Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

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Liu, Ou; Grant, Barth D.

2015-01-01

The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions. PMID:26393361

Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis.

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Youhei Egami

Full Text Available Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

Contrasting patterns in the evolution of the Rab GTPase family in Archaeplastida

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Romana Petrželková

2014-12-01

Full Text Available Rab GTPases are a vast group of proteins serving a role of master regulators in membrane trafficking in eukaryotes. Previous studies delineated some 23 Rab and Rab-like paralogs ancestral for eukaryotes and mapped their current phylogenetic distribution, but the analyses relied on a limited sampling of the eukaryotic diversity. Taking advantage of the recent growth of genome and transcriptome resources for phylogenetically diverse plants and algae, we reanalyzed the evolution of the Rab family in eukaryotes with the primary plastid, collectively constituting the presumably monophyletic supergroup Archaeplastida. Our most important novel findings are as follows: (i the ancestral set of Rabs in Archaeplastida included not only the paralogs Rab1, Rab2, Rab5, Rab6, Rab7, Rab8, Rab11, Rab18, Rab23, Rab24, Rab28, IFT27, and RTW (=Rabl2, as suggested previously, but also Rab14 and Rab34, because Rab14 exists in glaucophytes and Rab34 is present in glaucophytes and some green algae; (ii except in embryophytes, Rab gene duplications have been rare in Archaeplastida. Most notable is the independent emergence of divergent, possibly functionally novel, in-paralogs of Rab1 and Rab11 in several archaeplastidial lineages; (iii recurrent gene losses have been a significant factor shaping Rab gene complements in archaeplastidial species; for example, the Rab21 paralog was lost at least six times independently within Archaeplastida, once in the lineage leading to the “core” eudicots; (iv while the glaucophyte Cyanophora paradoxa has retained the highest number of ancestral Rab paralogs among all archaeplastidial species studied so far, rhodophytes underwent an extreme reduction of the Rab gene set along their stem lineage, resulting in only six paralogs (Rab1, Rab2, Rab6, Rab7, Rab11, and Rab18 present in modern red algae. Especially notable is the absence of Rab5, a virtually universal paralog essential for the endocytic pathway, suggesting that endocytosis

CED-10/Rac1 regulates endocytic recycling through the RAB-5 GAP TBC-2.

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Lin Sun

Full Text Available Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.

A role for Na+,K+-ATPase α1 in regulating Rab27a localisation on melanosomes.

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Antonia E G Booth

Full Text Available The mechanism(s by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1 as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP, is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.

Rab11-family of interacting protein 2 associates with chlamydial inclusions through its Rab-binding domain and promotes bacterial multiplication.

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Leiva, Natalia; Capmany, Anahí; Damiani, María Teresa

2013-01-01

Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named 'inclusion' and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11-Family of Interacting Proteins, presents at the C-terminus a Rab-binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP-tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11-Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab-binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab-binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion. © 2012 Blackwell Publishing Ltd.

Revealing localization and regulation of GTPase PmRab7 in lymphoid cells of Penaeus monodon after WSSV infection

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Amrendra Kumar

2016-10-01

Full Text Available Objective: To identify white spot syndrome virus (WSSV entry into the host-cells of the cultured shrimp Penaeus monodon, we have attempted to localize PmRab7 (Ras-related in brain which is playing a vital role in the WSSV internalization. Methods: In this study, we have cloned PmRab7 and expressed in Escherichia coli, further purified rPmRab7 was used for antibody production, isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein. Moreover, high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR. Results: 651 bp amplicon size gene was successfully amplified, ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel. Subcloned (pRSET-B 651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page, furthermore soluble fraction of rPmRab7 (26 kDa was purified by ni-NTA column. AntiPmRab7 antibody was received by Merk Pvt. Ltd., and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction. Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct = 1.0 to Ct = 8.5. Conclusions: Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation, other hand WSSV may follow the same route of entry. WSSV internalization has directly linked with regulation of PmRab7.

The late endocytic Rab39a GTPase regulates the interaction between multivesicular bodies and chlamydial inclusions.

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Gambarte Tudela, Julian; Capmany, Anahi; Romao, Maryse; Quintero, Cristian; Miserey-Lenkei, Stephanie; Raposo, Graca; Goud, Bruno; Damiani, Maria Teresa

2015-08-15

Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure that they have access to multiple host sources of essential lipids by interfering with vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi complex. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating the interaction between MVBs and chlamydial inclusions remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles - mainly MVBs - that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process that depends on the Rab39a GTP- or GDP-binding state. Interestingly, Rab39a participates in the delivery of MVBs and host sphingolipids to maturing chlamydial inclusions, thereby promoting inclusion growth and bacterial development. Taken together, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing that a late endocytic Rab GTPase is involved in chlamydial infection development. © 2015. Published by The Company of Biologists Ltd.

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

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Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

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Tsukamoto, Yuta; Katayama, Chisako [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Shinohara, Miki; Shinohara, Akira [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Maekawa, Shohei [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan)

2013-11-29

Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

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Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki; Shinohara, Akira; Maekawa, Shohei; Miyamoto, Masaaki

2013-01-01

Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions

Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

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Nayak, Ramesh C.; Keshava, Shiva; Esmon, Charles T.; Pendurthi, Usha R.; Rao, L. Vijaya Mohan

2013-01-01

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa. PMID:23555015

TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in Caenorhabditis elegans

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Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M.; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation. PMID:22654674

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

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Mandy Hannemann

Full Text Available Dense core vesicles (DCVs are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs. We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

The breast cancer antigen 5T4 interacts with Rab11, and is a target and regulator of Rab11 mediated trafficking.

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Harris, Janelle L; Dave, Keyur; Gorman, Jeffrey; Khanna, Kum Kum

2018-06-01

5T4 is a transmembrane glycoprotein with limited expression in normal adult tissues and expression in some solid tumours. It is unclear whether 5T4 is preferentially expressed by stem or differentiated cell types. Modes of 5T4 regulation are unknown despite its ongoing development as a cancer immunotherapy target. Our aims were to clarify the differentiation status of 5T4 expressing cells in breast cancer and to understand the mechanism underlying 5T4 membrane presentation. We analysed 5T4 expression in breast cancer cell populations by flow cytometery and found that 5T4 is highly expressed on differentiated cells, where it localizes to focal adhesions. Using immunoprecipitation and mass spectrometry, we identified interactions between 5T4 and the membrane trafficking proteins Rab11, Rab18 and ARF6. Mechanistically we found that Rab11 and Rab18 have oppositional roles in controlling expression and surface presentation of 5T4. 5T4 depletion stabilizes Rab11 protein expression with a consequent stimulation transferrin surface labelling, indicating that 5T4 represses endocytic activity. Successful immunotherapeutic targeting of 5T4 requires surface presentation and different immunotherapy strategies require surface presentation versus endocytosis. While breast cancer cells with high 5T4 surface expression and rapid cell surface turnover would be susceptible to antibody-drug conjugates that rely on intracellular release, 5T4 positive cells with lower expression or lower turnover may still be responsive to T-cell mediated approaches. We find that endocytosis of 5T4 is strongly Rab11 dependent and as such Rab11 activity could affect the success or failure of 5T4-targetted immunotherapy, particularly for antibody-drug conjugate approaches. In fact, 5T4 itself represses Rab11 expression. This newly uncovered relationship between Rab11 and 5T4 suggests that breast tumours with high 5T4 expression may not have efficient endocytic uptake of 5T4-targetted immunotherapeutics

Rab39a interacts with phosphatidylinositol 3-kinase and negatively regulates autophagy induced by lipopolysaccharide stimulation in macrophages.

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Shintaro Seto

Full Text Available Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K. Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34(th to 41(st in Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.

Rab27A mediated by NF-κB promotes the stemness of colon cancer cells via up-regulation of cytokine secretion.

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Feng, Feixue; Jiang, Yinghao; Lu, Huanyu; Lu, Xiaozhao; Wang, Shan; Wang, Lifeng; Wei, Mengying; Lu, Wei; Du, Zhichao; Ye, Zichen; Yang, Guodong; Yuan, Fang; Ma, Yanxia; Lei, Xiaoying; Lu, Zifan

2016-09-27

Recent evidences have unveiled critical roles of cancer stem cells (CSCs) in tumorigenicity, but how interactions between CSC and tumor environments help maintain CSC initiation remains obscure. The small GTPases Rab27A regulates autocrine and paracrine cytokines by monitoring exocytosis of extracellular vesicles, and is reported to promote certain tumor progression. We observe that overexpression of Rab27A increased sphere formation efficiency (SFE) by increasing the proportion of CD44+ and PKH26high cells in HT29 cell lines, and accelerating the growth of colosphere with higher percentage of cells at S phase. Mechanism study revealed that the supernatant derived from HT29 sphere after Rab27A overexpression was able to expand sphere numbers with elevated secretion of VEGF and TGF-β. In tumor implanting nude mice model, tumor initiation rates and tumor sizes were enhanced by Rab27A with obvious angiogenesis. As a contrast, knocking down Rab27A impaired the above effects. More importantly, the correlation between higher p65 level and Rab27A in colon sphere was detected, p65 was sufficient to induce up-regulation of Rab27A and a functional NF-κB binding site in the Rab27A promoter was demonstrated. Altogether, our findings reveal a unique mechanism that tumor environment related NF-κB signaling promotes various colon cancer stem cells (cCSCs) properties via an amplified paracrine mechanism regulated by higher Rab27A level.

Identification and characterization of a member of Rab subfamily, Rab8, from Clonorchis sinensis.

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Liang, Pei; He, Lei; Yu, Jinyun; Xie, Zhizhi; Chen, Xueqing; Mao, Qiang; Liang, Chi; Huang, Yan; Lu, Gang; Yu, Xinbing

2015-05-01

The Rabs act as a binary molecular switch that utilizes the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins. It regulates a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, and intracellular membrane trafficking in eukaryotes. The Rab8 from Clonorchis sinensis (CsRab8) was composed of 199 amino acids. The deduced amino acid sequence shared above 50% identities with other species from trematode, tapeworm, mammal, insecta, nematode, and reptile, respectively. The homologous analysis of sequences showed the conservative domains: G1 box (GDSGVGKS), G2 box (T), G3 box (DTAG), G4 box (GNKCDL), and G5 box. In addition, the structure modeling had also shown other functional domains: GTP/Mg(2+) binding sites, switch I region, and switch II region. A phylogenic tree analysis indicated that the CsRab8 was clustered with the Rab from Schistosoma japonicum, and trematode and tapeworm came from the same branch, which was different from an evolutional branch built by other species, such as mammal animal, insecta, nematode, and reptile. The recombinant CsRab8 protein was expressed in Escherichia coli and the purified protein was a soluble molecule by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. CsRab8 was identified as a component of excretory/secretory products of C. sinensis by western blot analysis. The transcriptional level of CsRab8 at metacercaria stage was the highest at the four stages and higher by 56.49-folds than that at adult worm, 1.23-folds than that at excysted metacercaria, and 2.69-folds than that at egg stage. Immunohistochemical localization analysis showed that CsRab8 was specifically distributed in the tegument, vitellarium, eggs, and testicle of adult worms, and detected on the vitellarium and tegument of metacercaria. Combined with the results, CsRab8 is indispensable for survival and development of parasites, especially for regulating

Rab GTPases in Immunity and Inflammation.

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Prashar, Akriti; Schnettger, Laura; Bernard, Elliott M; Gutierrez, Maximiliano G

2017-01-01

Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

Rab GTPases in Immunity and Inflammation

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Akriti Prashar

2017-09-01

Full Text Available Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

Rab23 is overexpressed in human astrocytoma and promotes cell migration and invasion through regulation of Rac1.

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Wang, Minghao; Dong, Qianze; Wang, Yunjie

2016-08-01

Rab23 overexpression has been implicated in several human cancers. However, its biological roles and molecular mechanism in astrocytoma have not been elucidated. The aim of this study is to explore clinical significance and biological roles of Rab23 in astrocytoma. We observed negative Rab23 staining in normal astrocytes and positive staining in 39 out of 86 (45 %) astrocytoma specimens using immunohistochemistry. The positive rate of Rab23 was higher in grades III and IV (56.5 %, 26/46) than grades I + II astrocytomas (32.5 %, 13/40, p Rac1 activity. Treatment of transfected cells with a Rac1 inhibitor decreased Rac1 activity and invasion. In conclusion, Rab23 serves as an important oncoprotein in human astrocytoma by regulating cell invasion and migration through Rac1 activity.

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

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Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

2001-09-18

It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

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Johnson, Jennifer L; Pacquelet, Sandrine; Lane, William S; Eam, Boreth; Catz, Sergio D

2005-08-01

Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

A RabGAP regulates life-cycle duration via trimeric G-protein cascades in Dictyostelium discoideum.

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Hidekazu Kuwayama

Full Text Available BACKGROUND: The life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In Dictyostelium discoideum, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified. METHODOLOGY/PRINCIPAL FINDINGS: Using yeast 2-hybrid library screening, we isolated a Dictyostelium discoideum RabGAP, Dd Rbg-3, as a candidate molecule by which the Dictyostelium Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of Dd rbg-3 exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner in vitro. Furthermore, both human and Caenorhabditis elegans rbg-3 homologs complemented the Dd rbg-3-deletion phenotype in D. discoideum, indicating that similar pathways may be generally conserved in multicellular organisms. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that Dd Rbg-3 acts as a key element regulating the duration of D. discoideum life-span potentially via trimeric G-protein cascades.

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

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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

Integration of two RAB5 groups during endosomal transport in plants

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Ebine, Kazuo; Choi, Seung-won; Ichinose, Sakura; Uemura, Tomohiro; Nakano, Akihiko

2018-01-01

RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells. PMID:29749929

Rab7: roles in membrane trafficking and disease.

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Zhang, Ming; Chen, Li; Wang, Shicong; Wang, Tuanlao

2009-06-01

The endocytosis pathway controls multiple cellular and physiological events. The lysosome is the destination of newly synthesized lysosomal hydrolytic enzymes. Internalized molecules or particles are delivered to the lysosome for degradation through sequential transport along the endocytic pathway. The endocytic pathway is also emerging as a signalling platform, in addition to the well-known role of the plasma membrane for signalling. Rab7 is a late endosome-/lysosome-associated small GTPase, perhaps the only lysosomal Rab protein identified to date. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome [or LRO (lysosome-related organelle)] and phagocytosis. These processes are closely related to substrates degradation, antigen presentation, cell signalling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neuropathy, cancer and lipid metabolism disease. Rab7 also plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses. Here, we give a brief review on the central role of Rab7 in endosomal traffic and summarize the studies focusing on the participation of Rab7 in disease pathogenesis. The underlying mechanism governed by Rab7 and its partners will also be discussed.

Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

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Li, Hewang; Yu, Peiying; Sun, Yuansheng; Felder, Robin A.; Periasamy, Ammasi; Jose, Pedro A.

2010-09-01

The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01+/-0.10 ns) or Rab7 (2.11+/-0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78+/-0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09+/-0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

Two Novel Rab2 Interactors Regulate Dense-core Vesicle Maturation

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Ailion, Michael; Hannemann, Mandy; Dalton, Susan; Pappas, Andrea; Watanabe, Shigeki; Hegermann, Jan; Liu, Qiang; Han, Hsiao-Fen; Gu, Mingyu; Goulding, Morgan Q.; Sasidharan, Nikhil; Schuske, Kim; Hullett, Patrick; Eimer, Stefan; Jorgensen, Erik M.

2014-01-01

Summary Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi, and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1 and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a new pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

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Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Vázquez-Chávez, Elena; Lasserre, Rémi; Agüera-González, Sonia; Cuche, Céline; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

2017-04-01

The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production. Copyright © 2017 by The American Association of Immunologists, Inc.

Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae.

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Chen, Ming-Chyuan; Cheng, Ying-Min; Sung, Ping-Jyun; Kuo, Cham-En; Fang, Lee-Shing

2003-08-29

The establishment and maintenance of the intracellular association between marine cnidarians and their symbiotic microalgae is essential to the well being of coral reef ecosystems; however, little is known concerning its underlying molecular mechanisms. In light of the critical roles of the small GTPase, Rab7, as a key regulator of vesicular trafficking, we cloned and characterized the Rab7 protein in the endosymbiosis system between the sea anemone, Aiptasia pulchella and its algal symbiont, Symbiodinium spp. The Aiptasia homologue of Rab7 proteins, ApRab7 is 88% identical to human Rab7 protein and contains all Rab-specific signature motifs. Results of EGFP reporter analysis, protein fractionation, and immunocytochemistry support that ApRab7 is located in late endocytic and phagocytic compartments and is able to promote their fusion. Significantly, the majority of phagosomes containing live symbionts that either have taken long residency in, or were newly internalized by Aiptasia digestive cells did not contain detectable levels of ApRab7, while most phagosomes containing either heat-killed or photosynthesis-impaired symbionts were positive for ApRab7 staining. Overall, our data suggest that live algal symbionts persist inside their host cells by actively excluding ApRab7 from their phagosomes, and thereby, establish and/or maintain an endosymbiotic relationship with their cnidarian hosts.

Rab7a modulates ER stress and ER morphology.

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Mateus, Duarte; Marini, Elettra Sara; Progida, Cinzia; Bakke, Oddmund

2018-05-01

The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called "ER matrices" together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22 N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells.

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Ahmad, Waqas; Li, Yingying; Guo, Yidi; Wang, Xinyu; Duan, Ming; Guan, Zhenhong; Liu, Zengshan; Zhang, Maolin

2017-06-01

Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID 50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.

Rice Rab11 is required for JA-mediated defense signaling

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Hong, Min Ji [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714 (Korea, Republic of); Lee, Yun mi [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); Son, Young Sim [Division of Applied Life Sciences (BK21), Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Im, Chak Han [Eco-Friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 660-360 (Korea, Republic of); Yi, Young Byung [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); Rim, Yeong Gil [Systems and Synthetic Agrobiotech Center, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Bahk, Jeong Dong, E-mail: jdbahk@gnu.ac.kr [Division of Applied Life Sciences (BK21), Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Heo, Jae Bok, E-mail: jbheo72@dau.ac.kr [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714 (Korea, Republic of)

2013-05-17

Highlights: •OsRab11 interacts with OsOPR8. •OsOPR8 is localized in the cytosol and peroxisome. •OsRab11 enhances the NADPH consumption by OsOPR8. •Transgenic Arabidopsis overexpressing OsRab11 represents a pathogen-resistant phenotype. -- Abstract: Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8.

Role of Rab family GTPases and their effectors in melanosomal logistics.

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Ohbayashi, Norihiko; Fukuda, Mitsunori

2012-04-01

Rab GTPases constitute a family of small GTPases that regulate a variety of membrane trafficking events in all eukaryotic cells by recruiting their specific effector molecules. Recent accumulating evidence indicates that members of the mammalian Rab small GTPase family are involved in certain physiological and pathological processes. In particular, functional impairments of specific Rab proteins, e.g. Rab38 and Rab27A, their regulators or their effectors cause pigmentation disorders in humans and coat colour variations in mice because such impairments cause defects in melanosomal logistics, i.e. defects in melanosome biogenesis and transport. Genetic and biochemical analyses of the gene products responsible for mammalian pigmentation disorders in the past decade have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin. In this article, we review current knowledge regarding melanosomal logistics, with particular focus on the roles of Rab small GTPases and their effectors.

Rab7b at the intersection of intracellular trafficking and cell migration.

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Distefano, Marita Borg; Kjos, Ingrid; Bakke, Oddmund; Progida, Cinzia

2015-01-01

Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting receptors. We recently identified the actin motor protein myosin II as a new interaction partner for Rab7b, and found that Rab7b transport is dependent on myosin II. Interestingly, we also discovered that Rab7b influences the phosphorylation state of myosin II by controlling the activation status of the small GTPase RhoA. Consequently, Rab7b is important for the remodeling of actin filaments in processes such as stress fiber formation, cell adhesion, polarization and cell migration. Our finding that Rab7b can control actomyosin reorganization reveals yet another important role for Rab proteins, in addition to their already established role as master regulators of intracellular transport. Here we discuss our findings and speculate how they can explain the importance of Rab7b in dendritic cells (DCs).

Rab proteins in the brain and corpus allatum of Bombyx mori.

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Uno, Tomohide; Furutani, Masayuki; Watanabe, Chihiro; Sakamoto, Katsuhiko; Uno, Yuichi; Kanamaru, Kengo; Yamagata, Hiroshi; Mizoguchi, Akira; Takeda, Makio

2016-07-01

In eukaryotic cells, Rab guanosine triphosphate-ases serve as key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab3, Rab6, and Rab27 control the regulatory secretory pathway of neuropeptides and neurotransmitters. The cDNAs of Rab3, Rab6, and Rab27 from B. mori were inserted into a plasmid, transformed into Escherichia coli, and then subsequently purified. We then produced antibodies against Rab3, Rab6, and Rab27 of Bombyx mori in rabbits and rats for use in western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue revealed a single band at approximately 26 kDa. Immunohistochemistry results revealed that Rab3, Rab6, and Rab27 expression was restricted to neurons in the pars intercerebralis and dorsolateral protocerebrum of the brain. Rab3 and Rab6 co-localized with bombyxin, an insect neuropeptide. However, there was no Rab that co-localized with prothoracicotropic hormone. The corpus allatum secretes neuropeptides synthesized in the brain into the hemolymph. Results showed that Rab3 and Rab6 co-localized with bombyxin in the corpus allatum. These findings suggest that Rab3 and Rab6 are involved in neurosecretion in B. mori. This study is the first to report a possible relationship between Rab and neurosecretion in the insect corpus allatum.

Structural plasticity mediates distinct GAP-dependent GTP hydrolysis mechanisms in Rab33 and Rab5.

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Majumdar, Soneya; Acharya, Abhishek; Prakash, Balaji

2017-12-01

The classical GTP hydrolysis mechanism, as seen in Ras, employs a catalytic glutamine provided in cis by the GTPase and an arginine supplied in trans by a GTPase activating protein (GAP). The key idea emergent from a large body of research on small GTPases is that GTPases employ a variety of different hydrolysis mechanisms; evidently, these variations permit diverse rates of GTPase inactivation, crucial for temporal regulation of different biological processes. Recently, we unified these variations and argued that a steric clash between active site residues (corresponding to positions 12 and 61 of Ras) governs whether a GTPase utilizes the cis-Gln or the trans-Gln (from the GAP) for catalysis. As the cis-Gln encounters a steric clash, the Rab GTPases employ the so-called dual finger mechanism where the interacting GAP supplies a trans-Gln for catalysis. Using experimental and computational methods, we demonstrate how the cis-Gln of Rab33 overcomes the steric clash when it is stabilized by a residue in the vicinity. In effect, this demonstrates how both cis-Gln- and trans-Gln-mediated mechanisms could operate in the same GTPase in different contexts, i.e. depending on the GAP that regulates its action. Interestingly, in the case of Rab5, which possesses a higher intrinsic GTP hydrolysis rate, a similar stabilization of the cis-Gln appears to overcome the steric clash. Taken together with the mechanisms seen for Rab1, it is evident that the observed variations in Rab and their GAP partners allow structural plasticity, or in other words, the choice of different catalytic mechanisms. © 2017 Federation of European Biochemical Societies.

Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

International Nuclear Information System (INIS)

Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri; Takegawa, Kaoru; Noguchi, Tetsuko; Miyamoto, Masaaki

2015-01-01

The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed

Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

Energy Technology Data Exchange (ETDEWEB)

Tsukamoto, Yuta [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Shimazu, Sayuri [Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Takegawa, Kaoru [Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Noguchi, Tetsuko [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan)

2015-03-20

The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

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

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

2015-01-01

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

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

Science.gov (United States)

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

2015-12-11

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

The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity.

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Matthias Stein

Full Text Available Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics.We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity.

Rab23 is a flagellar protein in Trypanosoma brucei

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Field Mark C

2011-06-01

Full Text Available Abstract Background Rab small GTPases are important mediators of membrane transport, and orthologues frequently retain similar locations and functions, even between highly divergent taxa. In metazoan organisms Rab23 is an important negative regulator of Sonic hedgehog signaling and is crucial for correct development and differentiation of cellular lineages by virtue of an involvement in ciliary recycling. Previously, we reported that Trypanosoma brucei Rab23 localized to the nuclear envelope 1, which is clearly inconsistent with the mammalian location and function. As T. brucei is unicellular the potential that Rab23 has no role in cell signaling was possible. Here we sought to further investigate the role(s of Rab23 in T. brucei to determine if Rab23 was an example of a Rab protein with divergent function in distinct taxa. Methods/major findings The taxonomic distribution of Rab23 was examined and compared with the presence of flagella/cilia in representative taxa. Despite evidence for considerable secondary loss, we found a clear correlation between a conventional flagellar structure and the presence of a Rab23 orthologue in the genome. By epitope-tagging, Rab23 was localized and found to be present at the flagellum throughout the cell cycle. However, RNAi knockdown did not result in a flagellar defect, suggesting that Rab23 is not required for construction or maintenance of the flagellum. Conclusions The location of Rab23 at the flagellum is conserved between mammals and trypanosomes and the Rab23 gene is restricted to flagellated organisms. These data may suggest the presence of a Rab23-mediated signaling mechanism in trypanosomes.

Identification and characterization of Iporin as a novel interaction partner for rab1

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Konczal Magdalena

2005-03-01

Full Text Available Abstract Background The small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport. Results Here, we report the characterization of Iporin, which is similar to KIAA0375 as a novel rab1-interacting protein. It was initially identified by yeast two-hybrid screening experiments with the active mutant of rab1b (rab1b Q67R as bait. Iporin contains a SH3 domain and two polyproline stretches, which are known to play a role in protein/protein interactions. In addition, Iporin encloses a RUN domain, which seems to be a major part of the rab1binding domain (R1BD. Iporin is ubiquitously expressed and immunofluorescence staining displays a cytosolic punctual distribution. Interestingly, we also show that Iporin interacts with another rab1 interacting partner, the GM130 protein. Conclusion Our results demonstrate that Iporin is a potential new interacting partner of rab1. Iporin is different from already identified rab1 interacting proteins concerning protein structure and cellular localization. We conclude that Iporin might function as a link between the targeting of ER derived vesicles, triggered by the rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions. The characterization of this novel intermolecular relation could help to elucidate how vesicles find their way from ER to the Golgi apparatus.

Characterization of a Rab11-like GTPase, EhRab11, of Entamoeba histolytica.

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McGugan, Glen C; Temesvari, Lesly A

2003-07-01

The Entamoeba histolytica Rab11 family of small molecular weight GTPases consists of three members, EhRab11, EhRab11B, and EhRab11C. The functions of these Rabs in Entamoeba have not been determined. Therefore, as an approach to elucidate the role of the Rab11 family of GTPases in Entamoeba, immunofluorescence microscopy was undertaken to define the subcellular localization of one member of this family, EhRab11. Under conditions of growth, EhRab11 displayed a punctate pattern in the cytoplasm of trophozoites. EhRab11 did not colocalize with markers for the Golgi apparatus, endoplasmic reticulum, pinosomes, phagosomes, or compartments formed by receptor-mediated endocytosis, suggesting that this Rab may not play a role in vesicle trafficking between these organelles. Under conditions of iron and serum starvation, EhRab11 was translocated to the periphery of the cell. The altered cellular localization was accompanied by multinucleation of the cells as well as the acquisition of detergent resistance by the cells, features that are characteristic of Entamoeba cysts. The translocation of EhRab11 to the periphery of the cell during iron and serum starvation was specific as the subcellular localizations of two other Rab GTPases, EhRab7 and EhRabA, were not altered under the same conditions. In addition, the formation of multinucleated cells by inhibition of cytokinesis was not sufficient to induce the translocation of EhRab11 to the cell periphery. Taken together, the data suggest that iron and serum starvation may induce encystation in E. histolytica and that EhRab11 may play a role in this process. Moreover, these studies are the first to describe a putative role for a Rab GTPase in encystation in Entamoeba sp.

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

The trypanosome Rab-related proteins RabX1 and RabX2 play no role in intracellular trafficking but may be involved in fly infectivity.

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Senthil Kumar A Natesan

2009-09-01

Full Text Available Rab GTPases constitute the largest subgroup of the Ras superfamily and are primarily involved in vesicle targeting. The full extent of Rab family function is unexplored. Several divergent Rab-like proteins are known but few have been characterized. In Trypanosoma brucei there are sixteen Rab genes, but RabX1, RabX2 and RabX3 are divergent within canonical sequence regions. Where known, trypanosome Rab functions are broadly conserved when orthologous relationships may be robustly established, but specific functions for RabX1, X2 and X3 have yet to be determined. RabX1 and RabX2 originated via tandem duplication and subcellular localization places RabX1 at the endoplasmic reticulum, while RabX2 is at the Golgi complex, suggesting distinct functions. We wished to determine whether RabX1 and RabX2 are involved in vesicle transport or other cellular processes.Using comparative genomics we find that RabX1 and RabX2 are restricted to trypanosomatids. Gene knockout indicates that RabX1 and RabX2 are non-essential. Simultaneous RNAi knockdown of both RabX1 and RabX2, while partial, was also non-lethal and may suggest non-redundant function, consistent with the distinct locations of the proteins. Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport. However, it was apparent that RabX1 and RabX2 knockout cells displayed somewhat enhanced survival within flies.RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments. These data extend the functional scope of Rab proteins in trypanosomes to include non-canonical roles in differentiation-associated processes in protozoa.

Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

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Saxena, Sunil K.; Horiuchi, Hisanori; Fukuda, Mitsunori

2006-01-01

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays with two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins

The GTPase Rab37 Participates in the Control of Insulin Exocytosis.

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Sanda Ljubicic

Full Text Available Rab37 belongs to a subclass of Rab GTPases regulating exocytosis, including also Rab3a and Rab27a. Proteomic studies indicate that Rab37 is associated with insulin-containing large dense core granules of pancreatic β-cells. In agreement with these observations, we detected Rab37 in extracts of β-cell lines and human pancreatic islets and confirmed by confocal microscopy the localization of the GTPase on insulin-containing secretory granules. We found that, as is the case for Rab3a and Rab27a, reduction of Rab37 levels by RNA interference leads to impairment in glucose-induced insulin secretion and to a decrease in the number of granules in close apposition to the plasma membrane. Pull-down experiments revealed that, despite similar functional effects, Rab37 does not interact with known Rab3a or Rab27a effectors and is likely to operate through a different mechanism. Exposure of insulin-secreting cells to proinflammatory cytokines, fatty acids or oxidized low-density lipoproteins, mimicking physiopathological conditions that favor the development of diabetes, resulted in a decrease in Rab37 expression. Our data identify Rab37 as an additional component of the machinery governing exocytosis of β-cells and suggest that impaired expression of this GTPase may contribute to defective insulin release in pre-diabetic and diabetic conditions.

Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption

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Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P.

2015-01-01

Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.—Patel, C., Douard, V., Yu, S., Gao, N., Ferraris, R. P. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption. PMID:26071406

Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes.

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Fu, Yulong; Zhu, Jun-Yi; Zhang, Fujian; Richman, Adam; Zhao, Zhanzheng; Han, Zhe

2017-06-01

The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Investigations of nephrocyte cell biological processes are fundamental to understanding the insect renal system. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. We analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. Our results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease. The Drosophila nephrocyte is emerging as a useful in vivo model system for molecular target identification and initial testing of therapeutic approaches in humans.

Rab11 family expression in the human placenta: Localization at the maternal-fetal interface

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Artemiuk, Patrycja A.; Hanscom, Sara R.; Lindsay, Andrew J.; Wuebbolt, Danielle; Breathnach, Fionnuala M.; Tully, Elizabeth C.; Khan, Amir R.; McCaffrey, Mary W.

2017-01-01

Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human

Rab4GTPase modulates CFTR function by impairing channel expression at plasma membrane

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Saxena, Sunil K.; Kaur, Simarna; George, Constantine

2006-01-01

Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the disruption of biosynthesis or the function of a membrane cAMP-activated chloride channel, CFTR. CFTR regulatory mechanisms include recruitment of channel proteins to the cell surface from intracellular pools and by protein-protein interactions. Rab proteins are small GTPases involved in regulated trafficking controlling vesicle docking and fusion. Rab4 controls recycling events from endosome to the plasma membrane, fusion, and degradation. The colorectal cell line HT-29 natively expresses CFTR and responds to cAMP stimulation with an increase in CFTR-mediated currents. Rab4 over-expression in HT-29 cells inhibits both basal and cAMP-stimulated CFTR-mediated currents. GTPase-deficient Rab4Q67L and GDP locked Rab4S22N both inhibit channel activity, which appears characteristically different. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. The pull-down and immunoprecipitation experiments suggest that Rab4 physically interacts with CFTR through protein-protein interaction. Biotinylation with cell impermeant NHS-Sulfo-SS-Biotin implies that Rab4 impairs CFTR expression at cell surface. The enhanced cytosolic CFTR indicates that Rab4 expression restrains CFTR appearance at the cell membrane. The study suggests that Rab4 regulates the channel through multiple mechanisms that include protein-protein interaction, GTP/GDP exchange, and channel protein trafficking. We propose that Rab4 is a dynamic molecule with a significant role in CFTR function

ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles.

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Shibata, Satoshi; Kawanai, Tsubasa; Hara, Takayuki; Yamamoto, Asuka; Chaya, Taro; Tokuhara, Yasunori; Tsuji, Chinami; Sakai, Manabu; Tachibana, Taro; Inagaki, Shinobu

2016-10-01

The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)-Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner. © 2016. Published by The Company of Biologists Ltd.

α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

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Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

2015-01-01

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

Rab32 is important for autophagy and lipid storage in Drosophila.

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Chao Wang

Full Text Available Lipids are essential components of all organisms. Within cells, lipids are mainly stored in a specific type of organelle, called the lipid droplet. The molecular mechanisms governing the dynamics of lipid droplets have been little explored. The protein composition of lipid droplets has been analyzed in numerous proteomic studies, and a large number of lipid droplet-associated proteins have been identified, including Rab small GTPases. Rab proteins are known to participate in many intracellular membranous events; however, their exact role in lipid droplets is largely unexplored. Here we systematically investigate the roles of Drosophila Rab family proteins in lipid storage in the larval adipose tissue, fat body. Rab32 and several other Rabs were found to affect the size of lipid droplets as well as lipid levels. Further studies showed that Rab32 and Rab32 GEF/Claret may be involved in autophagy, consequently affecting lipid storage. Loss-of-function mutants of several components in the autophagy pathway result in similar effects on lipid storage. These results highlight the potential functions of Rabs in regulating lipid metabolism.

RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization

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Zou, Wei; Yadav, Smita; DeVault, Laura; Jan, Yuh Nung; Sherwood, David R.

2015-01-01

Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth. PMID:26394140

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica.

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Liu, Zhao-liang; Luo, Cong; Dong, Long; Van Toan, Can; Wei, Peng-xiao; He, Xin-hua

2014-04-25

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984bp and contained an open reading frame of 600bp, which encoded a 200 amino acid protein with a molecular weight of 21.83kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant. Copyright © 2014 Elsevier B.V. All rights reserved.

Antagonistic control of lysosomal fusion by Rab14 and the Lyst-related protein LvsB

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Kypri, Elena; Falkenstein, Kristin; De Lozanne, Arturo

2013-01-01

While loss of the protein Lyst causes abnormal lysosomes in patients with Chediak-Higashi Syndrome, the contribution of Lyst to lysosome biology is not known. Previously we found that the Dictyostelium ortholog of Lyst, LvsB, is a cytosolic protein that associates with lysosomes and post-lysosomes to prevent their inappropriate fusion. Here we provide three lines of evidence that indicate that LvsB contributes to lysosome function by antagonizing the function of DdRab14, a protein that promot...

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor.

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Oesterlin, Lena K; Goody, Roger S; Itzen, Aymelt

2012-04-10

Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.

RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

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Bello, Oscar D.; Zanetti, M. Natalia; Mayorga, Luis S.; Michaut, Marcela A.

2012-01-01

Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: ► RIM and Munc13 are present in human sperm and localize to the acrosomal region. ► RIM and Munc13 are necessary for acrosomal exocytosis. ► RIM and Munc13 participate before the acrosomal calcium efflux. ► RIM, Munc13 and Rab3A interplay in human sperm acrosomal exocytosis. ► RIM and Rab3A

Rab21, a Novel PS1 Interactor, Regulates γ-Secretase Activity via PS1 Subcellular Distribution.

Science.gov (United States)

Sun, Zhenzhen; Xie, Yujie; Chen, Yintong; Yang, Qinghu; Quan, Zhenzhen; Dai, Rongji; Qing, Hong

2018-05-01

γ-Secretase has been a therapeutical target for its key role in cleaving APP to generate β-amyloid (Aβ), the primary constituents of senile plaques and a hallmark of Alzheimer's disease (AD) pathology. Recently, γ-secretase-associating proteins showed promising role in specifically modulating APP processing while sparing Notch signaling; however, the underlying mechanism is still unclear. A co-immunoprecipitation (Co-IP) coupled with mass spectrometry proteomic assay for Presenilin1 (PS1, the catalytic subunit of γ-secretase) was firstly conducted to find more γ-secretase-associating proteins. Gene ontology analysis of these results identified Rab21 as a potential PS1 interacting protein, and the interaction between them was validated by reciprocal Co-IP and immunofluorescence assay. Then, molecular and biochemical methods were used to investigate the effect of Rab21 on APP processing. Results showed that overexpression of Rab21 enhanced Aβ generation, while silencing of Rab21 reduced the accumulation of Aβ, which resulted due to change in γ-secretase activity rather than α- or β-secretase. Finally, we demonstrated that Rab21 had no effect on γ-secretase complex synthesis or metabolism but enhanced PS1 endocytosis and translocation to late endosome/lysosome. In conclusion, we identified a novel γ-secretase-associating protein Rab21 and illustrate that Rab21 promotes γ-secretase internalization and translocation to late endosome/lysosome. Moreover, silencing of Rab21 decreases the γ-secretase activity in APP processing thus production of Aβ. All these results open new gateways towards the understanding of γ-secretase-associating proteins in APP processing and make inhibition of Rab21 a promising strategy for AD therapy.

An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes.

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Katrin Kremer

2013-03-01

Full Text Available The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.

Drosophila Big bang regulates the apical cytocortex and wing growth through junctional tension.

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Tsoumpekos, Giorgos; Nemetschke, Linda; Knust, Elisabeth

2018-03-05

Growth of epithelial tissues is regulated by a plethora of components, including signaling and scaffolding proteins, but also by junctional tension, mediated by the actomyosin cytoskeleton. However, how these players are spatially organized and functionally coordinated is not well understood. Here, we identify the Drosophila melanogaster scaffolding protein Big bang as a novel regulator of growth in epithelial cells of the wing disc by ensuring proper junctional tension. Loss of big bang results in the reduction of the regulatory light chain of nonmuscle myosin, Spaghetti squash. This is associated with an increased apical cell surface, decreased junctional tension, and smaller wings. Strikingly, these phenotypic traits of big bang mutant discs can be rescued by expressing constitutively active Spaghetti squash. Big bang colocalizes with Spaghetti squash in the apical cytocortex and is found in the same protein complex. These results suggest that in epithelial cells of developing wings, the scaffolding protein Big bang controls apical cytocortex organization, which is important for regulating cell shape and tissue growth. © 2018 Tsoumpekos et al.

Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome

DEFF Research Database (Denmark)

Aligianis, Irene A; Johnson, Colin A; Gissen, Paul

2005-01-01

Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator...

Rab18 dynamics in adipocytes in relation to lipogenesis, lipolysis and obesity.

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Marina R Pulido

Full Text Available Lipid droplets (LDs are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K, the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed.

Alteraciones en el reclutamiento y activación de proteínas Rab durante la infección micobacteriana

OpenAIRE

Diana Castaño; Mauricio Rojas

2010-01-01

En el fagosoma, Mycobacterium spp. altera la activación y reclutamiento de diferentes proteínas "del gen Ras de cerebro de rata", comúnmente conocidas como Rab. En este manuscrito se revisa una serie de reportes que han demostrado que los fagosomas que contienen micobacterias tienen una expresión mayor y sostenida de Rab5, Rab11, Rab14 y Rab22a, y menor o ninguna expresión de Rab7, Rab9 y Rab6. Esto se correlaciona con aumento de la fusión de estos fagosomas con endosomas tempranos y de recic...

RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

Energy Technology Data Exchange (ETDEWEB)

Bello, Oscar D.; Zanetti, M. Natalia [Laboratorio de Biologia Celular y Molecular, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Mayorga, Luis S. [Laboratorio de Biologia Celular y Molecular, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Michaut, Marcela A., E-mail: mmichaut@fcm.uncu.edu.ar [Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Instituto de Ciencias Basicas, Universidad Nacional de Cuyo, Mendoza (5500) (Argentina)

2012-03-10

Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: Black-Right-Pointing-Pointer RIM and Munc13 are present in human sperm and localize to the acrosomal region. Black-Right-Pointing-Pointer RIM and Munc13 are necessary for acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Munc13 participate before the acrosomal calcium efflux. Black

Role of Rab5 in the formation of macrophage-derived foam cell.

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Chan, Lokwern; Hong, Jin; Pan, Junjie; Li, Jian; Wen, Zhichao; Shi, Haiming; Ding, Jianping; Luo, Xinping

2017-09-12

Foam cells play a key role in the occurrence and pathogenesis of atherosclerosis. Its formation starts with the ingestion of oxidized low-density lipoprotein (oxLDL). The process is associated with Ras related protein in brain 5 (Rab5) which plays a critical role in regulating endocytosis and early endosomal trafficking. Base on this, we presumed that Rab5 might participate in the maturation of foam cell. The aim of this study is to investigate the effect of Rab5 on macrophage cholesterol during the evolvement of macrophage when induced by oxLDL to the formation of foam cell. Immunohistochemistry was performed to analyze the distribution of macrophages and Rab5 in atherosclerotic plaque. RNA inteference study and transfection of inactive mutant (GFP-Rab5-S34N) and active mutant (GFP-Rab5-Q79L) in U937-derived macrophage were utilized to investigate the impact of Rab5 on the process of macrophage cholesterol, which could be detected by oil red O staining, determination of intracellular lipid content, filipin staining, nile red staining and the costaining of early endosome antigen-1 (EEA-1) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylin dicarbocyanine (Dil)-labelled oxLDL (Dil-oxLDL). Rab5 was found abundantly localized in macrophage rich areas of human atherosclerotic lesions. On the foam cell study, the expression of Rab5 was increased after the incubation of oxLDL. The inteference study indicated the depletion of Rab5 led to the decreases of oil red O staining areas, total cholesterol and cholesterol esters in U937-derived marophages. Moreover, the fluorescence intensity of filipin and nile red staining were lower in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L. The confocal study demonstrated less Dil-oxLDL was internalized in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L; the result showed also the decrease in colocalization of internalized Dil-oxLDL and EEA-1 for GFP-Rab5-S34N as compared with GFP-Rab5-Q79L. Rab5 plays an important role in modulating the

The GTPase Rab43 Controls the Anterograde ER-Golgi Trafficking and Sorting of GPCRs

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Chunman Li

2017-10-01

Full Text Available G-protein-coupled receptors (GPCRs constitute the largest superfamily of cell-surface signaling proteins. However, mechanisms underlying their surface targeting and sorting are poorly understood. Here, we screen the Rab family of small GTPases in the surface transport of multiple GPCRs. We find that manipulation of Rab43 function significantly alters the surface presentation and signaling of all GPCRs studied without affecting non-GPCR membrane proteins. Rab43 specifically regulates the transport of nascent GPCRs from the endoplasmic reticulum (ER to the Golgi. More interestingly, Rab43 directly interacts with GPCRs in an activation-dependent fashion. The Rab43-binding domain identified in the receptors effectively converts non-GPCR membrane protein transport into a Rab43-dependent pathway. These data reveal a crucial role for Rab43 in anterograde ER-Golgi transport of nascent GPCRs, as well as the ER sorting of GPCR members by virtue of its ability to interact directly.

Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors.

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Ojeda Naharros, Irene; Gesemann, Matthias; Mateos, José M; Barmettler, Gery; Forbes, Austin; Ziegler, Urs; Neuhauss, Stephan C F; Bachmann-Gagescu, Ruxandra

2017-12-01

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming

Integrins Regulate Apical Constriction via Microtubule Stabilization in the Drosophila Eye Disc Epithelium

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Vilaiwan M. Fernandes

2014-12-01

Full Text Available During morphogenesis, extracellular signals trigger actomyosin contractility in subpopulations of cells to coordinate changes in cell shape. To illuminate the link between signaling-mediated tissue patterning and cytoskeletal remodeling, we study the progression of the morphogenetic furrow (MF, the wave of apical constriction that traverses the Drosophila eye imaginal disc preceding photoreceptor neurogenesis. Apical constriction depends on actomyosin contractility downstream of the Hedgehog (Hh and bone morphogenetic protein (BMP pathways. We identify a role for integrin adhesion receptors in MF progression. We show that Hh and BMP regulate integrin expression, the loss of which disrupts apical constriction and slows furrow progression; conversely, elevated integrins accelerate furrow progression. We present evidence that integrins regulate MF progression by promoting microtubule stabilization, since reducing microtubule stability rescues integrin-mediated furrow acceleration. Thus, integrins act as a genetic link between tissue-level signaling events and morphological change at the cellular level, leading to morphogenesis and neurogenesis in the eye.

Control of MarRAB Operon in Escherichia coli via Autoactivation and Autorepression

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Prajapat, Mahendra Kumar; Jain, Kirti; Saini, Supreet

2015-01-01

Choice of network topology for gene regulation has been a question of interest for a long time. How do simple and more complex topologies arise? In this work, we analyze the topology of the marRAB operon in Escherichia coli, which is associated with control of expression of genes associated with conferring resistance to low-level antibiotics to the bacterium. Among the 2102 promoters in E. coli, the marRAB promoter is the only one that encodes for an autoactivator and an autorepressor. What advantages does this topology confer to the bacterium? In this work, we demonstrate that, compared to control by a single regulator, the marRAB regulatory arrangement has the least control cost associated with modulating gene expression in response to environmental stimuli. In addition, the presence of dual regulators allows the regulon to exhibit a diverse range of dynamics, a feature that is not observed in genes controlled by a single regulator. PMID:26445450

Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae.

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Chen, Ming-Chyuan; Cheng, Ying-Min; Hong, Min-Chang; Fang, Lee-Shing

2004-11-19

The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts.

Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae

International Nuclear Information System (INIS)

Chen, M.-C.; Cheng, Y.-M; Hong, M.-C.; Fang, L.-S.

2004-01-01

The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts

Molecular characterization and expression of Rab7 from Clonorchis sinensis and its potential role in autophagy.

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Jia, Feifei; Li, Ye; Huang, Yan; Chen, Tingjin; Li, Shan; Xu, Yanquan; Wu, Zhongdao; Li, Xuerong; Yu, Xinbing

2013-07-01

Accumulating evidences suggest that Rab7 GTPase is important for the normal progression of autophagy. However, the role of Rab7 GTPase in regulation of autophagy in Clonorchis sinensis is not known. In this study, a gene encoding Rab7 was isolated from C. sinensis adult cDNA. Recombinant CsRab7 was expressed and purified from Escherichia coli. CsRab7 transcripts were detected in the cDNA of adult worm, metacercaria, cercaria, and egg of C. sinensis, and were highly expressed in the metacercaria. Immunohistochemical localization results revealed that CsRab7 was specifically deposited on the vitellarium and eggs of adult worm. Furthermore, EGFP signal of CsRab7WT and the active mutant CsRab7Q67L were associated with autophagic vesicles in transiently transfected 293T cells. It is concluded from the present study that CsRab7 GTPase possibly contributes to the development of C. sinensis and that the autophagy pathway could be an important site of action with respect to the developmental role of CsRab7 in C. sinensis.

A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane.

Science.gov (United States)

Gidon, Alexandre; Bardin, Sabine; Cinquin, Bertrand; Boulanger, Jerome; Waharte, François; Heliot, Laurent; de la Salle, Henri; Hanau, Daniel; Kervrann, Charles; Goud, Bruno; Salamero, Jean

2012-06-01

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane. © 2012 John Wiley & Sons A/S.

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

Science.gov (United States)

Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

rab3 mediates cortical granule exocytosis in the sea urchin egg.

Science.gov (United States)

Conner, S; Wessel, G M

1998-11-15

Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel, G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking. Copyright 1998 Academic

Two Rab5 Homologs Are Essential for the Development and Pathogenicity of the Rice Blast Fungus Magnaporthe oryzae

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Cheng D. Yang

2017-05-01

Full Text Available The rice blast fungus, Magnaporthe oryzae, infects many economically important cereal crops, particularly rice. It has emerged as an important model organism for studying the growth, development, and pathogenesis of filamentous fungi. RabGTPases are important molecular switches in regulation of intracellular membrane trafficking in all eukaryotes. MoRab5A and MoRab5B are Rab5 homologs in M. oryzae, but their functions in the fungal development and pathogenicity are unknown. In this study, we have employed a genetic approach and demonstrated that both MoRab5A and MoRab5B are crucial for vegetative growth and development, conidiogenesis, melanin synthesis, vacuole fusion, endocytosis, sexual reproduction, and plant pathogenesis in M. oryzae. Moreover, both MoRab5A and MoRab5B show similar localization in hyphae and conidia. To further investigate possible functional redundancy between MoRab5A and MoRab5B, we overexpressed MoRAB5A and MoRAB5B, respectively, in MoRab5B:RNAi and MoRab5A:RNAi strains, but neither could rescue each other’s defects caused by the RNAi. Taken together, we conclude that both MoRab5A and MoRab5B are necessary for the development and pathogenesis of the rice blast fungus, while they may function independently.

The Rab2A GTPase Promotes Breast Cancer Stem Cells and Tumorigenesis via Erk Signaling Activation

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Man-Li Luo

2015-04-01

Full Text Available Proline-directed phosphorylation is regulated by the prolyl isomerase Pin1, which plays a fundamental role in driving breast cancer stem-like cells (BCSCs. Rab2A is a small GTPase critical for vesicle trafficking. Here, we show that Pin1 increases Rab2A transcription to promote BCSC expansion and tumorigenesis in vitro and in vivo. Mechanistically, Rab2A directly interacts with and prevents dephosphorylation/inactivation of Erk1/2 by the MKP3 phosphatase, resulting in Zeb1 upregulation and β-catenin nuclear translocation. In cancer cells, Rab2A is activated via gene amplification, mutation or Pin1 overexpression. Rab2A overexpression or mutation endows BCSC traits to primary normal human breast epithelial cells, whereas silencing Rab2A potently inhibits the expansion and tumorigenesis of freshly isolated BCSCs. Finally, Rab2A overexpression correlates with poor clinical outcome in breast cancer patients. Thus, Pin1/Rab2A/Erk drives BCSC expansion and tumorigenicity, suggesting potential drug targets.

The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses.

Science.gov (United States)

Takahama, Michihiro; Fukuda, Mitsunori; Ohbayashi, Norihiko; Kozaki, Tatsuya; Misawa, Takuma; Okamoto, Toru; Matsuura, Yoshiharu; Akira, Shizuo; Saitoh, Tatsuya

2017-09-19

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5), in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses

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Michihiro Takahama

2017-09-01

Full Text Available Cyclic GMP-AMP synthase (cGAS is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5, in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING, the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

The RabGAP TBC1D1 plays a central role in exercise-regulated glucose metabolism in skeletal muscle

DEFF Research Database (Denmark)

Stöckli, Jacqueline; Meoli, Christopher C; Hoffman, Nolan J

2015-01-01

Insulin and exercise stimulate glucose uptake into skeletal muscle via different pathways. Both stimuli converge on the translocation of the glucose transporter GLUT4 from intracellular vesicles to the cell surface. Two Rab guanosine triphosphatases-activating proteins (GAPs) have been implicated...... weight, insulin action, and exercise. TBC1D1(-/-) mice showed normal glucose and insulin tolerance, with no difference in body weight compared with wild-type littermates. GLUT4 protein levels were reduced by ∼40% in white TBC1D1(-/-) muscle, and TBC1D1(-/-) mice showed impaired exercise endurance...... together with impaired exercise-mediated 2-deoxyglucose uptake into white but not red muscles. These findings indicate that the RabGAP TBC1D1 plays a key role in regulating GLUT4 protein levels and in exercise-mediated glucose uptake in nonoxidative muscle fibers....

Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation.

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Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Lasserre, Rémi; Agüera-Gonzalez, Sonia; Cuche, Céline; Danckaert, Anne; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

2016-06-01

The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation. © 2016 The Authors.

Rab3a-Bound CD63 Is Degraded and Rab3a-Free CD63 Is Incorporated into HIV-1 Particles

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Yoshinao Kubo

2017-08-01

Full Text Available CD63, a member of the tetraspanin family, is involved in virion production by human immunodeficiency virus type 1 (HIV-1, but its mechanism is unknown. In this study, we showed that a small GTP-binding protein, Rab3a, interacts with CD63. When Rab3a was exogenously expressed, the amounts of CD63 decreased in cells. The Rab3a-mediated reduction of CD63 was suppressed by lysosomal and proteasomal inhibitors. The amount of CD63 was increased by reducing the endogenous Rab3a level using a specific shRNA. These results indicate that Rab3a binds to CD63 to induce the degradation of CD63. Rab3a is thought to be involved in exocytosis, but we found that another function of Rab3a affects the fate of CD63 in lysosomes. CD63 interacted with Rab3a and was incorporated into HIV-1 particles. However, Rab3a was not detected in HIV-1 virions, thereby indicating that Rab3a-free CD63, but not Rab3a-bound CD63, is incorporated into HIV-1 particles. Overexpression or silencing of Rab3a moderately reduced HIV-1 virion formation. Overexpression of Rab3a decreased CD63 levels, but did not affect the incorporation of CD63 into HIV-1 particles. This study showed that Rab3a binds to CD63 to induce the degradation of CD63, and only Rab3a-free CD63 is incorporated into HIV-1 particles.

The apical scaffold big bang binds to spectrins and regulates the growth of Drosophila melanogaster wing discs.

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Forest, Elodie; Logeay, Rémi; Géminard, Charles; Kantar, Diala; Frayssinoux, Florence; Heron-Milhavet, Lisa; Djiane, Alexandre

2018-03-05

During development, cell numbers are tightly regulated, ensuring that tissues and organs reach their correct size and shape. Recent evidence has highlighted the intricate connections between the cytoskeleton and the regulation of the key growth control Hippo pathway. Looking for apical scaffolds regulating tissue growth, we describe that Drosophila melanogaster big bang (Bbg), a poorly characterized multi-PDZ scaffold, controls epithelial tissue growth without affecting epithelial polarity and architecture. bbg -mutant tissues are smaller, with fewer cells that are less apically constricted than normal. We show that Bbg binds to and colocalizes tightly with the β-heavy-Spectrin/Kst subunit at the apical cortex and promotes Yki activity, F-actin enrichment, and the phosphorylation of the myosin II regulatory light chain Spaghetti squash. We propose a model in which the spectrin cytoskeleton recruits Bbg to the cortex, where Bbg promotes actomyosin contractility to regulate epithelial tissue growth. © 2018 Forest et al.

Localization and functional analysis of the insect-specific RabX4 in the brain of Bombyx mori.

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Uno, Tomohide; Furutani, Masayuki; Sakamoto, Katsuhiko; Uno, Yuichi; Kanamaru, Kengo; Mizoguchi, Akira; Hiragaki, Susumu; Takeda, Makio

2017-09-01

Rab proteins are small monomeric GTPases/GTP-binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect-specific Rab protein that has no close homolog in vertebrates. There is little information about insect-specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4-like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori. © 2017 Wiley Periodicals, Inc.

Recognition and stabilization of geranylgeranylated human Rab5 by the GDP Dissociation Inhibitor (GDI).

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Edler, Eileen; Stein, Matthias

2017-10-25

The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.

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Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

2002-10-01

Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His6-tagged ScRAB protein was expressed in E. coli, and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding.

The N-Myc down regulated Gene1 (NDRG1) Is a Rab4a effector involved in vesicular recycling of E-cadherin.

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Kachhap, Sushant K; Faith, Dennis; Qian, David Z; Shabbeer, Shabana; Galloway, Nathan L; Pili, Roberto; Denmeade, Samuel R; DeMarzo, Angelo M; Carducci, Michael A

2007-09-05

Cell to cell adhesion is mediated by adhesion molecules present on the cell surface. Downregulation of molecules that form the adhesion complex is a characteristic of metastatic cancer cells. Downregulation of the N-myc down regulated gene1 (NDRG1) increases prostate and breast metastasis. The exact function of NDRG1 is not known. Here by using live cell confocal microscopy and in vitro reconstitution, we report that NDRG1 is involved in recycling the adhesion molecule E-cadherin thereby stabilizing it. Evidence is provided that NDRG1 recruits on recycling endosomes in the Trans Golgi network by binding to phosphotidylinositol 4-phosphate and interacts with membrane bound Rab4aGTPase. NDRG1 specifically interacts with constitutively active Rab4aQ67L mutant protein and not with GDP-bound Rab4aS22N mutant proving NDRG1 as a novel Rab4a effector. Transferrin recycling experiments reveals NDRG1 colocalizes with transferrin during the recycling phase. NDRG1 alters the kinetics of transferrin recycling in cells. NDRG1 knockdown cells show a delay in recycling transferrin, conversely NDRG1 overexpressing cells reveal an increase in rate of transferrin recycling. This novel finding of NDRG1 as a recycling protein involved with recycling of E-cadherin will aid in understanding NDRG1 role as a metastasis suppressor protein.

The N-Myc down regulated Gene1 (NDRG1 Is a Rab4a effector involved in vesicular recycling of E-cadherin.

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Sushant K Kachhap

2007-09-01

Full Text Available Cell to cell adhesion is mediated by adhesion molecules present on the cell surface. Downregulation of molecules that form the adhesion complex is a characteristic of metastatic cancer cells. Downregulation of the N-myc down regulated gene1 (NDRG1 increases prostate and breast metastasis. The exact function of NDRG1 is not known. Here by using live cell confocal microscopy and in vitro reconstitution, we report that NDRG1 is involved in recycling the adhesion molecule E-cadherin thereby stabilizing it. Evidence is provided that NDRG1 recruits on recycling endosomes in the Trans Golgi network by binding to phosphotidylinositol 4-phosphate and interacts with membrane bound Rab4aGTPase. NDRG1 specifically interacts with constitutively active Rab4aQ67L mutant protein and not with GDP-bound Rab4aS22N mutant proving NDRG1 as a novel Rab4a effector. Transferrin recycling experiments reveals NDRG1 colocalizes with transferrin during the recycling phase. NDRG1 alters the kinetics of transferrin recycling in cells. NDRG1 knockdown cells show a delay in recycling transferrin, conversely NDRG1 overexpressing cells reveal an increase in rate of transferrin recycling. This novel finding of NDRG1 as a recycling protein involved with recycling of E-cadherin will aid in understanding NDRG1 role as a metastasis suppressor protein.

32 CFR 202.9 - Conducting RAB meetings.

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2010-07-01

... 32 National Defense 2 2010-07-01 2010-07-01 false Conducting RAB meetings. 202.9 Section 202.9... members. Group consensus is not a prerequisite for RAB input. Each member of the RAB may provide advice as an individual; however, when a RAB decides to vote or poll for consensus, only community members...

Rab3A Inhibition of Ca2+ -Dependent Dopamine Release From PC12 Cells Involves Interaction With Synaptotagmin I.

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Dai, Zhipan; Tang, Xia; Chen, Jia; Tang, Xiaochao; Wang, Xianchun

2017-11-01

Rab3 and synaptotagmin have been suggested to play important roles in the regulation of neurotransmitter release and, however, the molecular mechanism has not been completely clear. Here, we studied the effects of Rab3A and synaptotagmin I (Syt I) on dopamine release using PC12 cells as a model system. Rab3A was demonstrated to have effects on both Ca 2+ -independent and Ca 2+ -dependent dopamine releases from the PC12 cells. Application of Rab3A (up to 2500 nM) gradually decreased the amount of Ca 2+ -dependently released dopamine, indicating that Rab3A is a negative modulator that was further supported by the increase in dopamine release caused by Rab3A knockdown. Syt I knockdown weakened the Ca 2+ -dependent dopamine release, suggesting that Syt I plays a positive regulatory role in the cellular process. Treatment of the Syt I-knocked down PC12 cells with Rab3A further decreased Ca 2+ -dependent dopamine release and, however, the decrease magnitude was significantly reduced compared with that before Syt I knockdown, thus for the first time demonstrating that the inhibitory effect of Rab3A on Ca 2+ -dependent dopamine release involves the interaction with Syt I. This work has shed new light on the molecular mechanism for Rab3 and synaptotamin regulation of neurotransmitter release. J. Cell. Biochem. 118: 3696-3705, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Membrane localization and dynamics of geranylgeranylated Rab5 hypervariable region.

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Edler, Eileen; Schulze, Eric; Stein, Matthias

2017-08-01

The small GTPase Rab5 is a key regulator of endosomal trafficking processes and a marker for the early endosome. The C-terminal hypervariable region (HVR) of Rab5 is post-translationally modified at residues Cys 212 and Cys 213 to accommodate two geranylgeranyl anchors (C20 carbon chain length) in order to associate Rab5 with the membrane. The structural role of the HVR regarding protein-early endosome membrane recruitment is not resolved due to its high degree of flexibility and lack of crystallographic information. Here, full-atomistic and coarse-grained molecular dynamics simulations of the truncated Rab5 HVR 206-215 in three model membranes of increasing complexity (pure phospholipid bilayer, ternary membrane with cholesterol, six-component early endosome) were performed. Specific electrostatic interactions between the HVR 206-215 Arg 209 residue and the phosphate group of the inositol ring of PI(3)P were detected. This shows that PI(3)P acts as a first contact site of protein recruitment to the early endosome. The free energy change of HVR 206-215 extraction from the bilayer was largest for the physiological negatively charged membrane. 5μs coarse-grained simulations revealed an active recruitment of PI(3)P to the HVR 206-215 supporting the formation of Rab5- and PI(3)P enriched signaling platforms. Copyright © 2017 Elsevier B.V. All rights reserved.

MUC1 intra-cellular trafficking is clathrin, dynamin, and rab5 dependent

International Nuclear Information System (INIS)

Liu Xiaolong; Yuan Zhenglong; Chung, Maureen

2008-01-01

MUC1, a transmembrane glycoprotein, is abnormally over-expressed in most human adenocarcinomas. MUC1 association with cytoplasmic cell signal regulators and nuclear accumulation are important for its tumor related activities. Little is known about how MUC1 translocates from the cell membrane to the cytoplasm. In this study, live cell imaging was used to study MUC1 intracellular trafficking. The interaction between EGFR and MUC1 was mapped by FRET analysis and EGF stimulated MUC1 endocytosis was observed directly through live cell imaging. MUC1-CT endocytosis was clathrin and dynamin dependent. Rab5 over-expression resulted in decreased cell membrane localization of MUC1, with accumulation of MUC1 endocytic vesicles in the peri-nuclear region. Conversely, over-expression of a Rab5 dominant negative mutant (S34N) resulted in redistribution of MUC1 from the peri-nuclear region to the cytoplasm. Collectively, these results indicated that MUC1 intra-cellular trafficking occurs through a regulated process that was stimulated by direct EGFR and MUC1 interaction, mediated by clathrin coated pits that were dynamin dependent and regulated by Rab5

Rab3a is critical for trapping alpha-MSH granules in the high Ca²⁺-affinity pool by preventing constitutive exocytosis.

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Simon Sedej

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

Rab22a enhances CD147 recycling and is required for lung cancer cell migration and invasion.

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Zhou, Yang; Wu, Bo; Li, Jiang-Hua; Nan, Gang; Jiang, Jian-Li; Chen, Zhi-Nan

2017-08-01

Rab22a is a member of the Ras-related small GTPase family, which plays a key role in regulating the recycling of cargo proteins entering cells through clathrin-independent endocytosis (CIE). Rab22a is overexpressed in different cancer types, including liver cancer, malignant melanoma, ovarian cancer and osteosarcoma. However, its oncogenic role remains unknown. In this study, we found that silencing of Rab22a suppressed the migration and invasion of lung cancer cells. Furthermore, Rab22a interacts with CD147, and knockdown of Rab22a blocks CD147 recycling and promotes CD147 degradation. Taken together, our findings indicate that Rab22a enhances recycling of CD147, which is required for lung cancer cell migration and invasion,and targeting CD147 recycling may be a rational strategy for lung cancer therapy. Copyright © 2017. Published by Elsevier Inc.

A novel marRAB operon contributes to the rifampicin resistance in Mycobacterium smegmatis.

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Zhang, Haiwei; Gao, Long; Zhang, Jiaoling; Li, Weihui; Yang, Min; Zhang, Hua; Gao, Chunhui; He, Zheng-Guo

2014-01-01

The multiple-antibiotic resistance regulator (MarR) plays an important role in modulating bacterial antibiotic resistance. However, the regulatory model of the marRAB operon in mycobacteria remains to be characterized. Here we report that a MarR, encoded by Ms6508, and its marRAB operon specifically contribute to rifampicin (RIF) resistance in Mycobacterium smegmatis. We show that the MarR recognizes a conserved 21-bp palindromic motif and negatively regulates the expression of two ABC transporters in the operon, encoded by Ms6509-6510. Unlike other known drug efflux pumps, overexpression of these two ABC transporters unexpectedly increased RIF sensitivity and deletion of these two genes increased mycobacterial resistance to the antibiotic. No change can be detected for the sensitivity of recombinant mycobacterial strains to three other anti-TB drugs. Furthermore, HPLC experiments suggested that Ms6509-Ms6510 could pump RIF into the mycobacterial cells. These findings indicated that the mycobacterial MarR functions as a repressor and constitutively inhibits the expression of the marRAB operon, which specifically contributes to RIF resistance in M. smegmatis. Therefore, our data suggest a new regulatory mechanism of RIF resistance and also provide the new insight into the regulatory model of a marRAB operon in mycobacteria.

Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting.

Science.gov (United States)

Moeller, Hanne B; Fuglsang, Cecilia Hvitfeldt; Pedersen, Cecilie Nøhr; Fenton, Robert A

2018-01-01

Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2. Copyright © 2017 Elsevier Inc. All rights reserved.

Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7

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Vanessa M. D’Costa

2015-09-01

Full Text Available Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

阴道毛滴虫Rab11鸟苷三磷酸酶cDNA克隆和序列分析%Molecular Cloning and Sequence Analysis of Rab11 GTPase in Trichomonas vaginalis

Institute of Scientific and Technical Information of China (English)

张仁利; 许铭炎; 许锦阶; 高世同; 黄达娜; 耿艺介; 傅玉才

2006-01-01

Objective Rab11 GTPases play an essential role in regulating membrane trafficking pathways in eukaryotic cells. Nonetheless, there has been little work done on characterizing the transport machinery of Trichomonas. The aim of this study is to clone and characterize a Rab11 gene of Trichomonas vaginalis.Methods A cDNA expression library was constructed with T. vaginalis total RNA. A cDNA clone, which showed a high degree of homology with Rab proteins of different species, was isolated and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST and ClustalW programs. The genomic DNA corresponding to the cDNA sequence was amplified using PCR techniques and following by sequencing. Results cDNA with a length of 710 base pairs and an open reading frame of 636 bp was obtained. The deduced amino acid sequence from the open reading frame was found to possess 211 residuals. Sequence analysis demonstrated that this cDNA clone was homologous to the Rab11 subfamily of different species (60% identity and 79% similarity with Arabidopsis thaliana Rab11c, 58% identity and 78% similarity with human Rab11b), and that the amino acid sequence contains all the well known conserved sequence elements of Rab family. Specific Rab motifs were also detected in the deduced amino acid sequence. Phylogenetic analysis showed that its closest homologues are Rab11 proteins from other species. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA sequence encompassing the putative 5'-ATG and 3'-stop codon is identical to the cDNA sequence.Conclusion A cDNA clone corresponding to the T. vaginalis Rab11 gene was obtained.The function of this gene in regulating membrane trafficking pathways of the parasitic protist is still under investigation.%目的近年研究表明Rab11�

Isolation and functional characterisation of two new bZIP maize regulators of the ABA responsive gene rab28.

Science.gov (United States)

Nieva, Claudia; Busk, Peter K; Domínguez-Puigjaner, Eva; Lumbreras, Victoria; Testillano, Pilar S; Risueño, Maria-Carmen; Pagès, Montserrat

2005-08-01

The plant hormone abscisic acid regulates gene expression in response to growth stimuli and abiotic stress. Previous studies have implicated members of the bZIP family of transcription factors as mediators of abscisic acid dependent gene expression through the ABRE cis-element. Here, we identify two new maize bZIP transcription factors, EmBP-2 and ZmBZ-1 related to EmBP-1 and OsBZ-8 families. They are differentially expressed during embryo development; EmBP-2 is constitutive, whereas ZmBZ-1 is abscisic acid-inducible and accumulates during late embryogenesis. Both factors are nuclear proteins that bind to ABREs and activate transcription of the abscisic acid-inducible gene rab28 from maize. EmBP-2 and ZmBZ-1 are phosphorylated by protein kinase CK2 and phosphorylation alters their DNA binding properties. Our data suggest that EmBP-2 and ZmBZ-1 are involved in the expression of abscisic acid inducible genes such as rab28 and their activity is modulated by ABA and by phosphorylation.

Rab5 GTPase controls chromosome alignment through Lamin disassembly and relocation of the NuMA-like protein Mud to the poles during mitosis

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Capalbo, Luisa; D'Avino, Pier Paolo; Archambault, Vincent; Glover, David M.

2011-01-01

The small GTPase Rab5 is a conserved regulator of membrane trafficking; it regulates the formation of early endosomes, their transport along microtubules, and the fusion to the target organelles. Although several members of the endocytic pathway were recently implicated in spindle organization, it is unclear whether Rab5 has any role during mitosis. Here, we describe that Rab5 is required for proper chromosome alignment during Drosophila mitoses. We also found that Rab5 associated in vivo with nuclear Lamin and mushroom body defect (Mud), the Drosophila counterpart of nuclear mitotic apparatus protein (NuMA). Consistent with this finding, Rab5 was required for the disassembly of the nuclear envelope at mitotic entry and the accumulation of Mud at the spindle poles. Furthermore, Mud depletion caused chromosome misalignment defects that resembled the defects of Rab5 RNAi cells, and double-knockdown experiments indicated that the two proteins function in a linear pathway. Our results indicate a role for Rab5 in mitosis and reinforce the emerging view of the contributions made by cell membrane dynamics to spindle function. PMID:21987826

Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize.

Science.gov (United States)

Busk, P K; Jensen, A B; Pagès, M

1997-06-01

The rab17 gene from maize is transcribed in late embryonic development and is responsive to abscisic acid and water stress in embryo and vegetative tissues. In vivo footprinting and transient transformation of rab17 were performed in embryos and vegetative tissues to characterize the cis-elements involved in regulation of the gene. By in vivo footprinting, protein binding was observed to nine elements in the promoter, which correspond to five putative ABREs (abscisic acid responsive elements) and four other sequences. The footprints indicated that distinct proteins interact with these elements in the two developmental stages. In transient transformation, six of the elements were important for high level expression of the rab17 promoter in embryos, whereas only three elements were important in leaves. The cis-acting sequences can be divided in embryo-specific, ABA-specific and leaf-specific elements on the basis of protein binding and the ability to confer expression of rab17. We found one positive, new element, called GRA, with the sequence CACTGGCCGCCC. This element was important for transcription in leaves but not in embryos. Two other non-ABRE elements that stimulated transcription from the rab17 promoter resemble previously described abscisic acid and drought-inducible elements. There were differences in protein binding and function of the five ABREs in the rab17 promoter. The possible reasons for these differences are discussed. The in vivo data obtained suggest that an embryo-specific pathway regulates transcription of the rab genes during development, whereas another pathway is responsible for induction in response to ABA and drought in vegetative tissues.

Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs

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Bello, Oscar Daniel; Cappa, Andrea Isabel; Paola, Matilde de; Zanetti, María Natalia [Instituto de Histología y Embriología, CONICET – Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza (Argentina); Fukuda, Mitsunori [Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578 (Japan); Fissore, Rafael A. [Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, 661 North Pleasant Street, Amherst, MA 01003 (United States); Mayorga, Luis S. [Instituto de Histología y Embriología, CONICET – Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza (Argentina); Michaut, Marcela A., E-mail: mmichaut@gmail.com [Instituto de Histología y Embriología, CONICET – Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza (Argentina); Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo (Argentina)

2016-09-10

Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs. - Highlights: • Rab3A has a similar migration pattern to cortical granules in mouse oocytes. • Rab3A can be a marker of

IL4/PGE2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

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Wainszelbaum, Marisa J.; Proctor, Brandon M.; Pontow, Suzanne E.; Stahl, Philip D.; Barbieri, M. Alejandro

2006-01-01

The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E 2 (PGE 2 ) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells

Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.

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Pavarotti, Martín; Capmany, Anahí; Vitale, Nicolas; Colombo, María Isabel; Damiani, María Teresa

2012-02-01

Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCβII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCβII but not PKCβI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCβII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

The functional interplay of Rab11, FIP3 and Rho proteins on the endosomal recycling pathway controls cell shape and symmetry.

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Bouchet, Jérôme; McCaffrey, Mary W; Graziani, Andrea; Alcover, Andrés

2018-07-04

Several families of small GTPases regulate a variety of fundamental cellular processes, encompassing growth factor signal transduction, vesicular trafficking and control of the cytoskeleton. Frequently, their action is hierarchical and complementary, but much of the detail of their functional interactions remains to be clarified. It is well established that Rab family members regulate a variety of intracellular vesicle trafficking pathways. Moreover, Rho family GTPases are pivotal for the control of the actin and microtubule cytoskeleton. However, the interplay between these 2 types of GTPases has been rarely reported. We discuss here our recent findings showing that Rab11, a key regulator of endosomal recycling, and Rac1, a central actin cytoskeleton regulator involved in lamellipodium formation and cell migration, interplay on endosomes through the Rab11 effector FIP3. In the context of the rapidly reactive T lymphocytes, Rab11-Rac1 endosomal functional interplay is important to control cell shape changes and cell symmetry during lymphocyte spreading and immunological synapse formation and ultimately modulate T cell activation.

Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

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Hu, Dong; Wu, Jing; Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan; Xiao, Jian; Hu, Fengyu; Yang, Yabo; Zhang, Rongbo

2015-01-01

The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy

Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

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Hu, Dong, E-mail: austhudong@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wu, Jing, E-mail: wujing8008@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Hu, Fengyu; Yang, Yabo [Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Zhang, Rongbo, E-mail: lory456@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China)

2015-05-29

The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy.

Rab27 GTPases Distribute Extracellular Nanomaps for Invasive Growth and Metastasis: Implications for Prognosis and Treatment

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Olivier De Wever

2013-05-01

Full Text Available The Rab27 family of small GTPases regulates exocytosis of distinct vesicle types including multivesicular endosomes, which results in the release of exosomes. Exosomes are nanometer-sized membrane vesicles that enclose soluble factors such as proteins and nucleic acids within a lipid bilayer and can travel toward distant tissues to influence multiple aspects of cell behavior. In our view that tumors are endocrine organs producing exosomes, Rab27 GTPases and their effector proteins are critical determinants for invasive growth and metastasis. Rab27 proteins and their effectors may serve as prognostic biomarkers or as targets for patient-tailored therapy.

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium.

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Zuo, Wu-Lin; Li, Sheng; Huang, Jie-Hong; Yang, Deng-Liang; Zhang, Geng; Chen, Si-Liang; Ruan, Ye-Chun; Ye, Ke-Nan; Cheng, Christopher H K; Zhou, Wen-Liang

2011-01-01

The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

miR-184 and miR-150 promote renal glomerular mesangial cell aging by targeting Rab1a and Rab31.

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Liu, Xiujuan; Fu, Bo; Chen, Dapeng; Hong, Quan; Cui, Jing; Li, Jin; Bai, Xueyuan; Chen, Xiangmei

2015-08-15

The molecular mechanism of kidney aging is not well understood, but the abnormal expression of miRNAs with aging is considered to be an important contributor. miR-184 and miR-150 were screened using a miRNA microarray and qRT-PCR and found to be significantly upregulated in 24-month-old rats. Rat renal primary glomerular mesangial cells (GMCs) were isolated from 3-month and 24-month-old rats for the in vitro analysis of the roles of miR-184 and miR-150 in kidney aging. Bioinformatics analyses suggested that Rab1a and Rab31, which are associated with cell autophagy, were targeted by both miR-184 and miR-150. miR-184 and miR-150 were increased significantly in aging GMCs versus young cells, while Rab1a and Rab31 were significantly lower in aging cells. Furthermore, dual luciferase reporter assays revealed that miR-184 and miR-150 bound to the 3'-UTR of Rab1a and Rab31 mRNAs. Transfection of miR-184 and miR-150 mimics into young GMCs suppressed the expression of Rab1a and Rab31. Transfected cells showed lower autophagy activities and higher levels of cellular oxidative products, leading to the aging of young GMCs. However, miR-184 and miR-150 inhibitors promoted autophagy and reduced oxidative damage by upregulating Rab1a and Rab31 in old GMCs. In conclusion, miR-184 and miR-150 inhibited autophagy, promoting GMC aging. Copyright © 2015 Elsevier Inc. All rights reserved.

Extensive in silico analysis of Mimivirus coded Rab GTPase homolog suggests a possible role in virion membrane biogenesis

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Amrutraj eZade

2015-09-01

Full Text Available Rab GTPases are the key regulators of intracellular membrane trafficking in eukaryotes. Many viruses and intracellular bacterial pathogens have evolved to hijack the host Rab GTPase functions, mainly through activators and effector proteins, for their benefit. Acanthamoeba polyphaga mimivirus (APMV is one of the largest viruses and belongs to the monophyletic clade of nucleo-cytoplasmic large DNA viruses (NCLDV. The inner membrane lining is integral to the APMV virion structure. APMV assembly involves extensive host membrane modifications, like vesicle budding and fusion, leading to the formation of a membrane sheet that is incorporated into the virion. Intriguingly, APMV and all group I members of the Mimiviridae family code for a putative Rab GTPase protein. APMV is the first reported virus to code for a Rab GTPase (encoded by R214 gene. Our thorough in silico analysis of the subfamily specific (SF region of Mimiviridae Rab GTPase sequences suggests that they are related to Rab5, a member of the group II Rab GTPases, of lower eukaryotes. Because of their high divergence from the existing three isoforms, A, B and C of the Rab5-family, we suggest that Mimiviridae Rabs constitute a new isoform, Rab5D. Phylogenetic analysis indicated probable horizontal acquisition from a lower eukaryotic ancestor followed by selection and divergence. Furthermore, interaction network analysis suggests that vps34 (a Class III P13K homolog, coded by APMV L615, Atg-8 and dynamin (host proteins are recruited by APMV Rab GTPase during capsid assembly. Based on these observations, we hypothesize that APMV Rab plays a role in the acquisition of inner membrane during virion assembly.

阴道毛滴虫Rab1a重组蛋白的表达和细胞内定位%Expression of Recombinant TvRab1a and Intracellular Location of TvRab1a in Trichomonas vaginalis

Institute of Scientific and Technical Information of China (English)

徐晓园; 傅玉才; 许铭炎; 许锦阶; 张仁利

2006-01-01

目的 制备阴道毛滴虫(Trichomonas vaginalis,Tv)Rab1a重组蛋白及其多克隆抗体,并对TvRab1a蛋白进行阴道毛滴虫的细胞内定位.方法 将我们已构建的pQE80L/TvRab1a重组表达质粒转入大肠埃希菌E.coli M15,在IPTG诱导下表达重组蛋白,经Ni-NTA亲和柱层析后获得纯度较高的TvRab1a的重组蛋白;用经复性处理的重组蛋白免疫动物,获得TvRab1a重组蛋白抗血清,免疫印迹Western Blot鉴定抗血清.采用荧光免疫细胞化学对TvRab1a蛋白进行细胞内定位.结果 Western Blot分析显示,TvRab1a重组蛋白可与豚鼠的抗TvRab1a血清反应,同时该抗血清在滴虫提取蛋白中检测到与预测TvRab1a分子量一致的条带;免疫荧光化学检测发现TvRab1a分布于细胞核周围的高尔基复合体与内质网.结论 获得的抗TvRab1a蛋白的多抗血清可用于TvRab1a基因功能的研究;TvRab1a功能场所位于高尔基复合体与内质网.

Crystal structure of inactive form of Rab3B

International Nuclear Information System (INIS)

Zhang, Wei; Shen, Yang; Jiao, Ronghong; Liu, Yanli; Deng, Lingfu; Qi, Chao

2012-01-01

Highlights: ► This is the first structural information of human Rab3B. ► To provides a structural basis for the GDP/GTP switch in controlling the activity of Rab3. ► The charge distribution of Rab3B indicates its unique roles in vesicular trafficking. -- Abstract: Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium.

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Wu-Lin Zuo

Full Text Available The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+/HCO(3(- cotransporter in the pH regulation in rat epididymis.Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH solution, the intracellular pH (pHi recovery from NH(4Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+/H(+ exchanger (NHE. Immediately changing of the KH solution from HEPES buffered to HCO(3(- buffered would cause another pHi recovery. The pHi recovery in HCO(3(- buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, the inhibitor of HCO(3(- transporter or by removal of extracellular Na(+. The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH.The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

Crystal structure of inactive form of Rab3B

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Zhang, Wei [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China); Shen, Yang [Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, Ontario, Canada M5G 1L7 (Canada); Jiao, Ronghong [Department of Function Inspection, Hebei Provincial People' s Hospital, Shijiazhuang 050051 (China); Liu, Yanli; Deng, Lingfu [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China); Qi, Chao, E-mail: qichao@mail.ccnu.edu.cn [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China)

2012-02-24

Highlights: Black-Right-Pointing-Pointer This is the first structural information of human Rab3B. Black-Right-Pointing-Pointer To provides a structural basis for the GDP/GTP switch in controlling the activity of Rab3. Black-Right-Pointing-Pointer The charge distribution of Rab3B indicates its unique roles in vesicular trafficking. -- Abstract: Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 A resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

Crystallization and preliminary X-ray crystallographic studies of Rab6A′(Q72L): a GTP-locked form

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Shin, Young-Cheul; Jang, Tae-Ho; Yoon, Jong Hwan; Jeon, Ju-Hong; Park, Hyun Ho

2012-01-01

Rab6A′(Q72L), a constitutively active GTP-binding form of Rab6A, was purified and crystallized. The crystals were found to belong to space group P22 1 2 1 , with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The crystals were obtained at 293 K and diffracted to a resolution of 1.9 Å. Rab6A, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, cell differentiation and cell growth. Rab6A can exist in two isoforms termed Rab6A and Rab6A′. The substitution of Gln72 by Leu (Q72L) in the Rab6A family blocks GTP-hydrolysis activity, and this mutation usually causes the Rab6A protein to be in a constitutively active form. In this study, in order to understand the functional uniqueness of Rab6A′ and the molecular mechanism of the control of activity by GTP and GDP from the crystal structure, a Rab6A′(Q72L) mutant form was overexpressed in Escherichia coli with an engineered N-terminal His tag. Rab6A′(Q72L) was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 1.9 Å from a crystal belonging to space group P22 1 2 1 with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The asymmetric unit was estimated to contain two molecules

CSFV proliferation is associated with GBF1 and Rab2

Indian Academy of Sciences (India)

2016-12-29

Dec 29, 2016 ... 2.4 GBF1and Rab2 shRNA vector construction and cell ... CSFV RNA in ST cells at the earliest time point were selected as ... Real-time PCR for detection of GBF1 ..... pathways are also regulated by GBF1 and are involved in.

Wheat TaRab7 GTPase is part of the signaling pathway in responses to stripe rust and abiotic stimuli.

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Furong Liu

Full Text Available Small GTP-binding proteins function as regulators of specific intercellular fundamental biological processes. In this study, a small GTP-binding protein Rab7 gene, designated as TaRab7, was identified and characterized from a cDNA library of wheat leaves infected with Puccinia striiformis f. sp. tritici (Pst the wheat stripe rust pathogen. The gene was predicted to encode a protein of 206 amino acids, with a molecular mass of 23.13 KDa and an isoeletric point (pI of 5.13. Further analysis revealed the presence of a conserved signature that is characteristic of Rab7, and phylogenetic analysis demonstrated that TaRab7 has the highest similarity to a small GTP binding protein gene (BdRab7-like from Brachypodium distachyon. Quantitative real-time PCR assays revealed that the expression of TaRab7 was higher in the early stage of the incompatible interactions between wheat and Pst than in the compatible interaction, and the transcription level of TaRab7 was also highly induced by environmental stress stimuli. Furthermore, knocking down TaRab7 expression by virus induced gene silencing enhanced the susceptibility of wheat cv. Suwon 11 to an avirulent race CYR23. These results imply that TaRab7 plays an important role in the early stage of wheat-stripe rust fungus interaction and in stress tolerance.

Classifying the molecular functions of Rab GTPases in membrane trafficking using deep convolutional neural networks.

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Le, Nguyen-Quoc-Khanh; Ho, Quang-Thai; Ou, Yu-Yen

2018-06-13

Deep learning has been increasingly used to solve a number of problems with state-of-the-art performance in a wide variety of fields. In biology, deep learning can be applied to reduce feature extraction time and achieve high levels of performance. In our present work, we apply deep learning via two-dimensional convolutional neural networks and position-specific scoring matrices to classify Rab protein molecules, which are main regulators in membrane trafficking for transferring proteins and other macromolecules throughout the cell. The functional loss of specific Rab molecular functions has been implicated in a variety of human diseases, e.g., choroideremia, intellectual disabilities, cancer. Therefore, creating a precise model for classifying Rabs is crucial in helping biologists understand the molecular functions of Rabs and design drug targets according to such specific human disease information. We constructed a robust deep neural network for classifying Rabs that achieved an accuracy of 99%, 99.5%, 96.3%, and 97.6% for each of four specific molecular functions. Our approach demonstrates superior performance to traditional artificial neural networks. Therefore, from our proposed study, we provide both an effective tool for classifying Rab proteins and a basis for further research that can improve the performance of biological modeling using deep neural networks. Copyright © 2018 Elsevier Inc. All rights reserved.

Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes.

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Starling, Georgina P; Yip, Yan Y; Sanger, Anneri; Morton, Penny E; Eden, Emily R; Dodding, Mark P

2016-06-01

The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt-Hoge-Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri-nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi-associated small GTPase Rab34. Rab34-positive peri-nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34-induced peri-nuclear lysosome clustering. FLCN interacts directly via its C-terminal DENN domain with the Rab34 effector RILP Using purified recombinant proteins, we show that the FLCN-DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP We propose a model whereby starvation-induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34-positive peri-nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during Mammalian Spermiogenesis

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Ying-Hung Lin

2017-01-01

Full Text Available According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16. RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1, were identified using co-immunoprecipitation (co-IP and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS. We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning.

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Dodson, Mark W; Zhang, Ting; Jiang, Changan; Chen, Shengdi; Guo, Ming

2012-03-15

LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. Here, we show that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2(G2019S) allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD.

An auxin responsive CLE gene regulates shoot apical meristem development in Arabidopsis

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Hongyan eGuo

2015-05-01

Full Text Available Plant hormone auxin regulates most, if not all aspects of plant growth and development, including lateral root formation, organ pattering, apical dominance and tropisms. Peptide hormones are peptides with hormone activities. Some of the functions of peptide hormones in regulating plant growth and development are similar to that of auxin, however, the relationship between auxin and peptide hormones remains largely unknown. Here we report the identification of OsCLE48, a rice (Oryza sativa CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION gene, as an auxin response gene, and the functional characterization of OsCLE48 in Arabidopsis and rice. OsCLE48 encodes a CLE peptide hormone that is similar to Arabidopsis CLEs. RT-PCR analysis showed that OsCLE48 was induced by exogenously application of IAA (indole-3-acetic acid, a naturally occurred auxin. Expression of integrated OsCLE48p:GUS reporter gene in transgenic Arabidopsis plants was also induced by exogenously IAA treatment. These results indicate that OsCLE48 is an auxin responsive gene. Histochemical staining showed that GUS activity was detected in all the tissue and organs of the OsCLE48p:GUS transgenic Arabidopsis plants. Expression of OsCLE48 under the control of the 35S promoter in Arabidopsis inhibited shoot apical meristem development. Expression of OsCLE48 under the control of the CLV3 native regulatory elements almost completely complemented clv3-2 mutant phenotypes, suggesting that OsCLE48 is functionally similar to CLV3. On the other hand, expression of OsCLE48 under the control of the 35S promoter in Arabidopsis has little, if any effects on root apical meristem development, and transgenic rice plants overexpressing OsCLE48 are morphologically indistinguishable from wild type plants, suggesting that the functions of some CLE peptides may not be fully conserved in Arabidopsis and rice.

LRRK2 mediated Rab8a phosphorylation promotes lipid storage.

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Yu, Miao; Arshad, Muhammad; Wang, Wenmin; Zhao, Dongyu; Xu, Li; Zhou, Linkang

2018-02-27

Several mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson's disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability. It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Recent studies have shown that some Rab GTPases, especially Rab8, serve as substrates of LRRK2 and undergo phosphorylation in its switch II domain upon interaction. Current study was performed in order to find out the effects of the phosphorylation of Rab8 and its mutants on lipid metabolism and lipid droplets growth. The phosphorylation status of Rab8a was checked by phos-tag gel. Point mutant construct were generated to investigate the function of Rab8a. 3T3L1 cells were transfected with indicated plasmids and the lipid droplets were stained with Bodipy. Fluorescent microscopy experiments were performed to examine the sizes of lipid droplets. The interactions between Rab8a and Optineurin were determined by immunoprecipitation and western blot. Our assays demonstrated that Rab8a was phosphorylated by mutated LRRK2 that exhibits high kinase activity. Phosphorylation of Rab8a on amino acid residue T72 promoted the formation of large lipid droplets. T72D mutant of Rab8a had higher activity to promote the formation of large lipid droplets compared with wild type Rab8a, with increase in average diameter of lipid droplets from 2.10 μm to 2.46 μm. Moreover, phosphorylation of Rab8a weakened the interaction with its effector Optineurin. Y1699C mutated LRRK2 was able to phosphorylate Rab8a and phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplets. Taken together, our study suggests an indirect relationship between enhanced lipid storage capacity and PD pathogenesis.

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

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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...

Over-expression of a Rab family GTPase from phreatophyte Prosopis juliflora confers tolerance to salt stress on transgenic tobacco.

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George, Suja; Parida, Ajay

2011-03-01

Plant growth and productivity are adversely affected by various abiotic and biotic stress factors. In our previous study, we used Prosopis juliflora, an abiotic stress tolerant tree species of Fabaceae, as a model plant system for isolating genes functioning in abiotic stress tolerance. Here we report the isolation and characterization of a Rab family GTPase from P. juliflora (Pj Rab7) and the ability of this gene to confer salt stress tolerance in transgenic tobacco. Northern analysis for Pj Rab7 in P. juliflora leaf tissue revealed up-regulation of this gene under salt stress under the concentrations and time points analyzed. Pj Rab7 transgenic tobacco lines survived better under conditions of 150 mM NaCl stress compared to control un-transformed plants. Pj Rab7 transgenic plants were found to accumulate more sodium than control plants during salt stress. The results of our studies could be used as a starting point for generation of crop plants tolerant to abiotic stress.

Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase

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Isabelle Bouchez

2015-07-01

Full Text Available It has now been clearly shown that lipid droplets (LDs play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+ ATPase (V-ATPase. Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.

The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

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Szekeres, Ferenc; Chadt, Alexandra; Tom, Robby Z

2012-01-01

The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJL...... be explained partly by a 50% reduction in GLUT4 protein, since proximal signaling at the level of Akt, AMPK, and acetyl-CoA carboxylase (ACC) was unaltered. Paradoxically, in vivo insulin-stimulated 2-deoxyglucose uptake was increased in EDL and tibialis anterior muscle from TBC1D1-deficient mice......)) and wild-type littermates were studied. Glucose and insulin tolerance, glucose utilization, hepatic glucose production, and tissue-specific insulin-mediated glucose uptake were determined. The effect of insulin, AICAR, or contraction on glucose transport was studied in isolated skeletal muscle. Glucose...

Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

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Katherine Maringer

2014-08-01

Full Text Available Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA, and dominant negative (DN forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

High Rab27A expression indicates favorable prognosis in CRC.

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Shi, Chuanbing; Yang, Xiaojun; Ni, Yijiang; Hou, Ning; Xu, Li; Zhan, Feng; Zhu, Huijun; Xiong, Lin; Chen, Pingsheng

2015-06-13

Rab27A is a peculiar member in Rab family and has been suggested to play essential roles in the development of human cancers. However, the association between Rab27A expression and clinicopathological characteristics of colorectal cancer (CRC) has not been elucidated yet. One-step quantitative real-time polymerase chain reaction (qPCR) test with 18 fresh-frozen CRC samples and immunohistochemistry (IHC) analysis in 112 CRC cases were executed to evaluate the relationship between Rab27A expression and the clinicopathological features of CRC. Cox regression and Kaplan-Meier survival analyses were performed to identify the prognostic factors for 112 CRC patients. The results specified that the expression levels of Rab27A mRNA and protein were significantly higher in CRC tissues than that in matched non-cancerous tissues, in both qPCR test (p = 0.029) and IHC analysis (p = 0.020). The IHC data indicated that the Rab27A protein expression in CRC was statistically correlated with lymph node metastasis (p = 0.022) and TNM stage (p = 0.026). Cox multi-factor analysis and Kaplan-Meier method suggested Rab27A protein expression (p = 0.012) and tumor differentiation (p = 0.004) were significantly associated with the overall survival of CRC patients. The data indicated the differentiate expression of Rab27A in CRC tissues and matched non-cancerous tissues. Rab27A may be used as a valuable prognostic biomarker for CRC patients.

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

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Lucas, María; Gaspar, Andrew H.; Pallara, Chiara; Rojas, Adriana Lucely; Fernández-Recio, Juan; Machner, Matthias P.; Hierro, Aitor

2014-01-01

A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD–Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization. PMID:25114243

The monomeric GTPase RabA2 is required for progression and maintenance of membrane integrity of infection threads during root nodule symbiosis.

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Dalla Via, Virginia; Traubenik, Soledad; Rivero, Claudio; Aguilar, O Mario; Zanetti, María Eugenia; Blanco, Flavio Antonio

2017-04-01

Progression of the infection canal that conducts rhizobia to the nodule primordium requires a functional Rab GTPase located in Golgi/trans-Golgi that also participate in root hair polar growth. Common bean (Phaseolus vulgaris) symbiotically associates with its partner Rhizobium etli, resulting in the formation of root nitrogen-fixing nodules. Compatible bacteria can reach cortical cells in a tightly regulated infection process, in which the specific recognition of signal molecules is a key step to select the symbiotic partner. In this work, we show that RabA2, a monomeric GTPase from common bean, is required for the progression of the infection canal, referred to as the infection thread (IT), toward the cortical cells. Expression of miss-regulated mutant variants of RabA2 resulted in an increased number of abortive infection events, including bursting of ITs and a reduction in the number of nodules. Nodules formed in these plants were small and contained infected cells with disrupted symbiosome membranes, indicating either early senescence of these cells or defects in the formation of the symbiosome membrane during bacterial release. RabA2 localized to mobile vesicles around the IT, but mutations that affect GTP hydrolysis or GTP/GDP exchange modified this localization. Colocalization of RabA2 with ArfA1 and a Golgi marker indicates that RabA2 localizes in Golgi stacks and the trans-Golgi network. Our results suggest that RabA2 is part of the vesicle transport events required to maintain the integrity of the membrane during IT progression.

The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

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Emilie Fugier

2009-06-01

Full Text Available The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER-derived replicative organelle named the "Brucella-containing vacuole" (BCV. Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

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Michael G Connor

2015-10-01

Full Text Available Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.

Small G proteins in insulin action: Rab and Rho families at the crossroads of signal transduction and GLUT4 vesicle traffic.

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Ishikura, S; Koshkina, A; Klip, A

2008-01-01

Insulin stimulates glucose uptake into muscle and adipose tissues through glucose transporter 4 (GLUT4). GLUT4 cycles between the intracellular compartments and the plasma membrane. GLUT4 traffic-regulating insulin signals are largely within the insulin receptor-insulin receptor substrate-phosphatidylinositol 3-kinase (IR-IRS-PI3K) axis. In muscle cells, insulin signal bifurcates downstream of the PI3K into one arm leading to the activation of the Ser/Thr kinases Akt and atypical protein kinase C, and another leading to the activation of Rho family protein Rac1 leading to actin remodelling. Activated Akt inactivates AS160, a GTPase-activating protein for Rab family small G proteins. Here we review the roles of Rab and Rho proteins, particularly Rab substrates of AS160 and Rac1, in insulin-stimulated GLUT4 traffic. We discuss: (1) how distinct steps in GLUT4 traffic may be regulated by discrete Rab proteins, and (2) the importance of Rac1 activation in insulin-induced actin remodelling in muscle cells, a key element for the net gain in surface GLUT4.

Rab3 proteins involved in vesicle biogenesis and priming in embryonic mouse chromaffin cells

DEFF Research Database (Denmark)

Schonn, Jean-Sébastien; van Weering, Jan R T; Mohrmann, Ralf

2010-01-01

The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here we used a quadruple Rab3 knock-out (rab3a, rab3b, rab3c, rab3d null, here denoted ABCD(-/-)) mouse line to investigate Rab3 function in embryonic mouse adrena...

Distribution and function of HCN channels in the apical dendritic tuft of neocortical pyramidal neurons.

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Harnett, Mark T; Magee, Jeffrey C; Williams, Stephen R

2015-01-21

The apical tuft is the most remote area of the dendritic tree of neocortical pyramidal neurons. Despite its distal location, the apical dendritic tuft of layer 5 pyramidal neurons receives substantial excitatory synaptic drive and actively processes corticocortical input during behavior. The properties of the voltage-activated ion channels that regulate synaptic integration in tuft dendrites have, however, not been thoroughly investigated. Here, we use electrophysiological and optical approaches to examine the subcellular distribution and function of hyperpolarization-activated cyclic nucleotide-gated nonselective cation (HCN) channels in rat layer 5B pyramidal neurons. Outside-out patch recordings demonstrated that the amplitude and properties of ensemble HCN channel activity were uniform in patches excised from distal apical dendritic trunk and tuft sites. Simultaneous apical dendritic tuft and trunk whole-cell current-clamp recordings revealed that the pharmacological blockade of HCN channels decreased voltage compartmentalization and enhanced the generation and spread of apical dendritic tuft and trunk regenerative activity. Furthermore, multisite two-photon glutamate uncaging demonstrated that HCN channels control the amplitude and duration of synaptically evoked regenerative activity in the distal apical dendritic tuft. In contrast, at proximal apical dendritic trunk and somatic recording sites, the blockade of HCN channels decreased excitability. Dynamic-clamp experiments revealed that these compartment-specific actions of HCN channels were heavily influenced by the local and distributed impact of the high density of HCN channels in the distal apical dendritic arbor. The properties and subcellular distribution pattern of HCN channels are therefore tuned to regulate the interaction between integration compartments in layer 5B pyramidal neurons. Copyright © 2015 the authors 0270-6474/15/351024-14$15.00/0.

Essential role of RAB27A in determining constitutive human skin color.

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Yasuko Yoshida-Amano

Full Text Available Human skin color is predominantly determined by melanin produced in melanosomes within melanocytes and subsequently distributed to keratinocytes. There are many studies that have proposed mechanisms underlying ethnic skin color variations, whereas the processes involved from melanin synthesis in melanocytes to the transfer of melanosomes to keratinocytes are common among humans. Apart from the activities in the melanogenic rate-limiting enzyme, tyrosinase, in melanocytes and the amounts and distribution patterns of melanosomes in keratinocytes, the abilities of the actin-associated factors in charge of melanosome transport within melanocytes also regulate pigmentation. Mutations in genes encoding melanosome transport-related molecules, such as MYO5A, RAB27A and SLAC-2A, have been reported to cause a human pigmentary disease known as Griscelli syndrome, which is associated with diluted skin and hair color. Thus we hypothesized that process might play a role in modulating skin color variations. To address that hypothesis, the correlations of expression of RAB27A and its specific effector, SLAC2-A, to melanogenic ability were evaluated in comparison with tyrosinase, using human melanocytes derived from 19 individuals of varying skin types. Following the finding of the highest correlation in RAB27A expression to the melanogenic ability, darkly-pigmented melanocytes with significantly higher RAB27A expression were found to transfer significantly more melanosomes to keratinocytes than lightly-pigmented melanocytes in co-culture and in human skin substitutes (HSSs in vivo, resulting in darker skin color in concert with the difference observed in African-descent and Caucasian skins. Additionally, RAB27A knockdown by a lentivirus-derived shRNA in melanocytes concomitantly demonstrated a significantly reduced number of transferred melanosomes to keratinocytes in co-culture and a significantly diminished epidermal melanin content skin color intensity (�

Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent.

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Claudia A Bertuccio

Full Text Available The intermediate conductance, Ca2+-activated K+ channel (KCa3.1 targets to the basolateral (BL membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3

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

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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.

Characterization of the human RAB38 and RAB7 genes: exclusion of new major pathological loci for Japanese OCA.

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Suzuki, Tamio; Miyamura, Yoshinori; Inagaki, Katsuhiko; Tomita, Yasushi

2003-08-01

Oculocutaneous albinisms (OCAs) are due to various gene mutations that cause a disruption of melanogenesis in the melanocyte. Four different genes associated with human OCA have been reported, however, not all of OCA patients can be classified according to these four genes. We have sought to find a new major locus for Japanese OCA. Recently two genes, RAB38 and RAB7, were reported to play an important role in melanogenesis in the melanocyte, suggesting that these two genes could be good candidates for new OCA loci. To determine the structures of the human RAB38 and RAB7 genes, and examine if the two genes are new major loci for Japanese OCA. We screened mutations in these genes of 25 Japanese OCA patients who lacked mutations in the OCA1 and OCA2 genes with SSCP/heteroduplexes method. We determined the both genes, and their genomic organizations to design the primers for SSCP/heteroduplexes method. And then we screened mutations, but no mutation was detected. Neither of the genes is a new major locus for Japanese OCA.

Role of rab proteins in epithelial membrane traffic

NARCIS (Netherlands)

van Ijzendoorn, SCD; Mostov, KE; Hoekstra, D

2003-01-01

Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such

Insight into the molecular switch mechanism of human Rab5a from molecular dynamics simulations

Energy Technology Data Exchange (ETDEWEB)

Wang, Jing-Fang, E-mail: jfwang@gordonlifescience.org [Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shanghai Center for Bioinformation Technology, 100 Qinzhou Road, Shanghai 200235 (China); Gordon Life Science Institute, 13784 Torrey Del Mar Drive, San Diego, CA 92130 (United States); Chou, Kuo-Chen [Gordon Life Science Institute, 13784 Torrey Del Mar Drive, San Diego, CA 92130 (United States)

2009-12-18

Rab5a is currently a most interesting target because it is responsible for regulating the early endosome fusion in endocytosis and possibly the budding process. We utilized longtime-scale molecular dynamics simulations to investigate the internal motion of the wild-type Rab5a and its A30P mutant. It was observed that, after binding with GTP, the global flexibility of the two proteins is increasing, while the local flexibility in their sensitive sites (P-loop, switch I and II regions) is decreasing. Also, the mutation of Ala30 to Pro30 can cause notable flexibility variations in the sensitive sites. However, this kind of variations is dramatically reduced after binding with GTP. Such a remarkable feature is mainly caused by the water network rearrangements in the sensitive sites. These findings might be of use for revealing the profound mechanism of the displacements of Rab5a switch regions, as well as the mechanism of the GDP dissociation and GTP association.

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

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

International Nuclear Information System (INIS)

Mesa, Rosana; Magadan, Javier; Barbieri, Alejandro; Lopez, Cecilia; Stahl, Philip D.; Mayorga, Luis S.

2005-01-01

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN)

Rab1A is required for assembly of classical swine fever virus particle.

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Lin, Jihui; Wang, Chengbao; Liang, Wulong; Zhang, Jing; Zhang, Longxiang; Lv, Huifang; Dong, Wang; Zhang, Yanming

2018-01-15

Rab1A belongs to the small Rab GTPase family and is involved in the lifecycle of numerous viruses. Here, knockdown of Rab1A inhibited CSFV growth. Further study revealed that Rab1A depletion decreased intracellular and extracellular CSFV titers, but did not affect intracellular virus genome copies and E2 protein expression within a virus lifecycle, which suggested that Rab1A is required for CSFV particle assembly rather than for genome replication or virion release. This was proofed by blocking the spread of virus using neutralizing antibodies, through which the negative effects of Rab1A knockdown on multi-cycle replication of CSFV were eliminated. Moreover, co-immunoprecipitation and confocal microscopy assays showed that Rab1A bound to CSFV NS5A protein, indicating that Rab1A and viral NS5A proteins may work cooperatively during CSFV particle assembly. In conclusion, this study demonstrated for the first time that Rab1A is required for CSFV particle assembly and binds to viral particle assembly-related NS5A protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Fructose-induced increases in expression of intestinal fructolytic and gluconeogenic genes are regulated by GLUT5 and KHK

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Patel, Chirag; Douard, Veronique; Yu, Shiyan; Tharabenjasin, Phuntila; Gao, Nan

2015-01-01

Marked increases in fructose consumption have been tightly linked to metabolic diseases. One-third of ingested fructose is metabolized in the small intestine, but the underlying mechanisms regulating expression of fructose-metabolizing enzymes are not known. We used genetic mouse models to test the hypothesis that fructose absorption via glucose transporter protein, member 5 (GLUT5), metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein in brain 11a (Rab11a)-dependent endosomes are required for the regulation of intestinal fructolytic and gluconeogenic enzymes. Fructose feeding increased the intestinal mRNA and protein expression of these enzymes in the small intestine of adult wild-type (WT) mice compared with those gavage fed with lysine or glucose. Fructose did not increase expression of these enzymes in the GLUT5 knockout (KO) mice. Blocking intracellular fructose metabolism by KHK ablation also prevented fructose-induced upregulation. Glycolytic hexokinase I expression was similar between WT and GLUT5- or KHK-KO mice and did not vary with feeding solution. Gavage feeding with the fructose-specific metabolite glyceraldehyde did not increase enzyme expression, suggesting that signaling occurs before the hydrolysis of fructose to three-carbon compounds. Impeding GLUT5 trafficking to the apical membrane using intestinal epithelial cell-specific Rab11a-KO mice impaired fructose-induced upregulation. KHK expression was uniformly distributed along the villus but was localized mainly in the basal region of the cytosol of enterocytes. The feedforward upregulation of fructolytic and gluconeogenic enzymes specifically requires GLUT5 and KHK and may proactively enhance the intestine's ability to process anticipated increases in dietary fructose concentrations. PMID:26084694

Transcytosis of immunoglobulin A in the mouse enterocyte occurs through glycolipid raft- and rab17-containing compartments

DEFF Research Database (Denmark)

Hansen, Gert Helge; Niels-Christiansen, L L; Immerdal, Lissi

1999-01-01

BACKGROUND & AIMS: Glycolipid "rafts" have been shown to play a role in apical membrane trafficking in the enterocyte. The present study characterized the membrane compartments of the enterocyte involved in transepithelial transport of small intestinal immunoglobulin A (IgA). Methods: Immunogold...... electron microscopy and radioactive labeling of mouse small intestinal explants were performed. RESULTS: IgA and the polymeric immunoglobulin receptor/secretory component were present in a raft compartment. Raft association occurred posttranslationally within 30 minutes, preceding secretion...... and were also frequently seen associated with the same vesicular profiles of glycolipid rafts. Colocalization of IgA and rab17, a small guanosine triphosphatase involved in transcytosis, was seen mainly along the basolateral plasma membrane and over basolateral endosomes and vesicles, but also...

Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 regulation through the drought-responsive element in an ABA-dependent pathway.

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Kizis, Dimosthenis; Pagès, Montserrat

2002-06-01

The abscisic acid-responsive gene rab17 of maize is expressed during late embryogenesis, and is induced by ABA and desiccation in embryo and vegetative tissues. ABRE and DRE cis-elements are involved in regulation of the gene by ABA and drought. Using yeast one-hybrid screening, we isolated two cDNAs encoding two new DRE-binding proteins, designated DBF1 and DBF2, that are members of the AP2/EREBP transcription factor family. Analysis of mRNA accumulation profiles showed that DBF1 is induced during maize embryogenesis and after desiccation, NaCl and ABA treatments in plant seedlings, whereas the DBF2 mRNA is not induced. DNA-binding preferences of DBFs were analysed by electrophoretic mobility shift assays, and showed that both DBF1 and DBF2 bound to the wild-type DRE2 element, but not to the DRE2 mutant or to the DRE1 element which differs only in a single nucleotide. Transactivation activity using particle bombardment showed that DBF1 functioned as activator of DRE2-dependent transcription of rab17 promoter by ABA, whereas DBF2 overexpression had a repression action downregulating not only the basal promoter activity, but also the ABA effect. These results show that ABA plays a role in the regulation of DBF activity, and suggests the existence of an ABA-dependent pathway for the regulation of genes through the C-repeat/DRE element.

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.

Growth, 14C-sucrose uptake, and metabolites of starch synthesis in apical and basal kernels of corn (Zea mays L.)

International Nuclear Information System (INIS)

Greenberg, J.M.

1985-01-01

Developing field-grown kernels of corn (Zea mays L. cv. Cornell 175) from the base and apex of the ear were sampled from seven to 70 days after pollination (DAP) an compared with respect to dry weight, ability to take up 14 C-sucrose from solution in vitro, and content of sucrose, glucose, starch, glucose-1-P (G1P), glucose-6-P (G6P), fructose-6-P (F6P), ADP-glucose (ADPG), and UDP-glucose (UDPG). ADPG and UDPG were analyzed by HPLC. All other metabolites were analyzed enzymatically. Simultaneous hand-pollination of all ovaries in an ear did not reduce the difference between apical and basal kernels in dry weight, indicating that the latter fertilization of apical kernels was not responsible for their lesser mature dry weight. Detached kernels took up 14 C-sucrose (0.3-400 mM) and glucose (5-100 mM) at rates linearly proportional to the sugar concentration. Glucose, fructose, and sorbitol did not inhibit uptake of 14 C-sucrose. Uptake was not stimulated by 5 mM CaCl 2 or the addition of buffers (pH 4.5-6.7) to the medium. Sulfhydryl reagents (PCMBS, NEM) and metabolic inhibitors (TNBS, DNP, NaF) did not reduce uptake. These observations suggest that sucrose is taken up by a non-saturable, non-energy-requiring mechanism. Sucrose uptake increased throughout development, especially at the stage when basal kernels began to accumulate more dry weight than apical kernels (10-20 DAP in freely pollinated ears; 25 DAP in synchronously pollinated ears). Hydrolysis of incorporated sucrose increased from 87% at 14 DAP to 99% by 57 DAP

Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494

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Verma, Jitender Kumar; Rastogi, Ruchir

2017-01-01

Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment. PMID:28650977

Cloning and Expression of A Rab1a Gene from Trichomonas vaginalis%阴道毛滴虫Rab1a基因的cDNA克隆及重组表达

Institute of Scientific and Technical Information of China (English)

徐晓园; 傅玉才; 许铭炎; 史咏梅; 刘红

2006-01-01

目的克隆和分析阴道毛滴虫(Trichomonas vaginalis,Tv)Rab1a基因,构建Rab1a基因表达重组载体并表达其融合蛋白,以进一步探讨其功能.方法提取阴道毛滴虫基因组DNA为模板,扩增TvRab1a基因,用pQE80L载体与TvRab1a cDNA克隆构建原核表达重组体并表达融合蛋白,纯化表达产物并由SDS-PAGE鉴定.结果在阴道毛滴虫cDNA文库克隆中发现了Rab1a基因,序列分析显示Rab1a基因的基因组DNA序列含有一个25 bp大小的内含子.成功构建了pQE/Rab1a原核表达重组体,并表达出预期大小的重组蛋白质.结论分析表明TvRab1a基因是阴道毛滴虫Rab1鸟苷三磷酸酶同源基因,它含有一个25 bp的内含子.获得了该基因的重组蛋白,将对TvRab1a基因的功能进行进一步研究.

Site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia.

Science.gov (United States)

Wyroba, E; Kwaśniak, P; Miller, K; Kobyłecki, K; Osińska, M

2016-04-11

Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue - distinct from that of Rab7a directly involved in phagocytosis - was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14-]UDP-glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non- mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14-]UDP- glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.

Site‐directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia

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E. Wyroba

2016-04-01

Full Text Available Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post‐translational modifications (PTM in proper targeting of Paramecium Rab7b paralogue – distinct from that of Rab7a directly involved in phagocytosis ‐ was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14–]UDP‐glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non‐mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non‐ mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC‐MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14‐]UDP‐ glucose, the suggested composition of the adduct attached to Thr200 might be (Hex1(HexNAc1(Phos3 or (HexNAc1 (Deoxyhexose1 (Phos1 (HexA1. These data indicate that PTM of Thr200 located in the hypervariable C‐region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.   

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.

Sheep internal parasites on Rab and Pag

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Relja Beck

2010-01-01

Full Text Available The purpose of our research was to determine which groups and species of internal parasites endanger the health of sheep on the islands of Rab and Pag. The research was carried out in 10 flocks on both islands taking the fresh dung out of 30% of the total number of sheep in each flock. It was ascertained that the gastrointestinal parasites and protozoa of Eimeria genus are present in most flocks on both islands. The presence of the fluke Dicrocoelium dendriticum was ascertained in considerably larger number of flocks on the island of Rab than on the island of Pag. On the other hand, the presence of parasites of Moniezia and Nematodirus genus was ascertained in larger number of flocks on the island of Pag. In two flocks on Rab parasites of Protostrongylus genus were ascertained while on the island of Pag they were not found in any flock.

Disruption of endocytic trafficking protein Rab7 impairs invasiveness of cholangiocarcinoma cells.

Science.gov (United States)

Suwandittakul, Nantana; Reamtong, Onrapak; Molee, Pattamaporn; Maneewatchararangsri, Santi; Sutherat, Maleerat; Chaisri, Urai; Wongkham, Sopit; Adisakwattana, Poom

2017-09-07

Alterations and mutations of endo-lysosomal trafficking proteins have been associated with cancer progression. Identification and characterization of endo-lysosomal trafficking proteins in invasive cholangiocarcinoma (CCA) cells may benefit prognosis and drug design for CCA. To identify and characterize endo-lysosomal trafficking proteins in invasive CCA. A lysosomal-enriched fraction was isolated from a TNF-α induced invasive CCA cell line (KKU-100) and uninduced control cells and protein identification was performed with nano-LC MS/MS. Novel lysosomal proteins that were upregulated in invasive CCA cells were validated by real-time RT-PCR. We selected Rab7 for further studies of protein level using western blotting and subcellular localization using immunofluorescence. The role of Rab7 in CCA invasion was determined by siRNA gene knockdown and matrigel transwell assay. Rab7 mRNA and protein were upregulated in invasive CCA cells compared with non-treated controls. Immunofluorescence studies demonstrated that Rab7 was expressed predominantly in invasive CCA cells and was localized in the cytoplasm and lysosomes. Suppression of Rab7 translation significantly inhibited TNF-α-induced cell invasion compared to non-treated control (p= 0.044). Overexpression of Rab7 in CCA cells was associated with cell invasion, supporting Rab7 as a novel candidate for the development of diagnostic and therapeutic strategies for CCA.

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

Energy Technology Data Exchange (ETDEWEB)

Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); James, John; Prescott, Alan [Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Haga, Ismar R. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); Beard, Philippa M., E-mail: pip.beard@roslin.ed.ac.uk [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom)

2015-01-15

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton

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Sarah M. Carpanini

2014-06-01

Full Text Available Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM. Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic

A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton.

Science.gov (United States)

Carpanini, Sarah M; McKie, Lisa; Thomson, Derek; Wright, Ann K; Gordon, Sarah L; Roche, Sarah L; Handley, Mark T; Morrison, Harris; Brownstein, David; Wishart, Thomas M; Cousin, Michael A; Gillingwater, Thomas H; Aligianis, Irene A; Jackson, Ian J

2014-06-01

Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions. �

Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.

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Coreen Johnson

Full Text Available Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.

A Bacterial Pathogen Targets a Host Rab-Family GTPase Defense Pathway with a GAP.

Science.gov (United States)

Spanò, Stefania; Gao, Xiang; Hannemann, Sebastian; Lara-Tejero, María; Galán, Jorge E

2016-02-10

Cell-autonomous defense mechanisms are potent strategies that protect individual cells against intracellular pathogens. The Rab-family GTPase Rab32 was previously shown to restrict the intracellular human pathogen Salmonella Typhi, but its potential broader role in antimicrobial defense remains unknown. We show that Rab32 represents a general cell-autonomous, antimicrobial defense that is counteracted by two Salmonella effectors. Mice lacking Rab-32 or its nucleotide exchange factor BLOC-3 are permissive to S. Typhi infection and exhibit increased susceptibility to S. Typhimurium. S. Typhimurium counters this defense pathway by delivering two type III secretion effectors, SopD2, a Rab32 GAP, and GtgE, a specific Rab32 protease. An S. Typhimurium mutant strain lacking these two effectors exhibits markedly reduced virulence, which is fully restored in BLOC-3-deficient mice. These results demonstrate that a cell-autonomous, Rab32-dependent host defense pathway plays a central role in the defense against vacuolar pathogens and describe a mechanism evolved by a bacterial pathogen to counter it. Copyright © 2016 Elsevier Inc. All rights reserved.

Rab5 induces Rac-independent lamellipodia formation and cell migration

NARCIS (Netherlands)

Spaargaren, M.; Bos, J. L.

1999-01-01

Rab5 is a regulatory GTPase of vesicle docking and fusion that is involved in receptor-mediated endocytosis and pinocytosis. Introduction of active Rab5 in cells stimulates the rate of endocytosis and vesicle fusion, resulting in the formation of large endocytic vesicles, whereas dominant negative

A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK

International Nuclear Information System (INIS)

Abe, Yasuhito; Takeuchi, Takashi; Imai, Yoshinori; Murase, Ryuichi; Kamei, Yoshiaki; Fujibuchi, Taketsugu; Matsumoto, Suguru; Ueda, Norifumi; Ogasawara, Masahito; Shigemoto, Kazuhiro; Kito, Katsumi

2006-01-01

PRPK phosphorylates serine-15 residue of p53 and enhances transcriptional activity. PRPK possesses a bipartite nuclear localization signal and localizes in nucleus when over-expressed in cells. However, intrinsic PRPK localizes mainly in the cytosol in situ. While studying the mechanisms in the distribution of intrinsic PRPK, we identified a PRPK binding protein, an ubiquitously expressed Small Ras-like GTPase, Rab1c, also named Ray or Rab35. The over-expressed Ray was distributed in the nucleus, cytosol, and cell membrane. Both Ray wild type and GTP-restrictively binding mutant Ray-Q67L, but not guanine nucleotide unstable binding mutant Ray-N120I, partially distributed the over-expressed PRPK to the cytosol and also suppressed the PRPK-induced p53-transcriptional activity profoundly. A Small Ras-like GTPase protein Ray was thus indicated to modulate p53 transcriptional activity of PRPK

Molecular cloning, sequence characterization and expression pattern of Rab18 gene from watermelon (Citrullus lanatus).

Science.gov (United States)

Xinli, Xiao; Lei, Peng

2015-03-04

The complete mRNA sequence of watermelon Rab18 gene was amplified through the rapid amplification of cDNA ends (RACE) method. The full-length mRNA was 1010 bp containing a 645 bp open reading frame, which encodes a protein of 214 amino acids. Sequence analysis revealed that watermelon Rab18 protein shares high homology with the Rab18 of cucumber (99%), muskmelon (98%), Morus notabilis (90%), tomato (89%), wine grape (89%) and potato (88%). Phylogenetic analysis revealed that watermelon Rab18 gene has a closer genetic relationship with Rab18 gene of cucumber and muskmelon. Tissue expression profile analysis indicated that watermelon Rab18 gene was highly expressed in root, stem and leaf, moderately expressed in flower and weakly expressed in fruit.

Characterisation of SalRAB a salicylic acid inducible positively regulated efflux system of Rhizobium leguminosarum bv viciae 3841.

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Adrian J Tett

Full Text Available Salicylic acid is an important signalling molecule in plant-microbe defence and symbiosis. We analysed the transcriptional responses of the nitrogen fixing plant symbiont, Rhizobium leguminosarum bv viciae 3841 to salicylic acid. Two MFS-type multicomponent efflux systems were induced in response to salicylic acid, rmrAB and the hitherto undescribed system salRAB. Based on sequence similarity salA and salB encode a membrane fusion and inner membrane protein respectively. salAB are positively regulated by the LysR regulator SalR. Disruption of salA significantly increased the sensitivity of the mutant to salicylic acid, while disruption of rmrA did not. A salA/rmrA double mutation did not have increased sensitivity relative to the salA mutant. Pea plants nodulated by salA or rmrA strains did not have altered nodule number or nitrogen fixation rates, consistent with weak expression of salA in the rhizosphere and in nodule bacteria. However, BLAST analysis revealed seventeen putative efflux systems in Rlv3841 and several of these were highly differentially expressed during rhizosphere colonisation, host infection and bacteroid differentiation. This suggests they have an integral role in symbiosis with host plants.

The apical complex provides a regulated gateway for secretion of invasion factors in Toxoplasma.

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Nicholas J Katris

2014-04-01

Full Text Available The apical complex is the definitive cell structure of phylum Apicomplexa, and is the focus of the events of host cell penetration and the establishment of intracellular parasitism. Despite the importance of this structure, its molecular composition is relatively poorly known and few studies have experimentally tested its functions. We have characterized a novel Toxoplasma gondii protein, RNG2, that is located at the apical polar ring--the common structural element of apical complexes. During cell division, RNG2 is first recruited to centrosomes immediately after their duplication, confirming that assembly of the new apical complex commences as one of the earliest events of cell replication. RNG2 subsequently forms a ring, with the carboxy- and amino-termini anchored to the apical polar ring and mobile conoid, respectively, linking these two structures. Super-resolution microscopy resolves these two termini, and reveals that RNG2 orientation flips during invasion when the conoid is extruded. Inducible knockdown of RNG2 strongly inhibits host cell invasion. Consistent with this, secretion of micronemes is prevented in the absence of RNG2. This block, however, can be fully or partially overcome by exogenous stimulation of calcium or cGMP signaling pathways, respectively, implicating the apical complex directly in these signaling events. RNG2 demonstrates for the first time a role for the apical complex in controlling secretion of invasion factors in this important group of parasites.

Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues

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E Wyroba

2009-08-01

Full Text Available Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein and LAMP-2 (lysosomal membrane protein 2 as well as a7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2 in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100 000 x g of equal load were quantified by immunoblotting. LAMP-2 crossreacting polypeptide of ~106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The a7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILPrelated polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

Targeting eukaryotic Rab proteins: a smart strategy for chlamydial survival and replication.

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Damiani, María Teresa; Gambarte Tudela, Julián; Capmany, Anahí

2014-09-01

Chlamydia, an obligate intracellular bacterium which passes its entire lifecycle within a membrane-bound vacuole called the inclusion, has evolved a variety of unique strategies to establish an advantageous intracellular niche for survival. This review highlights the mechanisms by which Chlamydia subverts vesicular transport in host cells, particularly by hijacking the master controllers of eukaryotic trafficking, the Rab proteins. A subset of Rabs and Rab interacting proteins that control the recycling pathway or the biosynthetic route are selectively recruited to the chlamydial inclusion membrane. By interfering with Rab-controlled transport steps, this intracellular pathogen not only prevents its own degradation in the phagocytic pathway, but also creates a favourable intracellular environment for growth and replication. Chlamydia, a highly adapted and successful intracellular pathogen, has several redundant strategies to re-direct vesicles emerging from biosynthetic compartments that carry host molecules essential for bacterial development. Although current knowledge is limited, the latest findings have shed light on the role of Rab proteins in the course of chlamydial infections and could open novel opportunities for anti-chlamydial therapy. © 2014 John Wiley & Sons Ltd.

Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene.

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Amara, Imen; Capellades, Montserrat; Ludevid, M Dolors; Pagès, Montserrat; Goday, Adela

2013-06-15

Late Embryogenesis Abundant (LEA) proteins participate in plant stress responses and contribute to the acquisition of desiccation tolerance. In this report Rab28 LEA gene has been over-expressed in maize plants under a constitutive maize promoter. The expression of Rab28 transcripts led to the accumulation and stability of Rab28 protein in the transgenic plants. Native Rab28 protein is localized to nucleoli in wild type maize embryo cells; here we find by whole-mount immunocytochemistry that in root cells of Rab28 transgenic and wild-type plants the protein is also associated to nucleolar structures. Transgenic plants were tested for stress tolerance and resulted in sustained growth under polyethyleneglycol (PEG)-mediated dehydration compared to wild-type controls. Under osmotic stress transgenic seedlings showed increased leaf and root areas, higher relative water content (RWC), reduced chlorophyll loss and lower Malondialdehyde (MDA) production in relation to wild-type plants. Moreover, transgenic seeds exhibited higher germination rates than wild-type seeds under water deficit. Overall, our results highlight the presence of transgenic Rab28 protein in nucleolar structures and point to the potential of group 5 LEA Rab28 gene as candidate to enhance stress tolerance in maize plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

[6]-Gingerol, from Zingiber officinale, potentiates GLP-1 mediated glucose-stimulated insulin secretion pathway in pancreatic β-cells and increases RAB8/RAB10-regulated membrane presentation of GLUT4 transporters in skeletal muscle to improve hyperglycemia in Leprdb/db type 2 diabetic mice.

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Samad, Mehdi Bin; Mohsin, Md Nurul Absar Bin; Razu, Bodiul Alam; Hossain, Mohammad Tashnim; Mahzabeen, Sinayat; Unnoor, Naziat; Muna, Ishrat Aklima; Akhter, Farjana; Kabir, Ashraf Ul; Hannan, J M A

2017-08-09

[6]-Gingerol, a major component of Zingiber officinale, was previously reported to ameliorate hyperglycemia in type 2 diabetic mice. Endocrine signaling is involved in insulin secretion and is perturbed in db/db Type-2 diabetic mice. [6]-Gingerol was reported to restore the disrupted endocrine signaling in rodents. In this current study on Lepr db/db diabetic mice, we investigated the involvement of endocrine pathway in the insulin secretagogue activity of [6]-Gingerol and the mechanism(s) through which [6]-Gingerol ameliorates hyperglycemia. Lepr db/db type 2 diabetic mice were orally administered a daily dose of [6]-Gingerol (200 mg/kg) for 28 days. We measured the plasma levels of different endocrine hormones in fasting and fed conditions. GLP-1 levels were modulated using pharmacological approaches, and cAMP/PKA pathway for insulin secretion was assessed by qRT-PCR and ELISA in isolated pancreatic islets. Total skeletal muscle and its membrane fractions were used to measure glycogen synthase 1 level and Glut4 expression and protein levels. 4-weeks treatment of [6]-Gingerol dramatically increased glucose-stimulated insulin secretion and improved glucose tolerance. Plasma GLP-1 was found to be significantly elevated in the treated mice. Pharmacological intervention of GLP-1 levels regulated the effect of [6]-Gingerol on insulin secretion. Mechanistically, [6]-Gingerol treatment upregulated and activated cAMP, PKA, and CREB in the pancreatic islets, which are critical components of GLP-1-mediated insulin secretion pathway. [6]-Gingerol upregulated both Rab27a GTPase and its effector protein Slp4-a expression in isolated islets, which regulates the exocytosis of insulin-containing dense-core granules. [6]-Gingerol treatment improved skeletal glycogen storage by increased glycogen synthase 1 activity. Additionally, GLUT4 transporters were highly abundant in the membrane of the skeletal myocytes, which could be explained by the increased expression of Rab8 and Rab

BLOC-3 mutated in Hermansky-Pudlak syndrome is a Rab32/38 guanine nucleotide exchange factor.

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Gerondopoulos, Andreas; Langemeyer, Lars; Liang, Jin-Rui; Linford, Andrea; Barr, Francis A

2012-11-20

Hermansky-Pudlak syndrome (HPS) is a human disease characterized by partial loss of pigmentation and impaired blood clotting. These symptoms are caused by defects in the biogenesis of melanosomes and platelet dense granules, often referred to as lysosome-related organelles. Genes mutated in HPS encode subunits of the biogenesis of lysosome-related organelles complexes (BLOCs). BLOC-1 and BLOC-2, together with the AP-3 clathrin adaptor complex, act at early endosomes to sort components required for melanin formation and melanosome biogenesis away from the degradative lysosomal pathway toward early stage melanosomes. However the molecular functions of the Hps1-Hps4 complex BLOC-3 remain mysterious. Like other trafficking pathways, melanosome biogenesis and transport of enzymes involved in pigmentation involves specific Rab GTPases, in this instance Rab32 and Rab38. We now demonstrate that BLOC-3 is a Rab32 and Rab38 guanine nucleotide exchange factor (GEF). Silencing of the BLOC-3 subunits Hps1 and Hps4 results in the mislocalization of Rab32 and Rab38 and reduction in pigmentation. In addition, we show that BLOC-3 can promote specific membrane recruitment of Rab32/38. BLOC-3 therefore defines a novel Rab GEF family with a specific function in the biogenesis of lysosome-related organelles. Copyright © 2012 Elsevier Ltd. All rights reserved.

Evaluating algal growth performance and water use efficiency of pilot-scale revolving algal biofilm (RAB) culture systems.

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Gross, Martin; Mascarenhas, Vernon; Wen, Zhiyou

2015-10-01

A Revolving Algal Biofilm (RAB) growth system in which algal cells are attached to a flexible material rotating between liquid and gas phases has been developed. In this work, different configurations of RAB systems were developed at pilot-scale by retrofitting the attachment materials to a raceway pond (2000-L with 8.5 m(2) footprint area) and a trough reservoir (150 L with 3.5 m(2) footprint area). The algal growth performance and chemical composition, as well as the water evaporative loss and specific water consumption were evaluated over a period of nine months in a greenhouse environment near Boone, Iowa USA. Additionally a raceway pond was run in parallel, which served as a control. On average the raceway-based RAB and the trough-based RAB outperformed the control pond by 309% and 697%, respectively. A maximum productivity of 46.8 g m(-2) day(-1) was achieved on the trough-based RAB system. The evaporative water loss of the RAB system was modeled based on an energy balance analysis and was experimentally validated. While the RAB system, particularly the trough-based RAB, had higher water evaporative loss, the specific water consumption per unit of biomass produced was only 26% (raceway-based RAB) and 7% (trough-based RAB) of that of the control pond. Collectively, this research shows that the RAB system is an efficient algal culture system and has great potential to commercially produce microalgae with high productivity and efficient water use. © 2015 Wiley Periodicals, Inc.

Breaking into the epithelial apical-junctional complex--news from pathogen hackers.

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Vogelmann, Roger; Amieva, Manuel R; Falkow, Stanley; Nelson, W James

2004-02-01

The epithelial apical-junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical-junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical-junctional complex of the Ig superfamily--junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor--are important regulators of junction structure and function and represent critical targets of microbial virulence gene products.

Deficient Rab11 activity underlies glucose hypometabolism in primary neurons of Huntington’s disease mice

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Li, Xueyi; Valencia, Antonio; McClory, Hollis; Sapp, Ellen; Kegel, Kimberly B.; DiFiglia, Marian

2012-01-01

Highlights: ► Primary Huntington’s disease neurons are impaired in taking up glucose. ► Rab11 modulates glucose uptake in neurons. ► Increasing Rab11 activity attenuates the glucose uptake defect in disease neurons. ► We provide a novel mechanism for glucose hypometabolism in Huntington’s disease. -- Abstract: Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Positron emission tomography studies have revealed a decline in glucose metabolism in the brain of patients with HD by a mechanism that has not been established. We examined glucose utilization in embryonic primary cortical neurons of wild-type (WT) and HD knock-in mice, which have 140 CAG repeats inserted in the endogenous mouse huntingtin gene (HD 140Q/140Q ). Primary HD 140Q/140Q cortical neurons took up significantly less glucose than did WT neurons. Expression of permanently inactive and permanently active forms of Rab11 correspondingly altered glucose uptake in WT neurons, suggesting that normal activity of Rab11 is needed for neuronal uptake of glucose. It is known that Rab11 activity is diminished in HD 140Q/140Q neurons. Expression of dominant active Rab11 to enhance the activity of Rab11 normalized glucose uptake in HD 140Q/140Q neurons. These results suggest that deficient activity of Rab11 is a novel mechanism for glucose hypometabolism in HD.

Expression profile of Rab5, Rab7, tryptophan aspartate-containing coat protein, leprae lipoarabinomannan, and phenolic glycolipid-1 on the failure of the phagolysosome process in macrophages of leprosy patients as a viability marker of Mycobacterium leprae.

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Prakoeswa, Cita Rosita Sigit; Wahyuni, Ratna; Iswahyudi; Adriaty, Dinar; Yusuf, Irawan; Sutjipto; Agusni, Indropo; Izumi, Shinzo

2016-06-01

Phagolysosome process in macrophage of leprosy patients' is important in the early phase of eliminating Mycobacterium leprae invasion. This study was to clarify the involvement of Rab5, Rab7, and trytophan aspartate-containing coat protein (TACO) from host macrophage and leprae lipoarabinomannan (Lep-LAM) and phenolic glycolipid-1 (PGL-1) from M. leprae cell wall as the reflection of phagolysosome process in relation to 16 subunit ribosomal RNA (16S rRNA) M. leprae as a marker of viability of M. leprae. Using a cross sectional design study, skin biopsies were obtained from 47 newly diagnosed, untreated leprosy at Dr Soetomo Hospital, Surabaya, Indonesia. RNA isolation and complementary DNA synthesis were performed. Samples were divided into two groups: 16S rRNA M. leprae-positive and 16S rRNA M. leprae-negative. The expressions of Rab5, Rab7, TACO, Lep-LAM, and PGL-1 were assessed with an immunohistochemistry technique. Using Mann-Whitney U analysis, a significant difference in the expression profile of Rab5, Rab7, Lep-LAM, and PGL-1 was found (p.05). Spearman analysis revealed that there was a significant correlation between the score of Rab5, Rab7, Lep-LAM, and PGL-1 and the score of 16S rRNA M. leprae (pleprae infection, Rab5, Rab7, and Lep-LAM play important roles in the failure of phagolysosome process via a membrane trafficking pathway, while PGL-1 plays a role via blocking lysosomal activities. These inventions might be used for the development of an early diagnostic device in the future. Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

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

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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

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

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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.

3D time-lapse analysis of Rab11/FIP5 complex: spatiotemporal dynamics during apical lumen formation.

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Mangan, Anthony; Prekeris, Rytis

2015-01-01

Fluorescent imaging of fixed cells grown in two-dimensional (2D) cultures is one of the most widely used techniques for observing protein localization and distribution within cells. Although this technique can also be applied to polarized epithelial cells that form three-dimensional (3D) cysts when grown in a Matrigel matrix suspension, there are still significant limitations in imaging cells fixed at a particular point in time. Here, we describe the use of 3D time-lapse imaging of live cells to observe the dynamics of apical membrane initiation site (AMIS) formation and lumen expansion in polarized epithelial cells.

Herpesviruses in asymptomatic apical periodontitis lesions: an immunohistochemical approach.

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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.

The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

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Cecilia Bucci

2014-10-01

Full Text Available Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC and p75NTR, a member of the tumor necrosis factor (TNF receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways.

The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

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Bucci, Cecilia; Alifano, Pietro; Cogli, Laura

2014-01-01

Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and p75NTR, a member of the tumor necrosis factor (TNF) receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways. PMID:25295627

Expression of Rab25 in non-small cell lung cancer and its clinical significance

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Pu ZHOU

2014-03-01

Full Text Available Objective　To assess the expression of Rab25 protein in non-small cell lung cancer (NSCLC, and explore the correlation of its expression with tumor proliferation and metastasis. Methods　Sixty-one cases of NSCLC specimens (31 cases of squamous cell carcinoma, 26 cases of adenocarcinoma, and 4 cases of adenosquamous carcinoma undergone surgical treatment, and 40 specimens of adjacent normal lung tissues were obtained from Jan. 2009 to Jun. 2010 at Xingqiao Hospital of Third Military Medical University. Immunochemistry method of MaxVision was used to detect the expression of Rab25 in the specimens, and then the correlation of the expression with the clinicopathological parameters (patients' sex, age, smoking history, tumor type, differentiation, volume, TNM stage, lymph metastasis, etc. was analyzed using statistical software SPSS 21.0. Results 　Rab25 protein was mainly expressed in cytoplasm and cell membrane. The positive rate of Rab25 in NSCLC was 93.4%, which was significantly higher than that in adjacent normal tissues (27.5%, P＜0.01. The expression of Rab25 protein was significantly associated with the TNM stage and tumor size (P＜0.05. Conclusions　The expression of Rab25 is obviously higher in NSCLC than in the adjacent normal tissues, and the expression is associated with TNM stage and tumor size. Moreover, the later of the NSCLC stage, the larger of tumor size, and the higher of Rab25 expression will be in the NSCLC tissue. DOI: 10.11855/j.issn.0577-7402.2014.02.16

Strain-Specific Altered Regulatory Response of Rab7a and Tau in Creutzfeldt-Jakob Disease and Alzheimer's Disease.

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Zafar, Saima; Younas, Neelam; Correia, Susana; Shafiq, Mohsin; Tahir, Waqas; Schmitz, Matthias; Ferrer, Isidre; Andréoletti, Olivier; Zerr, Inga

2017-01-01

There is an increasing demand for the understanding of pathophysiology on neurodegeneration diseases at early stages. Changes in endocytic machinery and the cytoskeleton-associated response are the first alterations observed in Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease AD brain. In this study, we performed a targeted search for endocytic pathway proteins in the different regions of the brain. We found late endosome marker Rab7a which was significantly upregulated in the frontal cortex region in the rapid progressive CJD form (MM1) and rapid progressive AD (rpAD) forms. However, Rab9 expression was significantly downregulated only in CJD-MM1 brain frontal cortex region. In the cerebellum, Rab7a expression showed significant upregulation in both subtype MM1 and VV2 CJD forms, in contrast to Rab9 which showed significant downregulation in both subtype MM1 and VV2 CJD forms at terminal stage of the disease. To check regulatory response at pre-symptomatic stage of the disease, we checked the regulatory interactive response of Rab7a, Rab9, and known biomarkers PrP C and tau forms in frontal cortex at pre-symptomatic stage of the disease in tg340 mice expressing about fourfold of human PrP-M129 with PrP-null background that had been inoculated with human sCJD MM1 brain tissue homogenates (sCJD MM1 mice). In addition, we analyzed 5XFAD mice, exhibiting five mutations in the APP and presenilin genes related to familial Alzheimer's disease (FAD), to validate specific regulatory response of Rab7a, Rab9, tau, and phosphorylated form of tau by immunostaining 5XFAD mice in comparison with the wild-type age-matched mice brain. The cortical region of 5XFAD mice brain showed accumulated form of Rab7a in puncta that co-label for p-Tau, indicating colocalization by using confocal laser-scanning microscopy and was confirmed by using reverse co-immunoprecipitation. Furthermore, synthetic RNA (siRNA) against the Rab7a gene decreased expression of Rab7a protein, in cortical

C-reactive protein expression is up-regulated in apical lesions of endodontic origin in association with interleukin-6.

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Garrido, Mauricio; Dezerega, Andrea; Bordagaray, María José; Reyes, Montserrat; Vernal, Rolando; Melgar-Rodríguez, Samantha; Ciuchi, Pía; Paredes, Rodolfo; García-Sesnich, Jocelyn; Ahumada-Montalva, Pablo; Hernández, Marcela

2015-04-01

C-reactive protein (CRP) is the prototype component of acute-phase proteins induced ultimately by interleukin (IL)-6 in the liver, but it is unknown whether periradicular tissues locally express CRP. The present study aimed to identify whether CRP messenger RNA synthesis occurs in situ within apical lesions of endodontic origin (ALEOs) and healthy periodontal ligament and its association with IL-6 and to determine their protein levels and tissue localization. Patients with asymptomatic apical periodontitis and healthy volunteers presenting at the School of Dentistry, University of Chile, Santiago, Chile, were enrolled. ALEOs and healthy teeth were obtained and processed for either immunohistochemistry and double immunofluorescence to assess IL-6 and CRP tissue localization, whereas healthy periodontal ligaments were processed as controls for real-time reverse-transcription polymerase chain reaction for their RNA expression levels and multiplex assay to determine their protein levels. Statistic analysis was performed using the unpaired t test or Mann-Whitney test according to data distribution and Pearson correlation. IL-6 and CRP were synthesized in ALEOs, whereas their RNA expression and protein levels were significantly higher when compared with healthy periodontal ligament. IL-6 and CRP immunolocalized to the inflammatory cells, vascular endothelial cells, and mesenchymal cells. Both, IL-6 and CRP colocalized in ALEOs, and a positive correlation was found between their expression levels (P periodontal ligament and up-regulated in ALEOs along with higher protein levels. Given their pleiotropic effects, IL-6 and CRP protein levels in apical tissues might partially explain the development and progression of ALEOs as well as potentially asymptomatic apical periodontitis-associated systemic low-grade inflammation. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Spontaneous CD8 T cell responses against the melanocyte differentiation antigen RAB38/NY-MEL-1 in melanoma patients.

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Walton, Senta M; Gerlinger, Marco; de la Rosa, Olga; Nuber, Natko; Knights, Ashley; Gati, Asma; Laumer, Monika; Strauss, Laura; Exner, Carolin; Schäfer, Niklaus; Urosevic, Mirjana; Dummer, Reinhard; Tiercy, Jean-Marie; Mackensen, Andreas; Jaeger, Elke; Lévy, Frédéric; Knuth, Alexander; Jäger, Dirk; Zippelius, Alfred

2006-12-01

The melanocyte differentiation Ag RAB38/NY-MEL-1 was identified by serological expression cloning (SEREX) and is expressed in the vast majority of melanoma lesions. The immunogenicity of RAB38/NY-MEL-1 has been corroborated previously by the frequent occurrence of specific Ab responses in melanoma patients. To elucidate potential CD8 T cell responses, we applied in vitro sensitization with overlapping peptides spanning the RAB38/NY-MEL-1 protein sequence and the reverse immunology approach. The identified peptide RAB38/NY-MEL-1(50-58) exhibited a marked response in ELISPOT assays after in vitro sensitization of CD8 T cells from HLA-A *0201(+) melanoma patients. In vitro digestion assays using purified proteasomes provided evidence of natural processing of RAB38/NY-MEL-1(50-58) peptide. Accordingly, monoclonal RAB38/NY-MEL-1(50-58)-specific T cell populations were capable of specifically recognizing HLA-A2(+) melanoma cell lines expressing RAB38/NY-MEL-1. Applying fluorescent HLA-A2/RAB38/NY-MEL-1(50-58) multimeric constructs, we were able to document a spontaneously developed memory/effector CD8 T cell response against this peptide in a melanoma patient. To elucidate the Ag-processing pathway, we demonstrate that RAB38/NY-MEL-1(50-58) is produced efficiently by the standard proteasome and the immunoproteasome. In addition to the identification of a RAB38/NY-MEL-1-derived immunogenic CD8 T cell epitope, this study is instrumental for both the onset and monitoring of future RAB38/NY-MEL-1-based vaccination trials.

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

Deficient Rab11 activity underlies glucose hypometabolism in primary neurons of Huntington's disease mice

Energy Technology Data Exchange (ETDEWEB)

Li, Xueyi, E-mail: xli12@partners.org [Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129 (United States); Valencia, Antonio; McClory, Hollis; Sapp, Ellen; Kegel, Kimberly B. [Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129 (United States); DiFiglia, Marian, E-mail: difiglia@helix.mgh.harvard.edu [Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129 (United States)

2012-05-18

Highlights: Black-Right-Pointing-Pointer Primary Huntington's disease neurons are impaired in taking up glucose. Black-Right-Pointing-Pointer Rab11 modulates glucose uptake in neurons. Black-Right-Pointing-Pointer Increasing Rab11 activity attenuates the glucose uptake defect in disease neurons. Black-Right-Pointing-Pointer We provide a novel mechanism for glucose hypometabolism in Huntington's disease. -- Abstract: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Positron emission tomography studies have revealed a decline in glucose metabolism in the brain of patients with HD by a mechanism that has not been established. We examined glucose utilization in embryonic primary cortical neurons of wild-type (WT) and HD knock-in mice, which have 140 CAG repeats inserted in the endogenous mouse huntingtin gene (HD{sup 140Q/140Q}). Primary HD{sup 140Q/140Q} cortical neurons took up significantly less glucose than did WT neurons. Expression of permanently inactive and permanently active forms of Rab11 correspondingly altered glucose uptake in WT neurons, suggesting that normal activity of Rab11 is needed for neuronal uptake of glucose. It is known that Rab11 activity is diminished in HD{sup 140Q/140Q} neurons. Expression of dominant active Rab11 to enhance the activity of Rab11 normalized glucose uptake in HD{sup 140Q/140Q} neurons. These results suggest that deficient activity of Rab11 is a novel mechanism for glucose hypometabolism in HD.

REGULATED VESICULAR TRAFFICKING OF SPECIFIC PCDH15 AND VLGR1 VARIANTS IN AUDITORY HAIR CELLS

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Zallocchi, Marisa; Delimont, Duane; Meehan, Daniel T.; Cosgrove, Dominic

2012-01-01

Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal co-localization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular sub-pools. The apically trafficked pool co-localized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associates with membrane microdomains and SNAP25. Moreover, co-immunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions. PMID:23035094

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

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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

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

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Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex

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.

Clinical presentation of Griscelli syndrome type 2 and spectrum of RAB27A mutations

DEFF Research Database (Denmark)

Meeths, Marie; Bryceson, Yenan T; Rudd, Eva

2010-01-01

Griscelli syndrome type 2 (GS2) is an autosomal-recessive immunodeficiency caused by mutations in RAB27A, clinically characterized by partial albinism and haemophagocytic lymphohistocytosis (HLH). We evaluated the frequency of RAB27A mutations in 21 unrelated patients with haemophagocytic syndromes...

Periapical fluid RANKL and IL-8 are differentially regulated in pulpitis and apical periodontitis.

Science.gov (United States)

Rechenberg, Dan-K; Bostanci, Nagihan; Zehnder, Matthias; Belibasakis, Georgios N

2014-09-01

The dental pulp space can become infected due to a breach in the surrounding hard tissues. This leads to inflammation of the pulp (pulpitis), soft tissue breakdown, and finally to bone loss around the root apex (apical periodontitis). The succession of the molecular events leading to apical periodontitis is currently not known. The main inflammatory mediator associated with neutrophil chemotaxis is interleukin-8 (IL-8), and with bone resorption the dyad of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). The levels of RANKL, OPG and IL-8 were studied in periapical tissue fluid of human teeth (n = 48) diagnosed with symptomatic irreversible pulpitis (SIP) and asymptomatic apical periodontitis (AAP). SIP represents the starting point, and AAP an established steady state of the disease. Periapical tissue fluid samples were collected using paper points and then evaluated using enzyme-linked immunosorbent assays (ELISAs). Target protein levels per case were calibrated against the corresponding total protein content, as determined fluorometrically. RANKL was expressed at significantly higher levels in SIP compared to AAP (P apical periodontitis, periapical bone resorption signaling, as determined by RANKL, occurs prior to inflammatory cell recruitment signaling, as determined by IL-8. Copyright © 2014 Elsevier Ltd. All rights reserved.

32 CFR 202.4 - Composition of a RAB.

Science.gov (United States)

2010-07-01

... appropriate. At closing installations where BRAC Cleanup Teams (BCT) exist, representatives of the BCT may... installation and the other the community. Co-chairs shall be responsible for directing and managing the RAB...

Bioinformatic and Comparative Localization of Rab Proteins Reveals Functional Insights into the Uncharacterized GTPases Ypt10p and Ypt11p†

Science.gov (United States)

Buvelot Frei, Stéphanie; Rahl, Peter B.; Nussbaum, Maria; Briggs, Benjamin J.; Calero, Monica; Janeczko, Stephanie; Regan, Andrew D.; Chen, Catherine Z.; Barral, Yves; Whittaker, Gary R.; Collins, Ruth N.

2006-01-01

A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance. PMID:16980630

A novel missense mutation (G43S) in the switch I region of Rab27A causing Griscelli syndrome

DEFF Research Database (Denmark)

Westbroek, W.; Tuchman, M.; Tinloy, B.

2008-01-01

The autosomal recessive Griscelli syndrome type II (GSII) is caused by mutations in the RAB27A gene. Typical clinical features include immunological impairment, silver-gray scalp hair, eyelashes and eyebrows and hypomelanosis of the skin. Rabs help determine the specificity of membrane trafficking......-immunoprecipitation studies showed that Rab27A(G43S) fails to interact with its effector Melanophilin, indicating that the switch I region functions in the recruitment of Rab effector proteins Udgivelsesdato: 2008/6...

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

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.

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.

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.

Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages

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Youhei Egami

2015-01-01

Full Text Available Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.

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

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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.

Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin

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Chia, Pei Zhi Cheryl; Gasnereau, Isabelle; Lieu, Zi Zhao; Gleeson, Paul A.

2011-01-01

The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin. Using internalisation assays, we show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were required for efficient TGN retrieval of furin. By contrast, TGN38 trafficking was independent of Rab9 and GCC185. To identify the sorting signals for the early endosome-to-TGN pathway, the trafficking of furin–TGN38 chimeras was investigated. The diversion of furin from the Rab9-dependent late-endosome-to-TGN pathway to the retromer-dependent early-endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. We present evidence to suggest that the length of the transmembrane domain is a contributing factor in endosomal sorting. Overall, these data show that furin uses the Rab9-dependent pathway from late endosomes and that retrograde transport directly from early endosomes is dependent on both the transmembrane domain and the cytoplasmic tail. PMID:21693586

Reversible effects of acute hypertension on proximal tubule sodium transporters

DEFF Research Database (Denmark)

Zhang, Y; Magyar, C E; Norian, J M

1998-01-01

Acute hypertension provokes a rapid decrease in proximal tubule sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of membranes containing apical membrane sodium/hydrogen exchangers (NHE3) [Y. Zhang, A. K. Mircheff, C. B....... Renal cortex lysate was fractionated on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase activity (but not subunit immunoreactivity) decreased one-third to one-half after BP was elevated and recovered after BP was normalized. After BP was elevated, 55% of the apical NHE3 immunoreactivity......, smaller fractions of sodium-phosphate cotransporter immunoreactivity, and apical alkaline phosphatase and dipeptidyl-peptidase redistributed to membranes of higher density enriched in markers of the intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5), whereas density distributions...

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

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

High RAB25 expression is associated with good clinical outcome in patients with locally advanced head and neck squamous cell carcinoma

International Nuclear Information System (INIS)

Téllez-Gabriel, Marta; Arroyo-Solera, Irene; León, Xavier; Gallardo, Alberto; López, Montserrat; Céspedes, Maria V; Casanova, Isolda; López-Pousa, Antonio; Quer, Miquel; Mangues, Maria A; Barnadas, Agustí; Mangues, Ramón; Pavón, Miguel A

2013-01-01

Currently there are no molecular markers able to predict clinical outcome in locally advanced head and neck squamous cell carcinoma (HNSCC). In a previous microarray study, RAB25 was identified as a potential prognostic marker. The aim of this study was to analyze the association between RAB25 expression and clinical outcome in patients with locally advanced HNSCC treated with standard therapy. In a retrospective immunohistochemical study (n = 97), we observed that RAB25-negative tumors had lower survival (log-rank, P = 0.01) than patients bearing positive tumors. In an independent prospective mRNA study (n = 117), low RAB25 mRNA expression was associated with poor prognosis. Using classification and regression tree analysis (CART) we established two groups of patients according to their RAB25 mRNA level and their risk of death. Low mRNA level was associated with poor local recurrence-free (log-rank, P = 0.005), progression-free (log-rank, P = 0.002) and cancer-specific (log-rank, P < 0.001) survival. Multivariate Cox model analysis showed that low expression of RAB25 was an independent poor prognostic factor for survival (hazard ratio: 3.84, 95% confidence interval: 1.93–7.62, P < 0.001). Patients whose tumors showed high RAB25 expression had a low probability of death after treatment. We also found lower RAB25 expression in tumors than in normal tissue (Mann–Whitney U, P < 0.001). Moreover, overexpression of RAB25 in the UM-SCC-74B HNSCC cell line increased cisplatin sensitivity, and reduced cell migration and invasion. Our findings support a tumor suppressor role for RAB25 in HNSCC and its potential use to identify locally advanced patients with a high probability of survival after genotoxic treatment

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

Smart I’rab: Smart Aplicasion for Arabic Grammar Learning

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Syd. Ali Zein Farmadi

2013-12-01

Full Text Available Arabic grammar, known as nahwu, is necessary to comprehend the Holy Qur’an that is completely written in Arabic. However, many people get trouble to study this skill because there are various kinds of word formation and sentences that may be created from a single verb, noun, adjective, subject, predicate, object, adverb or another formation. This research proposes a new approach to identify the position and word function in Arabic sentence. The approach creates smart process that employs Natural Language Processing (NLP and expert system with modeling based on knowledge and inference engine in determining the word position. The knowledge base determines the part of speech while the inference engine shows the word function in the sentence. On processing, the system uses 82 templates consisting of 34 verb templates, 34 subject pronouns, 14 pronouns for object or possessive word. All the templates are in the form of char array for harakat (vowel and letters which become the comparators for determining the part of speech from input word sentence. Output from the system is an i’rab (the explanation of word function in sentence written in Arabic. The system has been tested for 159 times to examine word and sentence. The examination for word that is done 117 times has not made any error except for the word that is really like another word. While the detection for word function in sentence that is done 42 times experiment, there is no error too. An error happens when the part of speech from the word being examined is not included in the system yet, influencing the following word function detection. Keywords: I’rab, Arabic grammar, NLP, expert system, knowledge base, inference engine

Symptomatic and asymptomatic apical periodontitis associated with red complex bacteria: clinical and microbiological evaluation.

Science.gov (United States)

Buonavoglia, Alessio; Latronico, Francesca; Pirani, Chiara; Greco, Maria Fiorella; Corrente, Marialaura; Prati, Carlo

2013-01-01

In this study, the association of red complex (RC) bacteria that include Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis with acute, exacerbated or chronic apical periodontitis was evaluated. Seventy-one patients with periapical disease were evaluated by clinical examination and microbiological samples obtained from the root canals were analyzed by a polymerase chain reaction assay. Twenty-one (29.6%) samples were positive for RC bacteria, with T. denticola, T. forsythia and P. gingivalis being detected in 14 (19.7%), 10 (14.1%) and 6 (8.5%) samples, respectively. RC bacteria were mainly associated with acute apical periodontitis (29.2%) and phoenix abscess (63.2%), while they were only sporadically detected (7.1%) in patients with chronic apical periodontitis. Generally, RC bacteria were associated with pain and a higher frequency of intracanalar/intrasulcular pus drainage. Involvement of RC bacteria in symptomatic periapical disease should be suspected in the presence of particularly severe clinical pain and pus drainage.

Association among oral health, apical periodontitis, CD14 polymorphisms, and coronary heart disease in middle-aged adults.

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Pasqualini, Damiano; Bergandi, Loredana; Palumbo, Luigi; Borraccino, Alberto; Dambra, Valentina; Alovisi, Mario; Migliaretti, Giuseppe; Ferraro, Gaetana; Ghigo, Dario; Bergerone, Serena; Scotti, Nicola; Aimetti, Mario; Berutti, Elio

2012-12-01

There is evidence to suggest that an association exists between oral infections and coronary heart disease (CHD). Subjects presenting lesions of endodontic origin (LEOs) or pulpal inflammation had an increased risk of developing CHD. However, findings concerning systemic manifestations of apical periodontitis (AP) remain controversial. An association between CD14 gene polymorphisms and atherosclerosis-associated diseases has been shown, but there are no data regarding an association between CD14 polymorphism and AP. This study evaluated associations between clinical oral health status, CD14 polymorphisms, and CHD. A case-controlled clinical trial was designed to compare middle-aged adults with acute myocardial infarction or unstable angina (n = 51) within 12 months of the acute event defined as first manifestation with healthy controls (n = 49). Participants were matched for age, sex, and socioeconomic status. Indicators of oral disease and compliance were evaluated. CD14 polymorphisms were analyzed by restriction fragment length polymorphism-polymerase chain reaction. CHD subjects had a higher prevalence of oral diseases and lower compliance to oral preventive strategies than healthy controls. Multivariate analysis showed a positive association between missing teeth (odds ratio [OR] = 1.37; 95% confidence interval [CI], 1.02-1.85), the number of LEOs (OR = 4.37; 95% CI, 1.69-11.28), chronic periodontitis (OR = 5.87; 95% CI, 1.17-29.4), and CHD. No statistically significant association emerged between the CD14 C(-260)T and the CD14 C(-159)T polymorphism, endodontic or periodontal disease, and CHD. Chronic oral diseases may increase the risk of CHD and may be an unconventional risk factor for CHD. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Liver X receptors balance lipid stores in hepatic stellate cells through Rab18, a retinoid responsive lipid droplet protein.

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O'Mahony, Fiona; Wroblewski, Kevin; O'Byrne, Sheila M; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S; Beaven, Simon W

2015-08-01

Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ(-/-) mice have increased lipid droplet (LD) size, but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ(-/-) and wild-type mice were profiled by gene array during in vitro activation. Lipid content was quantified by high-performance liquid chromatography and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with small interfering RNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ(-/-) HSCs have increased cholesterol and retinyl esters. The retinoid increase drives intrinsic retinoic acid receptor signaling, and activation occurs more rapidly in Lxrαβ(-/-) HSCs. We identify Rab18 as a novel retinoic acid-responsive, LD-associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 guanosine triphosphatase activity and isoprenylation are required for stellate cell LD loss and induction of activation markers. These phenomena are accelerated in Lxrαβ(-/-) HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards LD loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Retinoid and cholesterol metabolism are linked in stellate cells by the LD-associated protein Rab18. Retinoid overload helps explain the profibrotic phenotype of Lxrαβ(-/-) mice, and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. © 2015 by the American Association for the Study of Liver Diseases.

Liver X Receptors Balance Lipid Stores in Hepatic Stellate Cells via Rab18, a Retinoid Responsive Lipid Droplet Protein

Science.gov (United States)

O’Mahony, Fiona; Wroblewski, Kevin; O’Byrne, Sheila M.; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S.; Beaven, Simon W.

2014-01-01

Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ−/− mice have increased lipid droplet (LD) size but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ−/− and wild-type (WT) mice were profiled by gene array during in vitro activation. Lipid content was quantified by HPLC and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with siRNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ−/− HSCs have increased cholesterol and retinyl esters (CEs & REs). The retinoid increase drives intrinsic retinoic acid receptor (RAR) signaling and activation occurs more rapidly in Lxrαβ−/− HSCs. We identify Rab18 as a novel retinoic acid responsive, lipid droplet associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 GTPase activity and isoprenylation are required for stellate cell lipid droplet loss and induction of activation markers. These phenomena are accelerated in the Lxrαβ−/− HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards lipid droplet loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Conclusion Retinoid and cholesterol metabolism are linked in stellate cells by the LD associated protein, Rab18. Retinoid overload helps explain the pro-fibrotic phenotype of Lxrαβ−/− mice and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. PMID:25482505

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...

Structural basis for Rab1 de-AMPylation by the Legionella pneumophila effector SidD.

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Yang Chen

Full Text Available The covalent attachment of adenosine monophosphate (AMP to proteins, a process called AMPylation (adenylylation, has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.

The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs.

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Levine, Timothy P; Daniels, Rachel D; Gatta, Alberto T; Wong, Louise H; Hayes, Matthew J

2013-02-15

Fronto-temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS, also called motor neuron disease, MND) are severe neurodegenerative diseases that show considerable overlap at the clinical and cellular level. The most common single mutation in families with FTD or ALS has recently been mapped to a non-coding repeat expansion in the uncharacterized gene C9ORF72. Although a plausible mechanism for disease is that aberrant C9ORF72 mRNA poisons splicing, it is important to determine the cellular function of C9ORF72, about which nothing is known. Sensitive homology searches showed that C9ORF72 is a full-length distant homologue of proteins related to Differentially Expressed in Normal and Neoplasia (DENN), which is a GDP/GTP exchange factor (GEF) that activates Rab-GTPases. Our results suggest that C9ORF72 is likely to regulate membrane traffic in conjunction with Rab-GTPase switches, and we propose to name the gene and its product DENN-like 72 (DENNL72).

Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles

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María Milagros López de Armentia

2016-03-01

Full Text Available Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila. The bacteria described in this review often use secretion systems to control the host’s response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections.

Functional Characterization of Rare RAB12 Variants and Their Role in Musician’s and Other Dystonias

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Eva Hebert

2017-10-01

Full Text Available Mutations in RAB (member of the Ras superfamily genes are increasingly recognized as cause of a variety of disorders including neurological conditions. While musician’s dystonia (MD and writer’s dystonia (WD are task-specific movement disorders, other dystonias persistently affect postures as in cervical dystonia. Little is known about the underlying etiology. Next-generation sequencing revealed a rare missense variant (c.586A>G; p.Ile196Val in RAB12 in two of three MD/WD families. Next, we tested 916 additional dystonia patients; 512 Parkinson’s disease patients; and 461 healthy controls for RAB12 variants and identified 10 additional carriers of rare missense changes among dystonia patients (1.1% but only one carrier in non-dystonic individuals (0.1%; p = 0.005. The detected variants among index patients comprised p.Ile196Val (n = 6; p.Ala174Thr (n = 3; p.Gly13Asp; p.Ala148Thr; and p.Arg181Gln in patients with MD; cervical dystonia; or WD. Two relatives of MD patients with WD also carried p.Ile196Val. The two variants identified in MD patients (p.Ile196Val; p.Gly13Asp were characterized on endogenous levels in patient-derived fibroblasts and in two RAB12-overexpressing cell models. The ability to hydrolyze guanosine triphosphate (GTP, so called GTPase activity, was increased in mutants compared to wildtype. Furthermore, subcellular distribution of RAB12 in mutants was altered in fibroblasts. Soluble Transferrin receptor 1 levels were reduced in the blood of all three tested p.Ile196Val carriers. In conclusion, we demonstrate an enrichment of missense changes among dystonia patients. Functional characterization revealed altered enzyme activity and lysosomal distribution in mutants suggesting a contribution of RAB12 variants to MD and other dystonias.

A Rab-centric perspective of bacterial pathogen-occupied vacuoles.

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Sherwood, Racquel Kim; Roy, Craig R

2013-09-11

The ability to create and maintain a specialized organelle that supports bacterial replication is an important virulence property for many intracellular pathogens. Living in a membrane-bound vacuole presents inherent challenges, including the need to remodel a plasma membrane-derived organelle into a novel structure that will expand and provide essential nutrients to support replication, while also having the vacuole avoid membrane transport pathways that target bacteria for destruction in lysosomes. It is clear that pathogenic bacteria use different strategies to accomplish these tasks. The dynamics by which host Rab GTPases associate with pathogen-occupied vacuoles provide insight into the mechanisms used by different bacteria to manipulate host membrane transport. In this review we highlight some of the strategies bacteria use to maintain a pathogen-occupied vacuole by focusing on the Rab proteins involved in biogenesis and maintenance of these novel organelles. Copyright © 2013 Elsevier Inc. All rights reserved.

Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex

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Jihui Lin

2017-08-01

Full Text Available Classical swine fever virus (CSFV is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B protein is one of the most important viral replicase proteins. Rab5, a member of the small Rab GTPase family, is involved in infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation, glutathione S-transferase pulldown and laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B protein to facilitate the NS4B related complexes formation.

p600 regulates spindle orientation in apical neural progenitors and contributes to neurogenesis in the developing neocortex

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Camille Belzil

2014-05-01

Full Text Available Apical neural progenitors (aNPs drive neurogenesis by means of a program consisting of self-proliferative and neurogenic divisions. The balance between these two manners of division sustains the pool of apical progenitors into late neurogenesis, thereby ensuring their availability to populate the brain with terminal cell types. Using knockout and in utero electroporation mouse models, we report a key role for the microtubule-associated protein 600 (p600 in the regulation of spindle orientation in aNPs, a cellular event that has been associated with cell fate and neurogenesis. We find that p600 interacts directly with the neurogenic protein Ndel1 and that aNPs knockout for p600, depleted of p600 by shRNA or expressing a Ndel1-binding p600 fragment all display randomized spindle orientation. Depletion of p600 by shRNA or expression of the Ndel1-binding p600 fragment also results in a decreased number of Pax6-positive aNPs and an increased number of Tbr2-positive basal progenitors destined to become neurons. These Pax6-positive aNPs display a tilted mitotic spindle. In mice wherein p600 is ablated in progenitors, the production of neurons is significantly impaired and this defect is associated with microcephaly. We propose a working model in which p600 controls spindle orientation in aNPs and discuss its implication for neurogenesis.

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking.

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Ruxandra Bachmann-Gagescu

2015-10-01

Full Text Available Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary

Charcot Marie Tooth 2B Peripheral Sensory Neuropathy: How Rab7 Mutations Impact NGF Signaling?

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Harry Liu

2017-02-01

Full Text Available Charcot-Marie-Tooth 2B peripheral sensory neuropathy (CMT2B is a debilitating autosomal dominant hereditary sensory neuropathy. Patients with this disease lose pain sensation and frequently need amputation. Axonal dysfunction and degeneration of peripheral sensory neurons is a major clinical manifestation of CMT2B. However, the cellular and molecular pathogenic mechanisms remain undefined. CMT2B is caused by missense point mutations (L129F, K157N, N161T/I, V162M in Rab7 GTPase. Strong evidence suggests that the Rab7 mutation(s enhances the cellular levels of activated Rab7 proteins, thus resulting in increased lysosomal activity and autophagy. As a consequence, trafficking and signaling of neurotrophic factors such as nerve growth factor (NGF in the long axons of peripheral sensory neurons are particularly vulnerable to premature degradation. A “gain of toxicity” model has, thus, been proposed based on these observations. However, studies of fly photo-sensory neurons indicate that the Rab7 mutation(s causes a “loss of function”, resulting in haploinsufficiency. In the review, we summarize experimental evidence for both hypotheses. We argue that better models (rodent animals and human neurons of CMT2B are needed to precisely define the disease mechanisms.

Charcot Marie Tooth 2B Peripheral Sensory Neuropathy: How Rab7 Mutations Impact NGF Signaling?

Science.gov (United States)

Liu, Harry; Wu, Chengbiao

2017-02-04

Charcot-Marie-Tooth 2B peripheral sensory neuropathy (CMT2B) is a debilitating autosomal dominant hereditary sensory neuropathy. Patients with this disease lose pain sensation and frequently need amputation. Axonal dysfunction and degeneration of peripheral sensory neurons is a major clinical manifestation of CMT2B. However, the cellular and molecular pathogenic mechanisms remain undefined. CMT2B is caused by missense point mutations (L129F, K157N, N161T/I, V162M) in Rab7 GTPase. Strong evidence suggests that the Rab7 mutation(s) enhances the cellular levels of activated Rab7 proteins, thus resulting in increased lysosomal activity and autophagy. As a consequence, trafficking and signaling of neurotrophic factors such as nerve growth factor (NGF) in the long axons of peripheral sensory neurons are particularly vulnerable to premature degradation. A "gain of toxicity" model has, thus, been proposed based on these observations. However, studies of fly photo-sensory neurons indicate that the Rab7 mutation(s) causes a "loss of function", resulting in haploinsufficiency. In the review, we summarize experimental evidence for both hypotheses. We argue that better models (rodent animals and human neurons) of CMT2B are needed to precisely define the disease mechanisms.

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

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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

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)

Bioinformatic and Comparative Localization of Rab Proteins Reveals Functional Insights into the Uncharacterized GTPases Ypt10p and Ypt11p†

OpenAIRE

Buvelot Frei, Stéphanie; Rahl, Peter B.; Nussbaum, Maria; Briggs, Benjamin J.; Calero, Monica; Janeczko, Stephanie; Regan, Andrew D.; Chen, Catherine Z.; Barral, Yves; Whittaker, Gary R.; Collins, Ruth N.

2006-01-01

A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising ...

阴道毛滴虫TvRab11C基因的克隆及原核表达%Cloning and prokaryotic expression of TvRab11C gene of Trichomonas vaginalis

Institute of Scientific and Technical Information of China (English)

刘畅; 丁鹤; 宫鹏涛; 李建华; 李淑红; 李赫; 张国才; 张西臣

2012-01-01

目的 克隆并原核表达阴道毛滴虫TvRab11C(G3 Ras-related protein Rab11C)基因.方法 利用PCR技术扩增阴道毛滴虫TvRab1 1C基因,与原核表达载体pET-28a连接,构建重组原核表达质粒pET-28a-TvRab1 1C,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析表达产物的可溶性,Western blot分析表达产物的反应原性.结果 重组原核表达质粒pET-28a-TvRab11C经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为30000,主要以包涵体形式存在,可被抗阴道毛滴虫多克隆抗体识别.结论 成功克隆了阴道毛滴虫TvRab11C基因,并在E.coli BL21 (DE3)中获得了表达,为进一步研究TvRas基因和G蛋白与阴道毛滴虫寄生能力和致病性的关系奠定了基础.%Objective To clone the TvRabllC gene of Trichomonas vaginalis and express in prokaryotic cells. Methods The TvRabllC gene was amplified by PCR from T. vaginalis and inserted into prokaryotic expression vector pET-28a. The constructed recombinant plasmid pET-28a-TvRabllC was transformed to E. coli BL21 (DE3) and induced by IPTG. The expressed product was analyzed for solubility by SDS-PAGE and for reactogenicity by Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-TvRabllC was constructed correctly. The expressed recombinant protein,with a relative molecular mass of about 30 000,mainly existed in a form of inclusion body,and was recognized by polyclonal antibody against T. vaginalis. Conclusion The TvRas gene of T. vaginalis was successfully cloned and expressed in E. coli BL21 (DE3),which laid a foundation of further study on relationship of TvRas gene and protein to the parasitic ability and pathogenicity of T. vaginalis.

Endocytic recycling via the TGN underlies the polarized hyphal mode of life.

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Hernández-González, Miguel; Bravo-Plaza, Ignacio; Pinar, Mario; de Los Ríos, Vivian; Arst, Herbert N; Peñalva, Miguel A

2018-04-01

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1μm/min) and the long intracellular distances (>100 μm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

International Nuclear Information System (INIS)

Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

2011-01-01

Highlights: ► APPL1 regulates the protein level of EGFR in response to EGF stimulation. ► Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. ► Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

Assessment of apical periodontitis by MRI. A feasibility study

Energy Technology Data Exchange (ETDEWEB)

Geibel, M.A. [Ulm Univ. (Germany). Oral and Maxillofacial Surgery; Schreiber, E.S.; Bracher, A.K.; Rasche, V. [Ulm Univ. (Germany). Internal Medicine II; Hell, E.; Ulrici, J. [Sirona Dental Systems GmbH, Bensheim (Germany). Dental Imaging; Sailer, L.K. [DOC Praxisklinik im Wiley, Neu-Ulm (Germany). MKG; Ozpeynirci, Y. [Ulm Univ. (Germany). Diagnostic and Interventional Radiology

2015-04-15

The purpose of this clinical feasibility study was to evaluate the applicability of magnetic resonance imaging (MRI) for the assessment of apical periodontitis in direct comparison with cone beam CT (CBCT). 19 consecutive patients (average age 43 ± 13 years) with 34 lesions in total (13 molars, 14 premolars and 7 front teeth) were enrolled in this feasibility study. Periapical lesions were defined as periapical radiolucencies (CBCT) or structural changes in the spongy bone signal (MRI), which were connected with the apical part of a root and with at least twice the width of the periodontal ligament space. The location and dimension of the lesions were compared between MRI and CBCT. While mainly mineralized tissue components such as teeth and bone were visible with CBCT, complimentary information of the soft tissue components was assessable with MRI. The MRI images provided sufficient diagnostic detail for the assessment of the main structures of interest. Heterogeneous contrast was observed within the lesion, with often a clear enhancement close to the apical foramen and the periodontal gap. No difference for lesion visibility was observed between MRI and CBCT. The lesion dimensions corresponded well, but were slightly but significantly overestimated with MRI. A heterogeneous lesion appearance was observed in several patients. Four patients presented with a well circumscribed hyperintense signal in the vicinity of the apical foramen. The MRI capability of soft tissue characterization may facilitate detailed analysis of periapical lesions. This clinical study confirms the applicability of multi-contrast MRI for the identification of periapical lesions.

Assessment of apical periodontitis by MRI. A feasibility study

International Nuclear Information System (INIS)

Geibel, M.A.; Schreiber, E.S.; Bracher, A.K.; Rasche, V.; Hell, E.; Ulrici, J.; Sailer, L.K.; Ozpeynirci, Y.

2015-01-01

The purpose of this clinical feasibility study was to evaluate the applicability of magnetic resonance imaging (MRI) for the assessment of apical periodontitis in direct comparison with cone beam CT (CBCT). 19 consecutive patients (average age 43 ± 13 years) with 34 lesions in total (13 molars, 14 premolars and 7 front teeth) were enrolled in this feasibility study. Periapical lesions were defined as periapical radiolucencies (CBCT) or structural changes in the spongy bone signal (MRI), which were connected with the apical part of a root and with at least twice the width of the periodontal ligament space. The location and dimension of the lesions were compared between MRI and CBCT. While mainly mineralized tissue components such as teeth and bone were visible with CBCT, complimentary information of the soft tissue components was assessable with MRI. The MRI images provided sufficient diagnostic detail for the assessment of the main structures of interest. Heterogeneous contrast was observed within the lesion, with often a clear enhancement close to the apical foramen and the periodontal gap. No difference for lesion visibility was observed between MRI and CBCT. The lesion dimensions corresponded well, but were slightly but significantly overestimated with MRI. A heterogeneous lesion appearance was observed in several patients. Four patients presented with a well circumscribed hyperintense signal in the vicinity of the apical foramen. The MRI capability of soft tissue characterization may facilitate detailed analysis of periapical lesions. This clinical study confirms the applicability of multi-contrast MRI for the identification of periapical lesions.

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

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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.

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

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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.

RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

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Hiroaki Kajiho

Full Text Available The Rab family of small guanosine triphosphatases (GTPases plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs. Ras and Rab interactor (or Ras interaction/interference-like (RINL, which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM domain-containing (Anks protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype.

Science.gov (United States)

Lamers, Ideke J C; Reijnders, Margot R F; Venselaar, Hanka; Kraus, Alison; Jansen, Sandra; de Vries, Bert B A; Houge, Gunnar; Gradek, Gyri Aasland; Seo, Jieun; Choi, Murim; Chae, Jong-Hee; van der Burgt, Ineke; Pfundt, Rolph; Letteboer, Stef J F; van Beersum, Sylvia E C; Dusseljee, Simone; Brunner, Han G; Doherty, Dan; Kleefstra, Tjitske; Roepman, Ronald

2017-11-02

The Rab GTPase family comprises ∼70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization. Copyright © 2017 American Society of Human Genetics. All rights reserved.

Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment.

Science.gov (United States)

Girada, Shravan Babu; Kuna, Ramya S; Bele, Shilpak; Zhu, Zhimeng; Chakravarthi, N R; DiMarchi, Richard D; Mitra, Prasenjit

2017-10-01

Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein-protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Ectopic expression of a vesicle trafficking gene, OsRab7, from Oryza ...

African Journals Online (AJOL)

Jane

2011-07-18

Jul 18, 2011 ... OsRab7, from Oryza sativa, confers tolerance to several ... Key Laboratory of Molecular Biology and Gene Engineering, College of Life Science, Nanchang University, ... The reverse transcription was performed using MMLV.

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.

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.

Proliferative effects of apical, but not basal, matrix metalloproteinase-7 activity in polarized MDCK cells

International Nuclear Information System (INIS)

Harrell, Permila C.; McCawley, Lisa J.; Fingleton, Barbara; McIntyre, J. Oliver; Matrisian, Lynn M.

2005-01-01

Matrix metalloproteinase-7 (MMP-7) is primarily expressed in glandular epithelium. Therefore, its mechanism of action may be influenced by its regulated vectorial release to either the apical and/or basolateral compartments, where it would act on its various substrates. To gain a better understanding of where MMP-7 is released in polarized epithelium, we have analyzed its pattern of secretion in polarized MDCK cells expressing stably transfected human MMP-7 (MDCK-MMP-7), and HCA-7 and Caco2 human colon cancer cell lines. In all cell lines, latent MMP-7 was secreted to both cellular compartments, but was 1.5- to 3-fold more abundant in the basolateral compartment as compared to the apical. However, studies in the MDCK system demonstrated that MMP-7 activity was 2-fold greater in the apical compartment of MDCK-MMP-7 HIGH -polarized monolayers, which suggests the apical co-release of an MMP-7 activator. In functional assays, MMP-7 over-expression increased cell saturation density as a result of increased cell proliferation with no effect on apoptosis. Apical MMP-7 activity was shown to be responsible for the proliferative effect, which occurred, as demonstrated by media transfer experiments, through cleavage of an apical substrate and not through the generation of a soluble factor. Taken together, our findings demonstrate the importance of MMP-7 secretion in relation to its mechanism of action when expressed in a polarized epithelium

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.

PKN1 Directs Polarized RAB21 Vesicle Trafficking via RPH3A and Is Important for Neutrophil Adhesion and Ischemia-Reperfusion Injury

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Qianying Yuan

2017-06-01

Full Text Available Polarized vesicle transport plays an important role in cell polarization, but the mechanisms underlying this process and its role in innate immune responses are not well understood. Here, we describe a phosphorylation-regulated polarization mechanism that is important for neutrophil adhesion to endothelial cells during inflammatory responses. We show that the protein kinase PKN1 phosphorylates RPH3A, which enhances binding of RPH3A to guanosine triphosphate (GTP-bound RAB21. These interactions are important for polarized localization of RAB21 and RPH3A in neutrophils, which leads to PIP5K1C90 polarization. Consistent with the roles of PIP5K1C90 polarization, the lack of PKN1 or RPH3A impairs neutrophil integrin activation, adhesion to endothelial cells, and infiltration in inflammatory models. Furthermore, myeloid-specific loss of PKN1 decreases tissue injury in a renal ischemia-reperfusion model. Thus, this study characterizes a mechanism for protein polarization in neutrophils and identifies a potential protein kinase target for therapeutic intervention in reperfusion-related tissue injury.

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

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Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

2016-11-01

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus.

Directory of Open Access Journals (Sweden)

Kai Xu

2016-10-01

Full Text Available Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs. The VRCs built by Tomato bushy stunt virus (TBSV are enriched with phosphatidylethanolamine (PE through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5-positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

Immune function of a Rab-related protein by modulating the JAK-STAT signaling pathway in the silkworm, Bombyx mori.

Science.gov (United States)

Chen, Chen; Eldein, Salah; Zhou, Xiaosan; Sun, Yu; Gao, Jin; Sun, Yuxuan; Liu, Chaoliang; Wang, Lei

2018-01-01

The Rab-family GTPases mainly regulate intracellular vesicle transport, and play important roles in the innate immune response in invertebrates. However, the function and signal transduction of Rab proteins in immune reactions remain unclear in silkworms. In this study, we analyzed a Rab-related protein of silkworm Bombyx mori (BmRABRP) by raising antibodies against its bacterially expressed recombinant form. Tissue distribution analysis showed that BmRABRP mRNA and protein were high expressed in the Malpighian tubule and fat body, respectively. However, among the different stages, only the fourth instar larvae and pupae showed significant BmRABRP levels. After challenge with four pathogenic microorganisms (Escherichia coli, BmNPV, Beauveria bassiana, Micrococcus luteus), the expression of BmRABRP mRNA in the fat body was significantly upregulated. In contrast, the BmRABRP protein was significantly upregulated after infection with BmNPV, while it was downregulated by E. coli, B. bassiana, and M. luteus. A specific dsRNA was used to explore the immune function and relationship between BmRABRP and the JAK-STAT signaling pathway. After BmRABRP gene interference, significant reduction in the number of nodules and increased mortality suggested that BmRABRP plays an important role in silkworm's response to bacterial challenge. In addition, four key genes (BmHOP, BmSTAT, BmSOCS2, and BmSOCS6) of the JAK-STAT signaling pathway showed significantly altered expressions after BmRABRP silencing. BmHOP and BmSOCS6 expressions were significantly decreased, while BmSTAT and BmSOCS2 were significantly upregulated. Our results suggested that BmRABRP is involved in the innate immune response against pathogenic microorganisms through the JAK-STAT signaling pathway in silkworm. © 2017 Wiley Periodicals, Inc.

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....

Class IA phosphatidylinositol 3-kinase p110α regulates phagosome maturation.

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Emily P Thi

Full Text Available Of the various phosphatidylinositol 3- kinases (PI3Ks, only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110α, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110α and contained PI3P and PI(3,4,5P3; however, p110α and PI(3,4,5P3 levels in phagosomes from p110α knockdown cells were decreased. Phagosomes from p110α knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, β-galactosidase. Phagosomes from p110α deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110α deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP and homotypic vacuole fusion and protein sorting (HOPs components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K.

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...

Definitions of apical vaginal support loss: a systematic review.

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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

Revascularization and Apical Plug in an Immature Molar

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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

The Spectrin cytoskeleton regulates the Hippo signalling pathway.

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Fletcher, Georgina C; Elbediwy, Ahmed; Khanal, Ichha; Ribeiro, Paulo S; Tapon, Nic; Thompson, Barry J

2015-04-01

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produces tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin binds to Ex and co-localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

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.

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.

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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.

Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.

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Tarafder, Abul K; Bolasco, Giulia; Correia, Maria S; Pereira, Francisco J C; Iannone, Lucio; Hume, Alistair N; Kirkpatrick, Niall; Picardo, Mauro; Torrisi, Maria R; Rodrigues, Inês P; Ramalho, José S; Futter, Clare E; Barral, Duarte C; Seabra, Miguel C

2014-04-01

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

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

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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.

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., ...

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.

A potential link between insulin signaling and GLUT4 translocation: Association of Rab10-GTP with the exocyst subunit Exoc6/6b

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Sano, Hiroyuki; Peck, Grantley R. [Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 (United States); Blachon, Stephanie [Hybrigenics Services SAS, 3-5 Impasse Reille, 75014 Paris (France); Lienhard, Gustav E., E-mail: gustav.e.lienhard@dartmouth.edu [Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 (United States)

2015-09-25

Insulin increases glucose transport in fat and muscle cells by stimulating the exocytosis of specialized vesicles containing the glucose transporter GLUT4. This process, which is referred to as GLUT4 translocation, increases the amount of GLUT4 at the cell surface. Previous studies have provided evidence that insulin signaling increases the amount of Rab10-GTP in the GLUT4 vesicles and that GLUT4 translocation requires the exocyst, a complex that functions in the tethering of vesicles to the plasma membrane, leading to exocytosis. In the present study we show that Rab10 in its GTP form binds to Exoc6 and Exoc6b, which are the two highly homologous isotypes of an exocyst subunit, that both isotypes are found in 3T3-L1 adipocytes, and that knockdown of Exoc6, Exoc6b, or both inhibits GLUT4 translocation in 3T3-L1 adipocytes. These results suggest that the association of Rab10-GTP with Exoc6/6b is a molecular link between insulin signaling and the exocytic machinery in GLUT4 translocation. - Highlights: • Insulin stimulates the fusion of vesicles containing GLUT4 with the plasma membrane. • This requires vesicular Rab10-GTP and the exocyst plasma membrane tethering complex. • We find that Rab10-GTP associates with the Exoc6 subunit of the exocyst. • We find that knockdown of Exoc6 inhibits fusion of GLUT4 vesicles with the membrane. • The interaction of Rab10-GTP with Exoc6 potentially links signaling to exocytosis.

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

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.

Drosophila VAMP7 regulates Wingless intracellular trafficking.

Science.gov (United States)

Gao, Han; He, Fang; Lin, Xinhua; Wu, Yihui

2017-01-01

Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. Here, we showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, we found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. We hypothesize that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling.

On the causes of persistent apical periodontitis: a review.

Science.gov (United States)

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.

Decreased expression of miR‑490‑3p in colorectal cancer predicts poor prognosis and promotes cell proliferation and invasion by targeting RAB14.

Science.gov (United States)

Wang, Bo; Yin, Mujun; Cheng, Cheng; Jiang, Hongpeng; Jiang, Kewei; Shen, Zhanlong; Ye, Yingjiang; Wang, Shan

2018-06-19

Growing evidence indicates a potential role for miR‑490‑3p in tumorigenesis. However, its function in colorectal carcinoma (CRC) remains undefined. In this study, miR‑490‑3p was markedly downregulated in fifty colorectal cancer tissue samples compared with the corresponding adjacent non‑cancerous specimens, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of miR‑490‑3p were closely associated with tumor differentiation and distant metastasis. In addition, both Kaplan-Meier and multivariate analyses indicated CRC patients with elevated miR‑490‑3p amounts had prolonged overall survival. Overexpression of miR‑490‑3p markedly reduced proliferation, colony formation and invasion in CRC cells by enhancing apoptosis and promoting G2/M phase arrest. Furthermore, ectopic expression of miR‑490‑3p resulted in decreased expression of RAB14, which was directly targeted by miR‑490‑3p, as shown by the dual-luciferase reporter gene assay. Finally, in a nude mouse model, miR‑490‑3p overexpression significantly suppressed the growth of CRC cells. The above results indicated that miR‑490‑3p might constitute a prognostic indicator and a novel molecular target for miRNA-based CRC therapy.

Arf6, Rab11 and transferrin receptor define distinct populations of recycling endosomes.

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Kobayashi, Hotaka; Fukuda, Mitsunori

2013-09-01

Recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane. To determine how recycling endosomes perform their functions, searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers. However, it remains unclear whether all the conventional markers have identical localizations. Here we report finding that three well-known recycling endosome markers, i.e., Arf6, Rab11 and transferrin receptor (TfR), have different intracellular localizations in PC12 cells. The results of immunofluorescence analyses showed that the signals of endogenous Arf6, Rab11 and TfR in nerve growth factor-stimulated PC12 cells generally differed, although there was some overlapping. Our findings provide new information about recycling endosome markers, and they highlight the heterogeneity of recycling endosomes.

A survey of small RNA population during FR-induced apical hook opening

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Ying eLi

2014-04-01

Full Text Available Photomorphogenesis is a mechanism employed by plants to regulate their architecture and developmental program in response to light conditions. As they emerge into light for the first time, dark-grown seedlings employ a rapid and finely-controlled photomorphogenic signaling network. Small RNAs have increasingly been revealed to play an important role in regulating multiple aspects of plant development, by modulating the stability of mRNAs. The rapid alteration of the mRNA transcriptome is a known hallmark of the de-etiolation response, thus we investigated the small RNA transcriptome during this process in specific seedling tissues. Here we describe a survey of the small RNA expression profile in four tissues of etiolated soybean seedlings, the cotyledons, hypocotyl and the convex and concave sides of the apical hook. We also investigate how this profile responds to a one-hour far-red light treatment. Our data suggests that miRNAs show a different global profile between these tissues and treatments, suggesting a possible role for tissue- and treatment-specific expression in the differential morphology of the seedling on de-etiolation. Further evidence for the role of miRNA in light-regulated development is given by the de-etiolation responses of a hypomorphic ago1 mutant, which displays reduced and delayed photomorphogenic responses in apical hook and cotyledon angle to far-red light.

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.

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

Science.gov (United States)

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes.

Science.gov (United States)

Marwaha, Rituraj; Arya, Subhash B; Jagga, Divya; Kaur, Harmeet; Tuli, Amit; Sharma, Mahak

2017-04-03

Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion. © 2017 Marwaha et al.

Mutations in the small GTPase gene RAB39B are responsible for X-linked mental retardation associated with autism, epilepsy, and macrocephaly.

Science.gov (United States)

Giannandrea, Maila; Bianchi, Veronica; Mignogna, Maria Lidia; Sirri, Alessandra; Carrabino, Salvatore; D'Elia, Errico; Vecellio, Matteo; Russo, Silvia; Cogliati, Francesca; Larizza, Lidia; Ropers, Hans-Hilger; Tzschach, Andreas; Kalscheuer, Vera; Oehl-Jaschkowitz, Barbara; Skinner, Cindy; Schwartz, Charles E; Gecz, Jozef; Van Esch, Hilde; Raynaud, Martine; Chelly, Jamel; de Brouwer, Arjan P M; Toniolo, Daniela; D'Adamo, Patrizia

2010-02-12

Human Mental Retardation (MR) is a common and highly heterogeneous pediatric disorder affecting around 3% of the general population; at least 215 X-linked MR (XLMR) conditions have been described, and mutations have been identified in 83 different genes, encoding proteins with a variety of function, such as chromatin remodeling, synaptic function, and intracellular trafficking. The small GTPases of the RAB family, which play an essential role in intracellular vesicular trafficking, have been shown to be involved in MR. We report here the identification of mutations in the small GTPase RAB39B gene in two male patients. One mutation in family X (D-23) introduced a stop codon seven amino acids after the start codon (c.21C > A; p.Y7X). A second mutation, in the MRX72 family, altered the 5' splice site (c.215+1G > A) and normal splicing. Neither instance produced a protein. Mutations segregate with the disease in the families, and in some family members intellectual disabilities were associated with autism spectrum disorder, epileptic seizures, and macrocephaly. We show that RAB39B, a novel RAB GTPase of unknown function, is a neuronal-specific protein that is localized to the Golgi compartment. Its downregulation leads to an alteration in the number and morphology of neurite growth cones and a significant reduction in presynaptic buttons, suggesting that RAB39B is required for synapse formation and maintenance. Our results demonstrate developmental and functional neuronal alteration as a consequence of downregulation of RAB39B and emphasize the critical role of vesicular trafficking in the development of neurons and human intellectual abilities. Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Predicting placebo response in adolescents with major depressive disorder: The Adolescent Placebo Impact Composite Score (APICS).

Science.gov (United States)

Nakonezny, Paul A; Mayes, Taryn L; Byerly, Matthew J; Emslie, Graham J

2015-09-01

The aim of this study was to construct a composite scoring system to predict the probability of placebo response in adolescents with Major Depressive Disorder (MDD). Participants of the current study were 151 adolescents (aged 12-17 years) who were randomized to the placebo arm (placebo transdermal patches) of a randomized controlled trial (RCT) comparing the selegiline transdermal patch with placebo (DelBello et al., 2014). The primary outcome of response was defined as a CGI-I score of 1 or 2 (very much or much improved) at week 12 (study-end) or exit. As a first step, a multiple logistic mixed model was used to estimate the odds of placebo response from each predictor in the model, including age, CDRS-R total at baseline (depressive symptom severity), history of recurrent depression (yes vs. no), sex (female vs. male), and race (non-Caucasian vs. Caucasian). On the basis of the initial logistic mixed model analysis, we then constructed an Adolescent Placebo Impact Composite Score (APICS) that became the sole predictor in a re-specified Bayesian logistic regression model to estimate the probability of placebo response. Finally, the AUC for the APICS was tested against a nominal area of 0.50 to evaluate how well the APICS discriminated placebo response status. Among the 151 adolescents, with a mean age of 14.6 years (SD = 1.6) and a mean baseline CDRS-R total of 60.6 (SD = 12.1), 68.2% were females, 50.3% was Caucasian, and 39.7% had a history of recurrent depression. Placebo response rate was 58.3%. Based on the logistic mixed model, the re-specified equation with the highest discriminatory ability to estimate the probability of placebo response was APICS = age + (0.32 × CDRS-R Total at baseline) + (-2.85 × if female) + (-5.50 × if history of recurrent depression) + (-5.85 × if non-Caucasian). The AUC for this model was 0.59 (p = .049). Within a Bayesian decision-theoretic framework, in 95.5% of the time, the 10,000 posterior Monte Carlo samples suggested

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

Science.gov (United States)

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

2017-02-01

was performed to identify variables independently associated with clinic Pelvic Organ Prolapse Quantification point C. In all, 52 women were included: 14 in the normal/normal group, 11 in the normal/prolapse group, and 27 in the prolapse/prolapse group. At 1 N of traction force in the operating room, 50% of women in the normal/prolapse group had cervix locations outside the normal range while 10% had apical support stiffness outside the normal range. Of women in the prolapse/prolapse group, 81% had cervix locations outside the normal range and 8% had apical support stiffness outside the normal range. Similar results for cervix locations were observed at 18 N of traction force; however the proportion of women with apical support stiffness outside the normal range increased to 50% in the normal/prolapse group and 59% in the prolapse/prolapse group. The prolapse/prolapse group had statistically lower apical support stiffness compared to the normal/normal group with increased traction from 1-18 N (0.47 ± 0.18 N/mm vs 0.63 ± 0.20 N/mm, P = .006), but all other comparisons were nonsignificant. After controlling for age, parity, body mass index, and apical support stiffness, cervix location at 1 N traction force remained an independent predictor of clinic Pelvic Organ Prolapse Quantification point C, but only in the prolapse/prolapse group. Approximately 50% of women with cystocele and/or rectocele but normal apical support in the clinic had cervix locations outside the normal range under intraoperative traction, while 19% of women with uterine prolapse had normal apical support. Identifying women whose apical support falls outside a defined normal range may be a more accurate way to identify those who truly need a hysterectomy and/or an apical support procedure and to spare those who do not. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Identification and biochemical analysis of Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein.

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Kuroda, Taruho S; Fukuda, Mitsunori

2005-01-01

Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

Transcriptomics insights into the genetic regulation of root apical meristem exhaustion and determinate primary root growth in Pachycereus pringlei (Cactaceae).

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Rodriguez-Alonso, Gustavo; Matvienko, Marta; López-Valle, Mayra L; Lázaro-Mixteco, Pedro E; Napsucialy-Mendivil, Selene; Dubrovsky, Joseph G; Shishkova, Svetlana

2018-06-04

Many Cactaceae species exhibit determinate growth of the primary root as a consequence of root apical meristem (RAM) exhaustion. The genetic regulation of this growth pattern is unknown. Here, we de novo assembled and annotated the root apex transcriptome of the Pachycereus pringlei primary root at three developmental stages, with active or exhausted RAM. The assembled transcriptome is robust and comprehensive, and was used to infer a transcriptional regulatory network of the primary root apex. Putative orthologues of Arabidopsis regulators of RAM maintenance, as well as putative lineage-specific transcripts were identified. The transcriptome revealed putative orthologues of most proteins involved in housekeeping processes, hormone signalling, and metabolic pathways. Our results suggest that specific transcriptional programs operate in the root apex at specific developmental time points. Moreover, the transcriptional state of the P. pringlei root apex as the RAM becomes exhausted is comparable to the transcriptional state of cells from the meristematic, elongation, and differentiation zones of Arabidopsis roots along the root axis. We suggest that the transcriptional program underlying the drought stress response is induced during Cactaceae root development, and that lineage-specific transcripts could contribute to RAM exhaustion in Cactaceae.

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.

[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.

Alteraciones en el reclutamiento y activación de proteínas Rab durante la infección micobacteriana

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Diana Castaño

2010-08-01

La expresión de mutantes constitutivamente activos de las Rab de endosomas tempranos impide la maduración de fagosomas que contienen esferas de látex o micobacterias inactivadas por calor. Mientras que su silenciamiento, mediante ARN de interferencia o mediante dominantes negativos, induce la maduración de fagosomas micobacterianos. Los mecanismos exactos por los que las micobacterias alteran la dinámica de expresión de estas GTPasas, afectando la maduración fagolisosómica, no se han establecido. El problema podría explicarse por defectos en el reclutamiento de las proteínas que interactúan con Rab, como la cinasa-3 del fosfatidilinositol y el antígeno endosómico temprano 1. La identificación de los mecanismos empleados por Mycobacterium spp. para interrumpir el ciclo de activación de las Rab, será esencial para comprender la fisiopatología de la infección micobacteriana y útil como posibles blancos farmacológicos.

A Role for EHD4 in the Regulation of Early Endosomal Transport

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Sharma, Mahak; Naslavsky, Naava; Caplan, Steve

2009-01-01

All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway. PMID:18331452

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

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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

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...

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.

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

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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

dOCRL maintains immune cell quiescence by regulating endosomal traffic.

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Steven J Del Signore

2017-10-01

Full Text Available Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome.

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...

Bony change of apical lesion healing process using fractal analysis

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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.

Nerve signaling regulates basal keratinocyte proliferation in the blastema apical epithelial cap in the axolotl (Ambystoma mexicanum).

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Satoh, Akira; Bryant, Susan V; Gardiner, David M

2012-06-15

The ability of adult vertebrates to repair tissue damage is widespread and impressive; however, the ability to regenerate structurally complex organs such as the limb is limited largely to the salamanders. The fact that most of the tissues of the limb can regenerate has led investigators to question and identify the barriers to organ regeneration. From studies in the salamander, it is known that one of the earliest steps required for successful regeneration involves signaling between nerves and the wound epithelium/apical epithelial cap (AEC). In this study we confirm an earlier report that the keratinocytes of the AEC acquire their function coincident with exiting the cell cycle. We have discovered that this unique, coordinated behavior is regulated by nerve signaling and is associated with the presence of gap junctions between the basal keratinocytes of the AEC. Disruption of nerve signaling results in a loss of gap junction protein, the reentry of the cells into the cell cycle, and regenerative failure. Finally, coordinated exit from the cell cycle appears to be a conserved behavior of populations of cells that function as signaling centers during both development and regeneration. Copyright © 2012 Elsevier Inc. All rights reserved.

Apical Revascularization after Delayed Tooth Replantation: An Unusual Case

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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.

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.

c-Yes regulates cell adhesion at the apical ectoplasmic specialization-blood-testis barrier axis via its effects on protein recruitment and distribution

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Xiao, Xiang; Mruk, Dolores D.

2013-01-01

During spermatogenesis, extensive restructuring takes place at the cell-cell interface since developing germ cells migrate progressively from the basal to the adluminal compartment of the seminiferous epithelium. Since germ cells per se are not motile cells, their movement relies almost exclusively on the Sertoli cell. Nonetheless, extensive exchanges in signaling take place between these cells in the seminiferous epithelium. c-Yes, a nonreceptor protein tyrosine kinase belonging to the Src family kinases (SFKs) and a crucial signaling protein, was recently shown to be upregulated at the Sertoli cell-cell interface at the blood-testis barrier (BTB) at stages VIII–IX of the seminiferous epithelial cycle of spermatogenesis. It was also highly expressed at the Sertoli cell-spermatid interface known as apical ectoplasmic specialization (apical ES) at stage V to early stage VIII of the epithelial cycle during spermiogenesis. Herein, it was shown that the knockdown of c-Yes by RNAi in vitro and in vivo affected both Sertoli cell adhesion at the BTB and spermatid adhesion at the apical ES, causing a disruption of the Sertoli cell tight junction-permeability barrier function, germ cell loss from the seminiferous epithelium, and also a loss of spermatid polarity. These effects were shown to be mediated by changes in distribution and/or localization of adhesion proteins at the BTB (e.g., occludin, N-cadherin) and at the apical ES (e.g., nectin-3) and possibly the result of changes in the underlying actin filaments at the BTB and the apical ES. These findings implicate that c-Yes is a likely target of male contraceptive research. PMID:23169788

Developmentally regulated GTP-binding protein 2 is required for stabilization of Rac1-positive membrane tubules.

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Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Yoon, Eun Hye; Lee, Byung Ju; Cho, Wha Ja; Park, Jeong Woo

2017-11-04

Previously we have reported that developmentally regulated GTP-binding protein 2 (DRG2) localizes on Rab5 endosomes and plays an important role in transferrin (Tfn) recycling. We here identified DRG2 as a key regulator of membrane tubule stability. At 30 min after Tfn treatment, DRG2 localized to membrane tubules which were enriched with phosphatidylinositol 4-monophosphate [PI(4)P] and did not contain Rab5. DRG2 interacted with Rac1 more strongly with GTP-bound Rac1 and tubular localization of DRG2 depended on Rac1 activity. DRG2 depletion led to destabilization of membrane tubules, while ectopic expression of DRG2 rescued the stability of the membrane tubules in DRG2-depleted cells. Our results reveal a novel mechanism for regulation of membrane tubule stability mediated by DRG2. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of a spatially specific enhancer element in the chicken Msx-2 gene that regulates its expression in the apical ectodermal ridge of the developing limb buds of transgenic mice.

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Sumoy, L; Wang, C K; Lichtler, A C; Pierro, L J; Kosher, R A; Upholt, W B

1995-07-01

Msx-2 is a member of the Msx family of homeobox-containing genes expressed in a variety of embryonic tissues involved in epithelial-mesenchymal interactions and pattern formation. In the developing chick limb bud, Msx-2 is expressed in the apical ectodermal ridge, which plays a crucial role in directing the growth and patterning of limb mesoderm. In addition, Msx-2 is expressed in the anterior nonskeletal-forming mesoderm of the limb bud, in the posterior necrotic zone, and in the interdigital mesenchyme. Studies of the altered expression patterns of Msx-2 in amelic and polydactylous mutant chick limbs have suggested that the apical ectodermal ridge and mesodermal domains of Msx-2 expression are independently regulated and that there might be separate cis-regulatory elements in the Msx-2 gene controlling its spatially distinct domains of expression. To test this hypothesis, we have isolated the chicken Msx-2 gene and have tested the ability of various regions of the gene to target expression of LacZ reporter gene to specific regions of the limbs of transgenic mice. A variety of these constructs are consistently expressed only in the apical ectodermal ridge and the ectoderm of the genital tubercle and are not expressed in the mesoderm of the limb bud or in other regions of the embryo where the endogenous Msx-2 gene is expressed. These results suggest the presence of spatially specific cis-regulatory elements in the Msx-2 gene. We identified a 348-bp region in the 5' flanking region of the Msx-2 gene which can act as an apical ectodermal ridge enhancer element when placed in reverse orientation in front of the reporter gene with transcription initiation directed by the minimal hsp68 promoter.

A Novel Wheat C-bZIP Gene, TabZIP14-B, Participates in Salt and Freezing Tolerance in Transgenic Plants

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Lina Zhang

2017-05-01

Full Text Available The group C-bZIP transcription factors (TFs are involved in diverse biological processes, such as the regulation of seed storage protein (SSP production and the responses to pathogen challenge and abiotic stress. However, our knowledge of the abiotic functions of group C-bZIP genes in wheat remains limited. Here, we present the function of a novel TabZIP14-B gene in wheat. This gene belongs to the group C-bZIP TFs and contains six exons and five introns; three haplotypes were identified among accessions of tetraploid and hexaploid wheat. A subcellular localization analysis indicated that TabZIP14-B was targeted to the nucleus of tobacco epidermal cells. A transactivation assay demonstrated that TabZIP14-B showed transcriptional activation ability and was capable of binding the abscisic acid (ABA responsive element (ABRE in yeast. RT-qPCR revealed that TabZIP14-B was expressed in the roots, stems, leaves, and young spikes and was up-regulated by exogenous ABA, salt, low-temperature, and polyethylene glycol (PEG stress treatments. Furthermore, Arabidopsis plants overexpressing TabZIP14-B exhibited enhanced tolerance to salt, freezing stresses and ABA sensitivity. Overexpression of TabZIP14-B resulted in increased expression of the AtRD29A, AtCOR47, AtRD20, AtGSTF6, and AtRAB18 genes and changes in several physiological characteristics. These results suggest that TabZIP14-B could function as a positive regulator in mediating the abiotic stress response.

Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

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Brejchova, Jana; Vosahlikova, Miroslava; Roubalova, Lenka; Parenti, Marco; Mauri, Mario; Chernyavskiy, Oleksandr; Svoboda, Petr

2016-08-01

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

Metabolism of [ 14 C]GA 19 and [ 14 C]GA 53 by ecotypes of Betula ...

African Journals Online (AJOL)

Continuing with this line of research, we studied the metabolism of 14C-labelled GA19 and GA53. [14C]GA19 and [14C] A53 were applied to the apices of the northern ecotype (67º N) and to the leaves of the southern ecotype (64º N) of Betula pendula Roth. under different photoperiods and at different times in order to ...

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

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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.

Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons.

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Lapatsina, Liudmila; Jira, Julia A; Smith, Ewan St J; Poole, Kate; Kozlenkov, Alexey; Bilbao, Daniel; Lewin, Gary R; Heppenstall, Paul A

2012-06-01

A complex of stomatin-family proteins and acid-sensing (proton-gated) ion channel (ASIC) family members participate in sensory transduction in invertebrates and vertebrates. Here, we have examined the role of the stomatin-family protein stomatin-like protein-3 (STOML3) in this process. We demonstrate that STOML3 interacts with stomatin and ASIC subunits and that this occurs in a highly mobile vesicle pool in dorsal root ganglia (DRG) neurons and Chinese hamster ovary cells. We identify a hydrophobic region in the N-terminus of STOML3 that is required for vesicular localization of STOML3 and regulates physical and functional interaction with ASICs. We further characterize STOML3-containing vesicles in DRG neurons and show that they are Rab11-positive, but not part of the early-endosomal, lysosomal or Rab14-dependent biosynthetic compartment. Moreover, uncoupling of vesicles from microtubules leads to incorporation of STOML3 into the plasma membrane and increased acid-gated currents. Thus, STOML3 defines a vesicle pool in which it associates with molecules that have critical roles in sensory transduction. We suggest that the molecular features of this vesicular pool may be characteristic of a 'transducosome' in sensory neurons.

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.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar CellsSummary

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Scott W. Messenger

2015-11-01

Full Text Available Background & Aims: Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI3P. PI(3,5P2 is necessary for maturation of early endosomes (EE to late endosomes (LE. Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Methods: Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1, and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8, bile acids, and cigarette toxin was determined. Results: PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini. Conclusions: These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

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Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

2015-11-01

Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

Ossifying fibroma misdiagnosed as chronic apical periodontitis.

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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.

RosettaAntibodyDesign (RAbD): A general framework for computational antibody design

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Adolf-Bryfogle, Jared; Kalyuzhniy, Oleks; Kubitz, Michael; Hu, Xiaozhen; Adachi, Yumiko; Schief, William R.

2018-01-01

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228–256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody–antigen complexes, using two design strategies—optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the

RosettaAntibodyDesign (RAbD): A general framework for computational antibody design.

Science.gov (United States)

Adolf-Bryfogle, Jared; Kalyuzhniy, Oleks; Kubitz, Michael; Weitzner, Brian D; Hu, Xiaozhen; Adachi, Yumiko; Schief, William R; Dunbrack, Roland L

2018-04-01

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228-256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody-antigen complexes, using two design strategies-optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native

Early stages of functional diversification in the Rab GTPase gene family revealed by genomic and localization studies in Paramecium species.

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Bright, Lydia J; Gout, Jean-Francois; Lynch, Michael

2017-04-15

New gene functions arise within existing gene families as a result of gene duplication and subsequent diversification. To gain insight into the steps that led to the functional diversification of paralogues, we tracked duplicate retention patterns, expression-level divergence, and subcellular markers of functional diversification in the Rab GTPase gene family in three Paramecium aurelia species. After whole-genome duplication, Rab GTPase duplicates are more highly retained than other genes in the genome but appear to be diverging more rapidly in expression levels, consistent with early steps in functional diversification. However, by localizing specific Rab proteins in Paramecium cells, we found that paralogues from the two most recent whole-genome duplications had virtually identical localization patterns, and that less closely related paralogues showed evidence of both conservation and diversification. The functionally conserved paralogues appear to target to compartments associated with both endocytic and phagocytic recycling functions, confirming evolutionary and functional links between the two pathways in a divergent eukaryotic lineage. Because the functionally diversifying paralogues are still closely related to and derived from a clade of functionally conserved Rab11 genes, we were able to pinpoint three specific amino acid residues that may be driving the change in the localization and thus the function in these proteins. © 2017 Bright et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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.

Pro-oxidant status and matrix metalloproteinases in apical lesions and gingival crevicular fluid as potential biomarkers for asymptomatic apical periodontitis and endodontic treatment response

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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.

HIV-1 Nef binds with human GCC185 protein and regulates mannose 6 phosphate receptor recycling

Energy Technology Data Exchange (ETDEWEB)

Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir; Tripathi, Raj Kamal, E-mail: rajkamalcdri@gmail.com

2016-05-20

HIV-1 Nef modulates cellular function that enhances viral replication in vivo which culminate into AIDS pathogenesis. With no enzymatic activity, Nef regulates cellular function through host protein interaction. Interestingly, trans-cellular introduction of recombinant Nef protein in Caenorhabditis elegans results in AIDS like pathogenesis which might share common pathophysiology because the gene sequence of C. elegans and humans share considerable homology. Therefore employing C. elegans based initial screen complemented with sequence based homology search we identified GCC185 as novel host protein interacting with HIV-1 Nef. The detailed molecular characterization revealed N-terminal EEEE{sub 65} acidic domain of Nef as key region for interaction. GCC185 is a tethering protein that binds with Rab9 transport vesicles. Our results show that Nef-GCC185 interaction disrupts Rab9 interaction resulting in delocalization of CI-MPR (cation independent Mannose 6 phosphate receptor) resulting in elevated secretion of hexosaminidase. In agreement with this, our studies identified novel host GCC185 protein that interacts with Nef EEEE65 acidic domain interfering GCC185-Rab9 vesicle membrane fusion responsible for retrograde vesicular transport of CI-MPR from late endosomes to TGN. In light of existing report suggesting critical role of Nef-GCC185 interaction reveals valuable mechanistic insights affecting specific protein transport pathway in docking of late endosome derived Rab9 bearing transport vesicle at TGN elucidating role of Nef during viral pathogenesis. -- Highlights: •Nef, an accessory protein of HIV-1 interacts with host factor and culminates into AIDS pathogenesis. •Using Caenorhabditis elegans based screen system, novel Nef interacting cellular protein GCC185 was identified. •Molecular characterization of Nef and human protein GCC185 revealed Nef EEEE{sub 65} key region interacted with full length GCC185. •Nef impeded the GCC185-Rab 9 interaction and

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 ...

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%)

Assessment of age of majority by measurement of open apices of the third molars using Cameriere's third molar maturity index.

Science.gov (United States)

Sharma, Preeti; Wadhwan, Vijay; Ravi Prakash, S M; Aggarwal, Pooja; Sharma, Neeraj

2017-01-01

Evaluation of biological age of a living subject around the legal cutoff age for adulthood has become a grave concern for forensic experts in India, mainly due to the consequences of criminal obligations in judicial proceedings. Thus, this study was planned to examine the open apices of third molars in discriminating between individuals who are aged 18 years or older and who are not 18 years or older and to assign a cutoff for estimation of the age of 18 years. Orthopantomographs of 1062 individuals (14 and 23 years) were assessed, to verify Cameriere's third molar maturity index (I 3M ). The apical ends of the roots of the left mandibular third molar were analyzed. If the apical ends of the roots are completely closed, then I 3M is zero; otherwise, it is calculated as the sum of the distances between the inner sides of the two open apices divided by the tooth length. The sensitivity of the test for 0.08 value was 74.7% for males and 66% for females. Specificity was 83.6% for males and 79.6% for females. The probability that an Indian individual with an I 3M age in Indian population.

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.

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.

Histopathological, Microbiological, and Radiographic Analysis of Antimicrobial Photodynamic Therapy for the Treatment of Teeth with Apical Periodontitis: A Study in Rats' Molars.

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Trindade, Alessandra Cesar; de Figueiredo, José Antônio Poli; de Oliveira, Sílvia Dias; Barth Junior, Valdir Cristóvão; Gallo, Stephanie Wagner; Follmann, Carina; Wolle, Carlos Frederico Brilhante; Steier, Liviu; Morgental, Renata Dornelles; Weber, João Batista Blessmann

2017-07-01

The aim of this study was to evaluate in vivo, by histological and radiographic analysis, the response of apical tissues of rats' teeth with experimentally induced apical periodontitis, after one- and two-session endodontic treatment with and without photodynamic therapy (PDT). A microbiological analysis was also performed to verify bacterial reduction after each treatment. Studies carried out in recent years highlighted the antibacterial potential of PDT when associated with conventional endodontic therapy in vitro. Although the antimicrobial effect of PDT is well-established, tissue response to PDT in teeth with apical periodontitis lacks studies. Thirty-two rats' root canals were assigned to four groups: one session/PDT-[chemomechanical preparation (CMP)+root canal filling (RCF)]; two sessions/PDT- [CMP+calcium hydroxide (CH) for 14 days+RCF]; one session/PDT+ [CMP+PDT+RCF], and two sessions/PDT+ [CMP+PDT+CH for 14 days+RCF]. For microbiological evaluation, samples were collected before and after proposed treatments. For radiographic and histological analysis, the animals were euthanized after 28 days and the mandibles surgically removed. PDT associated with conventional endodontic therapy was able to promote significant bacterial reduction in root canals with induced apical periodontitis, but this reduction was not significantly different to conventional endodontic therapy alone. Although radiographic evaluation showed no significant differences, histological analysis showed lower scores for neutrophils/eosinophils in PDT-treated groups and macrophages/giant cells in CH groups. The use of low-level laser as light source did not promote major improvement on radiographic and histological repair, but since the number of inflammatory cells slightly decreased, it may optimize repair by modulating inflammatory process. PDT may be indicated as an adjunct to conventional endodontic therapy for teeth with apical periodontitis, in association with an interappointment

Oral microbiota species in acute apical endodontic abscesses

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Noelle George; Erin Flamiatos; Kellie Kawasaki; Namgu Kim; Charles Carriere; Brian Phan; Raphael Joseph; Shay Strauss; Richie Kohli; Dongseok Choi; J. Craig Baumgartner; Christine Sedgley; Tom Maier; Curtis A. Machida

2016-01-01

Background and objectives: Acute apical abscesses are serious endodontic diseases resulting from pulpal infection with opportunistic oral microorganisms. The objective of this study was to identify and compare the oral microbiota in patients (N=18) exhibiting acute apical abscesses, originating from the demographic region in Portland, Oregon. The study hypothesis is that abscesses obtained from this demographic region may contain unique microorganisms not identified in specimens from other re...

Kinesin-73 is a processive motor that localizes to Rab5-containing organelles.

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Huckaba, Thomas M; Gennerich, Arne; Wilhelm, James E; Chishti, Athar H; Vale, Ronald D

2011-03-04

Drosophila Kinesin-73 (Khc-73), which plays a role in mitotic spindle polarity in neuroblasts, is a metazoan-specific member of the Kinesin-3 family of motors, which includes mammalian KIF1A and Caenorhabditis elegans Unc-104. The mechanism of Kinesin-3 motors has been controversial because some studies have reported that they transport cargo as monomers whereas other studies have suggested a dimer mechanism. Here, we have performed single-molecule motility and cell biological studies of Khc-73. We find that constructs containing the motor and the conserved short stretches of putative coiled-coil-forming regions are predominantly monomeric in vitro, but that dimerization allows for fast, processive movement and high force production (7 piconewtons). In Drosophila cell lines, we present evidence that Khc-73 can dimerize in vivo. We also show that Khc-73 is recruited specifically to Rab5-containing endosomes through its "tail" domain. Our results suggest that the N-terminal half of Khc-73 can undergo a monomer-dimer transition to produce a fast processive motor and that its C-terminal half possesses a specific Rab5-vesicle binding domain.

The Ubiquitin-Specific Protease 14 (USP14) Is a Critical Regulator of Long-Term Memory Formation

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Jarome, Timothy J.; Kwapis, Janine L.; Hallengren, Jada J.; Wilson, Scott M.; Helmstetter, Fred J.

2014-01-01

Numerous studies have suggested a role for ubiquitin-proteasome-mediated protein degradation in learning-dependent synaptic plasticity; however, very little is known about how protein degradation is regulated at the level of the proteasome during memory formation. The ubiquitin-specific protease 14 (USP14) is a proteasomal deubiquitinating enzyme…

Rab11-dependent Recycling of the Human Ether-a-go-go-related Gene (hERG) Channel*

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Chen, Jeffery; Guo, Jun; Yang, Tonghua; Li, Wentao; Lamothe, Shawn M.; Kang, Yudi; Szendrey, John A.; Zhang, Shetuan

2015-01-01

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr). A reduction in the hERG current causes long QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. We reported previously that hERG channels in the plasma membrane undergo vigorous internalization under low K+ conditions. In the present study, we addressed whether hERG internalization occurs under normal K+ conditions and whether/how internalized channels are recycled back to the plasma membrane. Using patch clamp, Western blot, and confocal imaging analyses, we demonstrated that internalized hERG channels can effectively recycle back to the plasma membrane. Low K+-enhanced hERG internalization is accompanied by an increased rate of hERG recovery in the plasma membrane upon reculture following proteinase K-mediated clearance of cell-surface proteins. The increased recovery rate is not due to enhanced protein synthesis, as hERG mRNA expression was not altered by low K+ exposure, and the increased recovery was observed in the presence of the protein biosynthesis inhibitor cycloheximide. GTPase Rab11, but not Rab4, is involved in the recycling of hERG channels. Interfering with Rab11 function not only delayed hERG recovery in cells after exposure to low K+ medium but also decreased hERG expression and function in cells under normal culture conditions. We concluded that the recycling pathway plays an important role in the homeostasis of plasma membrane-bound hERG channels. PMID:26152716

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.

The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28.

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Pla, M; Vilardell, J; Guiltinan, M J; Marcotte, W R; Niogret, M F; Quatrano, R S; Pagès, M

1993-01-01

The maize gene rab28 has been identified as ABA-inducible in embryos and vegetative tissues. It is also induced by water stress in young leaves. The proximal promoter region contains the conserved cis-acting element CCACGTGG (ABRE) reported for ABA induction in other plant genes. Transient expression assays in rice protoplasts indicate that a 134 bp fragment (-194 to -60 containing the ABRE) fused to a truncated cauliflower mosaic virus promoter (35S) is sufficient to confer ABA-responsiveness upon the GUS reporter gene. Gel retardation experiments indicate that nuclear proteins from tissues in which the rab28 gene is expressed can interact specifically with this 134 bp DNA fragment. Nuclear protein extracts from embryo and water-stressed leaves generate specific complexes of different electrophoretic mobility which are stable in the presence of detergent and high salt. However, by DMS footprinting the same guanine-specific contacts with the ABRE in both the embryo and leaf binding activities were detected. These results indicate that the rab28 promoter sequence CCACGTGG is a functional ABA-responsive element, and suggest that distinct regulatory factors with apparent similar affinity for the ABRE sequence may be involved in the hormone action during embryo development and in vegetative tissues subjected to osmotic stress.

A RAB3GAP1 SINE Insertion in Alaskan Huskies with Polyneuropathy, Ocular Abnormalities, and Neuronal Vacuolation (POANV Resembling Human Warburg Micro Syndrome 1 (WARBM1

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Michaela Wiedmer

2016-02-01

Full Text Available We observed a hereditary phenotype in Alaskan Huskies that was characterized by polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV. The affected dogs developed a progressive severe ataxia, which led to euthanasia between 8 and 16 months of age. The pedigrees were consistent with a monogenic autosomal recessive inheritance. We localized the causative genetic defect to a 4 Mb interval on chromosome 19 by a combined linkage and homozygosity mapping approach. Whole genome sequencing of one affected dog, an obligate carrier, and an unrelated control revealed a 218-bp SINE insertion into exon 7 of the RAB3GAP1 gene. The SINE insertion was perfectly associated with the disease phenotype in a cohort of 43 Alaskan Huskies, and it was absent from 541 control dogs of diverse other breeds. The SINE insertion induced aberrant splicing and led to a transcript with a greatly altered exon 7. RAB3GAP1 loss-of-function variants in humans cause Warburg Micro Syndrome 1 (WARBM1, which is characterized by additional developmental defects compared to canine POANV, whereas Rab3gap1-deficient mice have a much milder phenotype than either humans or dogs. Thus, the RAB3GAP1 mutant Alaskan Huskies provide an interesting intermediate phenotype that may help to better understand the function of RAB3GAP1 in development. Furthermore, the identification of the presumed causative genetic variant will enable genetic testing to avoid the nonintentional breeding of affected dogs.

Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5.

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Weir, Dawn L; Laing, Eric D; Smith, Ina L; Wang, Lin-Fa; Broder, Christopher C

2014-02-27

Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined. ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol. Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry. The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

Effect of four different intracanal medicaments on the apical seal of the root canal system: a dye extraction study.

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Tandan, Monika; Hegde, Mithra N; Hegde, Priyadarshini

2014-01-01

The aim was to determine the effect of four different intracanal medicaments on the apical seal of the root canal system in vitro. Fifty freshly extracted intact human permanent maxillary central incisors were collected, stored and disinfected. The root canals were prepared to a master apical size of number 50 using step back technique. Depending upon the intracanal medicament used, the teeth were divided randomly into five groups of 10 teeth each including one control group and four experimental groups. Group A: No intracanal medicament. Group B: Calcium hydroxide powder mixed with distilled water. Group C: Calcium hydroxide gutta percha points (calcium hydroxide points). Group D: 1% chlorhexidine gel (hexigel). Group E: Chlorhexidine gutta percha points (Roeko Activ Points). The medication was left in canals for 14 days. Following removal of the intracanal medicament, all the groups were obturated with lateral compaction technique. The apical leakage was then evaluated using dye extraction method with the help of a spectrophotometer. RESULTS were statistically analyzed using Kruskal-Wallis and Mann-Whitney U-test, which showed statistically significant difference among the five groups tested. It can be concluded from this study that the control group showed least amount of leakage, whereas the 1% chlorhexidine gel group showed maximum amount of leakage. Apical leakage was observed with all the experimental groups with little variations in between them. Under the parameters of this study, it can be concluded that use of intracanal medicaments during endodontic treatment has a definite impact on the apical seal of the root canal system.

Apical extrusion of debris in four different endodontic instrumentation systems: A meta-analysis.

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Western, J Sylvia; Dicksit, Daniel Devaprakash

2017-01-01

All endodontic instrumentation systems tested so far, promote apical extrusion of debris, which is one of the main causes of postoperative pain, flare ups, and delayed healing. Of this meta-analysis was to collect and analyze in vitro studies quantifying apically extruded debris while using Hand ProTaper (manual), ProTaper Universal (rotary), Wave One (reciprocating), and self-adjusting file (SAF; vibratory) endodontic instrumentation systems and to determine methods which produced lesser extrusion of debris apically. An extensive electronic database search was done in PubMed, Scopus, Cochrane, LILACS, and Google Scholar from inception until February 2016 using the key terms "Apical Debris Extrusion, extruded material, and manual/rotary/reciprocating/SAF systems." A systematic search strategy was followed to extract 12 potential articles from a total of 1352 articles. The overall effect size was calculated from the raw mean difference of weight of apically extruded debris. Statistically significant difference was seen in the following comparisons: SAF ProTaper. Apical extrusion of debris was invariably present in all the instrumentation systems analyzed. SAF system seemed to be periapical tissue friendly as it caused reduced apical extrusion compared to Rotary ProTaper and Wave One.

Molecular Analysis of Gli3, Ihh, Rab23, and Jag1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

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Gilbert, James R; Taylor, Gwen M; Losee, Joseph E; Mooney, Mark P; Cooper, Gregory M

2018-03-01

Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. The etiology of CS is complex and mutations in more than 50 distinct genes have been causally linked to the disorder. Many of the genes that have been associated with CS in humans play an essential role in tissue patterning and early craniofacial development. Among these genes are members of the Hedgehog (HH) and Notch signal transduction pathways, including the GLI family member Gli3, Indian Hedgehog ( Ihh), the RAS oncogene family member Rab23, and the Notch ligand JAGGED1 ( Jag1). We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if coding errors in Gli3, Ihh, Rab23, or Jag1 could be causally linked to craniosynostosis in this unique animal model. Sequencing of cDNA templates was performed using samples obtained from wild-type and craniosynostotic rabbits. Several nucleotide polymorphisms were identified in Gli3, Ihh, and Rab23, although these variants failed to segregate by phenotype. No nucleotide polymorphisms were identified in Jag1. These data indicate that the causal locus for heritable craniosynostosis in this rabbit model is not located within the protein coding regions of Gli3, Ihh, Rab23, or Jag1.

Influence of UV and blue-light sun radiation to developing of age-related macular degeneration on Croatian Island Rab

International Nuclear Information System (INIS)

Vojnikovic, Bozo; Coklo, Miran; Ranogajec-Komor, Maria

2008-01-01

Full text: Croatian island Rab has one of the highest solar radiation in geographical area of Europe. The aim of this clinical trial was to estimate correlation between incidence of age-related macular degeneration (AMD) and the exposure to sunlight in different population on island Rab. In the first group agriculturists and fishermen were collected, in the second group members of the urban population were involved. 1753 individuals were investigated. The age in this population was between 40 and 73 years. The incidence of AMD in the first group was 19% while in the urban population only 2% showed AMD. It can be concluded that a very significant incidence exists between chronic exposure to sunlight and appearance of age-related macular degeneration. (author)

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.

Immediate Implant Placement in Sockets with Asymptomatic Apical Periodontitis.

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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.

Respiration-dependent proton translocation in alkalophilic Bacillus firmus RAB and its non-alkalophilic mutant derivative.

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Lewis, R J; Krulwich, T A; Reynafarje, B; Lehninger, A L

1983-02-25

Obligately alkalophilic Bacillus firmus RAB had a higher molar growth yield on L-malate (Ymal = 38 mg, dry weight/mmol of L-malate) than its non-alkalophilic mutant derivative, strain RABN (Ymal = 12 mg, dry weight/mmol of L-malate). Measurements of respiration dependent proton translocation by the two strains in the presence of K+ and valinomycin showed that the alkalophile also has much higher H+/O stoichiometries (at pH 9.0) than does the mutant (at pH 7.0). H+/O ratios for B. firmus RAB at pH 9.0 were as high as 13, with a frequently observed value of 9. These high values were observed in the first phase of a set of biphasic curves for both oxygen consumption and proton ejection. At pH 7.0, both the wild type and the mutant exhibited H+/O ratios near 4 in a single phase of oxygen consumption and proton ejection. The results are consistent with suggestions that the alkalophilic respiratory chain is especially well adapted for effective energy transduction at alkaline but not neutral pH.

Dental Apical Papilla as Therapy for Spinal Cord Injury.

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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

A comparison of apical transportation between FlexMaster and Twisted Files rotary instruments.

Science.gov (United States)

Duran-Sindreu, Fernando; García, Marc; Olivieri, Juan Gonzalo; Mercadé, Montse; Morelló, Sergio; Roig, Miguel

2012-07-01

The aim of this study was to evaluate apical transportation in root canals after the use of Twisted Files (TF; SybronEndo, Orange, CA) and FlexMaster (VDW, Munich, Germany) #40/04 rotary files. A double-digital radiographic technique was used to compare apical transportation between the TF and FlexMaster systems. Each rotary system was used to instrument mesial canals from 80 extracted mandibular molars. The central axes of the file imaged before instrumentation (#15 K-file) and the master apical rotary file (#40/04) were superimposed digitally. AutoCAD 2008 (Autodesk Inc, San Rafael, CA) was used to measure apical transportation at 0.5 mm from the working length (WL). The data were analyzed using the Student's t test, and significance was set at P < .05. The mean amount of apical transportation at 0.5 mm from the WL was 0.17 ± 0.09 mm for the FlexMaster group and 0.19 ± 0.12 mm for the TF group. No statistically significant differences in apical transportation were found between the 2 groups. Under the conditions of the study, no statistically significant differences in apical transportation were observed between FlexMaster and TF rotary files. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

ROOT CANAL MICROORGANISMS PROFILES O F UPPER ANTERIOR TEETH WITH APICAL PERIODONTITIS

OpenAIRE

Tanumihardja, Maria; Riewpassa, Irene E; Mansjurnasir; Burhanuddin, DP

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 ch ronic api...

Effects of sodium hypochlorite associated with EDTA and etidronate on apical root transportation.

Science.gov (United States)

Silva e Souza, P A R; das Dores, R S E; Tartari, T; Pinheiro, T P S; Tuji, F M; Silva e Souza, M H

2014-01-01

To evaluate the influence of sodium hypochlorite associated with EDTA and etidronate on apical root transportation. Forty-five roots of human mandibular molars with curvatures of 15-25° were embedded in acrylic resin to allow standardized angulation of the initial and final radiographs. The pre-instrumentation radiographs of the mesiobuccal canal of each root were taken using a radiograph digital sensor with a size 15 K-file in the canal. The canals were prepared with the ProTaper Universal system (Dentsply Maillefer, Ballaigues, Switzerland), using one of the following irrigation regimens during the instrumentation (n = 15): G1 - irrigation with 20 mL of saline solution (control); G2 - alternating irrigation with 2.5% hypochlorite solution (NaOCl) (15 mL); and 17% ethylenediaminetetraacetic acid (EDTA) (5 mL). During instrumentation, the canal was filled with NaOCl and then between each exchange of instrument filled with EDTA for 1 min, and G3 - irrigation with 20 mL of 5% NaOCl and 18% etidronate solution (HEBP) mixed in equal parts. The postinstrumentation radiographs were made with a F3 instrument in the canal. The images were magnified and superposed with Adobe Photoshop software (Adobe Systems, Mountain View, CA, USA). Apical transportation was determined with AutoCAD 2012 software (Autodesk Inc., San Rafael, CA, USA) by measuring the distance in millimetres between the tips of the instruments. The results were subjected to the nonparametric statistical Kruskal-Wallis test (α < 0.05). The median transportation and interquartile range values were 0.00 ± 0.05 for G1, 0.08 ± 0.23 for G2 and 0.13 ± 0.14 for G3. Comparison between groups showed that apical transportation in G3 was significantly greater than in G1 (P < 0.05). The use of NaOCl associated with etidronate increased apical transportation in the canals of extracted teeth. © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Nonsurgical root canal therapy of large cyst-like inflammatory periapical lesions and inflammatory apical cysts.

Science.gov (United States)

Lin, Louis M; Ricucci, Domenico; Lin, Jarshen; Rosenberg, Paul A

2009-05-01

It is a general belief that large cyst-like periapical lesions and apical true cysts caused by root canal infection are less likely to heal after nonsurgical root canal therapy. Nevertheless, there is no direct evidence to support this assumption. A large cyst-like periapical lesion or an apical true cyst is formed within an area of apical periodontitis and cannot form by itself. Therefore, both large cyst-like periapical lesions and apical true cysts are of inflammatory and not of neoplastic origin. Apical periodontitis lesions, regardless of whether they are granulomas, abscesses, or cysts, fail to heal after nonsurgical root canal therapy for the same reason, intraradicular and/or extraradicular infection. If the microbial etiology of large cyst-like periapical lesions and inflammatory apical true cysts in the root canal is removed by nonsurgical root canal therapy, the lesions might regress by the mechanism of apoptosis in a manner similar to the resolution of inflammatory apical pocket cysts. To achieve satisfactory periapical wound healing, surgical removal of an apical true cyst must include elimination of root canal infection.

A comparative evaluation of gutta percha removal and extrusion of apical debris by rotary and hand files.

Science.gov (United States)

Chandrasekar; Ebenezar, A V Rajesh; Kumar, Mohan; Sivakumar, A

2014-11-01

The aim of this study was to evaluate the efficacy of Protaper retreatment files in comparison with RaCe, K3 and H-files for removal of gutta-percha and apically extruded debris using volumetric analysis. Forty extracted single rooted maxillary incisor teeth with straight canals and mature apices were selected for the study. After access cavity preparation, apical patency was confirmed with a size 10 K-file extending 1mm beyond the point at which it was first visible at the apical end. Working lengths were determined with the use of size 15 K-file. The canals were prepared in a step-back technique and the master apical file was size 30 for all teeth. 3% sodium hypochlorite was used as an irrigant after each instrumentation. Before final rinse, size 20 K-file was passed 1mm beyond the apex to remove any dentinal shaving plugs and maintain the apical patency. Then the canals were dried with paper points. The root canal was filled using standard gutta-percha points and zinc oxide eugenol sealer under lateral condensation technique. The teeth were then randomly divided into four groups of ten teeth each based on the instrument used for gutta percha removal. All the rotary instruments used in this study were rotated at 300rpm. The instruments used were: Group 1 - RaCe Files, Group 2 - ProTaper retreatment Files, Group 3 - K3 Files and Group 4 - H Files. The volume of the obturating material was calculated before and after removal using volumetric analysis with spiral CT. The removal efficacy with each instrument was calculated and statistically analysed. The results of the study show that the ProTaper retreatment files (Group 2) (97.4%) showed the highest efficiency in the removal of obturating material, which was followed by RaCe (95.74%), K3 (92.86%) and H files (90.14%) with the efficiency in the decreasing order. Similarly the mean apical extrusion in H files (0.000 ± 0.002) was significantly lower than all the rotary instruments. However, the difference among the

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

OpenAIRE

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

2017-01-01

Objectives 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. Materials and methods 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 m...

Increased Eps15 homology domain 1 and RAB11FIP3 expression regulate breast cancer progression via promoting epithelial growth factor receptor recycling.

Science.gov (United States)

Tong, Dandan; Liang, Ya-Nan; Stepanova, A A; Liu, Yu; Li, Xiaobo; Wang, Letian; Zhang, Fengmin; Vasilyeva, N V

2017-02-01

Recent research indicates that the C-terminal Eps15 homology domain 1 is associated with epithelial growth factor receptor-mediated endocytosis recycling in non-small-cell lung cancer. The aim of this study was to determine the clinical significance of Eps15 homology domain 1 gene expression in relation to phosphorylation of epithelial growth factor receptor expression in patients with breast cancer. Primary breast cancer samples from 306 patients were analyzed for Eps15 homology domain 1, RAB11FIP3, and phosphorylation of epithelial growth factor receptor expression via immunohistochemistry. The clinical significance was assessed via a multivariate Cox regression analysis, Kaplan-Meier curves, and the log-rank test. Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor were upregulated in 60.46% (185/306) and 53.92% (165/306) of tumor tissues, respectively, as assessed by immunohistochemistry. The statistical correlation analysis indicated that Eps15 homology domain 1 overexpression was positively correlated with the increases in phosphorylation of epithelial growth factor receptor ( r = 0.242, p breast cancer for the overall survival in the total, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. However, the use of combined expression of Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor markers is more effective for the disease-free survival in the overall population, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. Moreover, the combined markers are also significant prognostic markers of breast cancer in the human epidermal growth factor receptor 2 (+), estrogen receptor (+), and estrogen receptor (-) groups. Eps15 homology domain 1 has a tumor suppressor function, and the combined marker of Eps15 homology domain 1/phosphorylation of epithelial growth factor receptor expression was identified as a better prognostic marker in breast cancer diagnosis

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

Science.gov (United States)

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.

Spontaneous coronary artery dissection associated with apical hypertrophic cardiomyopathy

International Nuclear Information System (INIS)

Tuncer, M.; Gumrukcuoglu, H.A.; Ekim, H.; Gunes, Y.; Simsek, H.

2010-01-01

Apical hypertrophic cardiomyopathy (HCM) is a relatively uncommon inherited disease. Spontaneous coronary artery dissection (SCAD) is also uncommonly observed, which often occurs in pregnant or post partum women but is rare in men. This report describes a 38 years old man with apical hypertrophic cardiomyopathy who developed SCAD leading to acute inferior myocardial infarction. After emergent appendectomy operation at another hospital, he was immediately transferred to the Cardiology Department of our hospital due to acute myocardial infarction. He emergently underwent coronary angiography which showed a long dissection involving the right coronary. He underwent an emergent CABG with cardiopulmonary bypass. Postoperative recovery was uneventful and he was discharged. According to our knowledge, no case of spontaneous coronary artery dissection associated with apical hypertrophic cardiomyopathy unrelated to postpartum period or oral contraceptive use has been reported so far. (author)

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.

Micro-computed Tomographic Analysis of Apical Microcracks before and after Root Canal Preparation by Hand, Rotary, and Reciprocating Instruments at Different Working Lengths.

Science.gov (United States)

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

2017-07-01

This study aimed to compare apical microcrack formation after root canal shaping by hand, rotary, and reciprocating files at different working lengths using micro-computed tomographic analysis. Sixty mandibular incisors were randomly divided into 6 experimental groups (n = 10) according to the systems and working lengths used for the root canal preparation: ProTaper Universal for Hand Use (Dentsply Maillefer, Ballaigues, Switzerland), HyFlex CM (Coltene-Whaledent, Allstetten, Switzerland), and Reciproc (VDW, Munich, Germany) files working at the apical foramen (AF) and 1 mm short of the AF (AF - 1 mm). The teeth were imaged with micro-computed tomographic scanning at an isotropic resolution of 14 μm before and after root canal preparation, and the cross-sectional images generated were assessed to detect microcracks in the apical portion of the roots. Overall, 17 (28.3%) specimens presented microcracks before instrumentation. Apical microcracks were present in 1 (ProTaper Universal for Hand Use), 3 (Hyflex CM), and 2 (Reciproc) specimens when the instrumentation terminated at the AF. When instrumentation was terminated at AF - 1 mm, apical microcracks were detected in 3 (ProTaper Universal for Hand Use) and 4 (Hyflex CM and Reciproc) specimens. All these microcracks detected after root canal preparation were already present before instrumentation, and no new apical microcrack was visualized. For all groups, the number of slices presenting microcracks after root canal preparation was the same as before canal preparation. Root canal shaping with ProTaper Universal for Hand Use, HyFlex CM, and Reciproc systems, regardless of the working length, did not produce apical microcracks. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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

The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

Science.gov (United States)

Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

2016-09-01

Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

Participation of gibberellin in the control of apical dominance in soybean and redwood

Energy Technology Data Exchange (ETDEWEB)

Ruddat, M.; Pharis, R.P.

1966-01-01

Loss of apical dominance in soybeans and redwood was increased when the plants were treated with the growth retardant AMO-1618. Simultaneous application of gibberellin reduced the number of elongating buds and promoted growth of the first or second uppermost auxillary bud, thus restoring apical dominance. It is concluded that gibberellin participates in the expression of apical dominance. 30 references, 2 tables.

Viral-bacterial associations in acute apical abscesses.

Science.gov (United States)

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.

14 CFR 1310.11 - Regulations of the Office of Management and Budget.

Science.gov (United States)

2010-01-01

... Budget. (a) The regulations in this part supplement the regulations of the Office of Management and... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Regulations of the Office of Management and Budget. 1310.11 Section 1310.11 Aeronautics and Space AIR TRANSPORTATION SYSTEM STABILIZATION AIR...

Proliferation and osteo/odontogenic differentiation of stem cells from apical papilla regulated by Zinc fingers and homeoboxes 2: An in vitro study.

Science.gov (United States)

Wan, Fang; Gao, Lifen; Lu, Yating; Ma, Hongxin; Wang, Hongxing; Liang, Xiaohong; Wang, Yan; Ma, Chunhong

2016-01-15

In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression of osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs. Copyright © 2015 Elsevier Inc. All rights reserved.

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...

Apically-extruded debris using the ProTaper system.

Science.gov (United States)

Azar, Nasim Gheshlaghi; Ebrahimi, Gholamreza

2005-04-01

The purpose of this in vitro study was to determine the quantity of debris and irrigant extruded apically using the ProTaper system compared to ProFiles and K-Flexofiles. Thirty-six mesio-buccal root canals of human mandibular molars were selected and divided into three groups of twelve canals. Two groups were instrumented with ProFiles and ProTapers according to the manufacturer's instructions. The other group was instrumented with K-Flexofiles using the step-back technique. A standard amount of irrigant was used for each canal. Apically-extruded debris and irrigant was collected in pre-weighed vials. The mean weight of extruded debris and irrigant for each group was statistically analysed using Student's t-test and one-way ANOVA. All instrumentation techniques produced extruded debris and irrigant. Although the mean amount of extrusion with the step-back technique was higher than the two rotary systems, there was no significant difference between the three groups (p > 0.05). NiTi rotary systems were associated with less apical extrusion, but were not significantly better than hand file instrumentation. All techniques extruded debris.

Morphology of the Physiological Apical Foramen in Maxillary and Mandibular First Molars

OpenAIRE

Abarca, J.; Zaror, C.; Monardes, H.; Hermosilla, V.; Muñoz, C.; Cantin, M.

2014-01-01

Information regarding the anatomy of the physiological apical foramen is limited. Knowing its diameter and shapes contributes to clinical work, specifically to the cleaning and shaping of the apical third. The aim of this ex vivo study was to determine the minimum and maximum diameters and shape of the physiological apical foramen in the roots of maxillary and mandibular first molars. A descriptive study was conducted on 89 recently extracted first molars. Roots 3–5 mm from the apex were sect...

Rab9-dependent autophagy is required for the IGF-IIR triggering mitophagy to eliminate damaged mitochondria.

Science.gov (United States)

Huang, Chih-Yang; Kuo, Wei-Wen; Ho, Tsung-Jung; Chiang, Shu-Fen; Pai, Pei-Ying; Lin, Jing-Ying; Lin, Ding-Yu; Kuo, Chia-Hua; Huang, Chih-Yang

2018-03-25

Mitochondria dysfunction is the major characteristic of mitophagy, which is essential in mitochondrial quality control. However, excessive mitophagy contributes to cell death in a number of diseases, including ischemic stroke and hepatotoxicity. Insulin-like growth factor II (IGF-II) and its receptor (IGF-IIR) play vital roles in the development of heart failure during hypertension. We found that IGF-II triggers IGF-IIR receptor activation, causing mitochondria dysfunction, resulting in mitophagy, and cardiomyocyte cell death. These results indicated that IGF-IIR activation triggers mitochondria fragmentation, leading to autophagosome formation, and loss of mitochondria content. These results are associated with Parkin-dependent mitophagy. Additionally, autophagic proteins Atg5, and Atg7 deficiency did not suppress IGF-IIR-induced mitophagy. However, Rab9 knockdown reduced mitophagy and maintained mitochondrial function. These constitutive mitophagies through IGF-IIR activation trigger mitochondria loss and mitochondrial ROS accumulation for cardiomyocyte viability decrease. Together, our results indicate that IGF-IIR predominantly induces mitophagy through the Rab9-dependent alternative autophagy. © 2018 Wiley Periodicals, Inc.

C9orf72’s interaction with Rab GTPases - modulation of membrane traffic and autophagy

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Bor Luen Tang

2016-10-01

Full Text Available Hexanucleotide repeat expansion in an intron of Chromosome 9 open reading frame 72 (C9orf72 is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS and Frontotemporal Dementia (FTD. While functional haploinsufficiency of C9orf72 resulting from the mutation may play a role in ALS/FTD, the actual cellular role of the protein has been unclear. Recent findings have now shown that C9orf72 physically and functionally interacts with multiple members of the Rab small GTPases family, consequently exerting important influences on cellular membrane traffic and the process of autophagy. Loss of C9orf72 impairs endocytosis in neuronal cell lines, and attenuated autophagosome formation. Interestingly, C9orf72 could influence autophagy both as part of a Guanine nucleotide exchange factor (GEF complex, or as a Rab effector that facilitates transport of the Unc-51-like Autophagy Activating Kinase 1 (Ulk1 autophagy initiation complex. The cellular function of C9orf72 is discussed in the light of these recent findings

Micro‐CT analyses of apical enlargement and molar root canal complexity

DEFF Research Database (Denmark)

Markvart, M.; Darvann, Tron Andre; Larsen, P.

2012-01-01

Markvart M, Darvann TA, Larsen P, Dalstra M, Kreiborg S, Bjørndal L. Micro‐CT analyses of apical enlargement and molar root canal complexity. International Endodontic Journal, 45, 273–281, 2012. Aim To compare the effectiveness of two rotary hybrid instrumentation techniques with focus on apical...

Affinity between TBC1D4 (AS160 phosphotyrosine-binding domain and insulin-regulated aminopeptidase cytoplasmic domain measured by isothermal titration calorimetry

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SangYoun Park*, Keon Young Kim, Sunmin Kim & Young Seok Yu

2012-06-01

Full Text Available Uptake of circulating glucose into the cells happens via the insulin-mediated signalling pathway, which translocates the glucosetransporter 4 (GLUT4 vesicles from the intracellular compartmentto the plasma membrane. RabㆍGTPases are involvedin this vesicle trafficking, where RabㆍGTPase-activatingproteins (RabGAP enhance the GTP to GDP hydrolysis.TBC1D4 (AS160 and TBC1D1 are functional RabGAPs in theadipocytes and the skeletonal myocytes, respectively. Theseproteins contain two phosphotyrosine-binding domains (PTBsat the amino-terminus of the catalytic RabGAP domain. Thesecond PTB has been shown to interact with the cytoplasmicregion of the insulin-regulated aminopeptidase (IRAP of theGLUT4 vesicle. In this study, we quantitatively measured the∼μM affinity (KD between TBC1D4 PTB and IRAP using isothermaltitration calorimetry, and further showed that IRAP residues1-49 are the major region mediating this interaction. Wealso demonstrated that the IRAP residues 1-15 are necessarybut not sufficient for the PTB interaction.

Dental Pulp Revascularization of Necrotic Permanent Teeth with Immature Apices.

Science.gov (United States)

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.

Balance between Apical Membrane Growth and Luminal Matrix Resistance Determines Epithelial Tubule Shape

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Bo Dong

2014-05-01

Full Text Available The morphological stability of biological tubes is crucial for the efficient circulation of fluids and gases. Failure of this stability causes irregularly shaped tubes found in multiple pathological conditions. Here, we report that Drosophila mutants of the ESCRT III component Shrub/Vps32 exhibit a strikingly elongated sinusoidal tube phenotype. This is caused by excessive apical membrane synthesis accompanied by the ectopic accumulation and overactivation of Crumbs in swollen endosomes. Furthermore, we demonstrate that the apical extracellular matrix (aECM of the tracheal tube is a viscoelastic material coupled with the apical membrane. We present a simple mechanical model in which aECM elasticity, apical membrane growth, and their interaction are three vital parameters determining the stability of biological tubes. Our findings demonstrate a mechanical role for the extracellular matrix and suggest that the interaction of the apical membrane and an elastic aECM determines the final morphology of biological tubes independent of cell shape.

Balance between apical membrane growth and luminal matrix resistance determines epithelial tubule shape.

Science.gov (United States)

Dong, Bo; Hannezo, Edouard; Hayashi, Shigeo

2014-05-22

The morphological stability of biological tubes is crucial for the efficient circulation of fluids and gases. Failure of this stability causes irregularly shaped tubes found in multiple pathological conditions. Here, we report that Drosophila mutants of the ESCRT III component Shrub/Vps32 exhibit a strikingly elongated sinusoidal tube phenotype. This is caused by excessive apical membrane synthesis accompanied by the ectopic accumulation and overactivation of Crumbs in swollen endosomes. Furthermore, we demonstrate that the apical extracellular matrix (aECM) of the tracheal tube is a viscoelastic material coupled with the apical membrane. We present a simple mechanical model in which aECM elasticity, apical membrane growth, and their interaction are three vital parameters determining the stability of biological tubes. Our findings demonstrate a mechanical role for the extracellular matrix and suggest that the interaction of the apical membrane and an elastic aECM determines the final morphology of biological tubes independent of cell shape. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Gravity-induced buds formation from protonemata apical cells in the mosses

Science.gov (United States)

Kyyak, Natalia; Khorkavtsiv, Yaroslava

The acceleration of moss protonemata development after the exit it to light from darkness is important gravidependent morphogenetic manifestation of the moss protonemata. The accelerated development of mosses shows in transformation of apical protonemata cells into the gametophores buds (Ripetskyj et al., 1999). In order to establish, that such reaction on gravitation is general property of gravisensity species, or its typical only for single moss species, experiments with the following moss species - Bryum intermedium (Ludw.) Brig., Bryum caespiticium Hedw., Bryum argenteum Hedw., Dicranodontium denudatum (Brid.) Britt. were carried out. All these species in response to influence of gravitation were capable to form rich bunches of gravitropical protonemata in darkness, that testified to their gravisensity. After the transference of Petri dishes with gravitropical protonemata from darkness on light was revealed, that in 3 of the investigated species the gametophores buds were absent. Only B. argenteum has reacted to action of gravitation by buds formation from apical cells of the gravitropical protonemata. With the purpose of strengthening of buds formation process, the experiments with action of exogenous kinetin (in concentration of 10 (-6) M) were carried out. Kinetin essentially stimulated apical buds formation of B. argenteum. The quantity of apical buds has increased almost in three times in comparison with the control. Besides, on separate stolons a few (3-4) buds from one apical cell were formed. Experimentally was established, that the gametophores buds formation in mosses is controlled by phytohormones (Bopp, 1985; Demkiv et al., 1991). In conditions of gravity influence its essentially accelerated. Probably, gravity essentially strengthened acropetal transport of phytohormones and formation of attractive center in the protonemata apical cell. Our investigations have allowed to make the conclusion, that gravi-dependent formation of the apical buds is

Hypertrophic cardiomyopathy with mid-ventricular obstruction and apical aneurysm

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N.D. Oryshchyn

2016-11-01

Full Text Available A case report of apical left ventricular aneurysm in patient with hypertrophic cardiomyopathy with mid-ventricular obstruction (diagnosis and surgical treatment is presented. We revealed apical aneurysm and mid-ventricular obstruction during echocardiography and specified anatomical characteristics of aneurysm during computer tomography. There was no evidence of obstructive coronary artery disease during coronary angiography. Taking into consideration multiple cerebral infarcts, aneurysm resection and left ventricular plastics was performed. Electronic microscopy of myocardium confirmed the diagnosis of hypertrophic cardiomyopathy.

DRhoGEF2 regulates cellular tension and cell pulsations in the Amnioserosa during Drosophila dorsal closure.

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Dulce Azevedo

Full Text Available Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction.

Arg753gln and Arg677 Trp Polymorphisms of Toll-Like Receptor 2 In Acute Apical Abscess.

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Miri-Moghaddam, Ebrahim; Farhad Mollashahi, Narges; Naghibi, Nava; Garme, Yasaman; Bazi, Ali

2018-06-01

Genetic polymorphisms can alter immunity response against pathogens, which in turn influence individuals' susceptibility to certain infections. Our aim was to determine the association of Arg753Gln (rs5743708) and Arg677Trp (rs12191786) polymorphisms of toll like receptor-2 gene with the two clinical forms of apical periodontitis: acute apical abscess (AAA) and asymptomatic apical periodontitis (AAP). There were 50 patients with AAA as case group and 50 with AAP as control group. Genotyping was done using Tetra-ARMS (amplification refractory mutation system) PCR. Heterozygous genotype of Arg677Trp polymorphism was associated with risk of AAA (OR=1.9, 95% CI: 0.7-5.5, p = 0.05). Although statistically insignificant, Arg677Trp polymorphism promoted the risk of AAA in dominant model (OR=2.1, 95% CI: 0.7-5.9, p > 0.05). The frequency of mutant allele (T) of Arg677Trp polymorphism was higher in AAA (14%) than AAP (7%) subjects (OR=1.7, 95% CI: 0.6-4.7). For Arg753Gln polymorphism, wild homozygous (GG) represented the dominant genotype in both cases (96%) and controls (100%). Variant allele (A) of Arg753Gln polymorphism was identified in 2% of AAA, while no individual represented with this allele in AAP subjects. Individuals with Arg753Gln; Arg677Trp (GG; TC) combination showed an elevated risk of AAA (OR=1.6, 95% CI: 0.5- 4.2, p > 0.05). Arg677Trp polymorphism of TLR-2 rendered a higher risk for the development of abscesses in apical periodontitis. It is recommended to explore role of this polymorphism in other populations.

14 CFR 221.61 - Rules and regulations governing foreign air transportation.

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2010-01-01

... governing foreign air transportation. Instead of being included in the fares tariffs, the rules and regulations governing foreign air transportation required to be filed by §§ 221.20 and 221.30 and/or... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Rules and regulations governing foreign air...

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

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

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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...

Should Community College Be Free? Forum. "Education Next" Talks with Sara Goldrick-Rab and Andrew P. Kelly

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Goldrick-Rab, Sara; Kelly, Andrew P.

2016-01-01

In this article, "Education Next" talks with Sara Goldrick-Rab and Andrew Kelly. President Obama's proposal for tuition-free community college, seems to have laid down a marker for the Democratic Party. Vermont senator Bernie Sanders is touting his plan for free four-year public college on the primary trail; Massachusetts senator…

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

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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...

RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

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Alghanem, Ahmad F; Wilkinson, Emma L; Emmett, Maxine S; Aljasir, Mohammad A; Holmes, Katherine; Rothermel, Beverley A; Simms, Victoria A; Heath, Victoria L; Cross, Michael J

2017-08-01

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

Conservation of the TRAPPII-specific subunits of a Ypt/Rab exchanger complex

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Yoo Eunice

2007-02-01

Full Text Available Abstract Background Ypt/Rab GTPases and their GEF activators regulate intra-cellular trafficking in all eukaryotic cells. In S. cerivisiae, the modular TRAPP complex acts as a GEF for the Golgi gatekeepers: Ypt1 and the functional pair Ypt31/32. While TRAPPI, which acts in early Golgi, is conserved from fungi to animals, not much is known about TRAPPII, which acts in late Golgi and consists of TRAPPI plus three additional subunits. Results Here, we show a phylogenetic analysis of the three TRAPPII-specific subunits. One copy of each of the two essential subunits, Trs120 and Trs130, is present in almost every fully sequenced eukaryotic genome. Moreover, the primary, as well as the predicted secondary, structure of the Trs120- and Trs130-related sequences are conserved from fungi to animals. The mammalian orthologs of Trs120 and Trs130, NIBP and TMEM1, respectively, are candidates for human disorders. Currently, NIBP is implicated in signaling, and TMEM1 is suggested to have trans-membrane domains (TMDs and to function as a membrane channel. However, we show here that the yeast Trs130 does not function as a trans-membrane protein, and the human TMEM1 does not contain putative TMDs. The non-essential subunit, Trs65, is conserved only among many fungi and some unicellular eukaryotes. Multiple alignment analysis of each TRAPPII-specific subunit revealed conserved domains that include highly conserved amino acids. Conclusion We suggest that the function of both NIBP and TMEM1 in the regulation of intra-cellular trafficking is conserved from yeast to man. The conserved domains and amino acids discovered here can be used for functional analysis that should help to resolve the differences in the assigned functions of these proteins in fungi and animals.

Regulation of Kv1.4 potassium channels by PKC and AMPK kinases

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Andersen, Martin Nybo; Skibsbye, Lasse; Saljic, Arnela

2018-01-01

around the ubiquitin ligase Nedd4-2. In the present study we examined whether Kv1.4, constituting the cardiac Ito,s current, is subject to similar regulation. In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane...... targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK). In contrast, manipulating the activities of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serum and glucocorticoid......-regulated kinase 1 (SGK1) were without effect on channel localization. The PKC and AMPK-mediated downregulation of Kv1.4 membrane surface localization was confirmed by two-electrode voltage clamp in Xenopus laevis oocytes, where pharmacological activation of PKC and AMPK reduced Kv1.4 current levels. We further...

Prognostic factors in apical surgery with root-end filling: a meta-analysis

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von Arx, Thomas; Peñarrocha, Miguel; Jensen, Simon Storgård

2010-01-01

Apical surgery has seen continuous development with regard to equipment and surgical technique. However, there is still a shortage of evidence-based information regarding healing determinants. The objective of this meta-analysis was to review clinical articles on apical surgery with root-end fill...

Endodontic medicine: connections between apical periodontitis and systemic diseases.

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Segura-Egea, J J; Martín-González, J; Castellanos-Cosano, L

2015-10-01

The prevalence of apical periodontitis (AP) in Europe has been reported to affect 61% of individuals and 14% of teeth, and increase with age. Likewise, the prevalence of root canal treatment (RCT) in Europe is estimated to be around 30-50% of individuals and 2-9% of teeth with radiographic evidence of chronic persistent AP in 30-65% of root filled teeth (RFT). AP is not only a local phenomenon and for some time the medical and dental scientific community have analysed the possible connection between apical periodontits and systemic health. Endodontic medicine has developed, with increasing numbers of reports describing the association between periapical inflammation and systemic diseases. The results of studies carried out both in animal models and humans are not conclusive, but suggest an association between endodontic variables, that is AP and RCT, and diabetes mellitus (DM), tobacco smoking, coronary heart disease and other systemic diseases. Several studies have reported a higher prevalence of periapical lesions, delayed periapical repair, greater size of osteolityc lesions, greater likelihood of asymptomatic infections and poorer prognosis for RFT in diabetic patients. On the other hand, recent studies have found that a poorer periapical status correlates with higher HbA1c levels and poor glycaemic control in type 2 diabetic patients. However, there is no scientific evidence supporting a causal effect of periapical inflammation on diabetes metabolic control. The possible association between smoking habits and endodontic infection has also been investigated, with controversial results. The aim of this paper was to review the literature on the association between endodontic variables and systemic health (especially DM and smoking habits). © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Left ventricular apical ballooning syndrome

International Nuclear Information System (INIS)

Rahman, N.; Tai, J.; Soofi, A.

2007-01-01

The transient left ventricular apical ballooning syndrome, also known as Takotsubo cardiomyopathy, is characterized by transient left ventricular dysfunction in the absence of obstructive epicardial coronary disease. Although the syndrome has been reported in Japan since 1990, it is rare in other regions. Rapid recognition of the syndrome can modify the diagnostic and therapeutic attitude i.e. avoiding thrombolysis and performing catheterization in the acute phase. (author)

[Clinical characteristics and speech therapy of lingua-apical articulation disorder].

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Zhang, Feng-hua; Jin, Xing-ming; Zhang, Yi-wen; Wu, Hong; Jiang, Fan; Shen, Xiao-ming

2006-03-01

To explore the clinical characteristics and speech therapy of 62 children with lingua-apical articulation disorder. Peabody Picture Vocabulary Test (PPVT), Gesell development scales (Gesell), Wechsler Intelligence Scale for Preschool Children (WPPSI) and speech test were performed for 62 children at the ages of 3 to 8 years with lingua-apical articulation disorder. PPVT was used to measure receptive vocabulary skills. GESELL and WPPSI were utilized to represent cognitive and non-verbal ability. The speech test was adopted to assess the speech development. The children received speech therapy and auxiliary oral-motor functional training once or twice a week. Firstly the target sound was identified according to the speech development milestone, then the method of speech localization was used to clarify the correct articulation placement and manner. It was needed to change food character and administer oral-motor functional training for children with oral motor dysfunction. The 62 cases with the apical articulation disorder were classified into four groups. The combined pattern of the articulation disorder was the most common (40 cases, 64.5%), the next was apico-dental disorder (15 cases, 24.2%). The third was palatal disorder (4 cases, 6.5%) and the last one was the linguo-alveolar disorder (3 cases, 4.8%). The substitution errors of velar were the most common (95.2%), the next was omission errors (30.6%) and the last was absence of aspiration (12.9%). Oral motor dysfunction was found in some children with problems such as disordered joint movement of tongue and head, unstable jaw, weak tongue strength and poor coordination of tongue movement. Some children had feeding problems such as preference of eating soft food, keeping food in mouths, eating slowly, and poor chewing. After 5 to 18 times of therapy, the effective rate of speech therapy reached 82.3%. The lingua-apical articulation disorders can be classified into four groups. The combined pattern of the

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