammerer, Bernd; Delabar, Ursular; Salt, Alec N

2008-04-01

Local application of dexamethasone-21-dihydrogen-phosphate (Dex-P) to the round window (RW) membrane of guinea pigs produces a substantial basal-apical concentration gradient in scala tympani (ST) perilymph. In recent years, intratympanically applied glucocorticoids are increasingly being used for the treatment of inner ear disease. Although measurements of intracochlear concentrations after RW application exist, there is limited information on the distribution of these drugs in the inner ear fluids. It has been predicted from computer simulations that substantial concentration gradients will occur after RW application, with lower concentrations expected in apical turns. Concentration gradients of other substances along the cochlea have recently been confirmed using a sequential apical sampling method to obtain perilymph. Dexamethasone-21-dihydrogen-phosphate (10 mg/ml) was administered to the RW membrane of guinea pigs (n = 9) in vivo for 2 to 3 hours. Perilymph was then collected using a protocol in which 10 samples, each of approximately 1 mul, were taken sequentially from the cochlear apex into capillary tubes. Dexamethasone-21-dihydrogen-phosphate concentration of the samples was analyzed by high-performance liquid chromatography. Interpretation of sample data using a finite element model allowed the longitudinal gradients of Dex-P in ST to be quantified. The Dex-P content of the first sample in each experiment (dominated by perilymph from apical regions) was substantially lower than that of the third and fourth sample (dominated by basal turn perilymph). These findings qualitatively demonstrated the existence of a concentration gradient along ST. After detailed analysis of the measured sample concentrations using an established finite element computer model, the mean basal-apical concentration gradient was estimated to be 17,000. Both absolute concentrations of Dex-P in ST and the basal-apical gradients were found to vary substantially. The existence of

The antimicrobial effect of apical box versus apical cone preparation using iodine potassium iodide as root canal dressing

DEFF Research Database (Denmark)

Markvart, Merete; Dahlén, Gunnar; Reit, Claes-Erik

2013-01-01

Abstract Purpose. The purpose was to study the reduction of intra-canal microflora in premolars with apical periodontitis instrumented with either apical box or apical cone preparation and to provide measurements of intervention effects to allow proper power calculation in future clinical trials.......-week post-sampling, a power calculation revealed that over 900 patients are needed to show a difference of 9% between the two protocols tested. Conclusions. Future trials should be conducted using stringent protocols and as multi-centre trials for reaching the required information size....

Morfologia e biometria do ligamento apical do pênis de touros da raça Girolando Morphology and biometry of the apical ligament of the penis of Girolando race bulls

Directory of Open Access Journals (Sweden)

Júlio Roquete Cardoso

2010-08-01

Full Text Available Objetivou-se descrever a morfologia e biometria do ligamento apical do pênis de 32 touros da raça Girolando (Bos taurus indicus X Bos taurus taurus, Linnaeus - 1758, com idade de 36 a 48 meses e pesando de 480 a 540kg. As peças anatômicas foram obtidas em frigorífico e mantidas congeladas até dissecação. O ligamento originou-se a 15,1±2,9cm distalmente à curvatura caudal da flexura sigmoide e inseriu-se a 1,4±0,7cm proximalmente ao colo da glande, medindo 18,9±2,6cm de comprimento. Apresentou largura de 1,9±0,6mm na sua origem, 2,2±0,8mm na inserção e 35,2±10mm na altura da inserção da lâmina interna do prepúcio. A espessura média ao longo de sua extensão variou de 0,7 a 1,9mm. Próximo à coroa da glande, o ligamento apical se posiciona principalmente na superfície dorsolateral esquerda desse órgão, e a característica da sua fixação na albugínea apresentou variações ao longo de sua extensão. Em sua origem e inserção e na face esquerda do pênis, o ligamento apical é firmemente aderido à túnica albugínea, mas na face dorsal e direita do pênis essa união é realizada por meio de tecido conjuntivo frouxo. Verificou-se correlação média entre o comprimento e a circunferência do pênis com o comprimento do ligamento apical, mas a correlação entre essas variáveis do pênis e a largura do ligamento apical foi baixa.The aim of this study was to describe the morphology and biometry of the apical ligament of the penis of Girolando bulls. For this purpose, it was dissected 32 penis of Girolando bulls obtained from slaughterhouses and kept frozen until their dissection. The animals were 36 to 48 month old and weighted between 480 and 540kg. The origin of the apical ligament occurred at 15.1±2.9cm distally to the caudal loop of the sigmoid flexure and its insertion occurred at 1.4±0.7cm proximally to the neck of the glans. The length of the apical ligament was 19.9±2.6cm. The width was 1.9±0.6mm at its

Function and Dynamics of Tetraspanins during Antigen Recognition and Immunological Synapse Formation

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Vera eRocha-Perugini

2016-01-01

Full Text Available Tetraspanin-enriched microdomains (TEMs are specialized membrane platforms driven by protein-protein interactions that integrate membrane receptors and adhesion molecules. Tetraspanins participate in antigen recognition and presentation by antigen presenting cells (APCs through the organization of pattern recognition receptors (PRRs and their downstream induced-signaling, as well as the regulation of MHC-II-peptide trafficking. T lymphocyte activation is triggered upon specific recognition of antigens present on the APC surface during immunological synapse (IS formation. This dynamic process is characterized by a defined spatial organization involving the compartmentalization of receptors and adhesion molecules in specialized membrane domains that are connected to the underlying cytoskeleton and signaling molecules. Tetraspanins contribute to the spatial organization and maturation of the IS by controlling receptor clustering and local accumulation of adhesion receptors and integrins, their downstream signaling and linkage to the actin cytoskeleton. This review offers a perspective on the important role of TEMs in the regulation of antigen recognition and presentation, and in the dynamics of IS architectural organization.

Function and Dynamics of Tetraspanins during Antigen Recognition and Immunological Synapse Formation

Science.gov (United States)

Rocha-Perugini, Vera; Sánchez-Madrid, Francisco; Martínez del Hoyo, Gloria

2016-01-01

Tetraspanin-enriched microdomains (TEMs) are specialized membrane platforms driven by protein–protein interactions that integrate membrane receptors and adhesion molecules. Tetraspanins participate in antigen recognition and presentation by antigen-­presenting cells (APCs) through the organization of pattern-recognition receptors (PRRs) and their downstream-induced signaling, as well as the regulation of MHC-II–peptide trafficking. T lymphocyte activation is triggered upon specific recognition of antigens present on the APC surface during immunological synapse (IS) formation. This dynamic process is characterized by a defined spatial organization involving the compartmentalization of receptors and adhesion molecules in specialized membrane domains that are connected to the underlying cytoskeleton and signaling molecules. Tetraspanins contribute to the spatial organization and maturation of the IS by controlling receptor clustering and local accumulation of adhesion receptors and integrins, their downstream signaling, and linkage to the actin cytoskeleton. This review offers a perspective on the important role of TEMs in the regulation of antigen recognition and presentation and in the dynamics of IS architectural organization. PMID:26793193

Unpolarized release of vaccinia virus and HIV antigen by colchicine treatment enhances intranasal HIV antigen expression and mucosal humoral responses.

Directory of Open Access Journals (Sweden)

Yan Zhang

Full Text Available The induction of a strong mucosal immune response is essential to building successful HIV vaccines. Highly attenuated recombinant HIV vaccinia virus can be administered mucosally, but even high doses of immunization have been found unable to induce strong mucosal antibody responses. In order to solve this problem, we studied the interactions of recombinant HIV vaccinia virus Tiantan strain (rVTT-gagpol in mucosal epithelial cells (specifically Caco-2 cell layers and in BALB/c mice. We evaluated the impact of this virus on HIV antigen delivery and specific immune responses. The results demonstrated that rVTT-gagpol was able to infect Caco-2 cell layers and both the nasal and lung epithelia in BALB/c mice. The progeny viruses and expressed p24 were released mainly from apical surfaces. In BALB/c mice, the infection was limited to the respiratory system and was not observed in the blood. This showed that polarized distribution limited antigen delivery into the whole body and thus limited immune response. To see if this could be improved upon, we stimulated unpolarized budding of the virus and HIV antigens by treating both Caco-2 cells and BALB/c mice with colchicine. We found that, in BALB/c mice, the degree of infection and antigen expression in the epithelia went up. As a result, specific immune responses increased correspondingly. Together, these data suggest that polarized budding limits antigen delivery and immune responses, but unpolarized distribution can increase antigen expression and delivery and thus enhance specific immune responses. This conclusion can be used to optimize mucosal HIV vaccine strategies.

Five-year longitudinal assessment of the prognosis of apical microsurgery

DEFF Research Database (Denmark)

von Arx, Thomas; Jensen, Simon S; Hänni, Stefan

2012-01-01

Apical surgery is an important treatment option for teeth with post-treatment apical periodontitis. Knowledge of the long-term prognosis is necessary when weighing apical surgery against alternative treatments. This study assessed the 5-year outcome of apical surgery and its predictors in a cohor...

Unusual Self-Assembly of the Recombinant Chlamydia trachomatis Major Outer Membrane Protein-Based Fusion Antigen CTH522 Into Protein Nanoparticles

DEFF Research Database (Denmark)

Rose, Fabrice; Karlsen, Kasper; Jensen, Pernille

2018-01-01

Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic but is a c......Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic...... but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated......-defined secondary structural elements, and no thermal transitions were measurable. Chemical unfolding resulted monomers that upon removal of the denaturant self-assembled into higher order structures, comparable to the structure of the native protein. The conformation of CTH522 in nanoparticles is thus not entirely...

B-cell responses to pregnancy-restricted and -unrestricted Plasmodium falciparum erythrocyte membrane protein 1 antigens in Ghanaian women naturally exposed to malaria parasites

DEFF Research Database (Denmark)

Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F

2014-01-01

-linked immunosorbent assay (ELISA) and memory B-cell frequencies by enzyme-linked immunosorbent spot (ELISPOT) assay in a cohort of P. falciparum-exposed nonpregnant Ghanaian women. The antigens used were a VAR2CSA-type PfEMP1 (IT4VAR04) with expression restricted to parasites infecting the placenta, as well as two...... commonly recognized PfEMP1 proteins (HB3VAR06 and IT4VAR60) implicated in rosetting and not pregnancy restricted. This enabled, for the first time, a direct comparison in the same individuals of immune responses specific for a clinically important parasite antigen expressed only during well-defined periods...

Rac1 controls epithelial tube length through the apical secretion and polarity pathways

Directory of Open Access Journals (Sweden)

Kévin Sollier

2016-01-01

Full Text Available The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis.

Revascularization and Apical Plug in an Immature Molar

Science.gov (United States)

Roghanizadeh, Leyla; Fazlyab, Mahta

2018-01-01

Managing of necrotic permanent teeth with immature apices is a treatment challenges. Treatment of such teeth includes apexification, apical plug and more recently, revascularization technique with the probable advantage of continuation of root development. In the present case report the referred patient had discomfort with a necrotic immature mandibular first molar. Periapical radiography showed a rather large apical lesion around immature roots. Revascularization protocol using calcium-enriched mixture (CEM) cement was indicated for the mesial root. However, in distal canal apical plug technique was applied. At 2-year follow-up, both procedures were successful in relieving patient’s symptoms. Dentin formation and increase in length of the mesial root was obvious. Apical plug and revascularization technique proved to be successful in management of necrotic immature teeth; moreover, revascularization carried the advantage of continuation of root development. PMID:29692851

Cdk1 Activates Pre-Mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem cells

Science.gov (United States)

Baffet, Alexandre D.; Hu, Daniel J.; Vallee, Richard B.

2015-01-01

Summary Dynein recruitment to the nuclear envelope is required for pre-mitotic nucleus-centrosome interactions in nonneuronal cells, and for apical nuclear migration in neural stem cells. In each case, dynein is recruited to the nuclear envelope (NE) specifically during G2, via two nuclear pore-mediated mechanisms involving RanBP2-BicD2 and Nup133-CENP-F. The mechanisms responsible for cell cycle control of this behavior are unknown. We now find that Cdk1 serves as a direct master controller for NE dynein recruitment in neural stem cells and HeLa cells. Cdk1 phosphorylates conserved sites within RanBP2 and activates BicD2 binding and early dynein recruitment. Late recruitment is triggered by a Cdk1-induced export of CENP-F from the nucleus. Forced NE targeting of BicD2 overrides Cdk1 inhibition, fully rescuing dynein recruitment and nuclear migration in neural stem cells. These results reveal how NE dynein recruitment is cell cycle regulated, and identify the trigger mechanism for apical nuclear migration in the brain. PMID:26051540

Regulation of ion transport via apical purinergic receptors in intact rabbit airway epithelium

DEFF Research Database (Denmark)

Poulsen, Asser Nyander; Klausen, Thomas Levin; Pedersen, Peter Steen

2005-01-01

and unidirectional Cl- fluxes decreased significantly. The results suggest that nucleotides released to the airway surface liquid exert an autocrine regulation of epithelial NaCl absorption mainly by inhibiting the amiloride-sensitive epithelial Na+ channel (ENaC) and paracellular anion conductance via a P2Y......We investigated purinergic receptors involved in ion transport regulation in the intact rabbit nasal airway epithelium. Stimulation of apical membrane P2Y receptors with ATP or UTP (200 microM) induced transient increases in short-circuit current (Isc) of 13 and 6% followed by sustained inhibitions...

Effect of reciprocating systems and working lengths on apical microcrack development: a micro-CT Study

International Nuclear Information System (INIS)

Oliveira, Bruna Paloma de; Câmara, Andréa Cruz; Duarte, Daniel Amancio; Antonino, Antonio Celso Dantas; Aguiar, Carlos Menezes; Heck, Richard John

2017-01-01

The objective of this study was to evaluate the effect of root canal preparation with single-file reciprocating systems at different working lengths on the development of apical microcracks using micro-computed tomographic (micro-CT) imaging. Forty extracted human mandibular incisors were randomly assigned to 4 groups (n=10) according to the systems and working length used to prepare the root canals: Group A - WaveOne Gold at apical foramen (AF), Group B - WaveOne Gold 1 mm short of the AF (AF-1 mm), Group C - Unicone (AF) and Group D - Unicone (AF-1 mm). Micro-CT scanning was performed before and after root canal preparation at an isotropic resolution of 14 μm. Then, three examiners assessed the cross-sectional images generated to detect microcracks in the apical portion of the roots. Apical microcracks were visualized in 3, 1, 1, and 3 specimens in groups A, B, C, and D, respectively. All these microcracks observed after root canal preparation already existed prior to instrumentation, and no new apical microcrack was detected. For all groups, the number of slices presenting microcracks after root canal preparation was the same as before canal preparation. Root canal preparation with WaveOne Gold and Unicone, regardless of the working length, was not associated with apical microcrack formation. (author)

Effect of reciprocating systems and working lengths on apical microcrack development: a micro-CT Study

Energy Technology Data Exchange (ETDEWEB)

Oliveira, Bruna Paloma de; Câmara, Andréa Cruz; Duarte, Daniel Amancio; Antonino, Antonio Celso Dantas; Aguiar, Carlos Menezes, E-mail: bruna_paloma@msn.com [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil); Heck, Richard John [Department of Land Resource Science, University of Guelph (Canada)

2017-11-15

The objective of this study was to evaluate the effect of root canal preparation with single-file reciprocating systems at different working lengths on the development of apical microcracks using micro-computed tomographic (micro-CT) imaging. Forty extracted human mandibular incisors were randomly assigned to 4 groups (n=10) according to the systems and working length used to prepare the root canals: Group A - WaveOne Gold at apical foramen (AF), Group B - WaveOne Gold 1 mm short of the AF (AF-1 mm), Group C - Unicone (AF) and Group D - Unicone (AF-1 mm). Micro-CT scanning was performed before and after root canal preparation at an isotropic resolution of 14 μm. Then, three examiners assessed the cross-sectional images generated to detect microcracks in the apical portion of the roots. Apical microcracks were visualized in 3, 1, 1, and 3 specimens in groups A, B, C, and D, respectively. All these microcracks observed after root canal preparation already existed prior to instrumentation, and no new apical microcrack was detected. For all groups, the number of slices presenting microcracks after root canal preparation was the same as before canal preparation. Root canal preparation with WaveOne Gold and Unicone, regardless of the working length, was not associated with apical microcrack formation. (author)

Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag.

Science.gov (United States)

Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

2016-04-25

The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (L-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

Augmentation of antigen-specific immune responses using DNA-fusogenic liposome vaccine

International Nuclear Information System (INIS)

Yoshikawa, Tomoaki; Imazu, Susumu; Gao Jianqing; Hayashi, Kazuyuki; Tsuda, Yasuhiro; Shimokawa, Mariko; Sugita, Toshiki; Niwa, Takako; Oda, Atushi; Akashi, Mitsuru; Tsutsumi, Yasuo; Mayumi, Tadanori; Nakagawa, Shinsaku

2004-01-01

In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen

Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines.

Science.gov (United States)

Zheng, Song-yue; Yu, Bin; Zhang, Ke; Chen, Min; Hua, Yan-Hong; Yuan, Shuofeng; Watt, Rory M; Zheng, Bo-Jian; Yuen, Kwok-Yung; Huang, Jian-Dong

2012-09-26

Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines. To compare vaccine strains encoded with different antigen delivery and expression strategies, a series of recombinant Salmonella Typhimurium strains were constructed that expressed either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. The antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in either the cytoplasm or the outer membrane. Combinations of strategies were employed to evaluate the efficacy of combined delivery/expression approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities; the immunogenicity of the constructed recombinant Salmonella strains was evaluated in mice. Using the soluble model antigen EGFP, our results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, whilst eukaryotic expression or colonization with good construct stability was critical for T cell responses. For the insoluble model antigen HA, an outer membrane expression strategy induced better B cell and T cell responses than a cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited. Through systematically evaluating and comparing the immunogenicity of the constructed recombinant Salmonella strains in mice, we identified their respective advantages and deleterious or synergistic effects. Different construction strategies were optimally-required for soluble versus

Bile salt-induced increases in duodenal brush-border membrane proton permeability, fluidity, and fragility

International Nuclear Information System (INIS)

Zhao, D.L.; Hirst, B.H.

1990-01-01

Rabbit duodenal brush-border membrane vesicles were treated in vitro with deoxycholate, glycodeoxycholate, or taurodeoxycholate. Intravesicular [14C]glucose space at equilibrium, 0.54 microliters/mg protein, was reduced by exposure to the three bile salts in a concentration (0.1-5.0 mM)-dependent manner, equatable with increased membrane fragility. Net proton permeability (Pnet), determined by acridine orange fluorescence quenching, was increased from 6.3 x 10(-4) cm/sec in untreated vesicles, by approximately 120, 150, and 170%, by treatment with bile salts at 0.1, 0.5 and 1.0 mM, respectively. The three bile salts were equipotent. The increases in membrane fragility and Pnet were not accompanied by significant increases in membrane fluidity, as assessed from steady-state and time-resolved diphenylhexatriene fluorescence anisotropy. The data demonstrate direct effects of bile salts on duodenal apical membrane fragility and proton permeability that are likely to be early events in bile salt-induced mucosal damage

Ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) with reference to the apical groove and Flagellar apparatus.

Science.gov (United States)

Iwataki, Mitsunori; Hansen, Gert; Moestrup, Øjvind; Matsuoka, Kazumi

2010-01-01

The external and internal ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef has been examined with special reference to the apical groove and three-dimensional structure of the flagellar apparatus. The apical groove is U-shaped and connected to the anterior sulcal extension on the dorsal side of the epicone. The eyespot is located dorsally and composed of two layers of globules situated within the chloroplast. A narrow invagination of the plasma membrane is associated with the eyespot. The nuclear envelope has normal nuclear pores similar to other eukaryotes but different from the Gymnodinium group with diagnostic nuclear chambers. The longitudinal and transverse basal bodies are separated by approximately 0.5-1.0 microm and interconnected directly by a striated basal body connective and indirectly by microtubular and fibrous structures. Characteristic features of the flagellar apparatus are as follows: (1) a nuclear extension projects to the R1 (longitudinal microtubular root) and is connected to the root by thin fibrous material; (2) fibrillar structures are associated with the longitudinal and transverse flagellar canal; and (3) a striated ventral connective extends toward the posterior end of the cell along the longitudinal flagellar canal. We conclude, based on both morphological and molecular evidence, that Cochlodinium is only distantly related to Gymnodinium.

Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge

Science.gov (United States)

Blanc, Pascal; Moro-Sibilot, Ludovic; Barthly, Lucas; Jagot, Ferdinand; This, Sébastien; de Bernard, Simon; Buffat, Laurent; Dussurgey, Sébastien; Colisson, Renaud; Hobeika, Elias; Fest, Thierry; Taillardet, Morgan; Thaunat, Olivier; Sicard, Antoine; Mondière, Paul; Genestier, Laurent; Nutt, Stephen L.; Defrance, Thierry

2016-01-01

Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge. PMID:27924814

Healing of apical rarefaction of three nonvital open apex anterior teeth using a white portland cement apical plug

Directory of Open Access Journals (Sweden)

Amitabha Chakraborty

2012-01-01

Full Text Available The major challenge of performing root canal treatment in an open apex pulp-less tooth is to obtain a good apical seal. MTA has been successfully used to achieve a good apical seal, wherein the root canal obturation can be done immediately. MTA and White Portland Cement has been shown similarity in their physical, chemical and biological properties and has also shown similar outcome when used in animal studies and human trials. In our study, open apex of three non vital upper central incisors has been plugged using modified white Portland cement. 3 to 6 months follow up revealed absence of clinical symptoms and disappearance of peri-apical rarefactions. The positive clinical outcome may encourage the future use of white Portland cement as an apical plug material in case of non vital open apex tooth as much cheaper substitute of MTA.

Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus (FMDV serotype Asia1

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Alam SM

2013-08-01

Full Text Available SM Sabbir Alam,1 Ruhul Amin,1 Mohammed Ziaur Rahman,2 M Anwar Hossain,1 Munawar Sultana11Department of Microbiology, University of Dhaka, Dhaka, Bangladesh; 2International Centre for Diarrhoeal Disease Research, Dhaka, BangladeshAbstract: Foot and mouth disease virus (FMDV, with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes.Keywords: protein modeling, antigenic sites, sequence variation

Regeneration of okra ( Abelmoschus esculentus L.) via apical shoot ...

African Journals Online (AJOL)

Abelmoschus esculentus L. Monech) via apical shoot culture system. The study of apical shoot culture system was found effective for regeneration of apical shoots. The okra (A. esculentus L. Monech) N-550 line evolved at R&D, Nirmal Seeds Pvt. Ltd., ...

Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx.

Science.gov (United States)

van Hell, A J; Klymchenko, A; Gueth, D M; van Blitterswijk, W J; Koning, G A; Verheij, M

2014-09-01

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium. Copyright © 2014 Elsevier B.V. All rights reserved.

Does Increased Expression of the Plasma Membrane Calcium-ATPase Isoform 2 Confer Resistance to Apoptosis on Breast Cancer Cells?

National Research Council Canada - National Science Library

VanHouten, Joshua N

2008-01-01

The plasma membrane calcium ATPase isoform 2 (PMCA2) is highly expressed on the apical membrane of mammary epithelial cells during lactation, and is the predominant pump responsible for calcium transport into milk...

Computational Identification of Antigenicity-Associated Sites in the Hemagglutinin Protein of A/H1N1 Seasonal Influenza Virus.

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Xiaowei Ren

Full Text Available The antigenic variability of influenza viruses has always made influenza vaccine development challenging. The punctuated nature of antigenic drift of influenza virus suggests that a relatively small number of genetic changes or combinations of genetic changes may drive changes in antigenic phenotype. The present study aimed to identify antigenicity-associated sites in the hemagglutinin protein of A/H1N1 seasonal influenza virus using computational approaches. Random Forest Regression (RFR and Support Vector Regression based on Recursive Feature Elimination (SVR-RFE were applied to H1N1 seasonal influenza viruses and used to analyze the associations between amino acid changes in the HA1 polypeptide and antigenic variation based on hemagglutination-inhibition (HI assay data. Twenty-three and twenty antigenicity-associated sites were identified by RFR and SVR-RFE, respectively, by considering the joint effects of amino acid residues on antigenic drift. Our proposed approaches were further validated with the H3N2 dataset. The prediction models developed in this study can quantitatively predict antigenic differences with high prediction accuracy based only on HA1 sequences. Application of the study results can increase understanding of H1N1 seasonal influenza virus antigenic evolution and accelerate the selection of vaccine strains.

Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

International Nuclear Information System (INIS)

Chao, S.; Chao, L.; Chao, J.

1989-01-01

A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

Studies on the structures of the Tm, Sj, M1, Can, Sext and Hu blood group antigens.

Science.gov (United States)

Dahr, W; Knuppertz, G; Beyreuther, K; Moulds, J J; Moulds, M; Wilkinson, S; Capon, C; Fournet, B; Issitt, P D

1991-08-01

The Glycophorins (GPs = sialoglycoproteins) in erythrocyte membranes from various Black individuals, some of which exhibit the M1, Can, Sj, Tm, Sext and/or Hu antigens, and several Caucasian donors, including pooled fetal red cells, were studied. Using agglutination inhibition assays with GP fractions, GP fragments and chemically modified GPs as well as trypsin treatment of intact red cells, the antigens defined by anti-M1, anti-M+M1, anti-Can and anti-Tm sera were found to be located on the N-terminal tryptic peptide (T2, residues 1-31) of the major GP (GP A = MN sialoglycoprotein). Evidence was obtained that the N-terminal amino-acid residue, NeuNAc and/or (a) different sugar residue(s) are involved in the antigens. Amino-acid sequence and composition analyses excluded an amino-acid exchange within the N-terminal region (residues 1-31) of GP A. Carbohydrate analyses revealed the attachment of GlcNAc residues (up to about five, dependent on the strength of the above-mentioned antigens) to O-glycosidically linked oligosaccharides within the N-terminal portion (residues 1-31) of GP A. As judged from the carbohydrate compositions of peptides, the alteration of the O-glycosidic oligosaccharides is associated with a slight increase of the Gal and Fuc contents and a slight decrease of the NeuNAc level. Analyses of small, secondary cyanogen bromide and V8 proteinase peptides from the N-terminal region of GP A from Blacks, Caucasians and Caucasian fetal cells suggest that the variable attachment of small quantities of GlcNAc (about 0.03 to about 0.2 residues per peptide molecule) accounts, at least in part, for the polymorphisms detected by anti-Can and the original anti-Tm (serum Sheerin). Remarkably, the GlcNAc-containing O-glycosidic oligosaccharides occur only in small quantities, or not all at, within the positions 32-61 of GP A and the glycosylated domains of GP B and GP C.(ABSTRACT TRUNCATED AT 400 WORDS)

IN VIVO ANALYSIS OF SOME KEY CHARACTERISTICS OF THE APICAL ZONE IN TEETH WITH CHRONIC APICAL PERIODONTITIS.

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Angela Gusiyska

2014-11-01

Full Text Available Introduction: The pathogenesis of internal and external resorptive processes in the dental tissues and those of the periapical zone is not fully understood, but the main purpose, either in teeth with internal resorption or in teeth with periapical lesions, is decontamination of the endodontic space and subsequent three-dimensional obturation in order to isolate periapical and oral tissues and prevent reinfection. Purpose: The aim of this article is to analyze in vivo some key characteristics of the apical zone in teeth with chronic apical periodontitis. Material and Methods: To facilitate the clinical protocol after the radiographic analysis and assessment of patency, the working lengths of 153 root canals (n = 153 in 106 teeth were determined. The clinical widths of the apical narrowing were measured by using the last instrument (ISO 0.02 tapered file, which can move freely through the apical narrowing after electrometric determination of the working length (Raypex 5 /VDW, Germany/. Results and Discussion: Determination of working width and working length is important for realizing the first stage of decontamination – maximum instrumentation of the endodontic space and choice of a clinical protocol. The classification of root canals in a particular group according to the relative patency or lysed apical opening is important for the selection of obturation technique, which is essential for reducing the microleakage in the zone. Conclusion: Since the target of this work were teeth with CAP, in the majority of the cases with clinical findings of root canals with preexisting filling, radicular pins, obliteration, separated canal instruments, perforations at different levels, via falsa or thresholds, the access to the apical zone was not subjected to a closely observed instrumental clinical protocol. In the treatment of each case, however, the clinical principles of modern endodontic treatment were closely observed.

Influenza human monoclonal antibody 1F1 interacts with three major antigenic sites and residues mediating human receptor specificity in H1N1 viruses.

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Tshidi Tsibane

Full Text Available Most monoclonal antibodies (mAbs to the influenza A virus hemagglutinin (HA head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates. The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca(2. The 1F1 heavy chain also reaches into the receptor binding site (RBS and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.

Evaluation and Comparison of the Position of the Apical Constriction in Single-root and Multiple-root Teeth

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Alireza Farhad

2017-12-01

Full Text Available Introduction: Precise knowledge of the location of the apical constriction is essential to root canal treatment and long-term prognosis. Considering the differences in the apical constriction and size of the roots in single- and multiple-root teeth in various races, examination and comparison of the location of the apical constriction in single-root and multiple-root teeth are of paramount importance. The present studies aimed to measure and compare the distance of the apical constriction from the apical foramen and anatomical apex in single-root and multiple-root teeth. Materials and Methods: In this cross-sectional study, 60 roots of single-rooted teeth and 60 roots of multiple-rooted teeth were collected from the patients referring to the health centers in Isfahan, Iran. After cleansing and disinfecting the surface of the roots, the surface of the teeth was washed with hypochlorite. Based on the direction of the apical foramen, a longitudinal cut was made in the same direction, and the roots were examined microscopically at the magnification of 25. Following that, the distance of the apical constriction from the apical foramen and anatomical apex was measured using a digital camera. In addition, mean and standard deviation of the obtained distance values were determined. Distances in the single-root and multiple-root teeth were compared using independent t-test, at the significance level of Results: Mean distance between the apical constriction and apical foramen was 0.86±0.33 mm in the single-root teeth and 0.072±0.27 mm in the multiple-root teeth. Mean distance between the apical constriction and anatomical apex was 1.14±0.36 mm in the single-root teeth and 1.03±0.36 mm in the multiple-root teeth. Moreover, the results of independent t-test showed the distance of the apical constriction from the apical foramen to be significant between single-root and multiple-rooted teeth (P=0.013. However, the distance between the apical constriction

Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs.

Science.gov (United States)

Gonzalez-Vicente, Agustin; Garvin, Jeffrey L

2013-11-01

Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg(-1)·min(-1) compared with only 9.9 ± 1.1 AFU·mg(-1)·min(-1) in controls (P Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg(-1)·day(-1)) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

Engineering the chloroplast targeted malarial vaccine antigens in Chlamydomonas starch granules.

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David Dauvillée

2010-12-01

Full Text Available Malaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS, the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii.We describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species.This novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles designated herein as amylosomes, demonstrating that

Engineering the chloroplast targeted malarial vaccine antigens in Chlamydomonas starch granules.

Science.gov (United States)

Dauvillée, David; Delhaye, Stéphane; Gruyer, Sébastien; Slomianny, Christian; Moretz, Samuel E; d'Hulst, Christophe; Long, Carole A; Ball, Steven G; Tomavo, Stanislas

2010-12-15

Malaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS), the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii. We describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS) are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species. This novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles designated herein as amylosomes, demonstrating that efficient production

Apical pulmonary abscesses

International Nuclear Information System (INIS)

Mercado Ferrer, Cesar A; Serrano Vasquez, Francisco O

2004-01-01

We presented the case of a 54 year-old man with bilateral apical pulmonary abscess who consults due to fever and bronchorrhoea, isolating moraxella catharralis that is managed with ampicillin-sulbactam with an adequate clinical and radiological evolution

Immunochemical and autoantigenic properties of the globular domain of basement membrane collagen (type IV).

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von der Mark, H; Oberbäumer, I; Timpl, R; Kemler, R; Wick, G

1985-02-01

Polyclonal rabbit antibodies raised against the globular domain NC1 of collagen IV from human placenta and a mouse tumor react with conformational antigenic determinants present on the NC1 hexamers and also with the three major subunits obtained after dissociation. The antibodies recognized unique structures within basement membranes and showed a broad tissue reactivity but only limited species cross-reactivity. Using these antibodies, it was possible to detect small amounts of collagen IV antigens from cell cultures and in serum. Monoclonal rat antibodies against mouse NC1 revealed a similar reaction potential. Autoantibodies could be produced in mice against mouse NC1 which react with kidney and lung basement membranes in a pathological manner, mimicking Goodpasture syndrome.

The tip-link antigen, a protein associated with the transduction complex of sensory hair cells, is protocadherin-15.

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Ahmed, Zubair M; Goodyear, Richard; Riazuddin, Saima; Lagziel, Ayala; Legan, P Kevin; Behra, Martine; Burgess, Shawn M; Lilley, Kathryn S; Wilcox, Edward R; Riazuddin, Sheikh; Griffith, Andrew J; Frolenkov, Gregory I; Belyantseva, Inna A; Richardson, Guy P; Friedman, Thomas B

2006-06-28

Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formation.

The resection angle in apical surgery

DEFF Research Database (Denmark)

von Arx, Thomas; Janner, Simone F M; Jensen, Simon S

2016-01-01

OBJECTIVES: The primary objective of the present radiographic study was to analyse the resection angle in apical surgery and its correlation with treatment outcome, type of treated tooth, surgical depth and level of root-end filling. MATERIALS AND METHODS: In the context of a prospective clinical...... study, cone beam computed tomography (CBCT) scans were taken before and 1 year after apical surgery to measure the angle of the resection plane relative to the longitudinal axis of the root. Further, the surgical depth (distance from the buccal cortex to the most lingual/palatal point of the resection...... or with the retrofilling length. CONCLUSIONS: Statistically significant differences were observed comparing resection angles of different tooth groups. However, the angle had no significant effect on treatment outcome. CLINICAL RELEVANCE: Contrary to common belief, the resection angle in maxillary anterior teeth...

Membrane dynamics and the regulation of epithelial cell polarity

NARCIS (Netherlands)

van der Wouden, JM; Maier, O; van IJzendoorn, SCD; Hoekstra, D

2003-01-01

Plasma membranes of epithelial cells consist of two domains, an apical and a basolateral domain, the surfaces of which differ in composition. The separation of these domains by a tight junction and the fact that specific transport pathways exist for intracellular communication between these domains

The subapical compartment : a traffic center in membrane polarity development

NARCIS (Netherlands)

Hoekstra, D; Tyteca, D; van IJzendoorn, SCD

2004-01-01

Spatially separated apical and basolateral plasma membrane domains that have distinct functions and molecular compositions are a characteristic feature of epithelial cell polarity. The subapical compartment (SAC), also known as the common endosome (CE), where endocytic pathways from both surfaces

On the causes of persistent apical periodontitis: a review.

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Nair, P N R

2006-04-01

Apical periodontitis is a chronic inflammatory disorder of periradicular tissues caused by aetiological agents of endodontic origin. Persistent apical periodontitis occurs when root canal treatment of apical periodontitis has not adequately eliminated intraradicular infection. Problems that lead to persistent apical periodontitis include: inadequate aseptic control, poor access cavity design, missed canals, inadequate instrumentation, debridement and leaking temporary or permanent restorations. Even when the most stringent procedures are followed, apical periodontitis may still persist as asymptomatic radiolucencies, because of the complexity of the root canal system formed by the main and accessory canals, their ramifications and anastomoses where residual infection can persist. Further, there are extraradicular factors -- located within the inflamed periapical tissue -- that can interfere with post-treatment healing of apical periodontitis. The causes of apical periodontitis persisting after root canal treatment have not been well characterized. During the 1990s, a series of investigations have shown that there are six biological factors that lead to asymptomatic radiolucencies persisting after root canal treatment. These are: (i) intraradicular infection persisting in the complex apical root canal system; (ii) extraradicular infection, generally in the form of periapical actinomycosis; (iii) extruded root canal filling or other exogenous materials that cause a foreign body reaction; (iv) accumulation of endogenous cholesterol crystals that irritate periapical tissues; (v) true cystic lesions, and (vi) scar tissue healing of the lesion. This article provides a comprehensive overview of the causative factors of non-resolving periapical lesions that are seen as asymptomatic radiolucencies post-treatment.

Effect of diode laser irradiation on the apical sealing of MTA retrofillings Efeito da irradiação de laser de diodo no selamento apical em retrobturações com MTA

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Eliana Barbosa de Souza

2006-09-01

Full Text Available Apical sealing is essential for the success of paraendodontic surgery, so any procedure that may favor an adequate sealing of the apical remainder should be performed. The purpose of this study was to evaluate the influence of diode laser irradiation on the apical sealing of root-end cavities with MTA retrofillings. Root canals in twenty extracted human teeth were shaped with K-files and filled with gutta-percha. The apexes were cut off and root-end preparations were performed. The roots were divided randomly in 2 groups. Group 1 (ten specimens was retrofilled with MTA. Group 2 was irradiated with diode laser, with 1 W for 20 seconds, on the apical surface and root end cavity before retrofilling with MTA. The specimens had their external surfaces impermeabilized with cyanoacrylate, except for the apical surface, and were then immersed in 1% rhodamine B dye for 72 h and placed in plaster stone. After that, the specimens were submitted to longitudinal abrasion until half of the root remained. The linear dye leakage was observed in these mid-roots between the root canal wall and retrofilling. The linear dye leakage was measured with Image Lab software, and the results were statistically analyzed with Student's t test. There were no statistically significant differences between the two groups (p > 0.05. The diode laser irradiation did not improve the apical sealing of MTA retrofillings under the conditions of this in vitro study.O selamento apical é fundamental para o sucesso da cirurgia parendodôntica. Assim, procedimentos que melhorem o selamento do remanescente apical devem ser utilizados. O objetivo deste estudo foi verificar se a irradiação de laser de diodo poderia aumentar o selamento apical em cavidades retrógradas obturadas com MTA. Foram utilizadas 20 raízes de dentes humanos extraídos que, após preparo com lima tipo K, tiveram seus canais obturados com guta-percha. Os ápices foram cortados e sofreram preparo de cavidades retr

Intraoperative cervix location and apical support stiffness in women with and without pelvic organ prolapse.

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Swenson, Carolyn W; Smith, Tovia M; Luo, Jiajia; Kolenic, Giselle E; Ashton-Miller, James A; DeLancey, John O

2017-02-01

It is unknown how initial cervix location and cervical support resistance to traction, which we term "apical support stiffness," compare in women with different patterns of pelvic organ support. Defining a normal range of apical support stiffness is important to better understand the pathophysiology of apical support loss. The aims of our study were to determine whether: (1) women with normal apical support on clinic Pelvic Organ Prolapse Quantification, but with vaginal wall prolapse (cystocele and/or rectocele), have the same intraoperative cervix location and apical support stiffness as women with normal pelvic support; and (2) all women with apical prolapse have abnormal intraoperative cervix location and apical support stiffness. A third objective was to identify clinical and biomechanical factors independently associated with clinic Pelvic Organ Prolapse Quantification point C. We conducted an observational study of women with a full spectrum of pelvic organ support scheduled to undergo gynecologic surgery. All women underwent a preoperative clinic examination, including Pelvic Organ Prolapse Quantification. Cervix starting location and the resistance (stiffness) of its supports to being moved steadily in the direction of a traction force that increased from 0-18 N was measured intraoperatively using a computer-controlled servoactuator device. Women were divided into 3 groups for analysis according to their pelvic support as classified using the clinic Pelvic Organ Prolapse Quantification: (1) "normal/normal" was women with normal apical (C -5 cm and Ba and/or Bp ≥ 0 cm). Demographics, intraoperative cervix locations, and apical support stiffness values were then compared. Normal range of cervix location during clinic examination and operative testing was defined by the total range of values observed in the normal/normal group. The proportion of women in each group with cervix locations within and outside the normal range was determined. Linear regression

Apical surgery vs apical surgery with simultaneous orthograde retreatment: A prospective cohort clinical study of teeth affected by persistent periapical lesion

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Carlo Prati

2018-06-01

Full Text Available Aim: This prospective clinical study analyzed the 24-month outcome of conventional apical surgery retro-filled with calcium-silicate cement versus apical surgery with simultaneous orthograde retreatment by means of clinical and radiographic criteria. Materials and methods: This study included 83 teeth affected by persistent periapical lesions in 68 patients. Mean age was 52 years (median = 51 years; range 19–81 years. Twenty-eight cases were treated with apical surgery, 16 cases with apical surgery with simultaneous orthograde retreatment and 39 cases with orthograde retreatment in previously treated teeth established as control group. Periapical index score (PAI was used as radiographic criteria. Teeth were examined at 6 months, 1 and 2 years and classified as healed (without any symptoms and PAI ≤ 2, healing (without any symptoms and PAI = 3 or diseased (with symptoms or PAI ≥ 4 and not functional on the basis of radiographic and clinical criteria. At 24 months evaluation, healed and healing were considered as success and diseased and fracture as failure. Multilevel GLM model and an ordered logistic regression as statistical analysis was made with level of significance set at p < 0.05. Results: Total drop-out was 7% (n = 6. After 6–9 months, 6 teeth (3 from apical surgery, 2 from simultaneous treatment and 1 from orthograde retreatment were extracted for root fracture. Twenty-four-month success rate of apical surgery group was 78% (n = 17, apical surgery with simultaneous orthograde retreatment presented 81% (n = 10 and orthograde retreatment success was 80% (n = 24. There was no statistically difference between the groups at 24 months (p = 0.890. Conclusions: Both surgical techniques revealed a high percentage of healing, similar to that reported by previous studies. Apical surgery with simultaneous orthograde retreatment showed a faster healing after 12 months comparing to the control group. Riassunto: Scopo: In

Expression and distribution of leptospiral outer membrane components during renal infection of hamsters

NARCIS (Netherlands)

Barnett, J. K.; Barnett, D.; Bolin, C. A.; Summers, T. A.; Wagar, E. A.; Cheville, N. F.; Hartskeerl, R. A.; Haake, D. A.

1999-01-01

The outer membrane of pathogenic Leptospira species grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41. The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during

In Vitro Variant Surface Antigen Expression in Plasmodium falciparum Parasites from a Semi-Immune Individual Is Not Correlated with Var Gene Transcription

Science.gov (United States)

Tschan, Serena; Flötenmeyer, Matthias; Koch, Iris; Berger, Jürgen; Kremsner, Peter; Frank, Matthias

2016-01-01

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus var gene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific var gene primer set showed that the surface recognition signal was not correlated with transcription of individual var genes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections. PMID:27907004

T1-nerve root neuroma presenting with apical mass and Horner's syndrome

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Podnar Simon

2007-03-01

Full Text Available Abstract Background The appearance of dumbbell neuroma of the first thoracic root is extremely rare. The extradural component of a T1-dumbbell neuroma may present as an apical mass. The diagnosis of hand weakness is complex and may be delayed in T1-neuroma because of absence of the palpable cervical mass. One-stage removal of a T1-root neuroma and its intrathoracic extension demanded an extended posterior midline approach in the sitting position. Case presentation A 51-year old man had suffered a traumatic partial tendon rupture of his wrist flexor muscles 6 years ago. Since the incident he occasionally felt fullness and tenderness in the affected forearm with some tingling in his fingers bilaterally. During the last two years the hand weakness was continuous and hypotrophy of the medial flexor and intrinsic hand muscles had become apparent. Electrophysiological studies revealed an ulnar neuropathy in addition to mild median and radial nerve dysfunction, including a mild contralateral carpal tunnel syndrome. The diagnostic work-up for multiple mononeuropathy in the upper extremity was negative. Repeated electrophysiological studies revealed fibrillations in the C7 paravertebral muscles on the affected side. Chest x-ray revealed a large round apical mass on the affected side. A Horner's syndrome was noted at this point of diagnostic work-up. MRI of the cervical and thoracic spine revealed a dumbbell T1 neuroma enlarging the intervertebral foramen at T1-2 and a 5 cm large extradural tumor with extension into the apex of the ipsilateral lung. The patient underwent surgery in sitting position using a left dorsal midline approach. Although the T1 root could not be preserved, the patient's neurological condition was unchanged after the surgery. Conclusion Extended posterior midline exposure described here using hemilaminectomy, unilateral facetectomy and costo-transversectomy is efficient and safe for one-stage removal of dumbbell tumors at the T1

Revitalization of open apex teeth with apical periodontitis using a collagen-hydroxyapatite scaffold.

Science.gov (United States)

Nevins, Alan J; Cymerman, Jerome J

2015-06-01

An enhanced revision of the revitalization endodontic technique for immature teeth with apical periodontitis has been described. It includes the addition of collagen-hydroxyapatite scaffold to the currently practiced revascularization technique. Four cases treated in series are presented in this report, 1 case involving 2 teeth. Periapical diagnoses of immature teeth included "asymptomatic apical periodontitis," "symptomatic apical periodontitis," and "acute apical abscess." Additionally, 1 fully developed tooth that had undergone root canal treatment that failed had a periapical diagnosis of acute apical abscess. An established revascularization protocol was used for all teeth. In addition to stimulating blood clots, all teeth were filled with collagen-hydroxyapatite scaffolds. Periapical radiolucencies healed in all teeth, and diffuse radiopacity developed within the coronal portions of canal spaces. Root development with root lengthening occurred in the immature nonvital maxillary premolar that had not undergone prior treatment. The technique of adding a collagen-hydroxyapatite scaffold to the existing revitalization protocol has been described in which substantial hard tissue repair has occurred. This may leave teeth more fully developed and less likely to fracture. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Antigen entrapped in the escheriosomes leads to the generation of CD4(+) helper and CD8(+) cytotoxic T cell response.

Science.gov (United States)

Syed, Faisal M; Khan, Masood A; Nasti, Tahseen H; Ahmad, Nadeem; Mohammad, Owais

2003-06-02

In previous study, we demonstrated the potential of Escherichia coli (E. coli) lipid liposomes (escheriosomes) to undergo membrane-membrane fusion with cytoplasmic membrane of the target cells including professional antigen presenting cells. Our present study demonstrates that antigen encapsulated in escheriosomes could be successfully delivered simultaneously to the cytosolic as well as endosomal processing pathways of antigen presenting cells, leading to the generation of both CD4(+) T-helper and CD8(+) cytotoxic T cell response. In contrast, encapsulation of same antigen in egg phosphatidyl-choline (egg PC) liposomes, just like antigen-incomplete Freund's adjuvant (IFA) complex, has inefficient access to the cytosolic pathway of MHC I-dependent antigen presentation and failed to generate antigen-specific CD8(+) cytotoxic T cell response. However, both egg PC liposomes as well as escheriosomes-encapsulated antigen elicited strong humoral immune response in immunized animals but antibody titre was significantly higher in the group of animals immunized with escheriosomes-encapsulated antigen. These results imply usage of liposome-based adjuvant as potential candidate vaccine capable of eliciting both cell-mediated as well as humoral immune responses. Furthermore, antigen entrapped in escheriosomes stimulates antigen-specific CD4(+) T cell proliferation and also enhances the level of IL-2, IFN-gamma and IL-4 in the immunized animals.

Hormonal networks involved in apical hook development in darkness and their response to light

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Maria Agustina Mazzella

2014-02-01

Full Text Available In darkness, the dicot seedlings produces an apical hook as result of differential cell division and extension at opposite sides of the hypocotyl. This hook protects the apical meristem from mechanical damage during seedling emergence from the soil. In darkness, gibberellins act via the DELLA-PIF (PHYTOCHROME INTERACTING FACTORs pathway, and ethylene acts via the EIN3/EIL1 (ETHYLENE INSENSITIE 3/EIN3 like 1-HLS1 (HOOKLESS 1 pathway to control the asymmetric accumulation of auxin required for apical hook formation and maintenance. These core pathways form a network with multiple points of connection. Light perception by phytochromes and cryptochromes reduces the activity of PIFs and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1 --both required for hook formation in darkness--, lowers the levels of gibberellins, and triggers hook opening as a component of the switch between heterotrophic and photoautotrophic development. Apical hook opening is thus a suitable model to study the convergence of endogenous and exogenous signals on the control of cell division and cell growth.

Antigenic variation and the genetics and epigenetics of the PfEMP1 erythrocyte surface antigens in Plasmodium falciparum malaria

DEFF Research Database (Denmark)

Arnot, David E; Jensen, Anja T R

2011-01-01

. Sterile immunity is not achieved and chronic parasitization of apparently healthy adults is the norm. In this article, we analyse the best understood malaria "antigenic variation" system, that based on Plasmodium falciparum's PfEMP1-type cytoadhesion antigens, and critically review recent literature...

Herpesviruses in asymptomatic apical periodontitis lesions: an immunohistochemical approach.

Science.gov (United States)

Saboia-Dantas, C J; Coutrin de Toledo, L F; Sampaio-Filho, H R; Siqueira, J F

2007-10-01

Human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) have been recently detected in samples from apical periodontitis lesions by means of molecular biology techniques and a role in the pathogenesis of this disease has been suggested. The present study was designed to survey asymptomatic primary apical periodontitis lesions for the presence of HCMV- and/or EBV-infected cells by means of immunohistochemistry. Apical periodontitis lesions were obtained from 35 patients [26 human immunodeficiency virus (HIV) -seronegative patients and nine HIV-seropositive patients] after tooth extraction and subjected to immunohistochemical analysis using monoclonal antibodies specific for HCMV and EBV. Fifteen of the 35 apical periodontitis lesions were positive for the target herpesviruses. Overall, EBV was found in 31% of the samples and HCMV in 23%, with 14% of the lesions showing EBV and HCMV dual infection. No association was found between HCMV or EBV with any particular histopathological type of apical periodontitis (P > 0.05). HCMV was significantly more frequent in apical periodontitis lesions from HIV-positive patients (67%) than in lesions from HIV-negative patients (8%) (P = 0.001). EBV was detected in 44% of lesions from HIV-positive patients and in 27% of lesions from HIV-negative patients, but this difference was not significant (P = 0.91). Our results showed that cells infected by HCMV and EBV can be found in apical periodontitis lesions, with a higher prevalence in HIV-positive patients. The specific role that these viruses play in the pathogenesis of apical periodontitis remains to be described.

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

Science.gov (United States)

Paladino, Simona; Lebreton, Stéphanie; Lelek, Mickaël; Riccio, Patrizia; De Nicola, Sergio; Zimmer, Christophe; Zurzolo, Chiara

2017-12-01

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model. © 2017 The Author(s).

Cytoplasmic Dynein Regulation by Subunit Heterogeneity and Its Role in Apical Transport

Science.gov (United States)

Tai, Andrew W.; Chuang, Jen-Zen; Sung, Ching-Hwa

2001-01-01

Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin's cytoplasmic COOH-terminal tail, which encodes an apical targeting determinant in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that Tctex-1 and RP3 compete for binding to dynein intermediate chain and that overexpressed RP3 displaces endogenous Tctex-1 from dynein complexes in MDCK cells. Furthermore, replacement of Tctex-1 by RP3 selectively disrupts the translocation of rhodopsin to the MDCK apical surface. These results directly show that cytoplasmic dynein function can be regulated by its subunit composition and that cytoplasmic dynein is essential for at least one mode of apical transport in polarized epithelia. PMID:11425878

Role of interleukin-1 in antigen presentation by normal articular chondrocytes

International Nuclear Information System (INIS)

Tiku, M.L.; Liu, S.; Tiku, K.

1986-01-01

Recently the authors have described that normal articular chondrocytes of rabbits present antigen to immune T cells. In the present study the authors investigated the role of interleukin-1 (IL-1) on antigen presentation by chondrocytes. For these experiments the antigen pulsed chondroyctes were either untreated or fixed with paraformaldehyde and then co-cultured with immune T cells. T cell proliferation was measured by 3 H-thymidine incorporation. Pulsed non-fixed chondrocytes presented antigen, as expected, but pulsed and fixed cells failed to present antigen to T cells. The 3 H-TdR incorporation was partially restored by addition of purified human IL-1. Next, IL-1 activity was measured in primary chondrocyte culture supernatants stimulated with or without lipopolysaccharide (LPS) in comitogen thymocyte assay. No activity was detected in chondrocyte supernatants. Propagated chondrocyte culture supernatants also lacked IL-1 activity when stimulated with LPS in the presence of increasing concentration of indomethacin. On the other hand the authors observed that chondrocyte culture supernatants in a dose dependent manner inhibited human IL-1 induced 3 H-TdR incorporation of murine thymocytes. This suggested that these cells may produce an inhibitor of IL-1 and IL-1 production by chondrocytes may be essential for T cell proliferation by these cells. Inability to detect IL-1 in chondrocyte supernatants may be due to the presence of an inhibitor to IL-1. These findings may help in elucidating the immunological mechanisms in situations where chondrocytes and T cell interact, such as in arthritis

Love Wave Sensor for Prostate-Specific Membrane Antigen Detection Based on Hydrophilic Molecularly-Imprinted Polymer

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Pingping Tang

2018-05-01

Full Text Available Prostate-specific membrane antigen (PSMA is a biomarker for prostate cancer (PCa, and a specific and reliable detection technique of PSMA is urgently required for PCa early diagnosis. A Love wave sensor has been widely studied for real-time sensing and highly sensitive applications, but the sensing unit needs special handling for selective detection purpose. In this study, we prepared a versatile Love wave sensor functionalized with molecularly-imprinted polymers (MIP, PSMA as the template molecule. To enhance the specific template bindings of MIP in pure aqueous solutions, facile reversible addition/fragmentation chain transfer (RAFT precipitation polymerization (RAFTPP was used to produce surface hydrophilic polymer brushes on MIP. The presence of hydrophilic polymer brushes on MIP improved its surface hydrophilicity and significantly reduced their hydrophobic interactions with template molecules in pure aqueous media. In detection process, the acoustic delay-line is confederative to a microfluidic chip and inserted in an oscillation loop. The real-time resonance frequency of the MIP-based Love wave sensor to different concentrations of PSMA was investigated. The limit of detection (LOD for this Love SAW sensor was 0.013 ng mL−1, which demonstrates that this sensor has outstanding performance in terms of the level of detection.

Concentration of membrane antigens by forward transport and trapping in neuronal growth cones.

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Sheetz, M P; Baumrind, N L; Wayne, D B; Pearlman, A L

1990-04-20

Formation of the nervous system requires that neuronal growth cones follow specific paths and then stop at recognition signals, sensed at the growth cone's leading edge. We used antibody-coated gold particles viewed by video-enhanced differential interference contrast microscopy to observe the distribution and movement of two cell surface molecules, N-CAM and the 2A1 antigen, on growth cones of cultured cortical neurons. Gold particles are occasionally transported forward at 1-2 microns/s to the leading edge where they are trapped but continue to move. Concentration at the edge persists after cytochalasin D treatment or ATP depletion, but active movements to and along edges cease. We also observed a novel outward movement of small cytoplasmic aggregates at 1.8 microns/s in filopodia. We suggest that active forward transport and trapping involve reversible attachment of antigens to and transport along cytoskeletal elements localized to edges of growth cones.

Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite.

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Kriti Tyagi

Full Text Available Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to 'Pv-fam-a' family and some of them are potential drug/vaccine targets but their functional role(s largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7 and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle.

Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite

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Tyagi, Kriti; Hossain, Mohammad Enayet; Thakur, Vandana; Aggarwal, Praveen; Malhotra, Pawan; Mohmmed, Asif; Sharma, Yagya Dutta

2016-01-01

Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to ‘Pv-fam-a’ family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle. PMID:26954579

Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

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Wu, Lisa Y; Johnson, Jacqueline M; Simmons, Jessica K; Mendes, Desiree E; Geruntho, Jonathan J; Liu, Tiancheng; Dirksen, Wessel P; Rosol, Thomas J; Davis, William C; Berkman, Clifford E

2014-05-01

Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1)  mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50  = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly

Microbiology and Treatment of Acute Apical Abscesses

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Rôças, Isabela N.

2013-01-01

SUMMARY Acute apical abscess is the most common form of dental abscess and is caused by infection of the root canal of the tooth. It is usually localized intraorally, but in some cases the apical abscess may spread and result in severe complications or even mortality. The reasons why dental root canal infections can become symptomatic and evolve to severe spreading and sometimes life-threatening abscesses remain elusive. Studies using culture and advanced molecular microbiology methods for microbial identification in apical abscesses have demonstrated a multispecies community conspicuously dominated by anaerobic bacteria. Species/phylotypes commonly found in these infections belong to the genera Fusobacterium, Parvimonas, Prevotella, Porphyromonas, Dialister, Streptococcus, and Treponema. Advances in DNA sequencing technologies and computational biology have substantially enhanced the knowledge of the microbiota associated with acute apical abscesses and shed some light on the etiopathogeny of this disease. Species richness and abundance and the resulting network of interactions among community members may affect the collective pathogenicity and contribute to the development of acute infections. Disease modifiers, including transient or permanent host-related factors, may also influence the development and severity of acute abscesses. This review focuses on the current evidence about the etiology and treatment of acute apical abscesses and how the process is influenced by host-related factors and proposes future directions in research, diagnosis, and therapeutic approaches to deal with this disease. PMID:23554416

Inhibitory activities of microalgal extracts against Epstein-Barr Virus (EBV antigen expression in lymphoblastoid cells

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Koh Yih Yih

2014-01-01

Full Text Available The inhibitory activities of microalgal extracts against the expression of three EBV antigens, latent membrane protein (LMP1, Epstein-Barr nuclear antigen (EBNA1 and Z Epstein-Barr reactivation activator (ZEBRA were assessed by immunocytochemistry. The observation that the methanol extracts and their fractions from Ankistrodesmus convolutus, Synechococcus elongatus and Spirulina platensis exhibited inhibitory activity against EBV proteins in three Burkitt’s lymphoma cell lines at concentrations as low as 20 μg/ml suggests that microalgae could be a potential source of antiviral compounds against EBV.

A reappraisal of the role of abscisic acid and its interaction with auxin in apical dominance.

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Cline, Morris G; Oh, Choonseok

2006-10-01

Evidence from pea rms1, Arabidopsis max4 and petunia dad1 mutant studies suggest an unidentified carotenoid-derived/plastid-produced branching inhibitor which moves acropetally from the roots to the shoots and interacts with auxin in the control of apical dominance. Since the plant hormone, abscisic acid (ABA), known to inhibit some growth processes, is also carotenoid derived/plastid produced, and because there has been indirect evidence for its involvement with branching, a re-examination of the role of ABA in apical dominance is timely. Even though it has been determined that ABA probably is not the second messenger for auxin in apical dominance and is not the above-mentioned unidentified branching inhibitor, the similarity of their derivation suggests possible relationships and/or interactions. The classic Thimann-Skoog auxin replacement test for apical dominance with auxin [0.5 % naphthalene acetic acid (NAA)] applied both apically and basally was combined in similar treatments with 1 % ABA in Ipomoea nil (Japanese Morning Glory), Solanum lycopersicum (Better Boy tomato) and Helianthus annuus (Mammoth Grey-striped Sunflower). Auxin, apically applied to the cut stem surface of decapitated shoots, strongly restored apical dominance in all three species, whereas the similar treatment with ABA did not. However, when ABA was applied basally, i.e. below the lateral bud of interest, there was a significant moderate repression of its outgrowth in Ipomoea and Solanum. There was also some additive repression when apical auxin and basal ABA treatments were combined in Ipomoea. The finding that basally applied ABA is able partially to restore apical dominance via acropetal transport up the shoot suggests possible interactions between ABA, auxin and the unidentified carotenoid-derived branching inhibitor that justify further investigation.

Interleukin production by neonatal spleen cells during and as a result of antigen presentation: The effect of ultraviolet light

International Nuclear Information System (INIS)

Levin, D.; Gershon, H.

1989-01-01

Antigen presentation by neonatal murine spleen cells and the production of lymphokines and interleukins involved in the stimulation of a T-helper-2 (TH2) cell line (D10-G4.1) were studied as were the effects of ultra violet (UV)-irradiation on this system. Neonatal spleen cells are less capable than adult cells of performing the initial steps of the immune response required for antigen dependent activation of TH2 cells. These steps include soluble antigen processing and presentation and as a result reduced production of IL-4 and IL-1-Inducer Factor (IL-1-IF) by the T-helper cells and reduced production of IL-1 and IL-2 by the antigen presenting cell population. Spontaneous membrane IL-1 activity is low in the neonate, however, when exposed to IL-1-IF they can express adult levels. Ultraviolet (UV) irradiation of the antigen presenting population has a damaging effect on all the above mentioned processes. Antigen processing and presentation, induction of D10 IL-4 production and proliferation, and IL-2 production demonstrate two different age related patterns of UV-irradiation induced damage: a dose dependent inhibition when adult cells are irradiated and an inverse effect in which low doses of irradiation were more inhibitory than higher doses when neonatal cells are irradiated. However, the secretion and membrane expression of IL-1 by both age groups are directly and totally inhibited by the range of UV-irradiation doses used and cannot be reinduced with a supplement of a crude IL-1-IF. While the capacity to produced IL-1 is totally destroyed by UV-irradiation, the ability to produce IL-2 remains intact and remains responsive to an IL-2-Inducer activity during proper antigen presentation. The low responses of neonatal antigen presenting spleen cell populations and the damaging effect of UV on both neonatal and adult responses are not due to the induction of suppressor factors

Application of Hydroxyapatite Bioceramics and Oral Repair Membrane in the Surgery of Apical Syst%羟基磷灰石生物陶瓷及口腔修复膜在根尖囊肿手术中的应用

Institute of Scientific and Technical Information of China (English)

钱楚凡; 李小琳; 王栋

2015-01-01

Objective:To study the application of hydroxyapatite bioceramics and oral repair membrane in the surgery of apical syst and provide theory basis for jaw bone defect repair surgery of apical syst.Method:64 patients with apical syst in our hospital from June 2013 to May 2014 were selected and randomly divided into the control group and the observation group,with 32 cases in each group.The control group was treated with gelatin sponge filling repair,the observation group was treated with hydroxyapatite bioceramics filling and oral repair membrane for film repair.The clinical efficacy of the two groups was compared. Result:6 months after surgery,the height and thickness of bone graft and the bone growth effect in the observation group were better than those in the control group,the new bone density increased rate of the observation group was higher than that of the control group,the differences above were all statistically significant(P<0.05).Conclusion:Hydroxyapatite bioceramics and oral repair membrane applied in the jaw bone defect repair surgery of apical syst has a good effect,it is worth spreading.%目的：探讨羟基磷灰石生物陶瓷及口腔修复膜在根尖囊肿手术中的应用，为根尖囊肿颌骨缺损修复手术提供理论依据。方法：本研究选取2013年6月-2014年5月本院收治的64例根尖囊肿患者作为研究对象，采用随机数字表法将其分为对照组和观察组，每组32例，对照组采用明胶海棉填充修复，观察组采用羟基磷灰石生物陶瓷填充及口腔修复膜覆盖修复，比较两组患者的治疗效果。结果：手术后6个月，观察组植骨高度、厚度及骨生长效果均优于对照组，观察组新生骨密度明显增加比率高于对照组，比较差异均有统计学意义（P<0.05）。结论：羟基磷灰石生物陶瓷及口腔修复膜应用于根尖囊肿颌骨缺损修复手术具有良好效果，值得推广。

Bone resorptive activity in symptomatic and asymptomatic apical lesions of endodontic origin.

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Salinas-Muñoz, M; Garrido-Flores, M; Baeza, M; Huamán-Chipana, P; García-Sesnich, J; Bologna, R; Vernal, R; Hernández, M

2017-11-01

The aim of this study is to assess the levels and diagnostic accuracy of a set of bone resorption biomarkers, including TRAP-5, RANKL, and OPG in symptomatic and asymptomatic apical lesions and controls. Apical tissues from symptomatic and asymptomatic apical periodontitis patients and periodontal ligaments from healthy teeth extracted for orthodontic reasons were processed for tissue homogenization and the levels of TRAP-5, RANKL, and OPG were determined by multiplex assay. Marker levels were analyzed by Kruskal Wallis test, and diagnostic accuracy was analyzed with ROC curves. Higher levels of RANKL, OPG, and RANKL/OPG ratio were determined in both types of apical lesions compared to healthy periodontal ligament, whereas higher TRAP-5 levels were found only in symptomatic apical lesions (p apical lesions versus healthy controls (AUC = 0.69, p asymptomatic apical periodontitis (AUC = 0.71, p Apical lesions showed higher RANKL and OPG levels than healthy tissues. TRAP-5 levels were the highest in symptomatic apical lesions, suggesting that these represent a progressive state, and showed diagnostic potential. Clinically symptomatic apical periodontitis might represent biologically progressive apical lesions based on TRAP5 levels. TRAP5 has diagnostic potential to identify these lesions, representing a candidate prognostic biomarker.

Dental Pulp Revascularization of Necrotic Permanent Teeth with Immature Apices.

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El Ashiry, Eman A; Farsi, Najat M; Abuzeid, Sawsan T; El Ashiry, Mohamed M; Bahammam, Hammam A

The treatment of immature necrotic teeth with apical periodontitis presents challenges in endodontic and pediatric dentistry. Revascularization is a recent treatment for such cases as an alternative to conventional apexification. The purpose is to examine the effect of a pulpal revascularization procedure on immature necrotic teeth with apical periodontitis. Twenty patients were enrolled for pulp revascularization procedure by root canal disinfection using a triple antibiotic mixture for 1-2 weeks, followed by creating a blood clot, sealing the root canal orifice using white mineral trioxide aggregate and a coronal seal of composite resin. Patients were recalled periodically for up to 24 months. During follow-up, all patients were asymptomatic. Three cases of chronic apical periodontitis showed clinical disappearance of the sinus tract 2 weeks after treatment. Radiography revealed progressive periapical radiolucency resolution within the first 12 months. Within 12-24 months, the treated teeth showed progressive increases in dentinal wall thickness, root length and continued root development. Clinical and radiographic evidence showed successful revascularization treatments of immature necrotic permanent teeth with apical periodontitis. More studies are necessary to understand the underlying mechanisms and to perform histopathology of the pulp space contents after revascularization procedures.

[Apical resorption in pre-surgical orthodontics].

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Piasente, M; Merlini, C; Amelotti, C; Antonioli, M; Roghi, M

1991-07-15

Apical root resorption is a frequent phenomenon observed in pre-surgical orthodontic; the reason is double: we deal with adult patients and we often move the teeth in the opposite direction compared to the position obtained in previous inefficacious orthodontic treatments. Notwithstanding the amount of apical root resorption we couldn't record an hyper-mobility of the teeth and a long term evaluation of occlusal stability didn't show any significant change.

[A retrospective study of 180 cases of apical microsurgery].

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Wang, Hanguo; Li, Dan; Tian, Yu; Yu, Qing

2014-07-01

To evaluate the outcome and the potential prognostic factors of apical microsurgery. The teeth with persistent periapical diseases were treated by microsurgery using micro instruments, ultrasonic retrotips and mineral trioxide aggregate (MTA) under dental operate microscope. The procedure includes incision and flap retraction, osteotomy, apicoectomy, retro- preparation and retro- filling of root canal. Patients were recalled at 1, 3, 6, and 12- month intervals. The outcome was evaluated by clinical and radiographic examinations, and the potential prognostic factors were analyzed. One hundred and eighty cases (240 teeth), including 132 upper anterior teeth, 22 lower anterior teeth, 31 upper premolars, 18 lower premolars, 19 upper molars and 18 lower molars, were treated by microsurgery between July 2010 and December 2012. A total of 152 cases (207 teeth) were recalled. The application of the apical microsurgery included failure of previous endodontic treatment, periapical lesion with post, periapical cyst, calcified canals, separated instruments, overfilling, open apex, root facture, failure of previous apical surgery, apical fenestration, and special root canal system. The success rate was 90.8% (188/207). Age, sex, tooth position, type of periapical radiolucency, fistula and clinical application type appeared to have a negative effect on the outcome. Endo-perio lesion was a significant factor. Eighteen cases (19 teeth) failed mainly because of periodontally involved lesion and vertical root fracture. Apical microsurgery, which combines the magnification and illumination provided by the microscope with the proper use of micro instruments, can treat the teeth with persistent periapical diseases precisely and less traumatically with high success rate. Case selection and standardized operations play a key role for success.

Frequency and levels of candidate endodontic pathogens in acute apical abscesses as compared to asymptomatic apical periodontitis

OpenAIRE

Rôças, Isabela N.; Siqueira, José F.

2018-01-01

Introduction Acute apical abscess is caused by bacteria that leave the infected dental root canal to invade the periodontal tissues. Most species occurring in abscesses are also found in asymptomatic infections; therefore, the possibility exists that not only the presence of certain species but also their specific counts influence the appearance of symptoms. This molecular study compared the frequency and levels of several candidate endodontic pathogens in teeth with acute apical abscesses an...

Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome.

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Fumitaka Momose

Full Text Available Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs. Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM. However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD of Rab11 family interacting proteins (Rab11-FIPs. Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

Effects of loxoprofen on the apical root resorption during orthodontic tooth movement in rats.

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Yamamoto, Taeko; Kaku, Masato; Sumi, Hiromi; Yashima, Yuka; Izumino, Jin; Tanimoto, Kotaro

2018-01-01

Studies have revealed that severe apical root resorption during tooth movement is caused by the noninfective inflammatory reaction of apical root tissues. We hypothesized that loxoprofen can suppress apical root resorption during tooth movement. Cyclic tensile force (CTF) of 10 kPa was applied to the human pulp cells for 48 hours by the Flexcell Strain Unit. Loxoprofen (10 and 100 μM) was added to the culture cells, and expression of cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1β, receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and macrophage colony-stimulating factor (M-CSF) were examined. To determine the effects of loxoprofen sodium on apical root reabsorption during tooth movement, the upper first molars of 7-week-old rats were subjected to mesial movement by 10g force for 30 days with or without the oral administration of loxoprofen. Gene expression and protein concentration of COX-1, COX-2, IL-1β, TNF-α, RANKL and M-CSF were significantly higher in the CTF group than in the control group. However, these levels were decreased by loxoprofen administration. After orthodontic tooth movement, the expression of IL-1β, TNF-α, RANKL and M-CSF decreased in the loxoprofen group than in the control group by immunohistochemical staining. In comparison to control group, less number of odontoclasts and a decrease in the amount of apical root resorption was observed in the loxoprofen group. Many osteoclasts became visible on the pressure side of the alveolar bone in the both groups, and the amount of tooth movement did not show a significant difference. These findings demonstrate that severe apical root resorption may be suppressed by loxoprofen administration, without a disturbance of tooth movement.

Viral-bacterial associations in acute apical abscesses.

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Ferreira, Dennis C; Rôças, Isabela N; Paiva, Simone S M; Carmo, Flávia L; Cavalcante, Fernanda S; Rosado, Alexandre S; Santos, Kátia R N; Siqueira, José F

2011-08-01

Viral-bacterial and bacterial synergism have been suggested to contribute to the pathogenesis of several human diseases. This study sought to investigate the possible associations between 9 candidate endodontic bacterial pathogens and 9 human viruses in samples from acute apical abscesses. DNA extracts from purulent exudate aspirates of 33 cases of acute apical abscess were surveyed for the presence of 9 selected bacterial species using a 16S ribosomal RNA gene-based nested polymerase chain reaction (PCR) approach. Single or nested PCR assays were used for detection of the human papillomavirus (HPV) and herpesviruses types 1 to 8. Two-thirds of the abscess samples were positive for at least one of the target viruses. Specifically, the most frequently detected viruses were HHV-8 (54.5%); HPV (9%); and varicella zoster virus (VZV), Epstein-Barr virus (EBV), and HHV-6 (6%). Bacterial DNA was present in all cases and the most prevalent bacterial species were Treponema denticola (70%), Tannerella forsythia (67%), Porphyromonas endodontalis (67%), Dialister invisus (61%), and Dialister pneumosintes (57.5%). HHV-8 was positively associated with 7 of the target bacterial species and HPV with 4, but all these associations were weak. Several bacterial pairs showed a moderate positive association. Viral coinfection was found in 6 abscess cases, but no significant viral association could be determined. Findings demonstrated that bacterial and viral DNA occurred concomitantly in two-thirds of the samples from endodontic abscesses. Although this may suggest a role for viruses in the etiology of apical abscesses, the possibility also exists that the presence of viruses in abscess samples is merely a consequence of the bacterially induced disease process. Further studies are necessary to clarify the role of these viral-bacterial interactions, if any, in the pathogenesis of acute apical abscesses. Copyright © 2011 Mosby, Inc. All rights reserved.

Endothelial cell markers in vascular neoplasms: an immunohistochemical study comparing factor VIII-related antigen, blood group specific antigens, 6-keto-PGF1 alpha, and Ulex europaeus 1 lectin.

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Little, D; Said, J W; Siegel, R J; Fealy, M; Fishbein, M C

1986-06-01

Markers for endothelial cells including Ulex europaeus 1 lectin, blood group A, B, and H, and the prostaglandin metabolite 6-keto-PGF1 alpha were evaluated in paraffin secretions from formalin-fixed benign and malignant vascular neoplasms using a variety of immunohistochemical techniques, and results compared with staining for factor VIII-related antigen. Staining for Ulex appeared more sensitive than factor VIII-related antigen in identifying poorly differentiated neoplasms including haemangiosarcomas and spindle cell proliferations in Kaposi's sarcoma. Staining for blood group related antigens correlated with blood group in all cases. Ulex europaeus 1 lectin was the only marker for endothelial cells in lymphangiomas.

Cone beam computed tomography study of apical root resorption induced by Herbst appliance

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SCHWARTZ, João Paulo; RAVELI, Taísa Boamorte; ALMEIDA, Kélei Cristina de Mathias; SCHWARTZ-FILHO, Humberto Osvaldo; RAVELI, Dirceu Barnabé

2015-01-01

Objective This study evaluated the frequency of root resorption during the orthodontic treatment with Herbst appliance by Cone Beam Computed Tomography (CBCT). Material and Methods The sample comprised 23 patients (11 men, 12 women; mean ages 15.76±1.75 years) with Class II division 1 malocclusion, treated with Herbst appliance. CBCT was obtained before treatment (T0) and after Herbst treatment (T1). All the dental roots, except third molars, were evaluated, and apical root resorption was determined using the axial guided navigation method. Paired t-tests and Wilcoxon T Test were used to compare the dependent samples in parametric and nonparametric cases, respectively. Chi-Square Test with Yates’ correction was used to evaluate the relationship between apical root resorption and gender. Results were considered at a significance level of 5%. Results Apical resorption was detected by CBCT in 57.96% of 980 roots that underwent Herbst appliance treatment. All patients had minimal resorption and there was no statistical significance between the genders. Conclusion CBCT three-dimensional evaluation showed association between Herbst appliance and minimal apical root resorption, mostly in the anchoring teeth, without clinical significance. PMID:26537718

Cone beam computed tomography study of apical root resorption induced by Herbst appliance

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João Paulo SCHWARTZ

2015-10-01

Full Text Available Objective This study evaluated the frequency of root resorption during the orthodontic treatment with Herbst appliance by Cone Beam Computed Tomography (CBCT.Material and Methods The sample comprised 23 patients (11 men, 12 women; mean ages 15.76±1.75 years with Class II division 1 malocclusion, treated with Herbst appliance. CBCT was obtained before treatment (T0 and after Herbst treatment (T1. All the dental roots, except third molars, were evaluated, and apical root resorption was determined using the axial guided navigation method. Paired t-tests and Wilcoxon T Test were used to compare the dependent samples in parametric and nonparametric cases, respectively. Chi-Square Test with Yates’ correction was used to evaluate the relationship between apical root resorption and gender. Results were considered at a significance level of 5%.Results Apical resorption was detected by CBCT in 57.96% of 980 roots that underwent Herbst appliance treatment. All patients had minimal resorption and there was no statistical significance between the genders.Conclusion CBCT three-dimensional evaluation showed association between Herbst appliance and minimal apical root resorption, mostly in the anchoring teeth, without clinical significance.

Root Canal Microorganism Profiles on Upper Anterior Teeth of Apical Periodontitis

OpenAIRE

Riuwpassa, E. Irene

2013-01-01

Microorganisms are the main causative agents on the development of apical periodontitis. Microorganisms infecting the root canal system are colonized in communities as biofilm. These bacterial communities show distinct pattern related to the different forms of apical periodontitis which are determined by species richness and abundance.this study is aimed to examine the root canal microorganisms on upper anterior teeth of asymptomatic apical periodontitis and chronic apical abscess. Samples we...

Prostate cancer nodal oligometastasis accurately assessed using prostate-specific membrane antigen positron emission tomography-computed tomography and confirmed histologically following robotic-assisted lymph node dissection.

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O'Kane, Dermot B; Lawrentschuk, Nathan; Bolton, Damien M

2016-01-01

We herein present a case of a 76-year-old gentleman, where prostate-specific membrane antigen positron emission tomography-computed tomography (PSMA PET-CT) was used to accurately detect prostate cancer (PCa), pelvic lymph node (LN) metastasis in the setting of biochemical recurrence following definitive treatment for PCa. The positive PSMA PET-CT result was confirmed with histological examination of the involved pelvic LNs following pelvic LN dissection.

Prostate cancer nodal oligometastasis accurately assessed using prostate-specific membrane antigen positron emission tomography-computed tomography and confirmed histologically following robotic-assisted lymph node dissection

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Dermot B O′Kane

2016-01-01

Full Text Available We herein present a case of a 76-year-old gentleman, where prostate-specific membrane antigen positron emission tomography-computed tomography (PSMA PET-CT was used to accurately detect prostate cancer (PCa, pelvic lymph node (LN metastasis in the setting of biochemical recurrence following definitive treatment for PCa. The positive PSMA PET-CT result was confirmed with histological examination of the involved pelvic LNs following pelvic LN dissection.

Solution structure of a Plasmodium falciparum AMA-1/MSP 1 chimeric protein vaccine candidate (PfCP-2.9 for malaria

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Jin Changwen

2010-03-01

Full Text Available Abstract Background The Plasmodium falciparum chimeric protein PfCP-2.9 is a promising asexual-stage malaria vaccine evaluated in clinical trials. This chimeric protein consists of two cysteine-rich domains: domain III of the apical membrane antigen 1 (AMA-1 [III] and the C-terminal region of the merozoite surface protein 1 (MSP1-19. It has been reported that the fusion of these two antigens enhanced their immunogenicity and antibody-mediated inhibition of parasite growth in vitro. Methods The 15N-labeled and 13C/15N-labeled PfCP-2.9 was produced in Pichia pastoris for nuclear magnetic resonance (NMR structure analysis. The chemical shift assignments of PfCP-2.9 were compared with those previously reported for the individual domains (i.e., PfAMA-1(III or PfMSP 1-19. The two-dimensional spectra and transverse relaxation rates (R2 of the PfMSP1-19 alone were compared with that of the PfCP-2.9. Results Confident backbone assignments were obtained for 122 out of 241 residues of PfCP-2.9. The assigned residues in PfCP-2.9 were very similar to those previously reported for the individual domains. The conformation of the PfMSP1-19 in different constructs is essentially the same. Comparison of transverse relaxation rates (R2 strongly suggests no weak interaction between the domains. Conclusions These data indicate that the fusion of AMA-1(III and MSP1-19 as chimeric protein did not change their structures, supporting the use of the chimeric protein as a potential malaria vaccine.

Strategies to reduce the apical necrosis in vitro multiplication and rooting of Pistachio

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Mariela Cid

2016-07-01

Full Text Available The pistachio (Pistacia sp. is one of the least exploited fruit among the main causes is the high cost of plant material by the difficulties of propagation of the species. The propagation in vitro offers great potential for the industry of this species by multiplication scale of selected clones. The aim of this study was to control the apical necrosis of outbreaks in the in vitro propagation Pistachio. From young shoots of plants of this species kept in growing houses, proceeded to disinfection with sodium hypochlorite 1% for different times. The apical and axillary buds were established in the culture medium Murashige and Skoog (1962 modified and supplemented with 1 mg/L Metatopolina. Different types of bottles, number and type of explants and culture media formulations for two subcultures multiplication and rooting were evaluated. Glass flask of 200 ml capacity, and line proliferates DKW medium achieved the lowest values apical necrosis. In the multiplication phase values were low in all treatments tested no statistical difference between them compared to rooting where apical necrosis reached higher values. The average DKW and prolific lines obtained lower values. MS medium modified crop, favored the number of segments rooted and DKW (Driver and Kuniyuki, 1984 the number of segments that sprouted, the latter having lower incidence of apical necrosis.

Evidence for in vitro and in vivo expression of the conserved VAR3 (type 3) plasmodium falciparum erythrocyte membrane protein 1

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Wang, Christian W; Lavstsen, Thomas; Bengtsson, Dominique C

2012-01-01

ABSTRACT: BACKGROUND: Members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion antigen family are major contributors to the pathogenesis of P. falciparum malaria infections. The PfEMP1-encoding var genes are among the most diverse sequences in nature, but three genes......, var1, var2csa and var3 are found conserved in most parasite genomes. The most severe forms of malaria disease are caused by parasites expressing a subset of antigenically conserved PfEMP1 variants. Thus the ubiquitous and conserved VAR3 PfEMP1 is of particular interest to the research field. Evidence...... of VAR3 expression on the infected erythrocyte surface has never been presented, and var3 genes have been proposed to be transcribed and expressed differently from the rest of the var gene family members. METHODS: In this study, parasites expressing VAR3 PfEMP1 were generated using anti-VAR3 antibodies...

Microdomains in the membrane landscape shape antigen-presenting cell function.

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Zuidscherwoude, Malou; de Winde, Charlotte M; Cambi, Alessandra; van Spriel, Annemiek B

2014-02-01

The plasma membrane of immune cells is a highly organized cell structure that is key to the initiation and regulation of innate and adaptive immune responses. It is well-established that immunoreceptors embedded in the plasma membrane have a nonrandom spatial distribution that is important for coupling to components of intracellular signaling cascades. In the last two decades, specialized membrane microdomains, including lipid rafts and TEMs, have been identified. These domains are preformed structures ("physical entities") that compartmentalize proteins, lipids, and signaling molecules into multimolecular assemblies. In APCs, different microdomains containing immunoreceptors (MHC proteins, PRRs, integrins, among others) have been reported that are imperative for efficient pathogen recognition, the formation of the immunological synapse, and subsequent T cell activation. In addition, recent work has demonstrated that tetraspanin microdomains and lipid rafts are involved in BCR signaling and B cell activation. Research into the molecular mechanisms underlying membrane domain formation is fundamental to a comprehensive understanding of membrane-proximal signaling and APC function. This review will also discuss the advances in the microscopy field for the visualization of the plasma membrane, as well as the recent progress in targeting microdomains as novel, therapeutic approach for infectious and malignant diseases.

Assessment of Root Morphology and Apices of First and Second Maxillary Molars in Tehran Population

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Mandana Naseri

2015-12-01

Full Text Available Introduction: Objective: This study aimed to assess the possible variations in root canal anatomy and topography of the apices of first and second maxillary molars. Materials and methods: A total of 67 first and second maxillary permanent molars were collected. Access cavity was prepared and 2% methylene blue was injected. The teeth were demineralized by 5% nitric acid and cleared with methyl salicylate. Specimens were evaluated under stereomicroscopy and analyzed using the sample t-test. Results: Based on Vertucci’s classification, the mesiobuccal root of maxillary first molars was type I in 87.5% and type IV in 12.5% of the cases. The mesiobuccal root of second maxillary molars was type I in 60%, type II in 8.6%, type IV in 25.7% and type V in 5.7% of cases. In maxillary first and second molars, the distobuccal and palatal roots were type I in 100% of the cases. The distance of the apical constriction from the apical foramen was 0.21±0.09 mm, the distance from the apical constriction tothe anatomic apex was 0.44±0.19 mm and the distance of the apical foramen from the anatomic apex was 0.15±0.15 mm. The mean percentage of delta prevalence was 3.2% in both teeth. Conclusion: The mean distance of the apical foramen and apical constriction from the anatomic apex was less than 0.6 and 1.2 mm, respectively. In maxillary first and second molars, the mean distance of the apical constriction from the apical foramen and anatomic apex was 0.21 and 0.44, respectively and the mean distance of the apical foramen from the anatomic apex was 0.15 mm

Targeted Delivery of GP5 Antigen of PRRSV to M Cells Enhances the Antigen-Specific Systemic and Mucosal Immune Responses

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Luping Du

2018-01-01

Full Text Available Efficient delivery of antigens through oral immunization is a first and critical step for successful induction of mucosal immunity, which can provide protection against pathogens invading the mucosa. Membranous/microfold cells (M cells within the mucosa can transcytose internalized antigen without degradation and thus play an important role in initiating antigen-specific mucosal immune responses through inducing secretory IgA production. In this research, we modified poly (D, L-lactide-co-glycolide (PLGA nanoparticles (NPs with Ulex europaeus agglutinin 1 (UEA-1 and successfully prepared an oral vaccine delivery system, UEA-1/PLGA NPs. PLGA NPs were prepared using a standard double emulsion solvent evaporation technique, which can protect the entrapped PRRSV DNA vaccine [pcDNA3.1-SynORF5 (synthetic ORF5] or subunit vaccine ORF5-encoded glycoprotein (GP5 from exposure to the gastrointestinal (GI tract and release the plasmids in a controlled manner. With UEA-1 modification, the UEA-1/PLGA NPs can be effectively transported by M-cells. We investigated immune response induced by UEA-1/PLGA-SynORF5 or UEA-1/PLGA-GP5 following inoculation in mice and piglets. Compared with PLGA-SynORF5 or PLGA-GP5 NPs, UEA-1/PLGA-SynORF5, or UEA-1/PLGA-GP5 NPs stimulated significantly increased serum IgG levels and augmented intestinal IgA levels in mice and piglets (P < 0.05. Our findings indicate UEA-1/PLGA NPs can be applied as a promising and universally robust oral vaccine delivery system.

Targeted Delivery of GP5 Antigen of PRRSV to M Cells Enhances the Antigen-Specific Systemic and Mucosal Immune Responses

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Du, Luping; Yu, Zhengyu; Pang, Fengjiao; Xu, Xiangwei; Mao, Aihua; Yuan, Wanzhe; He, Kongwang; Li, Bin

2018-01-01

Efficient delivery of antigens through oral immunization is a first and critical step for successful induction of mucosal immunity, which can provide protection against pathogens invading the mucosa. Membranous/microfold cells (M cells) within the mucosa can transcytose internalized antigen without degradation and thus play an important role in initiating antigen-specific mucosal immune responses through inducing secretory IgA production. In this research, we modified poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) with Ulex europaeus agglutinin 1 (UEA-1) and successfully prepared an oral vaccine delivery system, UEA-1/PLGA NPs. PLGA NPs were prepared using a standard double emulsion solvent evaporation technique, which can protect the entrapped PRRSV DNA vaccine [pcDNA3.1-SynORF5 (synthetic ORF5)] or subunit vaccine ORF5-encoded glycoprotein (GP5) from exposure to the gastrointestinal (GI) tract and release the plasmids in a controlled manner. With UEA-1 modification, the UEA-1/PLGA NPs can be effectively transported by M-cells. We investigated immune response induced by UEA-1/PLGA-SynORF5 or UEA-1/PLGA-GP5 following inoculation in mice and piglets. Compared with PLGA-SynORF5 or PLGA-GP5 NPs, UEA-1/PLGA-SynORF5, or UEA-1/PLGA-GP5 NPs stimulated significantly increased serum IgG levels and augmented intestinal IgA levels in mice and piglets (P < 0.05). Our findings indicate UEA-1/PLGA NPs can be applied as a promising and universally robust oral vaccine delivery system. PMID:29423381

Evaluating Recombinant Antigen ROP1 Efficacy in Diagnosis of Toxoplasma Gondii Infection

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F Keshavarzi

2015-07-01

Full Text Available Introduction:Toxoplasma gondii is a ubiquitous obligate intracellular parasite with a relatively broad host range infecting both mammals and birds. Toxoplasma proteins are strong antigens that can begin strong immune reactions, among which Rhoptry protein 1 (ROP1 can be named discharging from rhoptry cell-organ. ROP1 is regarded as a competitor for recombinant vaccines against toxoplasmosis. Therefore, the main objective of the current study was to evaluate the cloning and expression of ROP1 Toxoplasma gondii in a cloning vector as well as to create this recombinant antigen in order to be applied for later uses. Methods:Genomic DNA of Toxoplasma gondii was removed and reproduced by PCR, then the PCR product was cloned into the EcoR1 and BamH1 sites of cloning vector, pUET1, and transformed into Escherichia coli BL21 plysS strain. Moreover, pcROP1 was sub-cloned into the HindIII and EcoRI sites of the pcDNA3 in order to produce recombining eukaryotic declaration vector. The cloned ROP1 was verified by PCR, limitation enzymes (HindIII and BglΙ digestion and nucleotide sequencing. Then, this recombinant antigen was covered applying IgM and ELISAIgG. Results:The study results demonstrated that a fragment of 757 bp was separated. In addition, nucleotide sequence analysis of the ROP1 cloned in pUET1vector revealed high homology (96% with RH strain Gene Bank Accession (No. M71274. Conclusion:The recombinant ROP1 antigen in an IgM Rec-ELISA test can be replaced with the tachyzoite antigen in IgG and IgM serologic tests.

The C-terminal hypervariable domain targets Aradopsis ROP9 to the invaginated pollen tube plasma membrane

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Rop9 is a small GTPase of the Type II class, whereas the often studied type I Rops play roles during pollen tube growth. In pollen, Rop9 is located at the invaginated plasma membrane that surrounds the sperm cells, whereas type I Rops are located at the apical membrane of the pollen tube. The C-ter...

Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

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Laiko, Marina; Murtazina, Rakhilya; Malyukova, Irina [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Zhu, Chengru; Boedeker, Edgar C. [Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131 (United States); Gutsal, Oksana [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); O' Malley, Robert; Cole, Robert N. [Department of Biochemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Tarr, Phillip I. [Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110 (United States); Murray, Karen F. [Department of Pediatrics, Children' s Hospital and Regional Medical Center, Seattle, WA 98105 (United States); Kane, Anne [The Tufts New England Medical Center, Boston, MA 02111 (United States); Donowitz, Mark [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Kovbasnjuk, Olga, E-mail: okovbas1@jhmi.edu [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States)

2010-02-15

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.

Membrane type 1-matrix metalloproteinase/Akt signaling axis modulates TNF-α-induced procoagulant activity and apoptosis in endothelial cells.

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Hiroshi Ohkawara

Full Text Available Membrane type 1-matrix metalloproteinase (MT1-MMP functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt in tumor necrosis factor (TNF-α-induced signaling pathways of vascular responses, including tissue factor (TF procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs. TNF-α (10 ng/mL induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1-dependent signaling pathway and nuclear factor-kB (NF-kB activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs.

Proteomic study via a non-gel based approach of meningococcal outer membrane vesicle vaccine obtained from strain CU385: a road map for discovering new antigens.

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Gil, Jeovanis; Betancourt, L Zaro H; Sardiñas, Gretel; Yero, Daniel; Niebla, Olivia; Delgado, Maité; García, Darien; Pajón, Rolando; Sánchez, Aniel; González, Luis J; Padrón, Gabriel; Campa, Concepción; Sotolongo, Franklin; Barberó, Ramón; Guillén, Gerardo; Herrera, Luis; Besada, Vladimir

2009-05-01

This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatographymass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.

Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

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Cecilia González

Full Text Available The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs, which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua. High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

Human leukocyte antigen (HLA)-G during pregnancy part I

DEFF Research Database (Denmark)

Klitkou, Louise; Dahl, Mette; Hviid, Thomas Vauvert F

2015-01-01

Human leukocyte antigen (HLA)-G is a class Ib molecule with restricted tissue distribution expressed on trophoblast cells and has been proposed to have immunomodulatory functions during pregnancy. Soluble HLA-G1 (sHLA-G1) can be generated by the shedding of membrane-bound HLA-G molecules; however...... of importance for production of sHLA-G in the mother and child, or it may support the theory that sHLA-G in the pregnant woman and the fetus is partly derived from a "shared organ", the placenta....

An Innovative Regenerative Endodontic Procedure Using Leukocyte and Platelet-rich Fibrin Associated with Apical Surgery: A Case Report.

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Pinto, Nelson; Harnish, Alexandra; Cabrera, Carolina; Andrade, Catherine; Druttman, Tony; Brizuela, Claudia

2017-11-01

Regenerative endodontic procedures (REPs) associated with apical surgery could represent an alternative treatment strategy for patients whose teeth present incomplete root formation and extensive apical lesions. Leukocyte platelet-rich fibrin (L-PRF) has potential benefits in REPs; it could promote apical root formation and optimal bone healing. The aim of this case report was to describe innovative regenerative endodontic therapy using L-PRF in the root canal and an extensive apical lesion in an immature tooth with dens invaginatus and asymptomatic apical periodontitis. A healthy 20-year-old woman was referred to the dental clinic of the Universidad de Los Andes, Santiago, Chile, for endodontic treatment in tooth # 22 with incomplete root development and an extensive apical lesion. The diagnosis was asymptomatic apical periodontitis associated with dens invaginatus type II. The patient was treated with an innovative approach using L-PRF in REPs associated with apical surgery. Follow-ups were performed at 6 months and 1 year later. They included periapical radiographs, cone-beam computed tomographic imaging, sensitivity, and vitality tests. The clinical evaluations performed at 6 months and 1 year revealed an absence of symptoms. The radiographic evaluations showed that the apical lesion was resolved. The cone-beam images indicated that the root length increased and the walls had thickened. The sensitivity tests were positive, and the laser Doppler flowmetry showed positive blood flow after 1 year. The success of the results in this case report indicate that L-PRF can be used as a complement in apical surgery and REPs and could provide an innovative alternative treatment strategy for complex clinical cases like these. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

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Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

1991-01-01

The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients. Images PMID:1774262

Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam.

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Takemae, Nobuhiro; Nguyen, Tung; Ngo, Long Thanh; Hiromoto, Yasuaki; Uchida, Yuko; Pham, Vu Phong; Kageyama, Tsutomu; Kasuo, Shizuko; Shimada, Shinichi; Yamashita, Yasutaka; Goto, Kaoru; Kubo, Hideyuki; Le, Vu Tri; Van Vo, Hung; Do, Hoa Thi; Nguyen, Dang Hoang; Hayashi, Tsuyoshi; Matsuu, Aya; Saito, Takehiko

2013-04-01

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.

Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

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Yukiko Kiniwa

Full Text Available Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+ T helper (Th cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1 as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+ T cell-mediated immunotherapy in melanoma.

Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

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Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E; Wang, Rong-Fu; Wang, Helen Y

2015-01-01

Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+) T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+) T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+) T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+) Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+) T cell-mediated immunotherapy in melanoma.

Structural Studies on Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Malaria Antigens Using Small Angle X-Ray Scattering (SAXS)

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Christoffersen, Stig

Chemistry (App I) [1]. VAR2CSA binds specifically to CSA in the placental tissue of pregnant women hereby causing severe malaria symptoms endangering both mother and child. The minimal VAR2CSA region required to effectively bind CSA was determined to be the N-terminal DBL domain, DBL2X which we locate......Infection with the pathogenic Plasmodium falciparum parasite causes the potentially deadly Malaria disease which leads to over 1 million fatalities each year according to the WHO (World Health Organization). Individuals subjected to multiple infections gradually become immune to the disease...... symptoms and vaccine research is focused on trying to mimic or advance this immune acquisition. Immunity is primarily caused by acquisition of antibodies directed against a family of Plasmodium protein antigens called PfEMP1s located on the surface of infected erythrocytes. The PfEMP1 proteins are adhesive...

Light Responsive Polymer Membranes: A Review

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Fiore Pasquale Nicoletta

2012-03-01

Full Text Available In recent years, stimuli responsive materials have gained significant attention in membrane separation processes due to their ability to change specific properties in response to small external stimuli, such as light, pH, temperature, ionic strength, pressure, magnetic field, antigen, chemical composition, and so on. In this review, we briefly report recent progresses in light-driven materials and membranes. Photo-switching mechanisms, valved-membrane fabrication and light-driven properties are examined. Advances and perspectives of light responsive polymer membranes in biotechnology, chemistry and biology areas are discussed.

The Immunogenicity of the Tumor-Associated Antigen α-Fetoprotein Is Enhanced by a Fusion with a Transmembrane Domain

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Lucile Tran

2012-01-01

Full Text Available Aim. To investigate the ability of recombinant modified vaccinia virus Ankara (rMVA vector to induce an immune response against a well-tolerated self-antigen. Methods. rMVA vectors expressing different form of α-fetoprotein (AFP were produced and characterized. Naïve mice were vaccinated with MVA vectors expressing the AFP antigen in either a secreted, or a membrane-bound, or an intracellular form. The immune response was monitored by an IFNΓ ELISpot assay and antibody detection. Results. Vaccination with the membrane-associated form of AFP induced a stronger CD8+ T-cell response compared to the ones obtained with the MVA encoding the secreted or the intracellular forms of AFP. Moreover, the vaccination with the membrane-bound AFP elicited the production of AFP-specific antibodies. Conclusions. The AFP transmembrane form is more immunogenic. Expressing a membrane-bound form in the context of an MVA vaccination could enhance the immunogenicity of a self-antigen.

Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism.

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Wani, Nissar Ahmad; Kaur, Jyotdeep

2011-03-01

We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high Km) for the substrate and by decreasing the number of transporter molecules (low Vmax) on the colon luminal surface. The decreased transport activity at the CAM was associated with down-regulation of the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism. Copyright © 2010 Wiley-Liss, Inc.

A lesão apical em cardiopatas chagásicos crônicos: estudo necroscópico Apical lesions in Chagas' heart disease patients: an autopsy study

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Cristina Brandt Friedrich Martin Gurgel

2010-12-01

Full Text Available INTRODUÇÃO: A lesão apical ventricular é típica da cardiopatia chagásica e sua presença representa risco de fenômenos tromboembólicos. O objetivo deste trabalho é avaliar a frequência de LA à necropsia de portadores de cardiopatia chagásica crônica. MÉTODOS: Análise retrospectiva de necropsias de chagásicos maiores que 17 anos. Efetuada análise estatística comparativa das variáveis clínicas e dos achados necroscópicos entre o grupo A (com lesão apical e o grupo B (ausência de lesão apical. RESULTADOS: Estudados 51 casos: 25 no grupo A (idade média de 53 anos, 64% do sexo masculinoe 26 no B.. A LA localizava-se no ventrículo esquerdo em 80% casos. No grupo B, a média de idade foi de 56 anos e 46,1% eram do sexo masculino. A forma clínica prevalente nos dois grupos foi a miopática, mas arritmia cardíaca também esteve presente em ambos (57,9% no grupo A e 32,1% no B. Foi constatada a presença de trombos em 60% dos casos do grupo A (53,3% localizados na LA e 30,7% no B; CONCLUSÕES: Houve predomínio da forma miopática nos casos com LA, com média de peso cardíaco maior em relação ao B. Em ambos os grupos observamos relação diretamente proporcional entre maior peso cardíaco e presença de tromboses. Houve predomínio do número de tromboses no grupo A, mais de 50% eram localizadas na lesão, cujo diferencial clínico principal consistiu na presença maior de arritmias. A miopatia (com aumento de peso acima de 500g foi primordial para aparecimento de tromboses.INTRODUCTION: The presence of an apical ventricular lesion increases the risk of intracardiac thrombosis and thromboembolic phenomena. The study evaluated the incidence of apical lesions and intracardiac thrombosis in Chagas' heart disease patients at autopsy. METHODS: A retrospective review of autopsies of Chagas' heart disease patients was conducted. Statistical analysis included comparison of clinical variables and autopsy findings between two groups

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane

OpenAIRE

Doudová, Lenka

2017-01-01

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane Yeast plasma membrane is divided into several different compartments. Membrane compartment of Can1 is specific for its protein and lipid composition, furthermore it creates furrow-like invaginations on the plasma membrane. These invaginations are made by multiprotein complexes called eisosomes, which are located in the cytosolic side of MCCs. It was established that this domain plays an importa...

Apical dominance and growth in vitro of Alstroemeria

NARCIS (Netherlands)

Pumisutapon, P.

2012-01-01

In Alstroemeria, micropropagation is achieved by axillary bud outgrowth. However, the multiplication rate is rather low (1.2–2.0 per cycle of 4 weeks) due to strong apical dominance. Even though several factors (i.e. culture media, growth regulators, and environmental conditions) have

Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture.

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Nakanishi, Takeo; Fukushi, Akimasa; Sato, Masanobu; Yoshifuji, Mayuko; Gose, Tomoka; Shirasaka, Yoshiyuki; Ohe, Kazuyo; Kobayashi, Masato; Kawai, Keiichi; Tamai, Ikumi

2011-12-05

Since in vitro cell culture models often show altered apical transporter expression, they are not necessarily suitable for the analysis of renal transport processes. Therefore, we aimed here to investigate the usefulness of primary-cultured rat proximal tubular cells (PTCs) for this purpose. After isolation of renal cortical cells from rat kidneys, PTCs were enriched and the gene expression and function of apical transporters were analyzed by means of microarray, RT-PCR and uptake experiments. RT-PCR confirmed that the major apical transporters were expressed in rat PTCs. Na(+)-dependent uptake of α-methyl-d-glucopyranoside (αMG), ergothioneine and carnitine by the PTCs suggests functional expression of Sglts, Octn1 and Octn2, respectively. Inhibition of pH-dependent glycylsarcosine uptake by low concentration of cephalexin, which is a β-lactam antibiotics recognized by Pepts, indicates a predominant role of high affinity type Pept2, but not low affinity type Pept1, in the PTCs. Moreover, the permeability ratio of [(14)C]αMG (apical to basolateral/basolateral to apical) across PTCs was 4.3, suggesting that Sglt-mediated reabsorptive transport is characterized. In conclusion, our results indicate that rat PTCs in primary culture are found to be a promising in vitro model to evaluate reabsorption processes mediated at least by Sglts, Pept2, Octn1 and Octn2.

WNK1 and p38-MAPK distribution in ionocytes and accessory cells of euryhaline teleost fish implies ionoregulatory function

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W. S. Marshall

2017-07-01

Full Text Available Ionocytes of euryhaline teleost fish secrete NaCl, under regulation by serine and threonine kinases, including with-no-lysine kinase (WNK1 and p38 mitogen-activated protein kinase (MAPK. Mummichogs (Fundulus heteroclitus L. were acclimated to freshwater (FW, full strength seawater (SW and hypersaline conditions (2SW. Immunocytochemistry of ionocytes in opercular epithelia of fish acclimated to SW and 2SW revealed that WNK1-anti-pT58 phosphoantibody localized strongly to accessory cells and was present in the cytosol of ionocytes, close to cystic fibrosis transmembrane conductance regulator (CFTR in the apical membrane and the sodium potassium 2 chloride cotransporter (NKCC in the basolateral membrane. In FW acclimated fish, WNK1 localized to a sub-apical zone, did not colocalize with apical membrane-located sodium chloride cotransporter (NCC, and typically was present in one cell of paired ionocytes and in some single ionocytes. Forskolin treatment (10 μM, 30 min increased WNK1 immunofluorescence in SW ionocytes only, while hypertonicity had little effect, compared to controls. Anti-p38-MAPK antibody localized to the cytosolic compartment. The distribution of WNK1 and p38MAPK is consistent with a proximal position in regulatory cascades, rather than directly affecting transporters. The strong staining of accessory cells by WNK1 phosphoantibody infers an osmoregulatory function for WNK.

Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI

Science.gov (United States)

Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing; Yang, Jenny J.

2016-06-01

Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 +/- 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 +/- 0.1 × 10-22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM-1 s-1 and r2 of 37.9 mM-1 s-1 per Gd (55.2 and 75.8 mM-1 s-1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM-1 s-1 per Gd (188.0 mM-1 s-1 per molecule) and r1 of 18.6 mM-1 s-1 per Gd (37.2 mM-1 s-1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high

Formulation of an inhibitor radiopharmaceutical of prostatic antigen of 177Lu-Glu-Nh-CO-Nh-Lys membrane

International Nuclear Information System (INIS)

Ortega S, D.

2015-01-01

The prostate specific membrane antigen (PSMA) is a zinc metalloenzyme that is expressed on the cell membrane and highly expressed in prostate cancer. Recently, it has been demonstrated that the peptide sequence Glu-Nh-CO-Nh-Lys inhibit PSMA activity through an electrostatic interaction with the Zn. Several theragnostic radiopharmaceuticals with base in 177 Lu have been developed for radiotherapy of specific molecular targets because gamma and beta emissions of the radionuclide (β = 0.498 MeV and γ= 0.133 MeV). However, there is currently no label a formulation for preparing a radiopharmaceutical of 177 Lu-Glu-Nh-CO-Nh-Lys useful treatment of prostate cancer. The aim of this research was to optimize and document the process of production of the radiopharmaceutical 177 Lu-Glu-Nh-CO-Nh-Lys for sanitary registration application before the Comision Federal para la Proteccion contra Riesgos Sanitarios (COFEPRIS). The optimization of the production process was assessed a factorial design of three variables with mixed levels (3 x 3 x 2) where the dependent variable is the radiochemical purity, the analytical method was validated by UV-Vis spectrophotometry. Next, process validation was carried out by labeling 3 lots of the optimized formulation of the radiopharmaceutical (5.55 GBq (2.16 μg) of 177 LuCl 3 , 90 mg peptide PSMA, 50 mg ascorbic acid and 150 μL of acetate buffer 1 M ph 5), long-term stability was performed by high resolution liquid chromatography) to determine its useful shelf life. 3 validation batches were prepared under protocols of Good Manufacturing Practice (GMP) in the Production Plant of Radiopharmaceuticals of the Instituto Nacional de Investigaciones Nucleares (ININ), meet specifications preset by obtaining a sterile and free development of bacterial endotoxin yields of labeled 100% and which retains its quality characteristics radiochemical purity greater than 90% for at least 15 days. (Author)

Deteksi Antigen pada Kriptokokosis

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Robiatul Adawiyah

2014-12-01

Full Text Available AbstrakKriptokokosis merupakan infeksi sistemik yang disebabkan Cryptococcus sp. Predileksi jamur tersebut adalah susunan saraf pusat dan selaput otak. Terdapat 5 spesies Cryptococcus sp. yang menyebabkan penyakit pada manusia; yang paling banyak adalah Cr. neoformans dan Cr. gattii. Diagnosis kriptokokosis ditegakkan berdasarkan gejala klinis, pemeriksaan laboratoris serta radiologis. Pemeriksaan laboratoris dilakukan dengan identifikasi morfologi, serologi danPCR. Pemeriksaan secara morfologi dengan tinta India positif  bila jumlah sel jamur 10  sel/ml spesimen. Kultur dilakukan di media sabouraud dextrose agar (SDA dan niger sheed agar (NSA, jamur tumbuh setelah 5-7 hari. Deteksi antigen dan antibodi dilakukan pada cairan tubuh dan tidak membutuhkan waktu lama. Deteksi antibodi Cr.neoformans memiliki kelemahan yaitu tidak menunjukkan hasil positif pada infeksi akut, IgA masih positif setelah 1-2 tahun fase penyembuhan, IgG dapat persisten, pada individu imunokompromis menunjukkan hasil yang sangat kompleks dan dalam menentukan diagnosis sering tidak konsisten. Polisakarida adalah komponen paling berperan dalam virulensi Cr. neoformans. Komponen polisakarida terutama glucuronoxylomannan merupakan petanda penting dalam diagnosis kriptokokosis secara serologis. Deteksi antigen Cr. neoformans memiliki kelebihan yaitu menunjukkan hasil positif pada infeksi akut/kronis, sensitivitas dan spesifisitas tinggi, dapat mendeteksi polisakarida hingga 10 ng/ml sehingga dengan kadarantigen yang minimal tetap dapat mendiagnosis kriptokokosis.Kata kunci: Cr. neoformans, glucuronoxylomannan, antigenAbstractCryptococcosis is systemic infection that caused by Cryptococcus sp. Predilection of this fungi is the central nervous system and brain membrane. There are 5 species of Cryptococcus sp. that cause cryptococcosis in human; but the majority are caused by Cr. neoformans and Cr. gattii. The diagnosis of cryptococcosis is made based on clinical symptoms

Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.

Science.gov (United States)

Kramers, C; Hylkema, M N; van Bruggen, M C; van de Lagemaat, R; Dijkman, H B; Assmann, K J; Smeenk, R J; Berden, J H

1994-01-01

Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis. Images PMID:8040312

Structural and antigenic variation among diverse clade 2 H5N1 viruses.

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David A Shore

Full Text Available Antigenic variation among circulating H5N1 highly pathogenic avian influenza A viruses mandates the continuous production of strain-specific pre-pandemic vaccine candidates and represents a significant challenge for pandemic preparedness. Here we assessed the structural, antigenic and receptor-binding properties of three H5N1 HPAI virus hemagglutinins, which were recently selected by the WHO as vaccine candidates [A/Egypt/N03072/2010 (Egypt10, clade 2.2.1, A/Hubei/1/2010 (Hubei10, clade 2.3.2.1 and A/Anhui/1/2005 (Anhui05, clade 2.3.4]. These analyses revealed that antigenic diversity among these three isolates was restricted to changes in the size and charge of amino acid side chains at a handful of positions, spatially equivalent to the antigenic sites identified in H1 subtype viruses circulating among humans. All three of the H5N1 viruses analyzed in this study were responsible for fatal human infections, with the most recently-isolated strains, Hubei10 and Egypt10, containing multiple residues in the receptor-binding site of the HA, which were suspected to enhance mammalian transmission. However, glycan-binding analyses demonstrated a lack of binding to human α2-6-linked sialic acid receptor analogs for all three HAs, reinforcing the notion that receptor-binding specificity contributes only partially to transmissibility and pathogenesis of HPAI viruses and suggesting that changes in host specificity must be interpreted in the context of the host and environmental factors, as well as the virus as a whole. Together, our data reveal structural linkages with phylogenetic and antigenic analyses of recently emerged H5N1 virus clades and should assist in interpreting the significance of future changes in antigenic and receptor-binding properties.

Paired Expression Analysis of Tumor Cell Surface Antigens

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Rimas J. Orentas

2017-08-01

Full Text Available Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19 or antibody-based therapy (anti-CD20 in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling’s T-squared test (Hotelling–Lawley multivariate analysis of variance for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues. We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK

Effect of radiation on the expression of carcinoembryonic antigen of human gastric adenocarcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Hareyama, M.; Imai, K.; Kubo, K.; Takahashi, H.; Koshiba, H.; Hinoda, Y.; Shidou, M.; Oouchi, A.; Yachi, A.; Morita, K. (Sapporo Medical College (Japan))

1991-05-01

The changes of antigenic expression of cultured human gastric adenocarcinoma MKN45 cells caused by irradiation were investigated to elucidate the immune responses to localized irradiation. The expression of carcinoembryonic antigen (CEA) showed remarkable increases in the culture supernatant and on the surface of the membrane of irradiated cells. The expression of major histocompatibility complex Class I antigen on the membrane also was enhanced by irradiation. In addition, the irradiated cell groups, when analyzed using a CEA-specific probe, showed remarkable increases in the CEA mRNA. These enhancements increased in the 10-Gy and 15-Gy irradiated populations compared with the 5-Gy irradiated population. These results suggest that the enhancement of expression of CEA by radiation takes place at the CEA gene expression (mRNA) level but not at the protein level.

Ossifying fibroma misdiagnosed as chronic apical periodontitis.

Science.gov (United States)

de Moraes Ramos-Perez, Flávia Maria; Soares, Ulysses Nicida; Silva-Sousa, Yara Teresinha Corrêa; da Cruz Perez, Danyel Elias

2010-03-01

Ossifying fibroma mimicking chronic apical periodontitis is extremely rare. This report describes a case of ossifying fibroma located in the periapical region of the mandibular right canine that was misdiagnosed as chronic apical periodontitis. A 40-year-old woman complained of slight pain in the right anterior mandibular region without mucosal abnormalities or swelling. Radiographically, a well-circumscribed, unilocular, radiolucent lesion was observed that was located in the periapical region of the mandibular right canine, which presented an endodontically treated root canal. Under local anesthesia, the lesion was fully excised. Microscopically, there was fibrocellular connective tissue associated with a mineralized component, which consisted of lamellar or trabecular and woven bone, compatible with the diagnosis of ossifying fibroma. Although it is very rare, ossifying fibroma should be considered in the differential diagnosis of unusual or persistent apical radiolucencies. Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

A 16-kilodalton lipoprotein of the outer membrane of Serpulina (Treponema) hyodysenteriae.

OpenAIRE

Thomas, W; Sellwood, R; Lysons, R J

1992-01-01

Serpulina (Treponema) hyodysenteriae P18A and VS1 were extracted by using the detergent Triton X-114 and separated into detergent and aqueous phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis confirmed that a membrane-associated 16-kDa antigen was hydrophobic, since it was found in the detergent phase. A 45-kDa antigen partitioned into the aqueous phase, suggesting that it was hydrophilic and may be of periplasmic origin. When spirochetes were g...

Prevalence of Weak D Antigen In Western Indian Population

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Tanvi Sadaria

2015-12-01

Full Text Available Introduction: Discovery of Rh antigens in 1939 by Landsteiner and Weiner was the revolutionary stage in blood banking. Of these antigens, D, which decides Rh positivity or negativity, is the most antigenic. A problem is encountered when an individual has a weakened expression of D (Du, i.e., fewer numbers of D antigens on red cell membrane. Aims and Objectives: To know the prevalence of weak D in Indian population because incidence varies in different population. To determine the risk of alloimmunization among Rh D negative patients who receives the blood of weak D positive donors. Material and Methods: Rh grouping of 38,962 donors who came to The Department of Immunohematology and Blood Transfusion of Civil Hospital, Ahmedabad from 1st January 2013 to 30th September 2014 was done using the DIAGAST (Automated Grouping. The samples that tested negative for D antigen were further analysed for weak D (Du by indirect antiglobulin test using blend of Ig G and Ig M Anti D. This was done using Column agglutination method in ID card (gel card. Results: The total number of donors studied was 38,962. Out of these 3360(8.6% were tested Rh D negative. All Rh D negative donors were tested for weak D (Du. 22 (0.056% of total donors and 0.65% of Rh negative donors turned out to be weak D (Du positive. Conclusion: The prevalence of weak D (Du in Western Indian population is 0.056 %, So the risk of alloimmunization in our setting due to weak D (Du antigen is marginal. But, testing of weak D antigen is necessary in blood bank because weak D antigen is immunogenic and can produce alloimmunization if transfused to Rh D negative subjects.

Identification of immunogenic outer membrane proteins of Haemophilus influenzae type b in the infant rat model system

International Nuclear Information System (INIS)

Hansen, E.J.; Frisch, C.F.; McDade, R.L. Jr.; Johnston, K.H.

1981-01-01

Outer membrane proteins of Haemophilus influenzae type b which are immunogenic in infant rats were identified by a radioimmunoprecipitation method. Intact cells of H. influenzae type b were radioiodinated by a lactoperoxidase-catalyzed procedure, and an outer membrane-containing fraction was prepared from these cells. These radioiodinated outer membranes were mixed with sera obtained from rats convalescing from systemic H. influenzae type b disease induced at 6 days of age, and the resultant (antibody-outer membrane protein antigen) complexes were extracted from these membranes by treatment with nonionic detergent and ethylenediaminetetraacetic acid. These soluble antibody-antigen complexes were isolated by means of adsorption to protein A-bearing staphylococci, and the radioiodinated protein antigens were identified by gel electrophoresis followed by autoradiography. Infant rats were shown to mount a readily detectable antibody response to several different proteins present in the outer membrane of H. influenzae type b. Individual infant rats were found to vary both qualitatively and quantitatively in their immune response to these immunogenic outer membrane proteins

Association analysis of clinical aspects and vitamin D receptor gene polymorphism with external apical root resorption in orthodontic patients.

Science.gov (United States)

Fontana, Maria Luiza S Simas Netta; de Souza, Cleber Machado; Bernardino, José Fabio; Hoette, Felix; Hoette, Maura Levi; Thum, Lotario; Ozawa, Terumi O; Capelozza Filho, Leopoldino; Olandoski, Marcia; Trevilatto, Paula Cristina

2012-09-01

Vitamin D is responsible for the regulation of certain genes at the transcription level, via interaction with the vitamin D receptor, and influences host immune responses and aspects of bone development, growth, and homeostasis. Our aim was to investigate the association of TaqI vitamin D receptor gene polymorphism with external apical root resorption during orthodontic treatment. Our subjects were 377 patients with Class II Division 1 malocclusion, divided into 3 groups: (1) 160 with external apical root resorption ≤1.43 mm, (2) 179 with external apical root resorption >1.43 mm), and (3) 38 untreated subjects. External apical root resorption of the maxillary incisors was evaluated on periapical radiographs taken before and after 6 months of treatment. After DNA collection and purification, vitamin D receptor TaqI polymorphism analysis was performed by polymerase chain reaction-restriction fragment length polymorphism. Univariate and multivariate analyses were performed to verify the association of clinical and genetic variables with external apical root resorption (P root resorption in orthodontically treated patients compared with the untreated subjects. In patients orthodontically treated, age higher than 14 years old, initial size of the maxillary incisor root superior to 30 mm, and premolar extraction were associated with increased external apical root resorption. Genotypes containing the C allele were weakly associated with protection against external apical root resorption (CC + CT × TT [odds ratio, 0.29; 95% confidence interval, 0.07-1.23; P = 0.091]) when treated orthodontic patients were compared to untreated individuals. Clinical factors and vitamin D receptor TaqI polymorphism were associated with external apical root resorption in orthodontic patients. Copyright © 2012 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Invasive breast cancer in Argentine women: association between risk and prognostic factors with antigens of a peptidic and carbohydrate nature

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Croce MV

2011-12-01

Full Text Available Sandra O Demichelis, Marina T Isla-Larrain, Luciano Cermignani, Cecilio G Alberdi, Amada Segal-Eiras, María Virginia CroceCentre of Basic and Applied Immunological Research, Faculty of Medical Sciences, National University of La Plata, La Plata, ArgentinaObjective: In breast cancer, several tumor markers have been identified. The marker most extensively associated with breast cancer is MUC1. The objective of the study was to analyze prognostic and risk factors in relation to tumor markers in order to clarify breast cancer biology. A total of 349 primary tumor samples and lymph nodes from breast cancer patients were studied. Risk and prognostic factors were considered. An immunohistochemical approach was applied and an extensive statistical analysis was performed, including frequency analysis and analysis of variance. Correlation among variables was performed with principal component analysis.Results: All the antigens showed an increased expression according to tumor size increment; moreover, sialyl Lewis x expression showed a significant increase in relation to disease stage, whereas Tn and TF presented a positive tendency. Vascular invasion was related to sialyl Lewis x expression and number of metastatic lymph nodes. Taking into account risk factors, when a patient had at least one child, Lewis antigens diminished their expression. In relation to breastfeeding, sialyl Lewis x expression diminished, although its apical expression increased.Conclusion: Associations between MUC1 and carbohydrate antigens and risk and prognostic factors show the complexity of the cellular biological behavior that these antigens modulate in breast cancer.Keywords: breast cancer, Argentine women, risk factors, prognostic factors, antigenic expression

Immunodiagnostic Value of Echinococcus Granulosus Recombinant B8/1 Subunit of Antigen B.

Science.gov (United States)

Savardashtaki, Amir; Sarkari, Bahador; Arianfar, Farzane; Mostafavi-Pour, Zohreh

2017-06-01

Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized. The sequence was synthesized and inserted into pGEX-4T-1 vector. Purification was performed with GST tag affinity column. Diagnostic performance of the produced recombinant antigen, native antigen B and a commercial ELISA kit were further evaluated in an ELISA system, using a panel of sera from CE patients and controls. SDS-PAGE demonstrated that the protein of interest had a high expression level and purity after GST tag affinity purification. Western blotting verified the immunoreactivity of the produced recombinant antigen with the sera of CE patients. In an ELISA system, the sensitivity and specificity (for human CE diagnosis) of the recombinant antigen, native antigen B and commercial kit were respectively 93% and 92%, 87% and 90% and 97% and 95%. The produced recombinant antigen showed a high diagnostic value which can be recommended for serodiagnosis of CE in Iran and other CE-endemic areas. Utilizing the combination of other subunits of AgB8 would improve the performance value of the introduced ELISA system.

Membrane Type 1–Matrix Metalloproteinase/Akt Signaling Axis Modulates TNF-α-Induced Procoagulant Activity and Apoptosis in Endothelial Cells

Science.gov (United States)

Ohkawara, Hiroshi; Ishibashi, Toshiyuki; Sugimoto, Koichi; Ikeda, Kazuhiko; Ogawa, Kazuei; Takeishi, Yasuchika

2014-01-01

Membrane type 1–matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs. PMID:25162582

Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children

DEFF Research Database (Denmark)

Dodoo, D; Staalsoe, T; Giha, H

2001-01-01

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand to endoth...

Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

International Nuclear Information System (INIS)

Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

1987-01-01

The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

Apical extrusion of debris in primary molar root canals using mechanical and manual systems.

Science.gov (United States)

Buldur, B; Hascizmeci, C; Aksoy, S; Nur Aydin, M; Guvendi, O N

2018-03-01

Apical extrusion of debris in primary root canal treatment has not been well elucidated. The purpose of this study is to compare the amount of apically extruded debris during the preparation of primary molar root canals using ProTaper, ProTaper Next, Self-adjusting File (SAF) and hand files. One hundred sixty extracted primary mandibular molar teeth were assigned to 2 groups: Group 1: Resorbed (n=80) and Group 2: Non-resorbed (n=80) and randomly to four subgroups (n=20 teeth for each subgroup) according to the instruments used, ProTaper, ProTaper Next, SAF, and hand file. The apically extruded debris was collected and dried in preweighed Eppendof tubes. The dry weight was calculated by subtracting the preoperative weight from the postoperative weight. Data were analysed statistically using the ANOVA and the Bonferroni post hoc t-test. The amount of apically extruded debris was significantly less for the non-resorbed group compared to the resorbed group (PProTaper Next and SAF extruded significantly less debris than did the ProTaper and hand files (PProTaper Next and SAF (P>0.05). All instruments caused apically extruded debris in primary teeth.

Solution Structure, Membrane Interactions, and Protein Binding Partners of the Tetraspanin Sm-TSP-2, a Vaccine Antigen from the Human Blood Fluke Schistosoma mansoni*

Science.gov (United States)

Jia, Xinying; Schulte, Leigh; Loukas, Alex; Pickering, Darren; Pearson, Mark; Mobli, Mehdi; Jones, Alun; Rosengren, Karl J.; Daly, Norelle L.; Gobert, Geoffrey N.; Jones, Malcolm K.; Craik, David J.; Mulvenna, Jason

2014-01-01

The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument. PMID:24429291

Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke Schistosoma mansoni.

Science.gov (United States)

Jia, Xinying; Schulte, Leigh; Loukas, Alex; Pickering, Darren; Pearson, Mark; Mobli, Mehdi; Jones, Alun; Rosengren, Karl J; Daly, Norelle L; Gobert, Geoffrey N; Jones, Malcolm K; Craik, David J; Mulvenna, Jason

2014-03-07

The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.

The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors.

Directory of Open Access Journals (Sweden)

Luciana Galetto

Full Text Available Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP strain, and three others as non-vectors. Interactions between a labelled (recombinant CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

Single Versus Multi-visit Endodontic Treatment of Teeth with Apical ...

African Journals Online (AJOL)

2016 Annals of Medical and Health Sciences Research | Published by Wolters ... apical periodontitis: An in vivo study with 1-year evaluation. ... temporary filling and dressing during the interim period in ..... Financial support and sponsorship.

Monocyte chemotactic protein-3: possible involvement in apical periodontitis chemotaxis.

Science.gov (United States)

Dezerega, A; Osorio, C; Mardones, J; Mundi, V; Dutzan, N; Franco, M; Gamonal, J; Oyarzún, A; Overall, C M; Hernández, M

2010-10-01

To study the expression of monocyte chemotactic protein-3 (MCP-3, also known as chemokine CCL-7) in tissue from apical lesions (AL) and to associate MCP-3 expression with symptomatic or asymptomatic apical periodontitis. To determine the expression of MCP-3 in AL, biopsies obtained during tooth extraction procedures were fixed, subjected to routine processing and diagnosed as apical granuloma (AG) (n = 7) or radicular cyst (RC) (n = 5). As controls, apical periodontal ligament (PDL) specimens from healthy premolars extracted for orthodontics reasons were included (n = 7). All specimens were immunostained for MCP-3 and examined under a light microscope. In addition, homogenates from AL (n = 14) and healthy PDL samples (n = 7) were studied through immunowestern blot. Finally, periapical exudates samples were collected from root canals of teeth having diagnosis of symptomatic (n = 14) and asymptomatic apical periodontitis (n = 14) during routine endodontic treatments and analysed by immunowestern blot and densitometry.   MCP-3 was detected in AG and RC and localized mainly to inflammatory leucocytes, whereas no expression was observed in healthy PDLs. MCP-3 was also detected in periapical exudate, and its levels were significantly higher in symptomatic than in asymptomatic apical periodontitis. MCP-3 was expressed in AL and its levels associated with clinical symptoms. MCP-3 might play a role in disease pathogenesis, possibly by stimulating mononuclear chemotaxis. © 2010 International Endodontic Journal.

Study of CD69 antigen expression and integrity of leukocyte cellular membrane in stored platelet concentrates following irradiation and treatment with Mirasol® PRT System.

Science.gov (United States)

Lachert, Elżbieta; Woźniak, Jolanta; Antoniewicz-Papis, Jolanta; Krzywdzińska, Agnieszka; Kubis, Jolanta; Mikołowska, Agata; Letowska, Magdalena

2017-01-01

Leukocytes in transfused blood components, particularly residual lymphocytes, have been shown to contribute to the occurrence of various adverse reactions. One of the most severe is transfusionassociated graft versus host disease (TA-GvHD) following transfusion of blood components contaminated with immunocompetent T lymphocytes. Irradiation is a routine method for protection against TA-GvHD. According to the literature, some pathogen reduction methods have also been proven effective for the inactivation of T lymphocytes, and so they may be considered as an alternative to irradiation. Comparison of CD69 antigen expression and the integrity of the leukocyte cellular membrane in stored platelet concentrates (PCs) following irradiation with the Gammacell 3000 Elan (Nordion Inc., Ottawa, Canada) and treatment with the Mirasol® Pathogen Reduction Technology (PRT) System (Terumo BCT, Lakewood, USA). The study included seven experiments. For each experiment we used 3 PCs, for Mirasol® PRT System treatment (M), for Gammacell 3000 Elan irradiation (R), and for the control (C). 7-amino-actinomycin D (7-AAD, Becton Dickinson, Franklin Lakes, USA) permeability was used to determine lymphocyte viability while CD69 antigen expression was the marker of lymphocyte activation. Analyses of 7-AAD and CD69 antigen expression were performed in a FACS Canto I flow cytometer (Becton Dickinson, USA). During 6 storage days, viable lymphocyte count decreased to 28% (p = 0.001) in the Mirasol® PRT System treated PCs and to 65% (p = 0.004) in the irradiated PCs. A statistically significant increase in CD69 expression in the irradiated PCs was observed; 1.3-fold on day 3 and 1.5-fold on day 6. In the Mirasol ® PRT System treated PCs, no statistically significant increase was observed. The in vitro results suggest that the Mirasol® PRT System is as effective as irradiation due to donor leukocyte inactivation capacity.

Pro-oxidant status and matrix metalloproteinases in apical lesions and gingival crevicular fluid as potential biomarkers for asymptomatic apical periodontitis and endodontic treatment response

Directory of Open Access Journals (Sweden)

Dezerega Andrea

2012-03-01

Full Text Available Abstract Background Oxidative stress and matrix metalloproteinases -9 and -2 are involved in periodontal breakdown, whereas gingival crevicular fluid has been reported to reflect apical status. The aim of this study was to characterize oxidant balance and activity levels of MMP -2 and -9 in apical lesions and healthy periodontal ligament; and second, to determine whether potential changes in oxidant balance were reflected in gingival crevicular fluid from asymptomatic apical periodontitis (AAP-affected teeth at baseline and after endodontic treatment. Methods Patients with clinical diagnosis of AAP and healthy volunteers having indication of tooth extraction were recruited. Apical lesions and healthy periodontal ligaments, respectively, were homogenized or processed to obtain histological tissue sections. Matrix metalloproteinase -9 and -2 levels and/or activity were analyzed by Immunowestern blot, zymography and consecutive densitometric analysis, and their tissue localization was confirmed by immunohistochemistry. A second group of patients with AAP and indication of endodontic treatment was recruited. Gingival crevicular fluid was extracted from AAP-affected teeth at baseline, after endodontic treatment and healthy contralateral teeth. Total oxidant and antioxidant status were determined in homogenized tissue and GCF samples. Statistical analysis was performed using STATA v10 software with unpaired t test, Mann-Whitney test and Spearman's correlation. Results Activity of MMP-2 and MMP-9 along with oxidant status were higher in apical lesions (p Conclusions Apical lesions display an oxidant imbalance along with increased activity of matrix metalloproteinase-2 and -9 and might contribute to AAP progression. Oxidant imbalance can also be reflected in GCF from AAP-affected teeth and was restored to normal levels after conservative endodontic treatment. These mediators might be useful as potential biomarkers for chair-side complementary diagnostic

Pro-oxidant status and matrix metalloproteinases in apical lesions and gingival crevicular fluid as potential biomarkers for asymptomatic apical periodontitis and endodontic treatment response.

Science.gov (United States)

Dezerega, Andrea; Madrid, Sonia; Mundi, Verónica; Valenzuela, María A; Garrido, Mauricio; Paredes, Rodolfo; García-Sesnich, Jocelyn; Ortega, Ana V; Gamonal, Jorge; Hernández, Marcela

2012-03-21

Oxidative stress and matrix metalloproteinases -9 and -2 are involved in periodontal breakdown, whereas gingival crevicular fluid has been reported to reflect apical status. The aim of this study was to characterize oxidant balance and activity levels of MMP -2 and -9 in apical lesions and healthy periodontal ligament; and second, to determine whether potential changes in oxidant balance were reflected in gingival crevicular fluid from asymptomatic apical periodontitis (AAP)-affected teeth at baseline and after endodontic treatment. Patients with clinical diagnosis of AAP and healthy volunteers having indication of tooth extraction were recruited. Apical lesions and healthy periodontal ligaments, respectively, were homogenized or processed to obtain histological tissue sections. Matrix metalloproteinase -9 and -2 levels and/or activity were analyzed by Immunowestern blot, zymography and consecutive densitometric analysis, and their tissue localization was confirmed by immunohistochemistry. A second group of patients with AAP and indication of endodontic treatment was recruited. Gingival crevicular fluid was extracted from AAP-affected teeth at baseline, after endodontic treatment and healthy contralateral teeth. Total oxidant and antioxidant status were determined in homogenized tissue and GCF samples. Statistical analysis was performed using STATA v10 software with unpaired t test, Mann-Whitney test and Spearman's correlation. Activity of MMP-2 and MMP-9 along with oxidant status were higher in apical lesions (p Apical lesions display an oxidant imbalance along with increased activity of matrix metalloproteinase-2 and -9 and might contribute to AAP progression. Oxidant imbalance can also be reflected in GCF from AAP-affected teeth and was restored to normal levels after conservative endodontic treatment. These mediators might be useful as potential biomarkers for chair-side complementary diagnostic of apical status in GCF.

Anti-Chol-1 antigen, GQ1bα, antibodies are associated with Alzheimer's disease.

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Toshio Ariga

Full Text Available The interaction of amyloid β-proteins (Aβ with membrane gangliosides has been reported to be an early event in Aβ fibril formation in Alzheimer's disease (AD. Neuronal degeneration in AD has been postulated to be associated with the presence of anti-ganglioside antibodies in patient sera. Using an enzyme-linked immunosorbent assay (ELISA and high-performance thin-layer chromatography (HPTLC immunostaining, sera from 27 individuals (10 with AD, 6 with vascular dementia (VD, and 11 non-demented age-matched pathological controls were examined in order to detect anti-glycosphingolipid (GSL antibodies, including anti-cholinergic-specific antigen (Chol-1α; GQ1bα antibodies. All sera had natural antibodies against ganglio-N-tetraosyl gangliosides (brain-type gangliosides. However, sera of demented patients with AD and VD had significantly higher titers of anti-GSL antibodies than those in age-matched pathological controls. Although most serum antibodies, including anti- GM1, -GT1b, -GQ1b, -GQ1bα, were of the IgM type, the presence of the IgG type antibodies was also significantly elevated in the sera of demented patients with AD. Anti-GT1b antibodies of the IgG type were elevated in AD (90%, 9 of 10 cases and VD (100%, respectively. Most surprisingly, anti-GQ1bα antibodies (IgM were found in 90% (9/10 and 100% (6/6 in the sera of patients with AD and VD, respectively. Since GQ1bα is present in the cerebral cortex and hippocampus, the presence of anti-GQ1bα antibodies may play an important role in disrupting cholinergic synaptic transmission and may participate in the pathogenesis of dementia. We conclude that elevated anti-GSL antibody titers may be useful as an aid for clinical diagnosis of those dementias.

Effect of Instrumentation Length and Instrumentation Systems: Hand Versus Rotary Files on Apical Crack Formation - An In vitro Study.

Science.gov (United States)

Devale, Madhuri R; Mahesh, M C; Bhandary, Shreetha

2017-01-01

Stresses generated during root canal instrumentation have been reported to cause apical cracks. The smaller, less pronounced defects like cracks can later propagate into vertical root fracture, when the tooth is subjected to repeated stresses from endodontic or restorative procedures. This study evaluated occurrence of apical cracks with stainless steel hand files, rotary NiTi RaCe and K3 files at two different instrumentation lengths. In the present in vitro study, 60 mandibular premolars were mounted in resin blocks with simulated periodontal ligament. Apical 3 mm of the root surfaces were exposed and stained using India ink. Preoperative images of root apices were obtained at 100x using stereomicroscope. The teeth were divided into six groups of 10 each. First two groups were instrumented with stainless steel files, next two groups with rotary NiTi RaCe files and the last two groups with rotary NiTi K3 files. The instrumentation was carried out till the apical foramen (Working Length-WL) and 1 mm short of the apical foramen (WL-1) with each file system. After root canal instrumentation, postoperative images of root apices were obtained. Preoperative and postoperative images were compared and the occurrence of cracks was recorded. Descriptive statistical analysis and Chi-square tests were used to analyze the results. Apical root cracks were seen in 30%, 35% and 20% of teeth instrumented with K-files, RaCe files and K3 files respectively. There was no statistical significance among three instrumentation systems in the formation of apical cracks (p=0.563). Apical cracks were seen in 40% and 20% of teeth instrumented with K-files; 60% and 10% of teeth with RaCe files and 40% and 0% of teeth with K3 files at WL and WL-1 respectively. For groups instrumented with hand files there was no statistical significance in number of cracks at WL and WL-1 (p=0.628). But for teeth instrumented with RaCe files and K3 files significantly more number of cracks were seen at WL than

Prevalence of ciliated epithelium in apical periodontitis lesions.

Science.gov (United States)

Ricucci, Domenico; Loghin, Simona; Siqueira, José F; Abdelsayed, Rafik A

2014-04-01

This article reports on the morphologic features and the frequency of ciliated epithelium in apical cysts and discusses its origin. The study material consisted of 167 human apical periodontitis lesions obtained consecutively from patients presenting for treatment during a period of 12 years in a dental practice operated by one of the authors. All of the lesions were obtained still attached to the root apices of teeth with untreated (93 lesions) or treated canals (74 lesions). The former were obtained by extraction and the latter by extraction or apical surgery. Specimens were processed for histopathologic and histobacteriologic analyses. Lesions were classified, and the type of epithelium, if present, was recorded. Of the lesions analyzed, 49 (29%) were diagnosed as cysts. Of these, 26 (53%) were found in untreated teeth, and 23 (47%) related to root canal-treated teeth. Ciliated columnar epithelium was observed partially or completely lining the cyst wall in 4 cysts, and all of them occurred in untreated maxillary molars. Three of these lesions were categorized as pocket cysts, and the other was a true cyst. Ciliated columnar epithelium-lined cysts corresponded to approximately 2% of the apical periodontitis lesions and 8% of the cysts of endodontic origin in the population studied. This epithelium is highly likely to have a sinus origin in the majority of cases. However, the possibility of prosoplasia or upgraded differentiation into ciliated epithelium from the typical cystic lining squamous epithelium may also be considered. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Apical extrusion of debris using reciprocating files and rotary ...

African Journals Online (AJOL)

Procedure: Sixty extracted human mandibular premolars were used. The root canals were instrumented using reciprocating (WaveOne, Reciproc, SafeSider) or rotary ... and cross‑sections, and kinematics, and this situation may influence the amount of apically extruded debris through the apical foramen.[15]. The aim of this ...

The role of CD4 in antigen-independent activation of isolated single T lymphocytes

DEFF Research Database (Denmark)

Kelso, A; Owens, T

1988-01-01

The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An ass...

Incidental apical disease at CT scanning

International Nuclear Information System (INIS)

McLoud, T.C.; Satoh, K.; Shepard, J.O.; Moore, E.H.; Kosiuk, J.P.

1990-01-01

Apical caps are commonly noted on standard radiographs. This paper determines how often abnormalities in the extreme apex of the lung could be identified on CT scans obtained for other reasons. A total of 158 consecutive CT scans were reviewed prospectively. Excluded were patients with obvious upper lobe pleural or parenchymal disease. Apical abnormalities were identified in 74 (46.8%) of the 158 cases. The prevalence increased with age (19% in the 8-39-year age group and 82% in patients older than 80 years). Opacities were unilateral in 44.5% and bilateral in 55.5%. The most common abnormality was linear opacities (95%)

Blood group antigen studies using CdTe quantum dots and flow cytometry

Directory of Open Access Journals (Sweden)

Cabral Filho PE

2015-07-01

Full Text Available Paulo E Cabral Filho,1 Maria IA Pereira,1 Heloise P Fernandes,2 Andre A de Thomaz,3 Carlos L Cesar,3 Beate S Santos,4 Maria L Barjas-Castro,2 Adriana Fontes1 1Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco, Recife, Pernambuco, 2Centro de Hematologia e Hemoterapia, Universidade Estadual de Campinas, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, São Paulo, 3Departamento de Eletrônica Quântica, Instituto de Física Gleb Wataghin, Universidade Estadual de Campinas, Campinas, São Paulo, 4Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife, PE, Brazil Abstract: New methods of analysis involving semiconductor nanocrystals (quantum dots [QDs] as fluorescent probes have been highlighted in life science. QDs present some advantages when compared to organic dyes, such as size-tunable emission spectra, broad absorption bands, and principally exceptional resistance to photobleaching. Methods applying QDs can be simple, not laborious, and can present high sensibility, allowing biomolecule identification and quantification with high specificity. In this context, the aim of this work was to apply dual-color CdTe QDs to quantify red blood cell (RBC antigen expression on cell surface by flow cytometric analysis. QDs were conjugated to anti-A or anti-B monoclonal antibodies, as well as to the anti-H (Ulex europaeus I lectin, to investigate RBCs of A1, B, A1B, O, A2, and Aweak donors. Bioconjugates were capable of distinguishing the different expressions of RBC antigens, both by labeling efficiency and by flow cytometry histogram profile. Furthermore, results showed that RBCs from Aweak donors present fewer amounts of A antigens and higher amounts of H, when compared to A1 RBCs. In the A group, the amount of A antigens decreased as A1 > A3 > AX = Ael, while H antigens were AX = Ael > A1. Bioconjugates presented stability and remained active for at least 6 months. In conclusion

Microdomains in the membrane landscape shape antigen-presenting cell function

NARCIS (Netherlands)

Zuidscherwoude, M.; Winde, C.M. de; Cambi, A.; Spriel, A.B. van

2014-01-01

The plasma membrane of immune cells is a highly organized cell structure that is key to the initiation and regulation of innate and adaptive immune responses. It is well-established that immunoreceptors embedded in the plasma membrane have a nonrandom spatial distribution that is important for

Human leukocyte antigen (HLA) polymorphism and type 1 diabetes ...

African Journals Online (AJOL)

Insulin-dependent diabetes mellitus or type 1 diabetes is an autoimmune multifactorial disease which has a great socio-economic impact. In Morocco, less is known about the contribution of Human leukocyte antigen (HLA) alleles to type 1 diabetes susceptibility. Our study focused on evaluating the distribution of class II ...

Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

Science.gov (United States)

Nováková, Zora; Foss, Catherine A; Copeland, Benjamin T; Morath, Volker; Baranová, Petra; Havlínová, Barbora; Skerra, Arne; Pomper, Martin G; Barinka, Cyril

2017-05-01

Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Frequency and levels of candidate endodontic pathogens in acute apical abscesses as compared to asymptomatic apical periodontitis

Science.gov (United States)

Rôças, Isabela N.

2018-01-01

Introduction Acute apical abscess is caused by bacteria that leave the infected dental root canal to invade the periodontal tissues. Most species occurring in abscesses are also found in asymptomatic infections; therefore, the possibility exists that not only the presence of certain species but also their specific counts influence the appearance of symptoms. This molecular study compared the frequency and levels of several candidate endodontic pathogens in teeth with acute apical abscesses and asymptomatic apical periodontitis. Methods Samples were taken from the root canals of teeth with asymptomatic apical periodontitis (n = 73) and by aspiration of purulent exudate from acute abscesses (n = 55). DNA was extracted from samples and bacterial identifications were performed by a closed-ended semi-quantitative reverse-capture checkerboard approach targeting 40 bacterial species/phylotypes. Results Bacterial DNA was detected in all cases. In abscesses, the most prevalent taxa were Fusobacterium nucleatum (60%), Porphyromonas endodontalis (53%), Parvimonas micra (51%), and Streptococcus species (45%). The most frequently detected taxa in asymptomatic teeth were P. endodontalis (63%), Dialister invisus (58%), Olsenella uli (56%), and F. nucleatum (51%). None of the targeted taxa were significantly associated with abscesses when only mere presence was evaluated (p>0.05). However, semi-quantitative data demonstrated that P. endodontalis, Prevotella baroniae, Treponema denticola and Streptococcus species were significantly more frequent at levels >105 in abscesses than in asymptomatic cases (pabscesses in terms of frequency. However, some taxa were significantly found in higher levels in abscesses. Presence of a potentially virulent pathogen in high counts may increase the collective pathogenicity of the bacterial community and give rise to symptoms. PMID:29293651

Frequency and levels of candidate endodontic pathogens in acute apical abscesses as compared to asymptomatic apical periodontitis.

Science.gov (United States)

Rôças, Isabela N; Siqueira, José F

2018-01-01

Acute apical abscess is caused by bacteria that leave the infected dental root canal to invade the periodontal tissues. Most species occurring in abscesses are also found in asymptomatic infections; therefore, the possibility exists that not only the presence of certain species but also their specific counts influence the appearance of symptoms. This molecular study compared the frequency and levels of several candidate endodontic pathogens in teeth with acute apical abscesses and asymptomatic apical periodontitis. Samples were taken from the root canals of teeth with asymptomatic apical periodontitis (n = 73) and by aspiration of purulent exudate from acute abscesses (n = 55). DNA was extracted from samples and bacterial identifications were performed by a closed-ended semi-quantitative reverse-capture checkerboard approach targeting 40 bacterial species/phylotypes. Bacterial DNA was detected in all cases. In abscesses, the most prevalent taxa were Fusobacterium nucleatum (60%), Porphyromonas endodontalis (53%), Parvimonas micra (51%), and Streptococcus species (45%). The most frequently detected taxa in asymptomatic teeth were P. endodontalis (63%), Dialister invisus (58%), Olsenella uli (56%), and F. nucleatum (51%). None of the targeted taxa were significantly associated with abscesses when only mere presence was evaluated (p>0.05). However, semi-quantitative data demonstrated that P. endodontalis, Prevotella baroniae, Treponema denticola and Streptococcus species were significantly more frequent at levels >105 in abscesses than in asymptomatic cases (p<0.05). None of the target species/phylotypes were associated with abscesses in terms of frequency. However, some taxa were significantly found in higher levels in abscesses. Presence of a potentially virulent pathogen in high counts may increase the collective pathogenicity of the bacterial community and give rise to symptoms.

Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera.

Science.gov (United States)

Hoelzle, Ludwig E; Hoelzle, Katharina; Wittenbrink, Max M

2004-10-05

Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.

Library of monoclonal antibodies against brush border membrane epithelial antigens

International Nuclear Information System (INIS)

Behar, M.; Katz, A.; Silverman, M.

1986-01-01

A purified fraction of proximal tubule brush border membranes (BBM) was prepared from dog kidney and used to immunize mice. The standard technique of hybridoma production was followed as described by Kohler and Milstein. Production of antibodies was detected by indirect immunofluorescence on dog kidney slices and by immunodot against the purified fraction on nitrocellulose. Five hybrids exhibited anti BBM activity. These were cloned twice and yielded stable cell lines producing IgG type monoclonal antibodies against BBM. They were designated A 1 , C 7 , D 3 , D 7 and H 4 . As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D 3 exhibits even broader specificity for epithelium reacting with bile canaliculi and choroid plexus. The authors have verified that at least 4/5 antibodies are directed against BBM protein as revealed by immunoprecipitation of solubilized BBM and detected by Coomassie blue staining or autoradiography of lactoperoxidase labelled BBM. Most interestingly all antibodies bind to the surface of LL CPK 1 cells, a continuous pig kidney cell line of undefined origin but exhibiting many characteristics of proximal tubule cells. The library of monoclonal antibodies obtained provide important probes with which to study membrane biogenesis and polarization in epithelial cells