WorldWideScience

Sample records for subcellular localization studies

  1. Studies on the subcellular localization of the porphycene CPO.

    Science.gov (United States)

    Kessel, David; Conley, Mary; Vicente, M Graça H; Reiners, John J

    2005-01-01

    This study was designed to provide more detailed information on the subcellular sites of binding of the porphycene, termed 9-capronyloxytetrakis (methoxyethyl) porphycene (CPO), with a fluorescence resonance energy transfer (FRET) technique. The proximity of CPO to two fluorescent probes was determined: nonyl acridine orange (NAO), a dye with specific affinity for the mitochondrial lipid cardiolipin, and dihexa-oxacarbocyanine iodide (DiOC6), an agent that labels the endoplasmic reticulum (ER). FRET spectra indicated energy transfer between DiOC6 and CPO but no significant transfer between NAO and CPO. These results confirm data obtained by fluorescence microscopy, suggesting a similar pattern of subcellular localization by CPO and DiOC6 but not by CPO and NAO. However, when cells containing CPO were irradiated and then loaded with NAO, FRET between the two fluorophores was observed. Hence, a relocalization of CPO can occur during irradiation. These data provide an explanation for recent studies on CPO-catalyzed photodamage to both ER and mitochondrial Bcl-2.

  2. Studies on the Subcellular Localization of the Porphycene CPO¶

    Science.gov (United States)

    Kessel, David; Conley, Mary; Vicente, M. Graça H.; Reiners, John J.

    2010-01-01

    This study was designed to provide more detailed information on the subcellular sites of binding of the porphycene, termed 9-capronyloxytetrakis (methoxyethyl) porphycene (CPO), with a fluorescence resonance energy transfer (FRET) technique. The proximity of CPO to two fluorescent probes was determined: nonyl acridine orange (NAO), a dye with specific affinity for the mitochondrial lipid cardiolipin, and dihexaoxacarbocyanine iodide (DiOC6), an agent that labels the endoplasmic reticulum (ER). FRET spectra indicated energy transfer between DiOC6 and CPO but no significant transfer between NAO and CPO. These results confirm data obtained by fluorescence microscopy, suggesting a similar pattern of subcellular localization by CPO and DiOC6 but not by CPO and NAO. However, when cells containing CPO were irradiated and then loaded with NAO, FRET between the two fluorophores was observed. Hence, a relocalization of CPO can occur during irradiation. These data provide an explanation for recent studies on CPO-catalyzed photodamage to both ER and mitochondrial Bcl-2. PMID:15745423

  3. Subcellular localization and function study of a secreted phospholipase C from Nocardia seriolae.

    Science.gov (United States)

    Xia, Liqun; Liang, Haiying; Xu, Liang; Chen, Jianlin; Bekaert, Michaël; Zhang, Honglian; Lu, Yishan

    2017-09-15

    Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen that causes it. The pathogenesis and virulence factors of N. seriolae are not fully understood. A phospholipase C (PLC), which is likely to be a secreted protein targeting host cell mitochondria, was found by a bioinformatics analysis of the whole genome sequence of N. seriolae. In order to determine the subcellular localization and study the preliminary function of PLC from N. seriolae (NsPLC), in this study gene cloning, secreted protein identification, subcellular localization in host cells and apoptosis detection of NsPLC were carried out. Mass spectrometry analysis of extracellular products from N. seriolae showed that NsPLC was a secreted protein. Subcellular localization of NsPLC-GFP fusion protein in fathead minnow (FHM) cells revealed that the green fluorescence exhibited a punctate distribution near the nucleus and did not co-localize with mitochondria. In addition, an apoptosis assay suggested that apoptosis was induced in FHM cells by the overexpression of NsPLC. This study may lay the foundations for further studies on the function of NsPLC and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Subcellular localization of leptin and leptin receptor in breast cancer detected in an electron microscopic study.

    Science.gov (United States)

    Al-Shibli, Saad M; Amjad, Nasser M; Al-Kubaisi, Muna K; Mizan, Shaikh

    2017-01-22

    Leptin (LEP) and leptin receptor (LEPR) have long been found associated with breast cancer. So far no high-resolution method such as electron microscopy has been used to investigate the subcellular localization of leptin and leptin receptor in breast cancer. We collected cancer and non-cancer breast tissues from 51 women with invasive ductal breast cancer. Leptin and leptin receptor in the tissues were estimated using immunohistochemistry (IHC). LEP and LEPR were localized at subcellular level by immunocytochemistry (ICC) using ultra-fine gold particle conjugated antibody, and visualized with transmission electron microscopy (TEM). IHC showed high presence of LEP and LEPR in 65% and 67% respectively of the breast cancer samples, 100% and 0% respectively of the adipose tissue samples, and no high presence in the non-cancer breast tissue samples. On TEM views both LEP and LEPR were found highly concentrated within the nucleus of the cancer cells, indicating that nucleus is the principal seat of action. However, presence of high concentration of LEP does not necessarily prove its over-expression, as often concluded, because LEP could be internalized from outside by LEPR in the cells. In contrast, LEPR is definitely over-expressed in the ductal breast cancer cells. Therefore, we hypothesize that over-expression of LEPR, rather than that of LEP has a fundamental role in breast carcinogenesis in particular, and probably for LEP-LEPR associated tumors in general. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Application of green fluorescent protein-labeled assay for the study of subcellular localization of Newcastle disease virus matrix protein.

    Science.gov (United States)

    Duan, Zhiqiang; Li, Qunhui; He, Liang; Zhao, Guo; Chen, Jian; Hu, Shunlin; Liu, Xiufan

    2013-12-01

    Green fluorescent protein (GFP) used as a powerful marker of gene expression in vivo has so far been applied widely in studying the localizations and functions of protein in living cells. In this study, GFP-labeled assay was used to investigate the subcellular localization of matrix (M) protein of different virulence and genotype Newcastle disease virus (NDV) strains. The M protein of ten NDV strains fused with GFP (GFP-M) all showed nuclear-and-nucleolar localization throughout transfection, whereas that of the other two strains were observed in the nucleus and nucleolus early in transfection but in the cytoplasm late in transfection. In addition, mutations to the previously defined nuclear localization signal in the GFP-M fusion protein were studied as well. Single changes at positions 262 and 263 did not affect nuclear localization of M, while changing both of these arginine residues to asparagine caused re-localization of M mainly to the cytoplasm. The GFP-M was validated as a suitable system for studying the subcellular localization of M protein and could be used to assist us in further identifying the signal sequences responsible for the nucleolar localization and cytoplasmic localization of M protein. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Expression and subcellular localization of antiporter regulating ...

    African Journals Online (AJOL)

    Expression and subcellular localization of antiporter regulating protein OsARP in rice induced by submergence, salt and drought stresses. Md Imtiaz Uddin, Maki Kihara, Lina Yin, Mst Farida Perveen, Kiyoshi Tanaka ...

  7. Systematic study of subcellular localization of Arabidopsis PPR proteins confirms a massive targeting to organelles.

    Science.gov (United States)

    Colcombet, Jean; Lopez-Obando, Mauricio; Heurtevin, Laure; Bernard, Clément; Martin, Karine; Berthomé, Richard; Lurin, Claire

    2013-01-01

    Four hundred and fifty-eight genes coding for PentatricoPeptide Repeat (PPR) proteins are annotated in the Arabidopsis thaliana genome. Over the past 10 years, numerous reports have shown that many of these proteins function in organelles to target specific transcripts and are involved in post-transcriptional regulation. Therefore, they are thought to be important players in the coordination between nuclear and organelle genome expression. Only four of these proteins have been described to be addressed outside organelles, indicating that some PPRs could function in post-transcriptional regulations of nuclear genes. In this work, we updated and improved our current knowledge on the localization of PPR proteins of Arabidopsis within the plant cell. We particularly investigated the subcellular localization of 166 PPR proteins whose targeting predictions were ambiguous, using a combination of high-throughput cloning and microscopy. Through systematic localization experiments and data integration, we confirmed that PPR proteins are largely targeted to organelles and showed that dual targeting to both the mitochondria and plastid occurs more frequently than expected. These results allow us to speculate that dual-targeted PPR proteins could be important for the fine coordination of gene expressions in both organelles.

  8. Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins.

    Science.gov (United States)

    Chanoca, Alexandra; Burkel, Brian; Kovinich, Nik; Grotewold, Erich; Eliceiri, Kevin W; Otegui, Marisa S

    2016-12-01

    Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both in vitro and in vivo conditions, using wild-type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime (τm ) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τm than the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and, potentially, for estimating vacuolar pH inside intact plant cells. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  9. Expression and subcellular localization of antiporter regulating ...

    African Journals Online (AJOL)

    Md. Imtiaz Uddin

    2012-02-14

    Feb 14, 2012 ... We examined the expression and subcellular localization of antiporter regulating protein OsARP in a submergence tolerant rice (Oryza sativa L.) cultivar FR13A. In the public databases, this protein was designated as putative Os02g0465900 protein. The cDNA containing the full-length sequence of OsARP.

  10. Differential subcellular targeting and activity-dependent subcellular localization of diacylglycerol kinase isozymes in transfected cells.

    Science.gov (United States)

    Kobayashi, Naoki; Hozumi, Yasukazu; Ito, Tsukasa; Hosoya, Takaaki; Kondo, Hisatake; Goto, Kaoru

    2007-08-01

    Diacylglycerol kinase (DGK) plays a pivotal role in cellular signal transduction through regulating levels of the second messenger diacylglycerol (DG). Previous studies have revealed that DGK is composed of a family of isozymes that show remarkable heterogeneity in terms of molecular structure, functional domains, tissue and cellular gene expression. Recently, it has been shown that DG is produced in various subcellular compartments including the plasma membrane, internal membranes, cytoskeleton, and nucleus. However, it remains unclear how DG is regulated at distinct subcellular sites. To address this point, we have used an epitope-tag expression system in cultured cells and investigated the subcellular localization of DGK isozymes under the same experimental conditions. We show here that DGK isozymes are targeted differentially to unique subcellular sites in transfected COS7 cells, including the cytoplasm, actin stress fibers, Golgi complex, endoplasmic reticulum, and nucleus. It is also shown that among the isozymes overexpression of DGKbeta causes fragmentation of actin stress fibers while a kinase-dead mutant of DGKbeta abolishes its colocalization with actin stress fibers. These data strongly suggest that each isozyme may be responsible for the metabolism of DG that is produced upon stimulation at a different and specific subcellular site and that DGKbeta activity might have effects on the reorganization of actin stress fibers in transfected COS7 cells.

  11. A set of GFP-based organelle marker lines combined with DsRed-based gateway vectors for subcellular localization study in rice (Oryza sativa L.).

    Science.gov (United States)

    Wu, Tsung-Meng; Lin, Ke-Chun; Liau, Wei-Shiang; Chao, Yun-Yang; Yang, Ling-Hung; Chen, Szu-Yun; Lu, Chung-An; Hong, Chwan-Yang

    2016-01-01

    In the post-genomic era, many useful tools have been developed to accelerate the investigation of gene functions. Fluorescent proteins have been widely used as protein tags for studying the subcellular localization of proteins in plants. Several fluorescent organelle marker lines have been generated in dicot plants; however, useful and reliable fluorescent organelle marker lines are lacking in the monocot model rice. Here, we developed eight different GFP-based organelle markers in transgenic rice and created a set of DsRed-based gateway vectors for combining with the marker lines. Two mitochondrial-localized rice ascorbate peroxidase genes fused to DsRed and successfully co-localized with mitochondrial-targeted marker lines verified the practical use of this system. The co-localization of GFP-fusion marker lines and DsRed-fusion proteins provide a convenient platform for in vivo or in vitro analysis of subcellular localization of rice proteins.

  12. Subcellular localization prediction through boosting association rules.

    Science.gov (United States)

    Yoon, Yongwook; Lee, Gary Geunbae

    2012-01-01

    Computational methods for predicting protein subcellular localization have used various types of features, including N-terminal sorting signals, amino acid compositions, and text annotations from protein databases. Our approach does not use biological knowledge such as the sorting signals or homologues, but use just protein sequence information. The method divides a protein sequence into short $k$-mer sequence fragments which can be mapped to word features in document classification. A large number of class association rules are mined from the protein sequence examples that range from the N-terminus to the C-terminus. Then, a boosting algorithm is applied to those rules to build up a final classifier. Experimental results using benchmark datasets show our method is excellent in terms of both the classification performance and the test coverage. The result also implies that the $k$-mer sequence features which determine subcellular locations do not necessarily exist in specific positions of a protein sequence. Online prediction service implementing our method is available at http://isoft.postech.ac.kr/research/BCAR/subcell.

  13. Tau regulates the subcellular localization of calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Barreda, Elena Gomez de [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Avila, Jesus, E-mail: javila@cbm.uam.es [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); CIBER de Enfermedades Neurodegenerativas, 28031 Madrid (Spain)

    2011-05-13

    Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in a change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.

  14. Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.

    Science.gov (United States)

    Rodríguez-Escudero, María; Cid, Víctor J; Molina, María; Schulze-Luehrmann, Jan; Lührmann, Anja; Rodríguez-Escudero, Isabel

    2016-01-01

    Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.

  15. Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803.

    Directory of Open Access Journals (Sweden)

    Lifang Zhang

    Full Text Available The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that "light" plasma membranes have a high carotenoid/protein ratio, when compared to "heavier" plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitope tagging and western blots. Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone.

  16. Validating subcellular localization prediction tools with mycobacterial proteins

    Directory of Open Access Journals (Sweden)

    Niño Luis F

    2009-05-01

    Full Text Available Abstract Background The computational prediction of mycobacterial proteins' subcellular localization is of key importance for proteome annotation and for the identification of new drug targets and vaccine candidates. Several subcellular localization classifiers have been developed over the past few years, which have comprised both general localization and feature-based classifiers. Here, we have validated the ability of different bioinformatics approaches, through the use of SignalP 2.0, TatP 1.0, LipoP 1.0, Phobius, PA-SUB 2.5, PSORTb v.2.0.4 and Gpos-PLoc, to predict secreted bacterial proteins. These computational tools were compared in terms of sensitivity, specificity and Matthew's correlation coefficient (MCC using a set of mycobacterial proteins having less than 40% identity, none of which are included in the training data sets of the validated tools and whose subcellular localization have been experimentally confirmed. These proteins belong to the TBpred training data set, a computational tool specifically designed to predict mycobacterial proteins. Results A final validation set of 272 mycobacterial proteins was obtained from the initial set of 852 mycobacterial proteins. According to the results of the validation metrics, all tools presented specificity above 0.90, while dispersion sensitivity and MCC values were above 0.22. PA-SUB 2.5 presented the highest values; however, these results might be biased due to the methodology used by this tool. PSORTb v.2.0.4 left 56 proteins out of the classification, while Gpos-PLoc left just one protein out. Conclusion Both subcellular localization approaches had high predictive specificity and high recognition of true negatives for the tested data set. Among those tools whose predictions are not based on homology searches against SWISS-PROT, Gpos-PLoc was the general localization tool with the best predictive performance, while SignalP 2.0 was the best tool among the ones using a feature

  17. Validating subcellular localization prediction tools with mycobacterial proteins

    Science.gov (United States)

    Restrepo-Montoya, Daniel; Vizcaíno, Carolina; Niño, Luis F; Ocampo, Marisol; Patarroyo, Manuel E; Patarroyo, Manuel A

    2009-01-01

    Background The computational prediction of mycobacterial proteins' subcellular localization is of key importance for proteome annotation and for the identification of new drug targets and vaccine candidates. Several subcellular localization classifiers have been developed over the past few years, which have comprised both general localization and feature-based classifiers. Here, we have validated the ability of different bioinformatics approaches, through the use of SignalP 2.0, TatP 1.0, LipoP 1.0, Phobius, PA-SUB 2.5, PSORTb v.2.0.4 and Gpos-PLoc, to predict secreted bacterial proteins. These computational tools were compared in terms of sensitivity, specificity and Matthew's correlation coefficient (MCC) using a set of mycobacterial proteins having less than 40% identity, none of which are included in the training data sets of the validated tools and whose subcellular localization have been experimentally confirmed. These proteins belong to the TBpred training data set, a computational tool specifically designed to predict mycobacterial proteins. Results A final validation set of 272 mycobacterial proteins was obtained from the initial set of 852 mycobacterial proteins. According to the results of the validation metrics, all tools presented specificity above 0.90, while dispersion sensitivity and MCC values were above 0.22. PA-SUB 2.5 presented the highest values; however, these results might be biased due to the methodology used by this tool. PSORTb v.2.0.4 left 56 proteins out of the classification, while Gpos-PLoc left just one protein out. Conclusion Both subcellular localization approaches had high predictive specificity and high recognition of true negatives for the tested data set. Among those tools whose predictions are not based on homology searches against SWISS-PROT, Gpos-PLoc was the general localization tool with the best predictive performance, while SignalP 2.0 was the best tool among the ones using a feature-based approach. Even though PA-SUB 2

  18. Predicting the subcellular localization of viral proteins within a mammalian host cell

    Directory of Open Access Journals (Sweden)

    Thomas DY

    2006-04-01

    Full Text Available Abstract Background The bioinformatic prediction of protein subcellular localization has been extensively studied for prokaryotic and eukaryotic organisms. However, this is not the case for viruses whose proteins are often involved in extensive interactions at various subcellular localizations with host proteins. Results Here, we investigate the extent of utilization of human cellular localization mechanisms by viral proteins and we demonstrate that appropriate eukaryotic subcellular localization predictors can be used to predict viral protein localization within the host cell. Conclusion Such predictions provide a method to rapidly annotate viral proteomes with subcellular localization information. They are likely to have widespread applications both in the study of the functions of viral proteins in the host cell and in the design of antiviral drugs.

  19. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  20. Predicting Subcellular Localization of Proteins by Bioinformatic Algorithms

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2015-01-01

    When predicting the subcellular localization of proteins from their amino acid sequences, there are basically three approaches: signal-based, global property-based, and homology-based. Each of these has its advantages and drawbacks, and it is important when comparing methods to know which approac...

  1. Protein subcellular localization prediction using artificial intelligence technology.

    Science.gov (United States)

    Nair, Rajesh; Rost, Burkhard

    2008-01-01

    Proteins perform many important tasks in living organisms, such as catalysis of biochemical reactions, transport of nutrients, and recognition and transmission of signals. The plethora of aspects of the role of any particular protein is referred to as its "function." One aspect of protein function that has been the target of intensive research by computational biologists is its subcellular localization. Proteins must be localized in the same subcellular compartment to cooperate toward a common physiological function. Aberrant subcellular localization of proteins can result in several diseases, including kidney stones, cancer, and Alzheimer's disease. To date, sequence homology remains the most widely used method for inferring the function of a protein. However, the application of advanced artificial intelligence (AI)-based techniques in recent years has resulted in significant improvements in our ability to predict the subcellular localization of a protein. The prediction accuracy has risen steadily over the years, in large part due to the application of AI-based methods such as hidden Markov models (HMMs), neural networks (NNs), and support vector machines (SVMs), although the availability of larger experimental datasets has also played a role. Automatic methods that mine textual information from the biological literature and molecular biology databases have considerably sped up the process of annotation for proteins for which some information regarding function is available in the literature. State-of-the-art methods based on NNs and HMMs can predict the presence of N-terminal sorting signals extremely accurately. Ab initio methods that predict subcellular localization for any protein sequence using only the native amino acid sequence and features predicted from the native sequence have shown the most remarkable improvements. The prediction accuracy of these methods has increased by over 30% in the past decade. The accuracy of these methods is now on par with

  2. Cellular and subcellular localization of Marlin-1 in the brain

    Directory of Open Access Journals (Sweden)

    Luján Rafael

    2009-04-01

    Full Text Available Abstract Background Marlin-1 is a microtubule binding protein that associates specifically with the GABAB1 subunit in neurons and with members of the Janus kinase family in lymphoid cells. In addition, it binds the molecular motor kinesin-I and nucleic acids, preferentially single stranded RNA. Marlin-1 is expressed mainly in the central nervous system but little is known regarding its cellular and subcellular distribution in the brain. Results Here we have studied the localization of Marlin-1 in the rodent brain and cultured neurons combining immunohistochemistry, immunofluorescence and pre-embedding electron microscopy. We demonstrate that Marlin-1 is enriched in restricted areas of the brain including olfactory bulb, cerebral cortex, hippocampus and cerebellum. Marlin-1 is abundant in dendrites and axons of GABAergic and non-GABAergic hippocampal neurons. At the ultrastructural level, Marlin-1 is present in the cytoplasm and the nucleus of CA1 neurons in the hippocampus. In the cytoplasm it associates to microtubules in the dendritic shaft and occasionally with the Golgi apparatus, the endoplasmic reticulum (ER and dendritic spines. In the nucleus, clusters of Marlin-1 associate to euchromatin. Conclusion Our results demonstrate that Marlin-1 is expressed in discrete areas of the brain. They also confirm the microtubule association at the ultrastructural level in neurons. Together with the abundance of the protein in dendrites and axons they are consistent with the emerging role of Marlin-1 as an intracellular protein linking the cytoskeleton and transport. Our study constitutes the first detailed description of the cellular and subcellular distribution of Marlin-1 in the brain. As such, it will set the basis for future studies on the functional implications of Marlin-1 in protein trafficking.

  3. Cellular and subcellular localization of Marlin-1 in the brain.

    Science.gov (United States)

    Vidal, René L; Valenzuela, José I; Luján, Rafael; Couve, Andrés

    2009-04-22

    Marlin-1 is a microtubule binding protein that associates specifically with the GABAB1 subunit in neurons and with members of the Janus kinase family in lymphoid cells. In addition, it binds the molecular motor kinesin-I and nucleic acids, preferentially single stranded RNA. Marlin-1 is expressed mainly in the central nervous system but little is known regarding its cellular and subcellular distribution in the brain. Here we have studied the localization of Marlin-1 in the rodent brain and cultured neurons combining immunohistochemistry, immunofluorescence and pre-embedding electron microscopy. We demonstrate that Marlin-1 is enriched in restricted areas of the brain including olfactory bulb, cerebral cortex, hippocampus and cerebellum. Marlin-1 is abundant in dendrites and axons of GABAergic and non-GABAergic hippocampal neurons. At the ultrastructural level, Marlin-1 is present in the cytoplasm and the nucleus of CA1 neurons in the hippocampus. In the cytoplasm it associates to microtubules in the dendritic shaft and occasionally with the Golgi apparatus, the endoplasmic reticulum (ER) and dendritic spines. In the nucleus, clusters of Marlin-1 associate to euchromatin. Our results demonstrate that Marlin-1 is expressed in discrete areas of the brain. They also confirm the microtubule association at the ultrastructural level in neurons. Together with the abundance of the protein in dendrites and axons they are consistent with the emerging role of Marlin-1 as an intracellular protein linking the cytoskeleton and transport. Our study constitutes the first detailed description of the cellular and subcellular distribution of Marlin-1 in the brain. As such, it will set the basis for future studies on the functional implications of Marlin-1 in protein trafficking.

  4. Evaluation and comparison of mammalian subcellular localization prediction methods

    Directory of Open Access Journals (Sweden)

    Fink J Lynn

    2006-12-01

    Full Text Available Abstract Background Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER, peroxisome, and lysosome. The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE

  5. MultiLoc2: integrating phylogeny and Gene Ontology terms improves subcellular protein localization prediction

    Directory of Open Access Journals (Sweden)

    Kohlbacher Oliver

    2009-09-01

    Full Text Available Abstract Background Knowledge of subcellular localization of proteins is crucial to proteomics, drug target discovery and systems biology since localization and biological function are highly correlated. In recent years, numerous computational prediction methods have been developed. Nevertheless, there is still a need for prediction methods that show more robustness and higher accuracy. Results We extended our previous MultiLoc predictor by incorporating phylogenetic profiles and Gene Ontology terms. Two different datasets were used for training the system, resulting in two versions of this high-accuracy prediction method. One version is specialized for globular proteins and predicts up to five localizations, whereas a second version covers all eleven main eukaryotic subcellular localizations. In a benchmark study with five localizations, MultiLoc2 performs considerably better than other methods for animal and plant proteins and comparably for fungal proteins. Furthermore, MultiLoc2 performs clearly better when using a second dataset that extends the benchmark study to all eleven main eukaryotic subcellular localizations. Conclusion MultiLoc2 is an extensive high-performance subcellular protein localization prediction system. By incorporating phylogenetic profiles and Gene Ontology terms MultiLoc2 yields higher accuracies compared to its previous version. Moreover, it outperforms other prediction systems in two benchmarks studies. MultiLoc2 is available as user-friendly and free web-service, available at: http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc2.

  6. ALG-2 oscillates in subcellular localization, unitemporally with calcium oscillations

    DEFF Research Database (Denmark)

    la Cour, Jonas Marstrand; Mollerup, Jens; Berchtold, Martin Werner

    2007-01-01

    discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate...... localization in an oscillatory fashion unitemporally with Ca2+ oscillations, whereas a Ca2+-binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG...

  7. Study of the localization of iron, ferritin, and hemosiderin in Alzheimer's disease hippocampus by analytical microscopy at the subcellular level.

    Science.gov (United States)

    Quintana, C; Bellefqih, S; Laval, J Y; Guerquin-Kern, J L; Wu, T D; Avila, J; Ferrer, I; Arranz, R; Patiño, C

    2006-01-01

    Previous studies of the structure of core nanocrystals of ferritin (Ft) in the brains of patients with Alzheimer's disease (AD) have shown differences in the mineral compound in comparison with physiological Ft. Both Ft cores have a polyphasic composition but whereas the major phase in physiological Ft is hexagonal ferric iron oxide (ferrihydrite), the major phases in brain AD Ft are two cubic mixed ferric-ferrous iron oxides (magnetite and wüstite). One of these (wüstite) is similar to what is detected in hemosiderin (Hm) cores in primary hemochromatosis (Quintana, C., Cowley, J.M, Marhic, C., 2004. Electron nanodiffraction and high resolution electron microscopy studies of the structure and composition of physiological and pathological ferritin. J. Struct. Biol. 147, 166-178). We have studied, herein, the distribution of iron, Ft, and Hm in sections of AD hippocampus using analytical microscopy. Iron present in Ft cores was directly mapped in a nanoSIMS microscope and the iron distribution has been correlated with the constituent elements N, P, and S. Ft and Hm cores were visualized at an ultrastructural level in an analytical transmission electron microscope. In senile plaques, Ft was observed in the coronal region associated with a non-beta-amyloid component and in the periphery of plaques, together with Hm, in sulfur-rich dense bodies of dystrophic neurites. Hm was also found in lysosomes and siderosomes of glial cells. Ft was observed in the cytoplasm and nucleus of oligodendrocytes. Ft was particularly abundant in myelinated axons in association with oligodendrocyte processes. These findings provide new arguments to support the hypothesis of a dysfunction of Ft (with eventual degradation to Hm) in AD resulting in an increase of toxic brain ferrous ions that may contribute to the production of free radicals that induce both cellular oxidative stress and aged-related myelin breakdown associated with cognitive decline and AD (Bartzokis, G., 2004. Age

  8. Subcellular Localization of Galloylated Catechins in Tea Plants (Camellia sinensis (L. O. Kuntze Assessed via Immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Huanhuan eXu

    2016-05-01

    Full Text Available Galloylated catechins, as the main secondary metabolites in the tea plant, including (--epigallocatechin-3-gallate and (--epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physiological functions. Classical histochemical localization staining reagents can not specifically detect galloylated catechins; thus, their subcellular localization remains controversial. In the present study, we generated a monoclonal antibody (mAb against galloylated catechins, which can be used for the subcellular localization of galloylated catechins in the tea plant by immunohistochemistry. Direct ELISA and ForteBio Octet Red 96 System assay indicated the mAb could recognize the galloylated catechins with high specificities and affinities. In addition, tea bud was ascertained as the optimal tissue for freezing microtomic sections for immunohistochemistry. What’s more, the high quality mAbs which exhibited excellent binding capability to galloylated catechins were utilised for the visualization of them via immunohistochemistry. Our findings demonstrated that vacuoles were the primary sites of localization of galloylated catechins at the subcellular level.

  9. Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa

    Directory of Open Access Journals (Sweden)

    Yasuhide Osaki

    2017-09-01

    Full Text Available Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein constructs. Particle bombardment delivers genes to the first layer of plant tissue. In leaves of higher plants, epidermal cells are the first cell layer. Many studies have used the epidermal cell layer of onion bulb (Allium cepa as the experimental tissue, because these cells are relatively large. However, onion epidermal cells lack developed plastids (i.e., chloroplasts, thereby precluding subcellular localization analysis of chloroplastic proteins. In this study, we developed a protocol for particle bombardment of the aquatic plant Egeria densa, and showed that it is a useful system for subcellular localization analysis of higher plant proteins. E. densa leaflets contain only two cell layers, and cells in the adaxial layer are sufficiently large for observation. The cells in both layers contain well-developed chloroplasts. We fused fluorescent proteins to conventional plant localization signals for the nucleus, cytosol, mitochondria, peroxisome, and chloroplast, and used particle bombardment to transiently express these fusion constructs in E. densa leaves. The plant subcellular localization signals functioned normally and displayed the expected distributions in transiently transformed E. densa cells, and even chloroplastic structures could be clearly visualized.

  10. Inducible control of subcellular RNA localization using a synthetic protein-RNA aptamer interaction.

    Directory of Open Access Journals (Sweden)

    Brian J Belmont

    Full Text Available Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis-elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs, which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically-inducible RNA-protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i the Tet Repressor protein (TetR genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii a target transcript containing genetically encoded TetR-binding RNA aptamers. TetR-fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.

  11. Dynamic subcellular localization of a respiratory complex controls bacterial respiration.

    Science.gov (United States)

    Alberge, François; Espinosa, Leon; Seduk, Farida; Sylvi, Léa; Toci, René; Walburger, Anne; Magalon, Axel

    2015-06-16

    Respiration, an essential process for most organisms, has to optimally respond to changes in the metabolic demand or the environmental conditions. The branched character of their respiratory chains allows bacteria to do so by providing a great metabolic and regulatory flexibility. Here, we show that the native localization of the nitrate reductase, a major respiratory complex under anaerobiosis in Escherichia coli, is submitted to tight spatiotemporal regulation in response to metabolic conditions via a mechanism using the transmembrane proton gradient as a cue for polar localization. These dynamics are critical for controlling the activity of nitrate reductase, as the formation of polar assemblies potentiates the electron flux through the complex. Thus, dynamic subcellular localization emerges as a critical factor in the control of respiration in bacteria.

  12. Gene ontology based transfer learning for protein subcellular localization

    Directory of Open Access Journals (Sweden)

    Zhou Shuigeng

    2011-02-01

    Full Text Available Abstract Background Prediction of protein subcellular localization generally involves many complex factors, and using only one or two aspects of data information may not tell the true story. For this reason, some recent predictive models are deliberately designed to integrate multiple heterogeneous data sources for exploiting multi-aspect protein feature information. Gene ontology, hereinafter referred to as GO, uses a controlled vocabulary to depict biological molecules or gene products in terms of biological process, molecular function and cellular component. With the rapid expansion of annotated protein sequences, gene ontology has become a general protein feature that can be used to construct predictive models in computational biology. Existing models generally either concatenated the GO terms into a flat binary vector or applied majority-vote based ensemble learning for protein subcellular localization, both of which can not estimate the individual discriminative abilities of the three aspects of gene ontology. Results In this paper, we propose a Gene Ontology Based Transfer Learning Model (GO-TLM for large-scale protein subcellular localization. The model transfers the signature-based homologous GO terms to the target proteins, and further constructs a reliable learning system to reduce the adverse affect of the potential false GO terms that are resulted from evolutionary divergence. We derive three GO kernels from the three aspects of gene ontology to measure the GO similarity of two proteins, and derive two other spectrum kernels to measure the similarity of two protein sequences. We use simple non-parametric cross validation to explicitly weigh the discriminative abilities of the five kernels, such that the time & space computational complexities are greatly reduced when compared to the complicated semi-definite programming and semi-indefinite linear programming. The five kernels are then linearly merged into one single kernel for

  13. DeepLoc: prediction of protein subcellular localization using deep learning.

    Science.gov (United States)

    Almagro Armenteros, José Juan; Sønderby, Casper Kaae; Sønderby, Søren Kaae; Nielsen, Henrik; Winther, Ole

    2017-11-01

    The prediction of eukaryotic protein subcellular localization is a well-studied topic in bioinformatics due to its relevance in proteomics research. Many machine learning methods have been successfully applied in this task, but in most of them, predictions rely on annotation of homologues from knowledge databases. For novel proteins where no annotated homologues exist, and for predicting the effects of sequence variants, it is desirable to have methods for predicting protein properties from sequence information only. Here, we present a prediction algorithm using deep neural networks to predict protein subcellular localization relying only on sequence information. At its core, the prediction model uses a recurrent neural network that processes the entire protein sequence and an attention mechanism identifying protein regions important for the subcellular localization. The model was trained and tested on a protein dataset extracted from one of the latest UniProt releases, in which experimentally annotated proteins follow more stringent criteria than previously. We demonstrate that our model achieves a good accuracy (78% for 10 categories; 92% for membrane-bound or soluble), outperforming current state-of-the-art algorithms, including those relying on homology information. The method is available as a web server at http://www.cbs.dtu.dk/services/DeepLoc. Example code is available at https://github.com/JJAlmagro/subcellular_localization. The dataset is available at http://www.cbs.dtu.dk/services/DeepLoc/data.php. jjalma@dtu.dk.

  14. Analysis of the subcellular localization of the human histone methyltransferase SETDB1

    Energy Technology Data Exchange (ETDEWEB)

    Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Gotoh, Eiko; Kawamata, Natsuko [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ishimoto, Kenji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Uchihara, Yoshie [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Iwanari, Hiroko [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sugiyama, Akira; Kawamura, Takeshi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032 (Japan); Mochizuki, Yasuhiro [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Tanaka, Toshiya [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sakai, Juro [Division of Metabolic Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Hamakubo, Takao [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Kodama, Tatsuhiko [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); and others

    2015-10-02

    SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.

  15. Enhancing membrane protein subcellular localization prediction by parallel fusion of multi-view features.

    Science.gov (United States)

    Yu, Dongjun; Wu, Xiaowei; Shen, Hongbin; Yang, Jian; Tang, Zhenmin; Qi, Yong; Yang, Jingyu

    2012-12-01

    Membrane proteins are encoded by ~ 30% in the genome and function importantly in the living organisms. Previous studies have revealed that membrane proteins' structures and functions show obvious cell organelle-specific properties. Hence, it is highly desired to predict membrane protein's subcellular location from the primary sequence considering the extreme difficulties of membrane protein wet-lab studies. Although many models have been developed for predicting protein subcellular locations, only a few are specific to membrane proteins. Existing prediction approaches were constructed based on statistical machine learning algorithms with serial combination of multi-view features, i.e., different feature vectors are simply serially combined to form a super feature vector. However, such simple combination of features will simultaneously increase the information redundancy that could, in turn, deteriorate the final prediction accuracy. That's why it was often found that prediction success rates in the serial super space were even lower than those in a single-view space. The purpose of this paper is investigation of a proper method for fusing multiple multi-view protein sequential features for subcellular location predictions. Instead of serial strategy, we propose a novel parallel framework for fusing multiple membrane protein multi-view attributes that will represent protein samples in complex spaces. We also proposed generalized principle component analysis (GPCA) for feature reduction purpose in the complex geometry. All the experimental results through different machine learning algorithms on benchmark membrane protein subcellular localization datasets demonstrate that the newly proposed parallel strategy outperforms the traditional serial approach. We also demonstrate the efficacy of the parallel strategy on a soluble protein subcellular localization dataset indicating the parallel technique is flexible to suite for other computational biology problems. The

  16. Subcellular Localization of Galloylated Catechins in Tea Plants [Camellia sinensis (L.) O. Kuntze] Assessed via Immunohistochemistry

    OpenAIRE

    Xu, Huanhuan; Wang, Ya; Chen, Yana; Zhang, Pan; Zhao, Yi; Huang, Yewei; Wang, Xuanjun; Sheng, Jun

    2016-01-01

    Galloylated catechins, as the main secondary metabolites in the tea plant, including (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physio...

  17. Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

    Science.gov (United States)

    Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

    2003-09-18

    The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

  18. Probing the subcellular localization of hopanoid lipids in bacteria using NanoSIMS.

    Directory of Open Access Journals (Sweden)

    David M Doughty

    Full Text Available The organization of lipids within biological membranes is poorly understood. Some studies have suggested lipids group into microdomains within cells, but the evidence remains controversial due to non-native imaging techniques. A recently developed NanoSIMS technique indicated that sphingolipids group into microdomains within membranes of human fibroblast cells. We extended this NanoSIMS approach to study the localization of hopanoid lipids in bacterial cells by developing a stable isotope labeling method to directly detect subcellular localization of specific lipids in bacteria with ca. 60 nm resolution. Because of the relatively small size of bacterial cells and the relative abundance of hopanoid lipids in membranes, we employed a primary (2H-label to maximize our limit of detection. This approach permitted the analysis of multiple stable isotope labels within the same sample, enabling visualization of subcellular lipid microdomains within different cell types using a secondary label to mark the growing end of the cell. Using this technique, we demonstrate subcellular localization of hopanoid lipids within alpha-proteobacterial and cyanobacterial cells. Further, we provide evidence of hopanoid lipid domains in between cells of the filamentous cyanobacterium Nostoc punctiforme. More broadly, our method provides a means to image lipid microdomains in a wide range of cell types and test hypotheses for their functions in membranes.

  19. ngLOC: software and web server for predicting protein subcellular localization in prokaryotes and eukaryotes

    Directory of Open Access Journals (Sweden)

    King Brian R

    2012-07-01

    Full Text Available Abstract Background Understanding protein subcellular localization is a necessary component toward understanding the overall function of a protein. Numerous computational methods have been published over the past decade, with varying degrees of success. Despite the large number of published methods in this area, only a small fraction of them are available for researchers to use in their own studies. Of those that are available, many are limited by predicting only a small number of organelles in the cell. Additionally, the majority of methods predict only a single location for a sequence, even though it is known that a large fraction of the proteins in eukaryotic species shuttle between locations to carry out their function. Findings We present a software package and a web server for predicting the subcellular localization of protein sequences based on the ngLOC method. ngLOC is an n-gram-based Bayesian classifier that predicts subcellular localization of proteins both in prokaryotes and eukaryotes. The overall prediction accuracy varies from 89.8% to 91.4% across species. This program can predict 11 distinct locations each in plant and animal species. ngLOC also predicts 4 and 5 distinct locations on gram-positive and gram-negative bacterial datasets, respectively. Conclusions ngLOC is a generic method that can be trained by data from a variety of species or classes for predicting protein subcellular localization. The standalone software is freely available for academic use under GNU GPL, and the ngLOC web server is also accessible at http://ngloc.unmc.edu.

  20. Cdc2/cyclin B1 regulates centrosomal Nlp proteolysis and subcellular localization.

    Science.gov (United States)

    Zhao, Xuelian; Jin, Shunqian; Song, Yongmei; Zhan, Qimin

    2010-11-01

    The formation of proper mitotic spindles is required for appropriate chromosome segregation during cell division. Aberrant spindle formation often causes aneuploidy and results in tumorigenesis. However, the underlying mechanism of regulating spindle formation and chromosome separation remains to be further defined. Centrosomal Nlp (ninein-like protein) is a recently characterized BRCA1-regulated centrosomal protein and plays an important role in centrosome maturation and spindle formation. In this study, we show that Nlp can be phosphorylated by cell cycle protein kinase Cdc2/cyclin B1. The phosphorylation sites of Nlp are mapped at Ser185 and Ser589. Interestingly, the Cdc2/cyclin B1 phosphorylation site Ser185 of Nlp is required for its recognition by PLK1, which enable Nlp depart from centrosomes to allow the establishment of a mitotic scaffold at the onset of mitosis . PLK1 fails to dissociate the Nlp mutant lacking Ser185 from centrosome, suggesting that Cdc2/cyclin B1 might serve as a primary kinase of PLK1 in regulating Nlp subcellular localization. However, the phosphorylation at the site Ser589 by Cdc2/cyclin B1 plays an important role in Nlp protein stability probably due to its effect on protein degradation. Furthermore, we show that deregulated expression or subcellular localization of Nlp lead to multinuclei in cells, indicating that scheduled levels of Nlp and proper subcellular localization of Nlp are critical for successful completion of normal cell mitosis, These findings demonstrate that Cdc2/cyclin B1 is a key regulator in maintaining appropriate degradation and subcellular localization of Nlp, providing novel insights into understanding on the role of Cdc2/cyclin B1 in mitotic progression.

  1. Alternative splicing and differential subcellular localization of the rat FGF antisense gene product

    Directory of Open Access Journals (Sweden)

    Casson Alan G

    2008-01-01

    Full Text Available Abstract Background GFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species including rat. In the present study we focused on elucidating the expression and subcellular distribution of alternatively spliced rat GFG isoforms. Results RT-PCR and immunohistochemistry revealed tissue-specific GFG mRNA isoform expression and subcellular distribution of GFG immunoreactivity in cytoplasm and nuclei of a wide range of normal rat tissues. FGF-2 and GFG immunoreactivity were co-localized in some, but not all, tissues examined. Computational analysis identified a mitochondrial targeting sequence (MTS in the N-terminus of three previously described rGFG isoforms. Confocal laser scanning microscopy and subcellular fractionation analysis revealed that all rGFG isoforms bearing the MTS were specifically targeted to mitochondria whereas isoforms and deletion mutants lacking the MTS were localized in the cytoplasm and nucleus. Mutation and deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization. Conclusion Previous findings strongly support a role for the FGF antisense RNA as a regulator of FGF2 expression. The present study demonstrates that the antisense RNA itself is translated, and that protein isoforms resulting form alternative RNA splicing are sorted to different subcellular compartments. FGF-2 and its antisense protein are co-expressed in many tissues and in some cases in the same cells. The strong conservation of sequence and genomic organization across animal species suggests important functional significance to the physical association of these transcript

  2. Subcellular localization of calcium deposits during zebrafish (Danio rerio) oogenesis.

    Science.gov (United States)

    Golpour, Amin; Pšenička, Martin; Niksirat, Hamid

    2016-01-01

    Calcium plays prominent roles in regulating a broad range of physiological events in reproduction. The aim of this study was to describe the subcellular distribution of calcium deposits during stages of oogenesis in zebrafish using a combined oxalate-pyroantimonate technique. The oocyte development of zebrafish was categorized into four stages: primary growth, cortical-alveolus, vitellogenic, and maturation, based on morphological criteria. Calcium deposits in the primary growth stage were detected in the cytoplasm, mitochondria, nucleus, and follicular cells. At the cortical-alveolus stage, calcium particles were transported from follicular cells and deposited in the cortical alveoli. In the vitellogenic stage, some cortical alveoli were compacted and transformed from flocculent electron-lucent to electron-dense objects with the progression of the stage. Calcium deposits were transformed from larger to smaller particles, coinciding with compaction of cortical alveoli. In the maturation stage, calcium deposits in all oocyte compartments decreased, with the exception of those in mitochondria. The proportion of area covered by calcium deposits in the mitochondria and cortical alveoli of oocytes at different stages of development was significantly different (poogenesis may contribute to better understanding of its role in oogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Subcellular localization of YKL-40 in normal and malignant epithelial cells of the breast

    DEFF Research Database (Denmark)

    Roslind, A.; Balslev, E.; Kruse, H.

    2008-01-01

    . YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial......YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy...

  4. Gram-positive and gram-negative subcellular localization using rotation forest and physicochemical-based features

    Science.gov (United States)

    2015-01-01

    Background The functioning of a protein relies on its location in the cell. Therefore, predicting protein subcellular localization is an important step towards protein function prediction. Recent studies have shown that relying on Gene Ontology (GO) for feature extraction can improve the prediction performance. However, for newly sequenced proteins, the GO is not available. Therefore, for these cases, the prediction performance of GO based methods degrade significantly. Results In this study, we develop a method to effectively employ physicochemical and evolutionary-based information in the protein sequence. To do this, we propose segmentation based feature extraction method to explore potential discriminatory information based on physicochemical properties of the amino acids to tackle Gram-positive and Gram-negative subcellular localization. We explore our proposed feature extraction techniques using 10 attributes that have been experimentally selected among a wide range of physicochemical attributes. Finally by applying the Rotation Forest classification technique to our extracted features, we enhance Gram-positive and Gram-negative subcellular localization accuracies up to 3.4% better than previous studies which used GO for feature extraction. Conclusion By proposing segmentation based feature extraction method to explore potential discriminatory information based on physicochemical properties of the amino acids as well as using Rotation Forest classification technique, we are able to enhance the Gram-positive and Gram-negative subcellular localization prediction accuracies, significantly. PMID:25734546

  5. Diverse subcellular localizations of the insect CMP-sialic acid synthetases.

    Science.gov (United States)

    Di, Wu; Fujita, Akiko; Hamaguchi, Kayo; Delannoy, Philippe; Sato, Chihiro; Kitajima, Ken

    2017-04-01

    The occurrence and biological importance of sialic acid (Sia) and its metabolic enzymes in insects have been studied using Drosophila melanogaster. The most prominent feature of D. melanogaster CMP-Sia synthetase (DmCSS) is its Golgi-localization, contrasted with nuclear localization of vertebrate CSSs. However, it remains unclear if the Golgi-localization is common to other insect CSSs and why it happens. To answer these questions, Aedes aegypti (mosquito) CSS (AaCSS) and Tribolium castaneum (beetle) CSS (TcCSS) were cloned and characterized for their activity and subcellular localization. Our new findings show: (1) AaCSS and TcCSS share a common overall structure with DmCSS in terms of evolutionarily conserved motifs and the absence of the C-terminal domain typical to vertebrate CSSs; (2) when expressed in mammalian and insect cells, AaCSS and TcCSS showed in vivo and in vitro CSS activities, similar to DmCSS. In contrast, when expressed in bacteria, they lacked CSS activity because the N-terminal hydrophobic region appeared to induce protein aggregation; (3) when expressed in Drosophila S2 cells, AaCSS and TcCSS were predominantly localized in the ER, but not in the Golgi. Surprisingly, DmCSS was mainly secreted into the culture medium, although partially detected in Golgi. Consistent with these results, the N-terminal hydrophobic regions of AaCSS and TcCSS functioned as a signal peptide to render them soluble in the ER, while the N-terminus of DmCSS functioned as a membrane-spanning region of type II transmembrane proteins whose cytosolic KLK sequence functioned as an ER export signal. Accordingly, the differential subcellular localization of insect CSSs are distinctively more diverse than previously recognized. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Osmotic stress changes the expression and subcellular localization of the Batten disease protein CLN3.

    Directory of Open Access Journals (Sweden)

    Amanda Getty

    Full Text Available Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm, achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm, human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress.

  7. ESLpred2: improved method for predicting subcellular localization of eukaryotic proteins

    Directory of Open Access Journals (Sweden)

    Raghava Gajendra PS

    2008-11-01

    Full Text Available Abstract Background The expansion of raw protein sequence databases in the post genomic era and availability of fresh annotated sequences for major localizations particularly motivated us to introduce a new improved version of our previously forged eukaryotic subcellular localizations prediction method namely "ESLpred". Since, subcellular localization of a protein offers essential clues about its functioning, hence, availability of localization predictor would definitely aid and expedite the protein deciphering studies. However, robustness of a predictor is highly dependent on the superiority of dataset and extracted protein attributes; hence, it becomes imperative to improve the performance of presently available method using latest dataset and crucial input features. Results Here, we describe augmentation in the prediction performance obtained for our most popular ESLpred method using new crucial features as an input to Support Vector Machine (SVM. In addition, recently available, highly non-redundant dataset encompassing three kingdoms specific protein sequence sets; 1198 fungi sequences, 2597 from animal and 491 plant sequences were also included in the present study. First, using the evolutionary information in the form of profile composition along with whole and N-terminal sequence composition as an input feature vector of 440 dimensions, overall accuracies of 72.7, 75.8 and 74.5% were achieved respectively after five-fold cross-validation. Further, enhancement in performance was observed when similarity search based results were coupled with whole and N-terminal sequence composition along with profile composition by yielding overall accuracies of 75.9, 80.8, 76.6% respectively; best accuracies reported till date on the same datasets. Conclusion These results provide confidence about the reliability and accurate prediction of SVM modules generated in the present study using sequence and profile compositions along with similarity search

  8. N-terminal acetylation by NatC is not a general determinant for substrate subcellular localization in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Henriette Aksnes

    Full Text Available N-terminal acetylation has been suggested to play a role in the subcellular targeting of proteins, in particular those acetylated by the N-terminal acetyltransferase complex NatC. Based on previous positional proteomics data revealing N-terminal acetylation status and the predicted NAT substrate classes, we selected 13 suitable NatC substrates for subcellular localization studies in Saccharomyces cerevisiae. Fluorescence microscopy analysis of GFP-tagged candidates in the presence or absence of the NatC catalytic subunit Naa30 (Mak3 revealed unaltered localization patterns for all 13 candidates, thus arguing against a general role for the N-terminal acetyl group as a localization determinant. Furthermore, all organelle-localized substrates indicated undisrupted structures, thus suggesting that absence of NatC acetylation does not have a vast effect on organelle morphology in yeast.

  9. Detrended cross-correlation coefficient: Application to predict apoptosis protein subcellular localization.

    Science.gov (United States)

    Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

    2016-12-01

    Apoptosis, or programed cell death, plays a central role in the development and homeostasis of an organism. Obtaining information on subcellular location of apoptosis proteins is very helpful for understanding the apoptosis mechanism. The prediction of subcellular localization of an apoptosis protein is still a challenging task, and existing methods mainly based on protein primary sequences. In this paper, we introduce a new position-specific scoring matrix (PSSM)-based method by using detrended cross-correlation (DCCA) coefficient of non-overlapping windows. Then a 190-dimensional (190D) feature vector is constructed on two widely used datasets: CL317 and ZD98, and support vector machine is adopted as classifier. To evaluate the proposed method, objective and rigorous jackknife cross-validation tests are performed on the two datasets. The results show that our approach offers a novel and reliable PSSM-based tool for prediction of apoptosis protein subcellular localization. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. PSI: a comprehensive and integrative approach for accurate plant subcellular localization prediction.

    Directory of Open Access Journals (Sweden)

    Lili Liu

    Full Text Available Predicting the subcellular localization of proteins conquers the major drawbacks of high-throughput localization experiments that are costly and time-consuming. However, current subcellular localization predictors are limited in scope and accuracy. In particular, most predictors perform well on certain locations or with certain data sets while poorly on others. Here, we present PSI, a novel high accuracy web server for plant subcellular localization prediction. PSI derives the wisdom of multiple specialized predictors via a joint-approach of group decision making strategy and machine learning methods to give an integrated best result. The overall accuracy obtained (up to 93.4% was higher than best individual (CELLO by ~10.7%. The precision of each predicable subcellular location (more than 80% far exceeds that of the individual predictors. It can also deal with multi-localization proteins. PSI is expected to be a powerful tool in protein location engineering as well as in plant sciences, while the strategy employed could be applied to other integrative problems. A user-friendly web server, PSI, has been developed for free access at http://bis.zju.edu.cn/psi/.

  11. Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly.

    Science.gov (United States)

    Becker, Jordan T; Sherer, Nathan M

    2017-03-15

    Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV

  12. Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome.

    Science.gov (United States)

    Jadot, Michel; Boonen, Marielle; Thirion, Jaqueline; Wang, Nan; Xing, Jinchuan; Zhao, Caifeng; Tannous, Abla; Qian, Meiqian; Zheng, Haiyan; Everett, John K; Moore, Dirk F; Sleat, David E; Lobel, Peter

    2017-02-01

    Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease

  13. Programmed subcellular release to study the dynamics of cell detachment

    Science.gov (United States)

    Wildt, Bridget

    Cell detachment is central to a broad range of physio-pathological changes however there are no quantitative methods to study this process. Here we report programmed subcellular release, a method for spatially and temporally controlled cellular detachment and present the first quantitative results of the detachment dynamics of 3T3 fibroblasts at the subcellular level. Programmed subcellular release is an in vitro technique designed to trigger the detachment of distinct parts of a single cell from a patterned substrate with both spatial and temporal control. Subcellular release is achieved by plating cells on an array of patterned gold electrodes created by standard microfabrication techniques. The electrodes are biochemically functionalized with an adhesion-promoting RGD peptide sequence that is attached to the gold electrode via a thiol linkage. Each electrode is electrically isolated so that a subcellular section of a single cell spanning multiple electrodes can be released independently. Upon application of a voltage pulse to a single electrode, RGD-thiol molecules on an individual electrode undergo rapid electrochemical desorption that leads to subsequent cell contraction. The dynamics of cell contraction are found to have characteristic induction and contraction times. This thesis presents the first molecular inhibition studies conducted using programmed subcellular release verifying that this technique can be used to study complex signaling pathways critical to cell motility. Molecular level dynamics of focal adhesion proteins and actin stress fibers provide some insight into the complexities associated with triggered cell detachment. In addition to subcellular release, the programmed release of alkanethiols provides a tool for to study the spatially and temporally controlled release of small molecules or particles from individually addressable gold electrodes. Here we report on experiments which determine the dynamics of programmed release using fluorophore

  14. Subcellular Localization of APE1/Ref-1 in Human Hepatocellular Carcinoma: Possible Prognostic Significance

    OpenAIRE

    Di Maso, Vittorio; Avellini, Claudio; Crocè, Lory Saveria; Rosso, Natalia; Quadrifoglio, Franco; Cesaratto, Laura; Codarin, Erika; Bedogni, Giorgio; Beltrami, Carlo Alberto; Tell, Gianluca; Tiribelli, Claudio

    2007-01-01

    APE1/Ref-1, normally localized in the nucleus, is a regulator of the cellular response to oxidative stress. Cytoplasmic localization has been observed in several tumors and correlates with a poor prognosis. Because no data are available on liver tumors, we investigated APE1/Ref-1 subcellular localization and its correlation with survival in 47 consecutive patients undergoing hepatocellular carcinoma (HCC) resection. APE1/Ref-1 expression was determined by immunohistochemistry in HCC and surro...

  15. Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle

    DEFF Research Database (Denmark)

    Høier, Birgitte; Prats Gavalda, Clara; Qvortrup, Klaus

    2013-01-01

    The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations...

  16. Subcellular localization of Bombyx mori ribosomal protein S3a and ...

    African Journals Online (AJOL)

    Subcellular localization of Bombyx mori ribosomal protein S3a and effect of its over-expression on BmNPV infection. Z Wu-song, B Xian-xun, X Jia-ping, Y Zheng-ying, Y Ying, W Hui-ling, W Wen-bing ...

  17. Geary autocorrelation and DCCA coefficient: Application to predict apoptosis protein subcellular localization via PSSM

    Science.gov (United States)

    Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

    2017-02-01

    Apoptosis is a fundamental process controlling normal tissue homeostasis by regulating a balance between cell proliferation and death. Predicting subcellular location of apoptosis proteins is very helpful for understanding its mechanism of programmed cell death. Prediction of apoptosis protein subcellular location is still a challenging and complicated task, and existing methods mainly based on protein primary sequences. In this paper, we propose a new position-specific scoring matrix (PSSM)-based model by using Geary autocorrelation function and detrended cross-correlation coefficient (DCCA coefficient). Then a 270-dimensional (270D) feature vector is constructed on three widely used datasets: ZD98, ZW225 and CL317, and support vector machine is adopted as classifier. The overall prediction accuracies are significantly improved by rigorous jackknife test. The results show that our model offers a reliable and effective PSSM-based tool for prediction of apoptosis protein subcellular localization.

  18. Beyond co-localization: inferring spatial interactions between sub-cellular structures from microscopy images

    Directory of Open Access Journals (Sweden)

    Paul Grégory

    2010-07-01

    Full Text Available Abstract Background Sub-cellular structures interact in numerous direct and indirect ways in order to fulfill cellular functions. While direct molecular interactions crucially depend on spatial proximity, other interactions typically result in spatial correlations between the interacting structures. Such correlations are the target of microscopy-based co-localization analysis, which can provide hints of potential interactions. Two complementary approaches to co-localization analysis can be distinguished: intensity correlation methods capitalize on pattern discovery, whereas object-based methods emphasize detection power. Results We first reinvestigate the classical co-localization measure in the context of spatial point pattern analysis. This allows us to unravel the set of implicit assumptions inherent to this measure and to identify potential confounding factors commonly ignored. We generalize object-based co-localization analysis to a statistical framework involving spatial point processes. In this framework, interactions are understood as position co-dependencies in the observed localization patterns. The framework is based on a model of effective pairwise interaction potentials and the specification of a null hypothesis for the expected pattern in the absence of interaction. Inferred interaction potentials thus reflect all significant effects that are not explained by the null hypothesis. Our model enables the use of a wealth of well-known statistical methods for analyzing experimental data, as demonstrated on synthetic data and in a case study considering virus entry into live cells. We show that the classical co-localization measure typically under-exploits the information contained in our data. Conclusions We establish a connection between co-localization and spatial interaction of sub-cellular structures by formulating the object-based interaction analysis problem in a spatial statistics framework based on nearest-neighbor distance

  19. Identification of an intrinsic determinant critical for maspin subcellular localization and function.

    Directory of Open Access Journals (Sweden)

    Sijana H Dzinic

    Full Text Available Maspin, a multifaceted tumor suppressor, belongs to the serine protease inhibitor superfamily, but only inhibits serine protease-like enzymes such as histone deacetylase 1 (HDAC1. Maspin is specifically expressed in epithelial cells and it is differentially regulated during tumor progression. A new emerging consensus suggests that a shift in maspin subcellular localization from the nucleus to the cytoplasm stratifies with poor cancer prognosis. In the current study, we employed a rational mutagenesis approach and showed that maspin reactive center loop (RCL and its neighboring sequence are critical for maspin stability. Further, when expressed in multiple tumor cell lines, single point mutation of Aspartate(346 (D(346 to Glutamate (E(346, maspin(D346E, was predominantly nuclear, whereas wild type maspin (maspin(WT was both cytoplasmic and nuclear. Evidence from cellular fractionation followed by immunological and proteomic protein identification, combined with the evidence from fluorescent imaging of endogenous proteins, fluorescent protein fusion constructs, as well as bimolecular fluorescence complementation (BiFC showed that the increased nuclear enrichment of maspin(D346E was, at least in part, due to its increased affinity to HDAC1. Maspin(D346E was also more potent than maspin(WT as an HDAC inhibitor. Taken together, our evidence demonstrates that D(346 is a critical cis-element in maspin sequence that determines the molecular context and subcellular localization of maspin. A mechanistic model derived from our evidence suggests a new window of opportunity for the development of maspin-based biologically competent HDAC inhibitors for cancer treatment.

  20. Fast subcellular localization by cascaded fusion of signal-based and homology-based methods

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2011-10-01

    Full Text Available Abstract Background The functions of proteins are closely related to their subcellular locations. In the post-genomics era, the amount of gene and protein data grows exponentially, which necessitates the prediction of subcellular localization by computational means. Results This paper proposes mitigating the computation burden of alignment-based approaches to subcellular localization prediction by a cascaded fusion of cleavage site prediction and profile alignment. Specifically, the informative segments of protein sequences are identified by a cleavage site predictor using the information in their N-terminal shorting signals. Then, the sequences are truncated at the cleavage site positions, and the shortened sequences are passed to PSI-BLAST for computing their profiles. Subcellular localization are subsequently predicted by a profile-to-profile alignment support-vector-machine (SVM classifier. To further reduce the training and recognition time of the classifier, the SVM classifier is replaced by a new kernel method based on the perturbational discriminant analysis (PDA. Conclusions Experimental results on a new dataset based on Swiss-Prot Release 57.5 show that the method can make use of the best property of signal- and homology-based approaches and can attain an accuracy comparable to that achieved by using full-length sequences. Analysis of profile-alignment score matrices suggest that both profile creation time and profile alignment time can be reduced without significant reduction in subcellular localization accuracy. It was found that PDA enjoys a short training time as compared to the conventional SVM. We advocate that the method will be important for biologists to conduct large-scale protein annotation or for bioinformaticians to perform preliminary investigations on new algorithms that involve pairwise alignments.

  1. Mutations in the C-terminal region affect subcellular localization of crucian carp herpesvirus (CaHV) GPCR.

    Science.gov (United States)

    Wang, Jun; Gui, Lang; Chen, Zong-Yan; Zhang, Qi-Ya

    2016-08-01

    G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture.

  2. Protein targeting to subcellular organelles via MRNA localization.

    Science.gov (United States)

    Weis, Benjamin L; Schleiff, Enrico; Zerges, William

    2013-02-01

    Cells have complex membranous organelles for the compartmentalization and the regulation of most intracellular processes. Organelle biogenesis and maintenance requires newly synthesized proteins, each of which needs to go from the ribosome translating its mRNA to the correct membrane for insertion or transclocation to an a organellar subcompartment. Decades of research have revealed how proteins are targeted to the correct organelle and translocated across one or more organelle membranes ro the compartment where they function. The paradigm examples involve interactions between a peptide sequence in the protein, localization factors, and various membrane embedded translocation machineries. Membrane translocation is either cotranslational or posttranslational depending on the protein and target organelle. Meanwhile research in embryos, neurons and yeast revealed an alternative targeting mechanism in which the mRNA is localized and only then translated to synthesize the protein in the correct location. In these cases, the targeting information is coded by the cis-acting sequences in the mRNA ("Zipcodes") that interact with localization factors and, in many cases, are transported by the molecular motors on the cytoskeletal filaments. Recently, evidence has been found for this "mRNA based" mechanism in organelle protein targeting to endoplasmic reticulum, mitochondria, and the photosynthetic membranes within chloroplasts. Here we review known and potential roles of mRNA localization in protein targeting to and within organelles. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  3. Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

    DEFF Research Database (Denmark)

    Nilsson, Tatjana; Bogdanovic, Nenad; Volkman, Inga

    2006-01-01

    The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD...

  4. Characterization of RanBPM Molecular Determinants that Control Its Subcellular Localization

    Science.gov (United States)

    Salemi, Louisa M.; Loureiro, Sandra O.; Schild-Poulter, Caroline

    2015-01-01

    RanBPM/RanBP9 is a ubiquitous, nucleocytoplasmic protein that is part of an evolutionary conserved E3 ubiquitin ligase complex whose function and targets in mammals are still unknown. RanBPM itself has been implicated in various cellular processes that involve both nuclear and cytoplasmic functions. However, to date, little is known about how RanBPM subcellular localization is regulated. We have conducted a systematic analysis of RanBPM regions that control its subcellular localization using RanBPM shRNA cells to examine ectopic RanBPM mutant subcellular localization without interference from the endogenously expressed protein. We show that several domains and motifs regulate RanBPM nuclear and cytoplasmic localization. In particular, RanBPM comprises two motifs that can confer nuclear localization, one proline/glutamine-rich motif in the extreme N-terminus which has a dominant effect on RanBPM localization, and a second motif in the C-terminus which minimally contributes to RanBPM nuclear targeting. We also identified a nuclear export signal (NES) which mutation prevented RanBPM accumulation in the cytoplasm. Likewise, deletion of the central RanBPM conserved domains (SPRY and LisH/CTLH) resulted in the relocalization of RanBPM to the nucleus, suggesting that RanBPM cytoplasmic localization is also conferred by protein-protein interactions that promote its cytoplasmic retention. Indeed we found that in the cytoplasm, RanBPM partially colocalizes with microtubules and associates with α-tubulin. Finally, in the nucleus, a significant fraction of RanBPM is associated with chromatin. Altogether, these analyses reveal that RanBPM subcellular localization results from the combined effects of several elements that either confer direct transport through the nucleocytoplasmic transport machinery or regulate it indirectly, likely through interactions with other proteins and by intramolecular folding. PMID:25659156

  5. PlantLoc: an accurate web server for predicting plant protein subcellular localization by substantiality motif

    OpenAIRE

    Tang, Shengnan; Li, Tonghua; Cong, Peisheng; Xiong, Wenwei; Wang, Zhiheng; Sun, Jiangming

    2013-01-01

    Knowledge of subcellular localizations (SCLs) of plant proteins relates to their functions and aids in understanding the regulation of biological processes at the cellular level. We present PlantLoc, a highly accurate and fast webserver for predicting the multi-label SCLs of plant proteins. The PlantLoc server has two innovative characters: building localization motif libraries by a recursive method without alignment and Gene Ontology information; and establishing simple architecture for rapi...

  6. LOCnet and LOCtarget: sub-cellular localization for structural genomics targets

    Science.gov (United States)

    Nair, Rajesh; Rost, Burkhard

    2004-01-01

    LOCtarget is a web server and database that predicts and annotates sub-cellular localization for structural genomics targets; LOCnet is one of the methods used in LOCtarget that can predict sub-cellular localization for all eukaryotic and prokaryotic proteins. Targets are taken from the central registration database for structural genomics, namely, TargetDB. LOCtarget predicts localization through a combination of four different methods: known nuclear localization signals (PredictNLS), homology-based transfer of experimental annotations (LOChom), inference through automatic text analysis of SWISS-PROT keywords (LOCkey) and de novo prediction through a system of neural networks (LOCnet). Additionally, we report predictions from SignalP. The final prediction is based on the method with the highest confidence. The web server can be used to predict sub-cellular localization of proteins from their amino acid sequence. The LOCtarget database currently contains localization predictions for all eukaryotic proteins from TargetDB and is updated every week. The server is available at http://www.rostlab.org/services/LOCtarget/. PMID:15215440

  7. Subcellular localization-dependent decrements in skeletal muscle glycogen and mitochondria content following short-term disuse in young and old men

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Suetta, Charlotte; Hvid, Lars G

    2010-01-01

    Previous studies have shown that skeletal muscle glycogen and mitochondria are distributed in distinct subcellular localizations, but the role and regulation of these subcellular localizations are unclear. In the present study, we used transmission electron microscopy to investigate the effect...... unchanged. A localization-dependent decrease (P = 0.03) in mitochondria content following immobilization was found in both age groups, where SS mitochondria decreased by 33% (P = 0.02), superficial IMF mitochondria decreased by 20% (P = 0.05), and central IMF mitochondria remained unchanged. In conclusion...

  8. Fast Fourier transform-based support vector machine for subcellular localization prediction using different substitution models.

    Science.gov (United States)

    Wang, Zhimeng; Jiang, Lin; Li, Menglong; Sun, Lina; Lin, Rongying

    2007-09-01

    There are approximately 10(9) proteins in a cell. A hotspot in bioinformatics is how to identify a protein subcellular localization, if its sequence is known. In this paper, a method using fast Fourier transform-based support vector machine is developed to predict the subcellular localization of proteins from their physicochemical properties and structural parameters. The prediction accuracies reached 83% in prokaryotic organisms and 84% in eukaryotic organisms with the substitution model of the c-p-v matrix (c, composition; p, polarity; and v, molecular volume). The overall prediction accuracy was also evaluated using the "leave-one-out" jackknife procedure. The influence of the substitution model on prediction accuracy has also been discussed in the work. The source code of the new program is available on request from the authors.

  9. Arginine methylation controls the subcellular localization and functions of the oncoprotein splicing factor SF2/ASF.

    Science.gov (United States)

    Sinha, Rahul; Allemand, Eric; Zhang, Zuo; Karni, Rotem; Myers, Michael P; Krainer, Adrian R

    2010-06-01

    Alternative splicing and posttranslational modifications (PTMs) are major sources of protein diversity in eukaryotic proteomes. The SR protein SF2/ASF is an oncoprotein that functions in pre-mRNA splicing, with additional roles in other posttranscriptional and translational events. Functional studies of SR protein PTMs have focused exclusively on the reversible phosphorylation of Ser residues in the C-terminal RS domain. We confirmed that human SF2/ASF is methylated at residues R93, R97, and R109, which were identified in a global proteomic analysis of Arg methylation, and further investigated whether these methylated residues regulate the properties of SF2/ASF. We show that the three arginines additively control the subcellular localization of SF2/ASF and that both the positive charge and the methylation state are important. Mutations that block methylation and remove the positive charge result in the cytoplasmic accumulation of SF2/ASF. The consequent decrease in nuclear SF2/ASF levels prevents it from modulating the alternative splicing of target genes, results in higher translation stimulation, and abrogates the enhancement of nonsense-mediated mRNA decay. This study addresses the mechanisms by which Arg methylation and the associated positive charge regulate the activities of SF2/ASF and emphasizes the significance of localization control for an oncoprotein with multiple functions in different cellular compartments.

  10. Subcellular localization of the delayed rectifier K(+) channels KCNQ1 and ERG1 in the rat heart

    DEFF Research Database (Denmark)

    Rasmussen, Hanne Borger; Møller, Morten; Knaus, Hans-Günther

    2003-01-01

    In the heart, several K(+) channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier K(+) currents. In the present study, the cellular and subcellular localization of the two delayed rectifier K(+) channels, KCNQ1 and ether......-a-go-go-related gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQ1 labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter....... Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERG1 channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQ1 was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERG1 displays a very restricted subcellular...

  11. Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor.

    Science.gov (United States)

    Bernabeu, M; Lopez, F J; Ferrer, M; Martin-Jaular, L; Razaname, A; Corradin, G; Maier, A G; Del Portillo, H A; Fernandez-Becerra, C

    2012-03-01

    The subcellular localization and function of variant subtelomeric multigene families in Plasmodium vivax remain vastly unknown. Among them, the vir superfamily is putatively involved in antigenic variation and in mediating adherence to endothelial receptors. In the absence of a continuous in vitro culture system for P. vivax, we have generated P. falciparum transgenic lines expressing VIR proteins to infer location and function. We chose three proteins pertaining to subfamilies A (VIR17), C (VIR14) and D (VIR10), with domains and secondary structures that predictably traffic these proteins to different subcellular compartments. Here, we showed that VIR17 remained inside the parasite and around merozoites, whereas VIR14 and VIR10 were exported to the membrane of infected red blood cells (iRBCs) in an apparent independent pathway of Maurer's clefts. Remarkably, VIR14 was exposed at the surface of iRBCs and mediated adherence to different endothelial receptors expressed in CHO cells under static conditions. Under physiological flow conditions, however, cytoadherence was only observed to ICAM-1, which was the only receptor whose adherence was specifically and significantly inhibited by antibodies against conserved motifs of VIR proteins. Immunofluorescence studies using these antibodies also showed different subcellular localizations of VIR proteins in P. vivax-infected reticulocytes from natural infections. These data suggest that VIR proteins are trafficked to different cellular compartments and functionally demonstrates that VIR proteins can specifically mediate cytoadherence to the ICAM-1 endothelial receptor. © 2011 Blackwell Publishing Ltd.

  12. Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope.

    Science.gov (United States)

    Mukai, Rie; Shirai, Yasuhito; Saito, Naoaki; Yoshida, Ken-Ichi; Ashida, Hitoshi

    2009-04-01

    Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning fluorescence microscope and mouse hepatoma Hepa-1c1c7 cells. Five flavonol aglycones showed autofluorescence in the cells under the conditions (Ex. 488 nm to Em. 515-535 nm), whereas three flavonol glycosides and eight compounds belonging to other flavonoid subclasses, i.e., flavones, flavanones, and catechins, did not. The autofluorescence of galangin and kaempferol appeared stronger in the nucleus than cytoplasm, suggesting that they are incorporated into the cells and accumulated in the nucleus. The proposed method provided evidence that flavonol aglycones are incorporated into, and accumulated in the nucleus of, hepatocytes.

  13. Subcellular Localization of Class I Histone Deacetylases in the Developing Xenopus tectum.

    Science.gov (United States)

    Guo, Xia; Ruan, Hangze; Li, Xia; Qin, Liming; Tao, Yi; Qi, Xianjie; Gao, Juanmei; Gan, Lin; Duan, Shumin; Shen, Wanhua

    2015-01-01

    Histone deacetylases (HDACs) are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis, and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus, and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus, and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2, and HDAC3 are transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12) remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA) or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

  14. Subcellular localization of class I histone deacetylases in the developing Xenopus tectum

    Directory of Open Access Journals (Sweden)

    Xia eGuo

    2016-01-01

    Full Text Available Histone deacetylases (HDACs are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2 and HDAC3 are transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12 remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

  15. CELLO2GO: a web server for protein subCELlular LOcalization prediction with functional gene ontology annotation.

    Directory of Open Access Journals (Sweden)

    Chin-Sheng Yu

    Full Text Available CELLO2GO (http://cello.life.nctu.edu.tw/cello2go/ is a publicly available, web-based system for screening various properties of a targeted protein and its subcellular localization. Herein, we describe how this platform is used to obtain a brief or detailed gene ontology (GO-type categories, including subcellular localization(s, for the queried proteins by combining the CELLO localization-predicting and BLAST homology-searching approaches. Given a query protein sequence, CELLO2GO uses BLAST to search for homologous sequences that are GO annotated in an in-house database derived from the UniProt KnowledgeBase database. At the same time, CELLO attempts predict at least one subcellular localization on the basis of the species in which the protein is found. When homologs for the query sequence have been identified, the number of terms found for each of their GO categories, i.e., cellular compartment, molecular function, and biological process, are summed and presented as pie charts representing possible functional annotations for the queried protein. Although the experimental subcellular localization of a protein may not be known, and thus not annotated, CELLO can confidentially suggest a subcellular localization. CELLO2GO should be a useful tool for research involving complex subcellular systems because it combines CELLO and BLAST into one platform and its output is easily manipulated such that the user-specific questions may be readily addressed.

  16. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family

    Science.gov (United States)

    Tanz, Sandra K.; Castleden, Ian; Hooper, Cornelia M.; Small, Ian; Millar, A. Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1–Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  17. The proprotein convertase SKI-1/S1P: alternate translation and subcellular localization.

    Science.gov (United States)

    Pullikotil, Philomena; Benjannet, Suzanne; Mayne, Janice; Seidah, Nabil G

    2007-09-14

    Subtilisin kexin isozyme-1 (SKI-1) represents the first mammalian member of secretory subtilisin-like processing enzymes that cleaves after nonbasic residues. It is synthesized as an inactive precursor that undergoes three sequential autocatalytic processing steps of its N-terminal prosegment and an ectodomain shedding at a site near the transmembrane domain. The various cellular functions of SKI-1 emphasize the need to understand the sites of its activation and shedding. We have previously shown that SKI-1 undergoes autocatalytic shedding at the sequence KHQKLL(953) downward arrow, resulting in a membrane-bound stump called St-1 (amino acids 954-1052). However, little is known about the cellular localization of SKI-1 or its shed forms. In the present study, we have further identified a smaller C-terminal fragment St-2 generated closer to the transmembrane domain. By sequencing and mass spectrometric analysis, the start site and the molecular mass of St-2 were determined. Site-directed mutagenesis revealed the critical amino acid involved in this novel process. Mutation of Met(990) to M990A, M990I, and M990L failed to generate St-2, suggesting an internal alternate translation event at Met(990), as confirmed by an in vitro transcription/translation assay. Confocal microscopy defined the subcellular localization of SKI-1 and its fragments. The data show that most of membrane-bound SKI-1 and its stumps St-1 and St-2 localize to the Golgi and can enter the endosomal/lysosomal compartments but do not sort to the cell surface. Deletion studies showed that the transmembrane domain of SKI-1 determines its trafficking. Finally, rSt-1 and rSt-2 seem to affect the processing of ATF6 by SKI-1, but cellular stress does not regulate the production of St-2.

  18. Direct imaging the subcellular localization of single-walled carbon nanotubes

    Science.gov (United States)

    Zhou, Feifan; Xing, Da; Chen, Wei R.

    2011-03-01

    The development of single-walled carbon nanotubes (SWNTs) for various biomedical applications is an area of great promise. However, the contradictory data on the interaction of single-walled carbon nanotubes with cells highlight the need to study their uptake and cytotoxic effects in cells. Here, we use confocal microscopy to image the translocation of single-walled carbon nanotubes into cells and localization on the subcellular organelle. We also observe that single-walled carbon nanotubes do not affect the cellular condition and mitochondrial membrane potential. One intrinsic property of single-walled carbon nanotubes is their strong optical absorbance in the near-infrared (NIR) region. It could be used to selectively increase the thermal destructions in the target tumors. A specific type of SWNT by the CoMoCAT method has an intense absorption band at 980 nm. When irradiated with a 980-nm laser, the single-walled carbon nanotubes affect the cellular oxidation and destroy the mitochondrial membrane potential, and induce cell apoptosis. Thus, the single-walled carbon nanotubes appear to enter the cytoplasm without cytotoxic effects in cells, and can be used as effective and selective nanomaterials for cancer photothermal therapy.

  19. Nanopipette-Based SERS Aptasensor for Subcellular Localization of Cancer Biomarker in Single Cells.

    Science.gov (United States)

    Hanif, Sumaira; Liu, Hai-Ling; Ahmed, Saud Asif; Yang, Jin-Mei; Zhou, Yue; Pang, Jie; Ji, Li-Na; Xia, Xing-Hua; Wang, Kang

    2017-09-19

    Single cell analysis is essential for understanding the heterogeneity, behaviors of cells, and diversity of target analyte in different subcellular regions. Nucleolin (NCL) is a multifunctional protein that is markedly overexpressed in most of the cancer cells. The variant expression levels of NCL in subcellular regions have a marked influence on cancer proliferation and treatments. However, the specificity of available methods to identify the cancer biomarkers is limited because of the high level of subcellular matrix effect. Herein, we proposed a novel technique to increase both the molecular and spectral specificity of cancer diagnosis by using aptamers affinity based portable nanopipette with distinctive surface-enhanced Raman scattering (SERS) activities. The aptamers-functionalized gold-coated nanopipette was used to capture target, while p-mercaptobenzonitrile (MBN) and complementary DNA modified Ag nanoparticles (AgNPs) worked as Raman reporter to produce SERS signal. The SERS signal of Raman nanotag was lost upon NCL capturing via modified DNA aptamers on nanoprobe, which further helped to verify the specificity of nanoprobe. For proof of concept, NCL protein was specifically extracted from different cell lines by aptamers modified SERS active nanoprobe. The nanoprobes manifested specifically good affinity for NCL with a dissociation constant Kd of 36 nM and provided a 1000-fold higher specificity against other competing proteins. Furthermore, the Raman reporter moiety has a vibrational frequency in the spectroscopically silent region (1800-2300 cm-1) with a negligible matrix effect from cell analysis. The subcellular localization and spatial distribution of NCL were successfully achieved in various types of cells, including MCF-7A, HeLa, and MCF-10A cells. This type of probing technique for single cell analysis could lead to the development of a new perspective in cancer diagnosis and treatment at the cellular level.

  20. The in vitro sub-cellular localization and in vivo efficacy of novel chitosan/GMO nanostructures containing paclitaxel.

    Science.gov (United States)

    Trickler, W J; Nagvekar, A A; Dash, A K

    2009-08-01

    To determine the in vitro sub-cellular localization and in vivo efficacy of chitosan/GMO nanostructures containing paclitaxel (PTX) compared to a conventional PTX treatment (Taxol). The sub-cellular localization of coumarin-6 labeled chitosan/GMO nanostructures was determined by confocal microscopy in MDA-MB-231 cells. The antitumor efficacy was evaluated in two separate studies using FOX-Chase (CB17) SCID Female-Mice MDA-MB-231 xenograph model. Treatments consisted of intravenous Taxol or chitosan/GMO nanostructures with or without PTX, local intra-tumor bolus of Taxol or chitosan/GMO nanostructures with or without PTX. The tumor diameter and animal weight was monitored at various intervals. Histopathological changes were evaluated in end-point tumors. The tumor diameter increased at a constant rate for all the groups between days 7-14. After a single intratumoral bolus dose of chitosan/GMO containing PTX showed significant reduction in tumor diameter on day 15 when compared to control, placebo and intravenous PTX administration. The tumor diameter reached a maximal decrease (4-fold) by day 18, and the difference was reduced to approximately 2-fold by day 21. Qualitatively similar results were observed in a separate study containing PTX when administered intravenously. Chitosan/GMO nanostructures containing PTX are safe and effective administered locally or intravenously. Partially supported by DOD Award BC045664.

  1. Effect heat stress on subcellular localization of Ca2+ in tomato fruits

    Directory of Open Access Journals (Sweden)

    Grażyna Garbaczewska

    2014-01-01

    Full Text Available The aim of this paper was to compare the fruit cell ultrastructure and subcellular localization of Ca2+ after heat stress with the use of the potassium antimonate method (Slocum and Roux 1982, Tretyn et al. 1992. The tomato plants Robin cv., relatively tolerant to heat stress, were grown under uncontrolled greenhouse conditions to the stage of fruiting. The plants were placed for 20h in two temperature regimes: 23oC (optimal temperature or 40oC (heat stress in darkness, under water vapour saturated atmosphere. Immediately after heat stress the fruits were harvested to estimate water soluble and insoluble calcium contents and subcellular localization of Ca2+. After heating the concentration of calcium in tomato fruits increased about twice. In both temperature treatments the water soluble fractions were lower than insoluble ones at smaller differences between insoluble and soluble fractions after heat stress. The shapes and localization of Ca2+ detected with the use of potassium antimonate method show that in fruits of control plants the precipitates were numerous, small and of oval shape. They were dispersed in cytosol or adjoined to endoplasmic reticulum or to external membrane of chloroplast. In the fruit of heated plants the precipitates were irregular in shape, amorphous and singly dispersed in the cytosol. We observed also some cytological changes in the structure of membranes and organelles of the plants of both experimental treatments. The heat induced increase of calcium content and the changes in subcellular localization of Ca2+ under heat stress suggest that calcium ions may be involved in avoiding heat injury. The problem requires more detailed further investigations.

  2. Detection and subcellular localization of dehydrin-like proteins in quinoa (Chenopodium quinoa Willd.) embryos.

    Science.gov (United States)

    Carjuzaa, P; Castellión, M; Distéfano, A J; del Vas, M; Maldonado, S

    2008-01-01

    The aim of this study was to characterize the dehydrin content in mature embryos of two quinoa cultivars, Sajama and Baer La Unión. Cultivar Sajama grows at 3600-4000 m altitude and is adapted to the very arid conditions characteristic of the salty soils of the Bolivian Altiplano, with less than 250 mm of annual rain and a minimum temperature of -1 degrees C. Cultivar Baer La Unión grows at sea-level regions of central Chile and is adapted to more humid conditions (800 to 1500 mm of annual rain), fertile soils, and temperatures above 5 degrees C. Western blot analysis of embryo tissues from plants growing under controlled greenhouse conditions clearly revealed the presence of several dehydrin bands (at molecular masses of approximately 30, 32, 50, and 55 kDa), which were common to both cultivars, although the amount of the 30 and 32 kDa bands differed. Nevertheless, when grains originated from their respective natural environments, three extra bands (at molecular masses of approximately 34, 38, and 40 kDa), which were hardly visible in Sajama, and another weak band (at a molecular mass of approximately 28 kDa) were evident in Baer La Unión. In situ immunolocalization microscopy detected dehydrin-like proteins in all axis and cotyledon tissues. At the subcellular level, dehydrins were detected in the plasma membrane, cytoplasm and nucleus. In the cytoplasm, dehydrins were found associated with mitochondria, rough endoplasmic reticulum cisternae, and proplastid membranes. The presence of dehydrins was also recognized in the matrix of protein bodies. In the nucleus, dehydrins were associated with the euchromatin. Upon examining dehydrin composition and subcellular localization in two quinoa cultivars belonging to highly contrasting environments, we conclude that most dehydrins detected here were constitutive components of the quinoa seed developmental program, but some of them (specially the 34, 38, and 40 kDa bands) may reflect quantitative molecular differences

  3. Role of the EHD2 unstructured loop in dimerization, protein binding and subcellular localization.

    Directory of Open Access Journals (Sweden)

    Kriti Bahl

    Full Text Available The C-terminal Eps 15 Homology Domain proteins (EHD1-4 play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2's homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2 might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting

  4. Role of the EHD2 unstructured loop in dimerization, protein binding and subcellular localization.

    Science.gov (United States)

    Bahl, Kriti; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2's homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2) might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting oligomerization.

  5. HybridGO-Loc: mining hybrid features on gene ontology for predicting subcellular localization of multi-location proteins.

    Directory of Open Access Journals (Sweden)

    Shibiao Wan

    Full Text Available Protein subcellular localization prediction, as an essential step to elucidate the functions in vivo of proteins and identify drugs targets, has been extensively studied in previous decades. Instead of only determining subcellular localization of single-label proteins, recent studies have focused on predicting both single- and multi-location proteins. Computational methods based on Gene Ontology (GO have been demonstrated to be superior to methods based on other features. However, existing GO-based methods focus on the occurrences of GO terms and disregard their relationships. This paper proposes a multi-label subcellular-localization predictor, namely HybridGO-Loc, that leverages not only the GO term occurrences but also the inter-term relationships. This is achieved by hybridizing the GO frequencies of occurrences and the semantic similarity between GO terms. Given a protein, a set of GO terms are retrieved by searching against the gene ontology database, using the accession numbers of homologous proteins obtained via BLAST search as the keys. The frequency of GO occurrences and semantic similarity (SS between GO terms are used to formulate frequency vectors and semantic similarity vectors, respectively, which are subsequently hybridized to construct fusion vectors. An adaptive-decision based multi-label support vector machine (SVM classifier is proposed to classify the fusion vectors. Experimental results based on recent benchmark datasets and a new dataset containing novel proteins show that the proposed hybrid-feature predictor significantly outperforms predictors based on individual GO features as well as other state-of-the-art predictors. For readers' convenience, the HybridGO-Loc server, which is for predicting virus or plant proteins, is available online at http://bioinfo.eie.polyu.edu.hk/HybridGoServer/.

  6. Subcellular localization of the five members of the human steroid 5α-reductase family

    Directory of Open Access Journals (Sweden)

    Antonella Scaglione

    2017-06-01

    We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

  7. DeepLoc: prediction of protein subcellular localization using deep learning

    DEFF Research Database (Denmark)

    Almagro Armenteros, Jose Juan; Sønderby, Casper Kaae; Sønderby, Søren Kaae

    2017-01-01

    current state-of-the-art algorithms, including those relying on homology information. The method is available as a web server at http://www.cbs.dtu.dk/services/DeepLoc . Example code is available at https://github.com/JJAlmagro/subcellular_localization . The dataset is available at http://www.cbs.dtu.dk/services/Deep...... knowledge databases. For novel proteins where no annotated homologues exist, and for predicting the effects of sequence variants, it is desirable to have methods for predicting protein properties from sequence information only. Here, we present a prediction algorithm using deep neural networks to predict...

  8. Subcellular localization and internalization of the vasopressin V1B receptor.

    Science.gov (United States)

    Kashiwazaki, Aki; Fujiwara, Yoko; Tsuchiya, Hiroyoshi; Sakai, Nobuya; Shibata, Katsushi; Koshimizu, Taka-aki

    2015-10-15

    Only limited information is available on agonist-dependent changes in the subcellular localization of vasopressin V1B receptors. Our radioligand binding study of membrane preparations and intact cells revealed that a large fraction of the V1B receptor is located in the cytoplasm in unstimulated CHO cells, which is in contrast to the plasma membrane localization of the V1A and V2 receptors. Moreover, when the affinity of radiolabeled arginine-vasopressin ([3H]AVP) was compared between membrane preparations and intact cells, the affinity of [3H]AVP to the cell surface V1B receptors, but not the V1A receptors, was significantly reduced. Although the number and affinity of cell surface V1B receptors decreased, they became extensively internalized upon binding with [3H]AVP. Approximately 87% of cell surface-bound [3H]AVP was internalized and became resistant to acid wash during incubation with 1 nM [3H]AVP. By contrast, less ligand (35%) was internalized in the cells expressing the V1A receptor. Extensive internalization of the V1B receptors was partially attenuated by inhibitors of cytoskeletal proteins, siRNA against β-arrestin 2, or the removal of sodium chloride from the extracellular buffer, indicating that this internalization involves clathrin-coated pits. Together, these results indicate that the mechanism that regulates the number and affinity of V1B receptors in the plasma membrane is markedly distinct from the corresponding mechanisms for the V1A and V2 receptors and plays a critical role under stress conditions, when vasopressin release is augmented. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Subcellular localization of a PhoE-LacZ fusion protein in E. coli by protease accessibility experiments reveals an inner-membrane-spanning form of the protein

    NARCIS (Netherlands)

    Tommassen, J.P.M.; Kroon, T. de

    1987-01-01

    Protease accessibility experiments were employed to localize a PhoE-LacZ hybrid protein, encompassing a large N-terminal fragment of the outer membrane PhoE protein of E. coli, fused to β-galactosidase, at the subcellular level. In previous studies, this protein was shown to co-fractionate with the

  10. Expression pattern, subcellular localization, and functional implications of ODAM in ameloblasts, odontoblasts, osteoblasts, and various cancer cells.

    Science.gov (United States)

    Lee, Hye-Kyung; Park, Su-Jin; Oh, Hyun-Jung; Kim, Jung-Wook; Bae, Hyun-Sook; Park, Joo-Cheol

    2012-01-01

    During tooth development and tumorigenesis, the odontogenic ameloblast-associated protein (ODAM) is involved in cellular differentiation and matrix protein production. However, the precise function of ODAM remains largely unknown. To suggest new functional roles of ODAM, we investigated the cellular expression and subcellular localization of ODAM in tooth and cancer cells. ODAM was expressed in ameloblasts, odontoblasts, and osteoblasts in vivo and in vitro. Furthermore, ODAM was localized in both the nucleus and cytoplasm of MMP-20 expressing ameloblasts and odontoblasts, but only in the cytoplasm of non-MMP-20 expressing osteoblasts. The extracellular secretion of ODAM was not observed in odontoblasts and osteoblasts, but was seen in ameloblasts. In addition, ODAM was discovered in the nucleus, cytoplasm, and extracellular matrix of various cancer cells. These results suggest that the expression pattern and subcellular localization of ODAM is highly variable and dependent on cell types and their differentiation states, and that functional correlations exist between ODAM and MMP-20. This study provides the first evidence for ODAM in multiple cellular compartments of differentiating odontogenic and cancer cell lines with important functional implications. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

    Full Text Available High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy.

  12. Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization

    DEFF Research Database (Denmark)

    Petersen, B O; Lukas, J; Sørensen, Claus Storgaard

    1999-01-01

    Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated...... by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo...... phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear...

  13. Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods.

    Science.gov (United States)

    Han, Mee-Jung; Yun, Hongseok; Lee, Jeong Wook; Lee, Yu Hyun; Lee, Sang Yup; Yoo, Jong-Shin; Kim, Jin Young; Kim, Jihyun F; Hur, Cheol-Goo

    2011-04-01

    Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum.

    Directory of Open Access Journals (Sweden)

    Wei Gao

    Full Text Available The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC imaging method for cadmium ions (Cd2+ was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

  15. A fast and effective determination of the biodistribution and subcellular localization of fluorescent immunoliposomes in freshly excised animal organs.

    Science.gov (United States)

    Tansi, Felista L; Rüger, Ronny; Kollmeier, Ansgar M; Böhm, Claudia; Kontermann, Roland E; Teichgraeber, Ulf K; Fahr, Alfred; Hilger, Ingrid

    2017-01-18

    Preclinical research implementing fluorescence-based approaches is inevitable for drug discovery and technology. For example, a variety of contrast agents developed for biomedical imaging are usually evaluated in cell systems and animal models based on their conjugation to fluorescent dyes. Biodistribution studies of excised organs are often performed by macroscopic imaging, whereas the subcellular localization though vital, is often neglected or further validated by histological procedures. Available systems used to define the subcellular biodistribution of contrast agents such as intravital microscopes or ex vivo histological analysis are expensive and not affordable by the majority of researchers, or encompass tedious and time consuming steps that may modify the contrast agents and falsify the results. Thus, affordable and more reliable approaches to study the biodistribution of contrast agents are required. We developed fluorescent immunoliposomes specific for human fibroblast activation protein and murine endoglin, and used macroscopic fluorescence imaging and confocal microscopy to determine their biodistribution and subcellular localization in freshly excised mice organs at different time points post intravenous injection. Near infrared fluorescence macroscopic imaging revealed key differences in the biodistribution of the respective immunoliposomes at different time points post injection, which correlated to the first-pass effect as well as the binding of the probes to molecular targets within the mice organs. Thus, a higher accumulation and longer retention of the murine endoglin immunoliposomes was seen in the lungs, liver and kidneys than the FAP specific immunoliposomes. Confocal microscopy showed that tissue autofluorescence enables detection of organ morphology and cellular components within freshly excised, non-processed organs, and that fluorescent probes with absorption and emission maxima beyond the tissue autofluorescence range can be easily

  16. Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

    Science.gov (United States)

    Foureau, E; Carqueijeiro, I; Dugé de Bernonville, T; Melin, C; Lafontaine, F; Besseau, S; Lanoue, A; Papon, N; Oudin, A; Glévarec, G; Clastre, M; St-Pierre, B; Giglioli-Guivarc'h, N; Courdavault, V

    2016-01-01

    Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform. © 2016 Elsevier Inc. All rights reserved.

  17. Prediction of essential proteins based on subcellular localization and gene expression correlation.

    Science.gov (United States)

    Fan, Yetian; Tang, Xiwei; Hu, Xiaohua; Wu, Wei; Ping, Qing

    2017-12-01

    Essential proteins are indispensable to the survival and development process of living organisms. To understand the functional mechanisms of essential proteins, which can be applied to the analysis of disease and design of drugs, it is important to identify essential proteins from a set of proteins first. As traditional experimental methods designed to test out essential proteins are usually expensive and laborious, computational methods, which utilize biological and topological features of proteins, have attracted more attention in recent years. Protein-protein interaction networks, together with other biological data, have been explored to improve the performance of essential protein prediction. The proposed method SCP is evaluated on Saccharomyces cerevisiae datasets and compared with five other methods. The results show that our method SCP outperforms the other five methods in terms of accuracy of essential protein prediction. In this paper, we propose a novel algorithm named SCP, which combines the ranking by a modified PageRank algorithm based on subcellular compartments information, with the ranking by Pearson correlation coefficient (PCC) calculated from gene expression data. Experiments show that subcellular localization information is promising in boosting essential protein prediction.

  18. Assessing the precision of high-throughput computational and laboratory approaches for the genome-wide identification of protein subcellular localization in bacteria

    Directory of Open Access Journals (Sweden)

    Brinkman Fiona SL

    2005-11-01

    Full Text Available Abstract Background Identification of a bacterial protein's subcellular localization (SCL is important for genome annotation, function prediction and drug or vaccine target identification. Subcellular fractionation techniques combined with recent proteomics technology permits the identification of large numbers of proteins from distinct bacterial compartments. However, the fractionation of a complex structure like the cell into several subcellular compartments is not a trivial task. Contamination from other compartments may occur, and some proteins may reside in multiple localizations. New computational methods have been reported over the past few years that now permit much more accurate, genome-wide analysis of the SCL of protein sequences deduced from genomes. There is a need to compare such computational methods with laboratory proteomics approaches to identify the most effective current approach for genome-wide localization characterization and annotation. Results In this study, ten subcellular proteome analyses of bacterial compartments were reviewed. PSORTb version 2.0 was used to computationally predict the localization of proteins reported in these publications, and these computational predictions were then compared to the localizations determined by the proteomics study. By using a combined approach, we were able to identify a number of contaminants and proteins with dual localizations, and were able to more accurately identify membrane subproteomes. Our results allowed us to estimate the precision level of laboratory subproteome studies and we show here that, on average, recent high-precision computational methods such as PSORTb now have a lower error rate than laboratory methods. Conclusion We have performed the first focused comparison of genome-wide proteomic and computational methods for subcellular localization identification, and show that computational methods have now attained a level of precision that is exceeding that of high

  19. Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds

    Directory of Open Access Journals (Sweden)

    Miguel A. Ibeas

    2017-12-01

    Full Text Available Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds.

  20. Subcellular localization of Arabidopsis arogenate dehydratases suggests novel and non-enzymatic roles

    Science.gov (United States)

    Bross, Crystal D.; Howes, Travis R.; Abolhassani Rad, Sara; Kljakic, Ornela

    2017-01-01

    Abstract Arogenate dehydratases (ADTs) catalyze the final step in phenylalanine biosynthesis in plants. The Arabidopsis thaliana genome encodes a family of six ADTs capable of decarboxylating/dehydrating arogenate into phenylalanine. Using cyan fluorescent protein (CFP)-tagged proteins, the subcellular localization patterns of all six A. thaliana ADTs were investigated in intact Nicotiana benthamiana and A. thaliana leaf cells. We show that A. thaliana ADTs localize to stroma and stromules (stroma-filled tubules) of chloroplasts. This localization pattern is consistent with the enzymatic function of ADTs as many enzymes required for amino acid biosynthesis are primarily localized to chloroplasts, and stromules are thought to increase metabolite transport from chloroplasts to other cellular compartments. Furthermore, we provide evidence that ADTs have additional, non-enzymatic roles. ADT2 localizes in a ring around the equatorial plane of chloroplasts or to a chloroplast pole, which suggests that ADT2 is a component of the chloroplast division machinery. In addition to chloroplasts, ADT5 was also found in nuclei, again suggesting a non-enzymatic role for ADT5. We also show evidence that ADT5 is transported to the nucleus via stromules. We propose that ADT2 and ADT5 are moonlighting proteins that play an enzymatic role in phenylalanine biosynthesis and a second role in chloroplast division or transcriptional regulation, respectively. PMID:28338876

  1. Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds.

    Science.gov (United States)

    Ibeas, Miguel A; Grant-Grant, Susana; Navarro, Nathalia; Perez, M F; Roschzttardtz, Hannetz

    2017-01-01

    Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds.

  2. Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

    Science.gov (United States)

    Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

    2017-03-01

    Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein. Copyright © 2017 by

  3. Subcellular localization of L-selectin ligand in the endometrium implies a novel function for pinopodes in endometrial receptivity

    Directory of Open Access Journals (Sweden)

    Nejatbakhsh Reza

    2012-06-01

    Full Text Available Abstract Background Apical surfaces of human endometrial epithelium and endothelium are key elements for the initiation of molecular interactions to capture the blastocyst or leukocyte, respectively. The L-selectin adhesion system has been strongly proposed to play an important role in the initial steps of trophoblast adhesion and promotion of integrin-dependent processes, ultimately culminating in the establishment of the embryo-maternal interface. On the basis of these facts, we hypothesized a novel role for pinopodes as the first embryo-fetal contact sites to contain the highest subcellular expression of L-selectin ligand suggesting its role in early adhesion as predicted. Thus, the objective of this study was therefore to determine the subcellular pattern of distribution of the L-selectin ligand (MECA-79 in human endometrial apical membrane region during the window of implantation. Methods Endometrial biopsies of secretory phases from fertile females ranging in age between 25 and 42years were studied using several approaches, including scanning electron microscopy (SEM, immunostaining for light microscopy and transmission electron microscopy (TEM, and immunoblotting as well as statistical analysis of the area-related numerical densities of immunoreactive MECA-79-bound nanogolds to detect the expression pattern and the subcellular distribution pattern of L-selectin ligand (MECA-79 in human endometrium during the window of implantation. Results The endometrial biopsies were scored according the dating criteria of Noyes et al. by an experienced histologist. The SEM images of the midluteal phase specimens revealed that fully developed pinopodes were abundant in our samples. HRP-immunostaining and immunofluorescent staining as well as immunoblotting revealed that MECA-79 was expressed in the midluteal phase specimens. The results of immunogold TEM illustrated the expression of MECA-79 in human pinopodes in the midluteal phase and a higher area

  4. [Expression, subcellular localization and nuclear translocation of transcription factor up stream stimulatory factor-1 in odontoblasts].

    Science.gov (United States)

    Wu, Li-An; Wen, Ling-Ying; Yang, Fu-Sheng; Wang, Xiao-Jing; Fang, Jun

    2007-09-01

    To examine the expression and subcellular localization of transcription factor USF1 in odontoblasts and investigate whether nuclear translocation occurs under stimuli. Odontoblasts MDPC-23 were cultured on coverslips and divided into 2 groups. Group 1 received no stimuli, and group 2 was stimulated by nicotine with various concentrations respectively for 1h. Then the mountings of odontoblasts were prepared and immunocytochemical staining was performed with specific USF1 antibody via SABC method. Hela cells were used as positive control. The staining was positive in the cytoplasm of odontoblasts in group 1, but in the nuclei of Hela cells and in 100 mg/L nicotine-stimulated odontoblasts in group 2. There exists USF1 protein in odontoblasts, which locates in the cytoplasm and could translocate into nuclei under the stimulation of nicotine.

  5. Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds 1

    Science.gov (United States)

    Swain, Elisabeth; Li, Chun Ping; Poulton, Jonathan E.

    1992-01-01

    In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and d-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two β-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. Images Figure 5 Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 PMID:16652960

  6. Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds.

    Science.gov (United States)

    Swain, E; Li, C P; Poulton, J E

    1992-09-01

    In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and d-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two beta-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase.

  7. Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

    Directory of Open Access Journals (Sweden)

    Gonzalo P Solis

    Full Text Available Analyses of cultured cells and transgenic mice expressing prion protein (PrP deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1 the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2 the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and

  8. Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

    Directory of Open Access Journals (Sweden)

    José Andrés Morgado-Díaz

    2005-07-01

    Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

  9. Comparison of expressed human and mouse sodium/iodide sym-porters reveals differences in transport properties and subcellular localization

    Energy Technology Data Exchange (ETDEWEB)

    Dayem, M.; Basquin, C.; Navarro, V.; Carrier, P.; Marsault, R.; Lindenthal, S.; Pourcher, T. [Univ Nice Sophia Antipolis, Sch Med, CEA, DSV, iBEB, SBTN, TIRO, F-06107 Nice (France); Chang, P. [CNRS, UPMC Biol Dev, UMR 7009, F-06230 Villefranche Sur Mer (France); Huc, S.; Darrouzet, E. [CEA Valrho, DSV, iBEB, SBTN, F-30207 Bagnols Sur Ceze (France)

    2008-07-01

    The active transport of iodide from the blood stream into thyroid follicular cells is mediated by the Na{sup +}/I{sup -} sym-porter (NIS). We studied mouse NIS (mNIS) and found that it catalyzes iodide transport into transfected cells more efficiently than human NIS (hNIS). To further characterize this difference,we compared {sup 125}I, uptake in the transiently transfected human embryonic kidney (HEK) 293 cells. We found that the Vmax for mNIS was four times higher than that for hNIS, and that the iodide transport constant (Km) was 2-5-fold lower for hNIS than mNIS. We also performed immuno-cyto-localization studies and observed that the subcellular distribution of the two ortho-logs differed. While the mouse protein was predominantly found at the plasma membrane, its human ortho-log was intracellular in {approx} 40% of the expressing cells. Using cell surface protein-labeling assays, we found that the plasma membrane localization frequency of the mouse protein was only 2-5-fold higher than that of the human protein, and therefore cannot alone account for,x values. We reasoned that the difference in the obtained Vmax the observed difference could also be caused by a higher turnover number for iodide transport in the mouse protein. We then expressed and analyzed chimeric proteins. The data obtained with these constructs suggest that the iodide recognition site could be located in the region extending from the N-terminus to transmembrane domain 8, and that the region between transmembrane domain 5 and the C-terminus could play a role in the subcellular localization of the protein. (authors)

  10. CoBaltDB: Complete bacterial and archaeal orfeomes subcellular localization database and associated resources

    Directory of Open Access Journals (Sweden)

    Lucchetti-Miganeh Céline

    2010-03-01

    Full Text Available Abstract Background The functions of proteins are strongly related to their localization in cell compartments (for example the cytoplasm or membranes but the experimental determination of the sub-cellular localization of proteomes is laborious and expensive. A fast and low-cost alternative approach is in silico prediction, based on features of the protein primary sequences. However, biologists are confronted with a very large number of computational tools that use different methods that address various localization features with diverse specificities and sensitivities. As a result, exploiting these computer resources to predict protein localization accurately involves querying all tools and comparing every prediction output; this is a painstaking task. Therefore, we developed a comprehensive database, called CoBaltDB, that gathers all prediction outputs concerning complete prokaryotic proteomes. Description The current version of CoBaltDB integrates the results of 43 localization predictors for 784 complete bacterial and archaeal proteomes (2.548.292 proteins in total. CoBaltDB supplies a simple user-friendly interface for retrieving and exploring relevant information about predicted features (such as signal peptide cleavage sites and transmembrane segments. Data are organized into three work-sets ("specialized tools", "meta-tools" and "additional tools". The database can be queried using the organism name, a locus tag or a list of locus tags and may be browsed using numerous graphical and text displays. Conclusions With its new functionalities, CoBaltDB is a novel powerful platform that provides easy access to the results of multiple localization tools and support for predicting prokaryotic protein localizations with higher confidence than previously possible. CoBaltDB is available at http://www.umr6026.univ-rennes1.fr/english/home/research/basic/software/cobalten.

  11. Ligand-binding properties and subcellular localization of maize cytokinin receptors

    Science.gov (United States)

    Lomin, Sergey N.; Yonekura-Sakakibara, Keiko; Romanov, Georgy A.; Sakakibara, Hitoshi

    2011-01-01

    The ligand-binding properties of the maize (Zea mays L.) cytokinin receptors ZmHK1, ZmHK2, and ZmHK3a have been characterized using cytokinin binding assays with living cells or membrane fractions. According to affinity measurements, ZmHK1 preferred N6-(Δ2-isopentenyl)adenine (iP) and had nearly equal affinities to trans-zeatin (tZ) and cis-zeatin (cZ). ZmHK2 preferred tZ and iP to cZ, while ZmHK3a preferred iP. Only ZmHK2 had a high affinity to dihydrozeatin (DZ). Analysis of subcellular fractions from leaves and roots of maize seedlings revealed specific binding of tZ in the microsome fraction but not in chloroplasts or mitochondria. In competitive binding assays with microsomes, tZ and iP were potent competitors of [3H]tZ while cZ demonstrated significantly lower affinity; adenine was almost ineffective. The binding specificities of microsomes from leaf and root cells for cytokinins were consistent with the expression pattern of the ZmHKs and our results on individual receptor properties. Aqueous two-phase partitioning and sucrose density-gradient centrifugation followed by immunological detection with monoclonal antibody showed that ZmHK1 was associated with the endoplasmic reticulum (ER). This was corroborated by observations of the subcellular localization of ZmHK1 fusions with green fluorescent protein in maize protoplasts. All these data strongly suggest that at least a part of cytokinin perception occurs in the ER. PMID:21778179

  12. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    NARCIS (Netherlands)

    Matthews, G.D.; Gur, N.; Koopman, W.J.H.; Pines, O.; Vardimon, L.

    2010-01-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS

  13. SPA Proteins Affect the Subcellular Localization of COP1 in the COP1/SPA Ubiquitin Ligase Complex during Photomorphogenesis.

    Science.gov (United States)

    Balcerowicz, Martin; Kerner, Konstantin; Schenkel, Christian; Hoecker, Ute

    2017-07-01

    The Arabidopsis ( Arabidopsis thaliana ) COP1/SPA ubiquitin ligase is a central repressor that suppresses light signaling in darkness by targeting positive regulators of the light response, mainly transcription factors, for degradation. Light inactivates COP1/SPA, in part by excluding COP1 from the nucleus. SPA proteins are essential cofactors of COP1, but their exact role in the COP1/SPA complex is thus far unknown. To unravel a potential role of SPA proteins in COP1 nucleocytoplasmic partitioning, we monitored the subcellular localization of COP1 in a spa1234 quadruple mutant ( spaQn ). We analyzed a YFP-COP1-expressing transgenic line and endogenous COP1 after subcellular fractionation. In dark-grown seedlings, both YFP-COP1 and endogenous COP1 accumulated in the nucleus in the absence and presence of SPA proteins, indicating that SPA proteins are not required for nuclear localization of COP1 in darkness. In contrast, in white light-grown seedlings, spaQn mutants failed to relocalize COP1 from the nucleus to the cytoplasm. Hence, SPA proteins are necessary for the light-controlled change in COP1 subcellular localization. We conclude that SPA proteins have a dual role: (1) they are required for light-responsiveness of COP1 subcellular localization, and (2) they promote COP1 activity in darkness in a fashion that is independent of the nuclear import/nuclear retention of COP1. © 2017 American Society of Plant Biologists. All Rights Reserved.

  14. Protein-protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery

    Directory of Open Access Journals (Sweden)

    Lynn eRichardson

    2011-06-01

    Full Text Available The Endosomal Sorting Complex Required for Transport (ESCRT consists of several multi-protein subcomplexes which assemble sequentially at the endosomal surface and function in multivesicular body (MVB biogenesis. While ESCRT has been relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Here we explored the yeast two-hybrid protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery. We show that Arabidopsis ESCRT interactome possess a number of protein-protein interactions that are either conserved in yeasts and mammals or distinct to plants. We show also that most of the Arabidopsis ESCRT proteins examined at least partially localize to MVBs in plant cells when ectopically expressed on their own or co-expressed with other interacting ESCRT proteins, and some also induce abnormal MVB phenotypes, consistent with their proposed functional roles in MVB biogenesis. Overall, our results help define the plant ESCRT machinery by highlighting both conserved and unique features when compared to ESCRT in other evolutionarily diverse organisms, providing a foundation for further exploration of ESCRT in plants.

  15. Subcellular localization of acyl-CoA binding protein in Aspergillus oryzae is regulated by autophagy machinery.

    Science.gov (United States)

    Kawaguchi, Kouhei; Kikuma, Takashi; Higuchi, Yujiro; Takegawa, Kaoru; Kitamoto, Katsuhiko

    2016-11-04

    In eukaryotic cells, acyl-CoA binding protein (ACBP) is important for cellular activities, such as in lipid metabolism. In the industrially important fungus Aspergillus oryzae, the ACBP, known as AoACBP, has been biochemically characterized, but its physiological function is not known. In the present study, although we could not find any phenotype of AoACBP disruptants in the normal growth conditions, we examined the subcellular localization of AoACBP to understand its physiological function. Using an enhanced green fluorescent protein (EGFP)-tagged AoACBP construct we showed that AoACBP localized to punctate structures in the cytoplasm, some of which moved inside the cells in a microtubule-dependent manner. Further microscopic analyses showed that AoACBP-EGFP co-localized with the autophagy marker protein AoAtg8 tagged with red fluorescent protein (mDsRed). Expression of AoACBP-EGFP in disruptants of autophagy-related genes revealed aggregation of AoACBP-EGFP fluorescence in the cytoplasm of Aoatg1, Aoatg4 and Aoatg8 disruptant cells. However, in cells harboring disruption of Aoatg15, which encodes a lipase for autophagic body, puncta of AoACBP-EGFP fluorescence accumulated in vacuoles, indicating that AoACBP is transported to vacuoles via the autophagy machinery. Collectively, these results suggest the existence of a regulatory mechanism between AoACBP localization and autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. MultiLoc: prediction of protein subcellular localization using N-terminal targeting sequences, sequence motifs and amino acid composition.

    Science.gov (United States)

    Höglund, Annette; Dönnes, Pierre; Blum, Torsten; Adolph, Hans-Werner; Kohlbacher, Oliver

    2006-05-15

    Functional annotation of unknown proteins is a major goal in proteomics. A key annotation is the prediction of a protein's subcellular localization. Numerous prediction techniques have been developed, typically focusing on a single underlying biological aspect or predicting a subset of all possible localizations. An important step is taken towards emulating the protein sorting process by capturing and bringing together biologically relevant information, and addressing the clear need to improve prediction accuracy and localization coverage. Here we present a novel SVM-based approach for predicting subcellular localization, which integrates N-terminal targeting sequences, amino acid composition and protein sequence motifs. We show how this approach improves the prediction based on N-terminal targeting sequences, by comparing our method TargetLoc against existing methods. Furthermore, MultiLoc performs considerably better than comparable methods predicting all major eukaryotic subcellular localizations, and shows better or comparable results to methods that are specialized on fewer localizations or for one organism. http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc/

  17. Correlation of N-myc downstream-regulated gene 1 subcellular localization and lymph node metastases of colorectal neoplasms

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yan [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014 (China); Lv, Liyang [Department of Health, Jinan Military Area Command, Jinan 250022 (China); Du, Juan; Yue, Longtao [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014 (China); Cao, Lili, E-mail: cllly22@163.com [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014 (China)

    2013-09-20

    Highlights: •We clarified NDRG1 subcellular location in colorectal cancer. •We found the changes of NDRG1 distribution during colorectal cancer progression. •We clarified the correlation between NDRG1 distribution and lymph node metastasis. •It is possible that NDRG1 subcellular localization may determine its function. •Maybe NDRG1 is valuable early diagnostic markers for metastasis. -- Abstract: In colorectal neoplasms, N-myc downstream-regulated gene 1 (NDRG1) is a primarily cytoplasmic protein, but it is also expressed on the cell membrane and in the nucleus. NDRG1 is involved in various stages of tumor development in colorectal cancer, and it is possible that the different subcellular localizations may determine the function of NDRG1 protein. Here, we attempt to clarify the characteristics of NDRG1 protein subcellular localization during the progression of colorectal cancer. We examined NDRG1 expression in 49 colorectal cancer patients in cancerous, non-cancerous, and corresponding lymph node tissues. Cytoplasmic and membrane NDRG1 expression was higher in the lymph nodes with metastases than in those without metastases (P < 0.01). Nuclear NDRG1 expression in colorectal neoplasms was significantly higher than in the normal colorectal mucosa, and yet the normal colorectal mucosa showed no nuclear expression. Furthermore, our results showed higher cytoplasmic NDRG1 expression was better for differentiation, and higher membrane NDRG1 expression resulted in a greater possibility of lymph node metastasis. These data indicate that a certain relationship between the cytoplasmic and membrane expression of NDRG1 in lymph nodes exists with lymph node metastasis. NDRG1 expression may translocate from the membrane of the colorectal cancer cells to the nucleus, where it is involved in lymph node metastasis. Combination analysis of NDRG1 subcellular expression and clinical variables will help predict the incidence of lymph node metastasis.

  18. F-box protein specificity for g1 cyclins is dictated by subcellular localization.

    Directory of Open Access Journals (Sweden)

    Benjamin D Landry

    Full Text Available Levels of G1 cyclins fluctuate in response to environmental cues and couple mitotic signaling to cell cycle entry. The G1 cyclin Cln3 is a key regulator of cell size and cell cycle entry in budding yeast. Cln3 degradation is essential for proper cell cycle control; however, the mechanisms that control Cln3 degradation are largely unknown. Here we show that two SCF ubiquitin ligases, SCF(Cdc4 and SCF(Grr1, redundantly target Cln3 for degradation. While the F-box proteins (FBPs Cdc4 and Grr1 were previously thought to target non-overlapping sets of substrates, we find that Cdc4 and Grr1 each bind to all 3 G1 cyclins in cell extracts, yet only Cln3 is redundantly targeted in vivo, due in part to its nuclear localization. The related cyclin Cln2 is cytoplasmic and exclusively targeted by Grr1. However, Cdc4 can interact with Cdk-phosphorylated Cln2 and target it for degradation when cytoplasmic Cdc4 localization is forced in vivo. These findings suggest that Cdc4 and Grr1 may share additional redundant targets and, consistent with this possibility, grr1Δ cdc4-1 cells demonstrate a CLN3-independent synergistic growth defect. Our findings demonstrate that structurally distinct FBPs are capable of interacting with some of the same substrates; however, in vivo specificity is achieved in part by subcellular localization. Additionally, the FBPs Cdc4 and Grr1 are partially redundant for proliferation and viability, likely sharing additional redundant substrates whose degradation is important for cell cycle progression.

  19. Functional Characterization and Subcellular Localization of Poplar (Populus trichocarpa × Populus deltoides) Cinnamate 4-Hydroxylase1

    Science.gov (United States)

    Ro, Dae Kyun; Mah, Nancy; Ellis, Brian E.; Douglas, Carl J.

    2001-01-01

    Cinnamic acid 4-hydroxylase (C4H), a member of the cytochrome P450 monooxygenase superfamily, plays a central role in phenylpropanoid metabolism and lignin biosynthesis and possibly anchors a phenylpropanoid enzyme complex to the endoplasmic reticulum (ER). A full-length cDNA encoding C4H was isolated from a hybrid poplar (Populus trichocarpa × P. deltoides) young leaf cDNA library. RNA-blot analysis detected C4H transcripts in all organs tested, but the gene was most highly expressed in developing xylem. C4H expression was also strongly induced by elicitor-treatment in poplar cell cultures. To verify the catalytic activity of the putative C4H cDNA, two constructs, C4H and C4H fused to the FLAG epitope (C4H::FLAG), were expressed in yeast. Immunoblot analysis showed that C4H was present in the microsomal fraction and microsomal preparations from strains expressing both enzymes efficiently converted cinnamic acid to p-coumaric acid with high specific activities. To investigate the subcellular localization of C4H in vivo, a chimeric C4H-green fluorescent protein (GFP) gene was engineered and stably expressed in Arabidopsis. Confocal laser microscopy analysis clearly showed that in Arabidopsis the C4H::GFP chimeric enzyme was localized to the ER. When expressed in yeast, the C4H::GFP fusion enzyme was also active but displayed significantly lower specific activity than either C4H or C4H::FLAG in in vitro and in vivo enzyme assays. These data definitively show that C4H is localized to the ER in planta. PMID:11351095

  20. iLoc-Animal: a multi-label learning classifier for predicting subcellular localization of animal proteins.

    Science.gov (United States)

    Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

    2013-04-05

    Predicting protein subcellular localization is a challenging problem, particularly when query proteins have multi-label features meaning that they may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing methods can only be used to deal with the single-label proteins. Actually, multi-label proteins should not be ignored because they usually bear some special function worthy of in-depth studies. By introducing the "multi-label learning" approach, a new predictor, called iLoc-Animal, has been developed that can be used to deal with the systems containing both single- and multi-label animal (metazoan except human) proteins. Meanwhile, to measure the prediction quality of a multi-label system in a rigorous way, five indices were introduced; they are "Absolute-True", "Absolute-False" (or Hamming-Loss"), "Accuracy", "Precision", and "Recall". As a demonstration, the jackknife cross-validation was performed with iLoc-Animal on a benchmark dataset of animal proteins classified into the following 20 location sites: (1) acrosome, (2) cell membrane, (3) centriole, (4) centrosome, (5) cell cortex, (6) cytoplasm, (7) cytoskeleton, (8) endoplasmic reticulum, (9) endosome, (10) extracellular, (11) Golgi apparatus, (12) lysosome, (13) mitochondrion, (14) melanosome, (15) microsome, (16) nucleus, (17) peroxisome, (18) plasma membrane, (19) spindle, and (20) synapse, where many proteins belong to two or more locations. For such a complicated system, the outcomes achieved by iLoc-Animal for all the aforementioned five indices were quite encouraging, indicating that the predictor may become a useful tool in this area. It has not escaped our notice that the multi-label approach and the rigorous measurement metrics can also be used to investigate many other multi-label problems in molecular biology. As a user-friendly web-server, iLoc-Animal is freely accessible to the public at the web-site .

  1. Characterization of subcellular localization and stability of a splice variant of G alphai2

    Directory of Open Access Journals (Sweden)

    Wedegaertner Philip B

    2002-05-01

    Full Text Available Abstract Background Alternative mRNA splicing of αi2, a heterotrimeric G protein α subunit, has been shown to produce an additional protein, termed sαi2. In the sαi2 splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of αi2. Whereas αi2 is found predominantly at cellular plasma membranes, sαi2 has been localized to intracellular Golgi membranes, and the unique 35 amino acids of sαi2 have been suggested to constitute a specific targeting signal. Results This paper proposes and examines an alternative hypothesis: disruption of the normal C-terminus of αi2 produces an unstable protein that fails to localize to plasma membranes. sαi2 is poorly expressed upon transfection of cultured cells; however, radiolabeling indicated that αi2 and sαi2 undergo myristoylation, a co-translational modification, equally well suggesting that protein stability rather than translation is affected. Indeed, pulse-chase analysis indicates that sαi2 is more rapidly degraded compared to αi2. Co-expression of βγ rescues PM localization and increases expression of sαi2. In addition, αi2A327S, a mutant previously shown to be unstable and defective in guanine-nucleotide binding, and αi2(1–331, in which the C-terminal 24 amino acids of αi2 are deleted, show a similar pattern of subcellular localization as sαi2 (i.e., intracellular membranes rather than plasma membranes. Finally, sαi2 displays a propensity to localize to potential aggresome-like structures. Conclusions Thus, instead of the novel C-terminus of sαi2 functioning as a specific Golgi targeting signal, the results presented here indicate that the disruption of the normal C-terminus of αi2 causes mislocalization and rapid degradation of sαi2.

  2. Point mutations in GLI3 lead to misregulation of its subcellular localization.

    Directory of Open Access Journals (Sweden)

    Sybille Krauss

    Full Text Available BACKGROUND: Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS, or Pallister-Hall-syndrome (PHS. Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A regulates the nuclear localization and transcriptional activity a of GLI3 function. PRINCIPAL FINDINGS: We have shown recently that protein phosphatase 2A (PP2A and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-alpha4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. CONCLUSIONS: The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and

  3. Characterization, sub-cellular localization and expression profiling of the isoprenylcysteine methylesterase gene family in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Ma Wujun

    2010-09-01

    Full Text Available Abstract Background Isoprenylcysteine methylesterases (ICME demethylate prenylated protein in eukaryotic cell. Until now, knowledge about their molecular information, localization and expression pattern is largely unavailable in plant species. One ICME in Arabidopsis, encoded by At5g15860, has been identified recently. Over-expression of At5g15860 caused an ABA hypersensitive phenotype in transgenic Arabidopsis plants, indicating that it functions as a positive regulator of ABA signaling. Moreover, ABA induced the expression of this gene in Arabidopsis seedlings. The current study extends these findings by examining the sub-cellular localization, expression profiling, and physiological functions of ICME and two other ICME-like proteins, ICME-LIKE1 and ICME-LIKE2, which were encoded by two related genes At1g26120 and At3g02410, respectively. Results Bioinformatics investigations showed that the ICME and other two ICME-like homologs comprise a small subfamily of carboxylesterase (EC 3.1.1.1 in Arabidopsis. Sub-cellular localization of GFP tagged ICME and its homologs showed that the ICME and ICME-like proteins are intramembrane proteins predominantly localizing in the endoplasmic reticulum (ER and Golgi apparatus. Semi-quantitative and real-time quantitative PCR revealed that the ICME and ICME-like genes are expressed in all examined tissues, including roots, rosette leaves, cauline leaves, stems, flowers, and siliques, with differential expression levels. Within the gene family, the base transcript abundance of ICME-LIKE2 gene is very low with higher expression in reproductive organs (flowers and siliques. Time-course analysis uncovered that both ICME and ICME-like genes are up-regulated by mannitol, NaCl and ABA treatment, with ICME showing the highest level of up-regulation by these treatments. Heat stress resulted in up-regulation of the ICME gene significantly but down-regulation of the ICME-LIKE1 and ICME-LIKE2 genes. Cold and dehydration

  4. Arginine Methylation Controls the Subcellular Localization and Functions of the Oncoprotein Splicing Factor SF2/ASF▿ †

    Science.gov (United States)

    Sinha, Rahul; Allemand, Eric; Zhang, Zuo; Karni, Rotem; Myers, Michael P.; Krainer, Adrian R.

    2010-01-01

    Alternative splicing and posttranslational modifications (PTMs) are major sources of protein diversity in eukaryotic proteomes. The SR protein SF2/ASF is an oncoprotein that functions in pre-mRNA splicing, with additional roles in other posttranscriptional and translational events. Functional studies of SR protein PTMs have focused exclusively on the reversible phosphorylation of Ser residues in the C-terminal RS domain. We confirmed that human SF2/ASF is methylated at residues R93, R97, and R109, which were identified in a global proteomic analysis of Arg methylation, and further investigated whether these methylated residues regulate the properties of SF2/ASF. We show that the three arginines additively control the subcellular localization of SF2/ASF and that both the positive charge and the methylation state are important. Mutations that block methylation and remove the positive charge result in the cytoplasmic accumulation of SF2/ASF. The consequent decrease in nuclear SF2/ASF levels prevents it from modulating the alternative splicing of target genes, results in higher translation stimulation, and abrogates the enhancement of nonsense-mediated mRNA decay. This study addresses the mechanisms by which Arg methylation and the associated positive charge regulate the activities of SF2/ASF and emphasizes the significance of localization control for an oncoprotein with multiple functions in different cellular compartments. PMID:20308322

  5. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    Science.gov (United States)

    Matthews, Gideon D; Gur, Noa; Koopman, Werner J H; Pines, Ophry; Vardimon, Lily

    2010-02-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS gene, it is not clear how tissue-specific subcellular localization is achieved. Here we show that in chicken, which utilizes the uricotelic system, the GS transcripts of liver and brain cells are identical and, consistently, there is no difference in the amino acid sequence of the protein. The N-terminus of GS, which constitutes a 'weak' mitochondrial targeting signal (MTS), is sufficient to direct a chimeric protein to the mitochondria in hepatocytes and to the cytoplasm in astrocytes. Considering that a weak MTS is dependent on a highly negative mitochondrial membrane potential (DeltaPsi) for import, we examined the magnitude of DeltaPsi in hepatocytes and astrocytes. Our results unexpectedly revealed that DeltaPsi in hepatocytes is considerably more negative than that of astrocytes and that converting the targeting signal into 'strong' MTS abolished the capability to confer tissue-specific subcellular localization. We suggest that evolutional selection of weak MTS provided a tool for differential targeting of an identical protein by taking advantage of tissue-specific differences in DeltaPsi.

  6. SLC30A3 (ZnT3 oligomerization by dityrosine bonds regulates its subcellular localization and metal transport capacity.

    Directory of Open Access Journals (Sweden)

    Gloria Salazar

    2009-06-01

    Full Text Available Non-covalent and covalent homo-oligomerization of membrane proteins regulates their subcellular localization and function. Here, we described a novel oligomerization mechanism affecting solute carrier family 30 member 3/zinc transporter 3 (SLC30A3/ZnT3. Oligomerization was mediated by intermolecular covalent dityrosine bonds. Using mutagenized ZnT3 expressed in PC12 cells, we identified two critical tyrosine residues necessary for dityrosine-mediated ZnT3 oligomerization. ZnT3 carrying the Y372F mutation prevented ZnT3 oligomerization, decreased ZnT3 targeting to synaptic-like microvesicles (SLMVs, and decreased resistance to zinc toxicity. Strikingly, ZnT3 harboring the Y357F mutation behaved as a "gain-of-function" mutant as it displayed increased ZnT3 oligomerization, targeting to SLMVs, and increased resistance to zinc toxicity. Single and double tyrosine ZnT3 mutants indicate that the predominant dimeric species is formed between tyrosine 357 and 372. ZnT3 tyrosine dimerization was detected under normal conditions and it was enhanced by oxidative stress. Covalent species were also detected in other SLC30A zinc transporters localized in different subcellular compartments. These results indicate that covalent tyrosine dimerization of a SLC30A family member modulates its subcellular localization and zinc transport capacity. We propose that dityrosine-dependent membrane protein oligomerization may regulate the function of diverse membrane protein in normal and disease states.

  7. Immunocytochemical localization and subcellular site of lectin synthesis in developing wheat embryos

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Peumans, W.J.

    1986-04-01

    Appearance of wheat germ agglutinin (WGA) during wheat germ embryogenesis was studied using indirect immunocytochemical methods at the light and electron microscope levels. Developing embryos were labelled with (/sup 35/S) cysteine in pulse and pulse-chase experiments to study the synthesis and transport of the lectin to protein bodies (PB). The radical, and coleorhiza first accumulated WGA around 10 days post anthesis (DPA), while WGA was found in the epiblast and coleoptile 15 and 20 DPA respectively. At the subcellular level WGA can be seen first in a small developing PB which later fused with larger ones. WGA was distributed evenly throughout the PB. When tissue was pulse-labelled, fractionated on an isopycnic sucrose gradient and exposed to detergent, the incorporated radioactivity of released lectin coincided with the position of the endoplasmic reticulum (ER) marker enzyme NADH-cytochrome c reductase. Both radioactivity and enzyme activity shifted to a higher density in the presence of 2 mM Mg acetate, indicating that radioactive lectin was associated with the rough ER.

  8. pLoc-mGneg: Predict subcellular localization of Gram-negative bacterial proteins by deep gene ontology learning via general PseAAC.

    Science.gov (United States)

    Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

    2017-10-06

    Information of the proteins' subcellular localization is crucially important for revealing their biological functions in a cell, the basic unit of life. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to develop computational tools for timely identifying their subcellular locations based on the sequence information alone. The current study is focused on the Gram-negative bacterial proteins. Although considerable efforts have been made in protein subcellular prediction, the problem is far from being solved yet. This is because mounting evidences have indicated that many Gram-negative bacterial proteins exist in two or more location sites. Unfortunately, most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions important for both basic research and drug design. In this study, by using the multi-label theory, we developed a new predictor called "pLoc-mGneg" for predicting the subcellular localization of Gram-negative bacterial proteins with both single and multiple locations. Rigorous cross-validation on a high quality benchmark dataset indicated that the proposed predictor is remarkably superior to "iLoc-Gneg", the state-of-the-art predictor for the same purpose. For the convenience of most experimental scientists, a user-friendly web-server for the novel predictor has been established at http://www.jci-bioinfo.cn/pLoc-mGneg/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Tissue distribution and subcellular localization of the cardiac sodium channel during mouse heart development.

    Science.gov (United States)

    Domínguez, Jorge N; de la Rosa, Angel; Navarro, Francisco; Franco, Diego; Aránega, Amelia E

    2008-04-01

    The aim of this study was to analyse the mRNA expression levels and protein distribution of the cardiac sodium channel Scn5a/Nav1.5 during mouse cardiogenesis. Scn5a mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E17.5 as well as postnatal and adult hearts. In addition, Scn5a protein (Nav1.5) distribution was analysed by immunohistochemistry and confocal microscopy. Scn5a mRNA levels displayed a peak at stage E11.5, decreased during the subsequent stages and then steadily increased from E17.5 onwards, and throughout the postnatal to the adult stages. Immunohistochemistry experiments revealed comparable distribution of Nav1.5 between the different cardiac chambers at early embryonic stages. During the foetal stages, Nav1.5 showed an enhanced expression in the trabeculated myocardium and in the bundle branches. At the subcellular level, Nav1.5 and Scn1b double-immunostaining analysis is consistent with the presence of both sodium channel subunits in the T-tubule system and the intercalated discs. Our results demonstrate that the cardiac sodium channel, Nav1.5, shows a dynamic expression pattern during mouse heart development, indicating that it could play an important role in the acquisition of a mature pattern of conduction and contraction during cardiogenesis.

  10. Subcellular Localization of Cadmium in Chlorella vulgaris Beijerinck Strain Bt-09

    Directory of Open Access Journals (Sweden)

    P.B. Lintongan

    2004-06-01

    Full Text Available Growth response curves of Chlorella vulgaris Beijerinck strain Bt-09 to sublethal concentrations of cadmium were evaluated. The growth responses of this microalgal isolate was determined through analysis of chlorophyll a levels. Cadmium was effectively taken up by the cells as determined by Flame Atomic Absorption Spectrophotometry (F-AAS. Subcellular fractionation was undertaken to locate sites that accumulate cadmium.

  11. PA-GOSUB: a searchable database of model organism protein sequences with their predicted Gene Ontology molecular function and subcellular localization

    OpenAIRE

    Lu, Paul; Szafron, Duane; Greiner, Russell; Wishart, David S.; Fyshe, Alona; Pearcy, Brandon; Poulin, Brett; Eisner, Roman; Ngo, Danny; Lamb, Nicholas

    2004-01-01

    PA-GOSUB (Proteome Analyst: Gene Ontology Molecular Function and Subcellular Localization) is a publicly available, web-based, searchable and downloadable database that contains the sequences, predicted GO molecular functions and predicted subcellular localizations of more than 107 000 proteins from 10 model organisms (and growing), covering the major kingdoms and phyla for which annotated proteomes exist (http://www.cs.ualberta.ca/~bioinfo/PA/GOSUB). The PA-GOSUB database effectively expands...

  12. Studying subcellular detail in fixed astrocytes: Dissociation of morphologically intact glial cells (DIMIGs

    Directory of Open Access Journals (Sweden)

    Julia eHaseleu

    2013-05-01

    Full Text Available Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs, since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from antigen in surrounding non-glial cells. Yet, PAPs frequently display antigens related to motility and glia-synaptic interaction. Here, we describe the dissociation of morphologically intact glial cells (DIMIGs, permitting unambiguous antigen localization using epifluorescence microscopy. Astrocytes are dissociated from juvenile (p13-15 mouse cortex by applying papain treatment and cytospin centrifugation to attach the cells to a slide. The cells and their complete processes including the PAPs is thus projected in 2D. The entire procedure takes 2½-3 hours. We show by morphometry that the diameter of DIMIGs, including the PAPs is similar to that of astrocytes in situ. In contrast to cell culture, results derived from this procedure allow for direct conclusions relating to (i the presence of an antigen in cortical astrocytes, (ii subcellular antigen distribution, in particular when localized in the PAPs. The detailed resolution is shown in an exemplary study of the organization of the astrocytic cytoskeleton components actin, ezrin, tubulin, and GFAP. The distribution of connexin 43 in relation to a single astrocyte’s process tree is also investigated.

  13. Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs).

    Science.gov (United States)

    Haseleu, Julia; Anlauf, Enrico; Blaess, Sandra; Endl, Elmar; Derouiche, Amin

    2013-01-01

    Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs), since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from antigen in surrounding non-glial cells. Yet, PAPs frequently display antigens related to motility and glia-synaptic interaction. Here, we describe the dissociation of morphologically intact glial cells (DIMIGs), permitting unambiguous antigen localization using epifluorescence microscopy. Astrocytes are dissociated from juvenile (p13-15) mouse cortex by applying papain treatment and cytospin centrifugation to attach the cells to a slide. The cells and their complete processes including the PAPs is thus projected in 2D. The entire procedure takes 2.5-3 h. We show by morphometry that the diameter of DIMIGs, including the PAPs is similar to that of astrocytes in situ. In contrast to cell culture, results derived from this procedure allow for direct conclusions relating to (1) the presence of an antigen in cortical astrocytes, (2) subcellular antigen distribution, in particular when localized in the PAPs. The detailed resolution is shown in an exemplary study of the organization of the astrocytic cytoskeleton components actin, ezrin, tubulin, and GFAP. The distribution of connexin 43 in relation to a single astrocyte's process tree is also investigated.

  14. mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging

    Directory of Open Access Journals (Sweden)

    Yadav Rahul B

    2013-01-01

    Full Text Available Abstract Background The mammalian target of rapamycin (mTOR signalling pathway has a key role in cellular regulation and several diseases. While it is thought that Rheb GTPase regulates mTOR, acting immediately upstream, while raptor is immediately downstream of mTOR, direct interactions have yet to be verified in living cells, furthermore the localisation of Rheb has been reported to have only a cytoplasmic cellular localization. Results In this study a cytoplasmic as well as a significant sub-cellular nuclear mTOR localization was shown , utilizing green and red fluorescent protein (GFP and DsRed fusion and highly sensitive single photon counting fluorescence lifetime imaging microscopy (FLIM of live cells. The interaction of the mTORC1 components Rheb, mTOR and raptor, tagged with EGFP/DsRed was determined using fluorescence energy transfer-FLIM. The excited-state lifetime of EGFP-mTOR of ~2400 ps was reduced by energy transfer to ~2200 ps in the cytoplasm and to 2000 ps in the nucleus when co-expressed with DsRed-Rheb, similar results being obtained for co-expressed EGFP-mTOR and DsRed-raptor. The localization and distribution of mTOR was modified by amino acid withdrawal and re-addition but not by rapamycin. Conclusions The results illustrate the power of GFP-technology combined with FRET-FLIM imaging in the study of the interaction of signalling components in living cells, here providing evidence for a direct physical interaction between mTOR and Rheb and between mTOR and raptor in living cells for the first time.

  15. Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Renard, C.; Vanderhaeghe, H.J.; Claes, P.J.; Zenebergh, A.; Tulkens, P.M.

    1987-03-01

    beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, /sup 14/C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of /sup 14/C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partly consistent with the model of de Duve et al., in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections.

  16. Molecular cloning, subcellular localization and characterization of two adenylate kinases from cassava, Manihot esculenta Crantz cv. KU50.

    Science.gov (United States)

    Boonrueng, Channarong; Tangpranomkorn, Surachat; Yazhisai, Uthaman; Sirikantaramas, Supaart

    2016-10-01

    Adenylate kinase (ADK) is a phosphotransferase that plays an important role in cellular energy homeostasis. Many isozymes located in different subcellular compartments have been reported. In this study, we focus on the characterization of cassava (Manihot esculenta) ADKs. We found 15 ADKs that are publicly available in the African cassava genome database. We cloned two ADKs, namely MeADK1 and MeADK2, which are phylogenetically grouped together with the plastidial ADK in potato. Both MeADK1 and MeADK2 showed 66% identity in the amino acid sequences with plastidial ADK in potato. However, we demonstrated that they are localized to mitochondria using GFP fusions of MeADK1 and MeADK2. The Escherichia coli-produced recombinant MeADK1 and MeADK2 preferred forward reactions that produce ATP. They exhibited similar specific activities. The semi-quantitative RT-PCR analysis showed that MeADK1 and MeADK2 in 2-month-old leaves have similar expression patterns under a diurnal light-dark cycle. However, MeADK2 transcripts were expressed at much higher levels than MeADK1 in 5-month-old leaves and roots. Thus, we conclude that MeADK2 might play a vital role in energy homeostasis in cassava mitochondria. Copyright © 2016 Elsevier GmbH. All rights reserved.

  17. Cellular and Subcellular Level Localization of Maize Lipids and Metabolites Using High-Spatial Resolution MALDI Mass Spectrometry Imaging.

    Science.gov (United States)

    Dueñas, Maria Emilia; Feenstra, Adam D; Korte, Andrew R; Hinners, Paige; Lee, Young Jin

    2018-01-01

    Recent technological advances have pushed the achievable spatial resolution for mass spectrometry imaging (MSI) to cellular and subcellular levels. Direct visualization of maize tissues by this tool has provided key insights into the localization of metabolites and lipids. This chapter outlines methodology for sample preparation, data acquisition, and data analysis of maize tissue sections using high-spatial resolution matrix-assisted laser desorption ionization (MALDI)-MSI, as well as the incorporation of a multi-resolution optical system, which allows for simple inter-conversion between different resolution setups (5, 10, and 50 μm imaging).

  18. Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut.

    Science.gov (United States)

    Rahner, C; Mitic, L L; Anderson, J M

    2001-02-01

    Paracellular transport varies widely among epithelia of the gastrointestinal tract. We determined whether members of the claudin family of tight junction proteins are differentially expressed consistent with a potential role in creating these variable properties. Rabbit polyclonal antibodies were produced against peptides from claudins 2 through 5. The distribution of individual claudins was detected by immunoblotting, and their cell type and subcellular localization were determined by immunofluorescence on cryosections of rat liver, pancreas, stomach, and small and large intestine. All antibodies detected single bands of the expected size on immunoblots and were monospecific based on peptide competition studies. Immunoblotting detected strong differences among tissues in the expression level of each claudin. Immunolocalization confirmed these differences and revealed striking variations in expression patterns. In the liver, claudin 2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, claudin 3 is uniformly expressed, claudin 4 is absent, and claudin 5 is only expressed in endothelial junctions. In the pancreas, claudin 2 is only detected in junctions of the duct epithelia, claudin 5 only in junctions of acinar cells, whereas claudin 3 and 4 are in both. Among differences in the gut are a crypt-to-villus decrease in claudin 2, a highly restricted expression of claudin 4 to colonic surface cells, and the finding that some claudins can be junctional, lateral, or show a gradient in junctional vs. lateral localization along the crypt-to-villus surface axis. Claudins have very different expression patterns among and within gastrointestinal tissues. We propose these patterns underlie differences in paracellular permeability properties, such as electrical resistance and ion selectivity that would complement known differences in transcellular transport.

  19. Subcellular localization of extracytoplasmic proteins in monoderm bacteria: rational secretomics-based strategy for genomic and proteomic analyses.

    Directory of Open Access Journals (Sweden)

    Sandra Renier

    Full Text Available Genome-scale prediction of subcellular localization (SCL is not only useful for inferring protein function but also for supporting proteomic data. In line with the secretome concept, a rational and original analytical strategy mimicking the secretion steps that determine ultimate SCL was developed for Gram-positive (monoderm bacteria. Based on the biology of protein secretion, a flowchart and decision trees were designed considering (i membrane targeting, (ii protein secretion systems, (iii membrane retention, and (iv cell-wall retention by domains or post-translocational modifications, as well as (v incorporation to cell-surface supramolecular structures. Using Listeria monocytogenes as a case study, results were compared with known data set from SCL predictors and experimental proteomics. While in good agreement with experimental extracytoplasmic fractions, the secretomics-based method outperforms other genomic analyses, which were simply not intended to be as inclusive. Compared to all other localization predictors, this method does not only supply a static snapshot of protein SCL but also offers the full picture of the secretion process dynamics: (i the protein routing is detailed, (ii the number of distinct SCL and protein categories is comprehensive, (iii the description of protein type and topology is provided, (iv the SCL is unambiguously differentiated from the protein category, and (v the multiple SCL and protein category are fully considered. In that sense, the secretomics-based method is much more than a SCL predictor. Besides a major step forward in genomics and proteomics of protein secretion, the secretomics-based method appears as a strategy of choice to generate in silico hypotheses for experimental testing.

  20. Mapping the subcellular localization of Fe3O4@TiO2 nanoparticles by X-ray Fluorescence Microscopy

    Science.gov (United States)

    Yuan, Y.; Chen, S.; Gleber, S. C.; Lai, B.; Brister, K.; Flachenecker, C.; Wanzer, B.; Paunesku, T.; Vogt, S.; Woloschak, G. E.

    2013-10-01

    The targeted delivery of Fe3O4@TiO2 nanoparticles to cancer cells is an important step in their development as nanomedicines. We have synthesized nanoparticles that can bind the Epidermal Growth Factor Receptor, a cell surface protein that is overexpressed in many epithelial type cancers. In order to study the subcellular distribution of these nanoparticles, we have utilized the sub-micron resolution of X-ray Fluorescence Microscopy to map the location of Fe3O4@TiO2 NPs and other trace metal elements within HeLa cervical cancer cells. Here we demonstrate how the higher resolution of the newly installed Bionanoprobe at the Advanced Photon Source at Argonne National Laboratory can greatly improve our ability to distinguish intracellular nanoparticles and their spatial relationship with subcellular compartments.

  1. The glycine hinge of transmembrane segment 2 modulates the subcellular localization and gating properties in TREK channels.

    Science.gov (United States)

    Zhuo, Ren-Gong; Peng, Peng; Zheng, Jian-Quan; Zhang, Yun-Long; Wen, Lei; Wei, Xiao-Li; Ma, Xiao-Yun

    2017-08-26

    TWIK-Related K+ channels (TREK), including TREK-1 and TREK-2, belong to the TREK/TRAAK subclass of two-pore domain K+ (K2P) family. The important functions of transmembrane segment 4 (M4)-glycine hinge in TREK channel gating have been characterized, but the roles of M2-hinge (the equivalent residue of M4-hinge) remain unclear. Here, by characterizing the macroscopic currents, subcellular localization and gating properties of their M2-hinge mutants (G166A for TREK-1 and G196A for TREK-2), we investigated the functions of M2-hinge. G166A displayed decreased whole-cell currents, whereas no current was produced by G196A. Subcellular analysis indicated that both mutants were aggregated near the perinuclear region, and most of them were retented within the endoplasmic reticulum (ER). Next, to explore the roles of M2-hinge in the gating mechanism, we tested the responses of the related M2-hinge mutants to 2-Aminoethoxydiphenyl borate (2-APB) and extracellular pH alteration (ΔpHo). TREK-1mut7-G166A displayed reduced sensitivity to 2-APB activation, but similar sensitivity to ΔpHo, when compared with TREK-1mut7. WT-ΔpCt, a TREK-2 tandom dimer, was used to assess the function of M2-hinge in the cis-type gating of TREK-2. The sensitivities of G196A-ΔpCt to both 2-APB and ΔpHo decreased compared with WT-ΔpCt. Taken together, our results reveal that the M2-hinge of TREK channels control their macroscopic current, subcellular localization and gating process. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. In silico identification of new putative pathogenic variants in the NEU1 sialidase gene affecting enzyme function and subcellular localization.

    Science.gov (United States)

    Bonardi, Dario; Ravasio, Viola; Borsani, Giuseppe; d'Azzo, Alessandra; Bresciani, Roberto; Monti, Eugenio; Giacopuzzi, Edoardo

    2014-01-01

    The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes that remove the terminal sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes: sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate variants tested, only two resulted in significantly lower levels of sialidase activity (pC and c.700G>A. These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.

  3. In silico identification of new putative pathogenic variants in the NEU1 sialidase gene affecting enzyme function and subcellular localization.

    Directory of Open Access Journals (Sweden)

    Dario Bonardi

    Full Text Available The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes that remove the terminal sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550, a severe lysosomal storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes: sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G and the NHLBI GO Exome Sequencing Project (ESP. Together these two datasets comprise a cohort of 7595 sequenced individuals, making it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate variants tested, only two resulted in significantly lower levels of sialidase activity (pC and c.700G>A. These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.

  4. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

    Directory of Open Access Journals (Sweden)

    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  5. The SubCons webserver: A user friendly web interface for state-of-the-art subcellular localization prediction.

    Science.gov (United States)

    Salvatore, M; Shu, N; Elofsson, A

    2017-09-13

    SubCons is a recently developed method that predicts the subcellular localization of a protein. It combines predictions from four predictors using a Random Forest classifier. Here, we present the user-friendly web-interface implementation of SubCons. Starting from a protein sequence, the server rapidly predicts the subcellular localizations of an individual protein. In addition, the server accepts the submission of sets of proteins either by uploading the files or programmatically by using command line WSDL API scripts. This makes SubCons ideal for proteome wide analyses allowing the user to scan a whole proteome in few days. From the web page, it is also possible to download precalculated predictions for several eukaryotic organisms. To evaluate the performance of SubCons we present a benchmark of LocTree3 and SubCons using two recent mass-spectrometry based datasets of mouse and drosophila proteins. The server is available at http://subcons.bioinfo.se/. © 2017 The Protein Society.

  6. Subcellular Localization of Matrin 3 Containing Mutations Associated with ALS and Distal Myopathy.

    Directory of Open Access Journals (Sweden)

    M Carolina Gallego-Iradi

    Full Text Available Mutations in Matrin 3 [MATR3], an RNA- and DNA-binding protein normally localized to the nucleus, have been linked to amyotrophic lateral sclerosis (ALS and distal myopathies. In the present study, we have used transient transfection of cultured cell lines to examine the impact of different disease-causing mutations on the localization of Matrin 3 within cells.Using CHO and human H4 neuroglioma cell models, we find that ALS/myopathy mutations do not produce profound changes in the localization of the protein. Although we did observe variable levels of Matrin 3 in the cytoplasm either by immunostaining or visualization of fluorescently-tagged protein, the majority of cells expressing either wild-type (WT or mutant Matrin 3 showed nuclear localization of the protein. When cytoplasmic immunostaining, or fusion protein fluorescence, was seen in the cytoplasm, the stronger intensity of staining or fluorescence was usually evident in the nucleus. In ~80% of cells treated with sodium arsenite (Ars to induce cytoplasmic stress granules, the nuclear localization of WT and F115C mutant Matrin 3 was not disturbed. Notably, over-expression of mutant Matrin 3 did not induce the formation of obvious large inclusion-like structures in either the cytoplasm or nucleus.Our findings indicate that mutations in Matrin 3 that are associated with ALS and myopathy do not dramatically alter the normal localization of the protein or readily induce inclusion formation.

  7. iLoc-Euk: a multi-label classifier for predicting the subcellular localization of singleplex and multiplex eukaryotic proteins.

    Directory of Open Access Journals (Sweden)

    Kuo-Chen Chou

    Full Text Available Predicting protein subcellular localization is an important and difficult problem, particularly when query proteins may have the multiplex character, i.e., simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular location predictor can only be used to deal with the single-location or "singleplex" proteins. Actually, multiple-location or "multiplex" proteins should not be ignored because they usually posses some unique biological functions worthy of our special notice. By introducing the "multi-labeled learning" and "accumulation-layer scale", a new predictor, called iLoc-Euk, has been developed that can be used to deal with the systems containing both singleplex and multiplex proteins. As a demonstration, the jackknife cross-validation was performed with iLoc-Euk on a benchmark dataset of eukaryotic proteins classified into the following 22 location sites: (1 acrosome, (2 cell membrane, (3 cell wall, (4 centriole, (5 chloroplast, (6 cyanelle, (7 cytoplasm, (8 cytoskeleton, (9 endoplasmic reticulum, (10 endosome, (11 extracellular, (12 Golgi apparatus, (13 hydrogenosome, (14 lysosome, (15 melanosome, (16 microsome (17 mitochondrion, (18 nucleus, (19 peroxisome, (20 spindle pole body, (21 synapse, and (22 vacuole, where none of proteins included has ≥25% pairwise sequence identity to any other in a same subset. The overall success rate thus obtained by iLoc-Euk was 79%, which is significantly higher than that by any of the existing predictors that also have the capacity to deal with such a complicated and stringent system. As a user-friendly web-server, iLoc-Euk is freely accessible to the public at the web-site http://icpr.jci.edu.cn/bioinfo/iLoc-Euk. It is anticipated that iLoc-Euk may become a useful bioinformatics tool for Molecular Cell Biology, Proteomics, System Biology, and Drug Development Also, its novel approach will further stimulate the development of

  8. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Bhaskar,; Kumari, Neeti [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India); Goyal, Neena, E-mail: neenacdri@yahoo.com [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  9. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Soledad Natalia Gonzalez

    Full Text Available The enzyme of the pentose phosphate pathway (PPP ribulose-5-phosphate-epimerase (RPE is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å. Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological

  10. Imaging of Caenorhabditis elegans samples and sub-cellular localization of new generation photosensitizers for photodynamic therapy, using non-linear microscopy

    Science.gov (United States)

    Filippidis, G.; Kouloumentas, C.; Kapsokalyvas, D.; Voglis, G.; Tavernarakis, N.; Papazoglou, T. G.

    2005-08-01

    Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structure and the function of tissues and individual cells. The extended application of this novel, innovative technique by the biological community is limited due to the high price of commercial multiphoton microscopes. In this study, a compact, inexpensive and reliable setup utilizing femtosecond pulses for excitation was developed for the TPEF and SHG imaging of biological samples. Specific cell types of the nematode Caenorhabditis elegans were imaged. Detection of the endogenous structural proteins of the worm, which are responsible for observation of SHG signals, was achieved. Additionally, the binding of different photosensitizers in the HL-60 cell line was investigated, using non-linear microscopy. The sub-cellular localization of photosensitizers of a new generation, very promising for photodynamic therapy (PDT), (Hypericum perforatum L. extracts) was achieved. The sub-cellular localization of these novel photosensitizers was linked with their photodynamic action during PDT, and the possible mechanisms for cell killing have been elucidated.

  11. A comparative antibody analysis of Pannexin1 expression in four rat brain regions reveals varying subcellular localizations

    Directory of Open Access Journals (Sweden)

    Angela C Cone

    2013-02-01

    Full Text Available Pannexin1 (Panx1 channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide-field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on

  12. Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

    Science.gov (United States)

    Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

    2017-10-01

    Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

  13. Optogenetic Tools for Subcellular Applications in Neuroscience.

    Science.gov (United States)

    Rost, Benjamin R; Schneider-Warme, Franziska; Schmitz, Dietmar; Hegemann, Peter

    2017-11-01

    The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. MNK1 expression increases during cellular senescence and modulates the subcellular localization of hnRNP A1

    Energy Technology Data Exchange (ETDEWEB)

    Ziaei, Samira, E-mail: ziaeisamira@gmail.com [City College of New York, City University of New York, New York, NY (United States); The Graduate School and University Center of CUNY, New York, NY (United States); Shimada, Naoko, E-mail: lensdev@yahoo.co.jp [City College of New York, City University of New York, New York, NY (United States); Kucharavy, Herman, E-mail: veterduy@yahoo.com [City College of New York, City University of New York, New York, NY (United States); Hubbard, Karen, E-mail: khubbard@sci.ccny.cuny.edu [City College of New York, City University of New York, New York, NY (United States); The Graduate School and University Center of CUNY, New York, NY (United States)

    2012-03-10

    Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence. -- Highlights: Black-Right-Pointing-Pointer MNK1 and not MAPKAPK2 phosphorylates hnRNP A1. Black-Right-Pointing-Pointer MNK1 has elevated levels in senescent cells, this has not been reported previously. Black-Right-Pointing-Pointer MNK1 activity induces cytoplasmic accumulation of hnRNP A1 in senescent cells. Black-Right-Pointing-Pointer Altered cytoplasmic localization of hnRNP A1 may alter gene expression patterns. Black-Right-Pointing-Pointer Our studies may increase our understanding of RNA metabolism during cellular aging.

  15. [Subcellular localization, purification, and various catalitic properties of aspartate aminotransferase from Spirodela polyrhiza].

    Science.gov (United States)

    Rakhmanova, T I; Popova, T N; Semenikhina, A V

    2006-01-01

    Intracellular distribution of aspartate aminotransferase (AAT) in Spirodela polyrhiza (Lemnaceae), strain SJ, has been studied by differential centrifugation. The bulk of the enzyme (73% of total cellular content) was localized in the cytoplasm and 24% activity was localized in chloroplasts. Purified cytoplasmic and chloroplastic isozymes differed by their affinity for substrates. The reaction balance was shifted towards direct and reverse transamination in the cytoplasm and chloroplast, respectively. Competitive inhibition of AAT by excessive substrates and enzyme affinity modulation by certain intermediates of the tricarboxylic acid cycle (isocitrate, succinate, and citrate) were observed. Possible involvement of AAT isozymes in the coordination of carbon and nitrogen metabolism through the regulation of 2-oxoglutarate synthesis and utilization in different cellular compartments is discussed.

  16. Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

    Science.gov (United States)

    Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

    2013-09-01

    Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  17. Arabinogalactan Glycosyltransferases: Enzyme Assay, Protein-Protein Interaction, Subcellular Localization, and Perspectives for Application

    Directory of Open Access Journals (Sweden)

    Naomi Geshi

    2014-01-01

    Full Text Available Arabinogalactan proteins (AGPs are abundant extracellular proteoglycans that are found in most plant species and involved in many cellular processes, such as cell proliferation and survival, pattern formation, and growth, and in plant microbe interaction. AGPs are synthesized by posttranslational O-glycosylation of proteins and attached glycan part often constitutes greater than 90% of the molecule. Subtle altered glycan structures during development have been considered to function as developmental markers on the cell surface, but little is known concerning the molecular mechanisms. My group has been working on glycosylation enzymes (glycosyltransferases of AGPs to investigate glycan function of the molecule. This review summarizes the recent findings from my group as for AtGalT31A, AtGlcAT14A-C, and AtGalT29A of Arabidopsis thaliana with a specific focus on the (i biochemical enzyme activities; (ii subcellular compartments targeted by the glycosyltransferases; and (iii protein-protein interactions. I also discuss application aspect of glycosyltransferase in improving AGP-based product used in industry, for example, gum arabic.

  18. Heat shock modulates the subcellular localization, stability, and activity of HIPK2

    Energy Technology Data Exchange (ETDEWEB)

    Upadhyay, Mamta; Bhadauriya, Pratibha; Ganesh, Subramaniam, E-mail: sganesh@iitk.ac.in

    2016-04-15

    The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress – such as hypoxia, oxidative stress, or UV damage – is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 and the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors.

  19. Subcellular localization and displacement by diuretics of the peripheral benzodiazepine binding site (PBS) from rat kidney

    Energy Technology Data Exchange (ETDEWEB)

    Lukeman, S.; Fanestil, D.

    1986-03-05

    Although the PBS has been identified in many organs, its function and cellular location are speculative. Using rapid filtration, binding of (/sup 3/H)RO 5-4864 (*RO) (.75 nM) was assessed in four subcellular fractions (.3 mg/ml) derived from depapillated rat kidney by differential centrifugation: N (450g x 2 min), O (13,000 x 10), P (105,000 x 30), and S. The binding distribution was: N-18%, O-74%, P-6%, and S-2%. Marker enzyme analysis revealed that O was enriched in mitochondria (M), lysosomes (L), peroxisomes (P), and endoplasmic reticulum (ER), but not plasma membrane, and that N contained small amounts (10-15%) of markers for the above. Repeated washing of O removed ER enzymes but preserved *RO binding. O was further fractionated with centrifugation (57,000g x 4 hr) on a linear sucrose gradient (18-65%); *RO binding then comigrated with M but not P and L markers. Centrifugation of isolated M (5500 x 10 min) on another linear sucrose gradient (37-65%) gave low and high density bands, which contained 65% and 35% of *RO binding activity, resp. *RO binding in O was specific, saturable, reversible, and inhibited by diuretics. Inhibitors with the highest potency were indacrinone (K/sub d/ = 35 ..mu..M), hydrochlorothiazide (100 ..mu..M), and ethacrynic acid (325 ..mu..M). Low potency inhibitors (K/sub d/ greater than or equal to 1 mM) included amiloride, triamterene, furosemide, bumetanide, and ozolinone.

  20. Expression patterns and subcellular localization of carbonic anhydrases are developmentally regulated during tooth formation.

    Science.gov (United States)

    Reibring, Claes-Göran; El Shahawy, Maha; Hallberg, Kristina; Kannius-Janson, Marie; Nilsson, Jeanette; Parkkila, Seppo; Sly, William S; Waheed, Abdul; Linde, Anders; Gritli-Linde, Amel

    2014-01-01

    Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or

  1. Expression patterns and subcellular localization of carbonic anhydrases are developmentally regulated during tooth formation.

    Directory of Open Access Journals (Sweden)

    Claes-Göran Reibring

    Full Text Available Carbonic anhydrases (CAs play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for

  2. Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway

    Science.gov (United States)

    The NITROGEN LIMITATION ADAPTATION (NLA) protein is a RING-type E3 ubiquitin ligase that plays an essential role in the regulation of nitrogen and phosphate homeostasis. NLA is localized to two distinct subcellular sites, the plasma membrane and nucleus, and contains four distinct domains: i) a RING...

  3. Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank, E-mail: fkempken@bot.uni-kiel.de

    2013-08-30

    Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

  4. Static magnetic fields inhibit proliferation and disperse subcellular localization of gamma complex protein3 in cultured C2C12 myoblast cells.

    Science.gov (United States)

    Kim, SeungChan; Im, Wooseok

    2010-05-01

    Magnetic fields may delay the rate of cell cycle progression, and there are reports that magnetic fields induce neurite outgrowth in cultured neuronal cells. To demonstrate whether magnetic field also effects on myoblast cells in cell growth, C2C12 cell lines were cultured and 2000G static magnetic field was applied. After 48 h of incubation, both the WST-1 assay (0.01 magnetic fields inhibit the proliferation of cultured C2C12 cells. Immunocytochemistry for alpha and tubulin gamma complex protein (TUBA and GCP3) was made and applying a static magnetic field-dispersed tubulin GCP3 formation, a intracellular apparatus for tubulin structuring in cell division. This protein expression was not altered by western blot. This study indicates that applying a static magnetic field alters the subcellular localizing of GCP3, and may delay the cell growth in cultured C2C12 myoblast cells.

  5. Subcellular Localization of a Plant Catalase-Phenol Oxidase, AcCATPO, from Amaranthus and Identification of a Non-canonical Peroxisome Targeting Signal.

    Science.gov (United States)

    Chen, Ning; Teng, Xiao-Lu; Xiao, Xing-Guo

    2017-01-01

    AcCATPO is a plant catalase-phenol oxidase recently identified from red amaranth. Its physiological function remains unexplored. As the starting step of functional analysis, here we report its subcellular localization and a non-canonical targeting signal. Commonly used bioinformatics programs predicted a peroxisomal localization for AcCATPO, but failed in identification of canonical peroxisomal targeting signals (PTS). The C-terminal GFP tagging led the fusion protein AcCATPO-GFP to the cytosol and the nucleus, but N-terminal tagging directed the GFP-AcCATPO to peroxisomes and nuclei, in transgenic tobacco. Deleting the tripeptide (PTM) at the extreme C-terminus almost ruled out the peroxisomal localization of GFP-AcCATPOΔ3, and removing the C-terminal decapeptide completely excluded peroxisomes as the residence of GFP-AcCATPOΔ10. Furthermore, this decapeptide as a targeting signal could import GFP-10aa to the peroxisome exclusively. Taken together, these results demonstrate that AcCATPO is localized to the peroxisome and the nucleus, and its peroxisomal localization is attributed to a non-canonical PTS1, the C-terminal decapeptide which contains an internal SRL motif and a conserved tripeptide P-S/T-I/M at the extreme of C-terminus. This work may further the study as to the physiological function of AcCATPO, especially clarify its involvement in betalain biosynthesis, and provide a clue to elucidate more non-canonic PTS.

  6. Subcellular Localization of a Plant Catalase-Phenol Oxidase, AcCATPO, from Amaranthus and Identification of a Non-canonical Peroxisome Targeting Signal

    Science.gov (United States)

    Chen, Ning; Teng, Xiao-Lu; Xiao, Xing-Guo

    2017-01-01

    AcCATPO is a plant catalase-phenol oxidase recently identified from red amaranth. Its physiological function remains unexplored. As the starting step of functional analysis, here we report its subcellular localization and a non-canonical targeting signal. Commonly used bioinformatics programs predicted a peroxisomal localization for AcCATPO, but failed in identification of canonical peroxisomal targeting signals (PTS). The C-terminal GFP tagging led the fusion protein AcCATPO-GFP to the cytosol and the nucleus, but N-terminal tagging directed the GFP-AcCATPO to peroxisomes and nuclei, in transgenic tobacco. Deleting the tripeptide (PTM) at the extreme C-terminus almost ruled out the peroxisomal localization of GFP-AcCATPOΔ3, and removing the C-terminal decapeptide completely excluded peroxisomes as the residence of GFP-AcCATPOΔ10. Furthermore, this decapeptide as a targeting signal could import GFP-10aa to the peroxisome exclusively. Taken together, these results demonstrate that AcCATPO is localized to the peroxisome and the nucleus, and its peroxisomal localization is attributed to a non-canonical PTS1, the C-terminal decapeptide which contains an internal SRL motif and a conserved tripeptide P-S/T-I/M at the extreme of C-terminus. This work may further the study as to the physiological function of AcCATPO, especially clarify its involvement in betalain biosynthesis, and provide a clue to elucidate more non-canonic PTS. PMID:28824680

  7. Subcellular Localization of a Plant Catalase-Phenol Oxidase, AcCATPO, from Amaranthus and Identification of a Non-canonical Peroxisome Targeting Signal

    Directory of Open Access Journals (Sweden)

    Ning Chen

    2017-08-01

    Full Text Available AcCATPO is a plant catalase-phenol oxidase recently identified from red amaranth. Its physiological function remains unexplored. As the starting step of functional analysis, here we report its subcellular localization and a non-canonical targeting signal. Commonly used bioinformatics programs predicted a peroxisomal localization for AcCATPO, but failed in identification of canonical peroxisomal targeting signals (PTS. The C-terminal GFP tagging led the fusion protein AcCATPO-GFP to the cytosol and the nucleus, but N-terminal tagging directed the GFP-AcCATPO to peroxisomes and nuclei, in transgenic tobacco. Deleting the tripeptide (PTM at the extreme C-terminus almost ruled out the peroxisomal localization of GFP-AcCATPOΔ3, and removing the C-terminal decapeptide completely excluded peroxisomes as the residence of GFP-AcCATPOΔ10. Furthermore, this decapeptide as a targeting signal could import GFP-10aa to the peroxisome exclusively. Taken together, these results demonstrate that AcCATPO is localized to the peroxisome and the nucleus, and its peroxisomal localization is attributed to a non-canonical PTS1, the C-terminal decapeptide which contains an internal SRL motif and a conserved tripeptide P-S/T-I/M at the extreme of C-terminus. This work may further the study as to the physiological function of AcCATPO, especially clarify its involvement in betalain biosynthesis, and provide a clue to elucidate more non-canonic PTS.

  8. Identification and subcellular localization analysis of two rubber elongation factor isoforms on Hevea brasiliensis rubber particles.

    Science.gov (United States)

    Dai, Longjun; Nie, Zhiyi; Kang, Guijuan; Li, Yu; Zeng, Rizhong

    2017-02-01

    Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis (the Para rubber tree) and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis. 16 BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE separations and mass spectrometric identification had revealed that two REF isoforms shared similar amino acid sequences and common C-terminal sequences. In this study, the gene sequences encoding these two REF isoforms (one is 23.6 kDa in size with 222 amino acid residues and the other is 27.3 kDa in size with 258 amino acid residues) were obtained. Their proteins were relatively enriched by sequential extraction of the rubber particle proteins and separated by 16 BAC/SDS-PAGE. The localization of these isoforms on the surfaces of rubber particles was further verified by western blotting and immunogold electron microscopy, which demonstrated that these two REF isoforms are mainly located on the surfaces of larger rubber particles and that they bind more tightly to rubber particles than the most abundant REF and SRPP (small rubber particle protein). Copyright © 2016. Published by Elsevier Masson SAS.

  9. Identification and subcellular localization of paracellin-1 (claudin-16) in human salivary glands.

    Science.gov (United States)

    Kriegs, Jan Ole; Homann, Veronika; Kinne-Saffran, Evamaria; Kinne, Rolf K H

    2007-07-01

    Salivary calcium plays an important role in the pathogenesis of dental caries and the bio-mineralization of dental enamel and exposed dentin. The cellular and molecular basis of calcium secretion by the human salivary glands is, however, poorly understood. Recently a transcellular transport of calcium by the acinus cells has been proposed. In this paper we looked for evidence for paracellular calcium transport by investigating the presence and cellular localization of paracellin-1 (claudin-16) that has been implied in paracellular magnesium and calcium transport in the kidney. At the mRNA level, using RT-PCR with primers of appropriate sequence, paracellin-1 mRNA could be found in human Glandula parotis, Glandula submandibularis, Glandula labialis and Glandula sublingualis samples. In addition, a splice variant was detected in three out of 15 glands consisting of exons one and five of the paracellin gene. In immunohistochemical studies paracellin-1 colocalised in the salivary excretory ducts with the tight junction proteins ZO-1 and occludin suggesting a potential role in paracellular calcium and magnesium transport. In the acini no such colocalisation was observed; paracellin was instead detected at the basal poles of the cells, between cells of the same acinus as well as between cells of neighboring acini. At this location paracellin-1 might act as selectivity filter for the paracellular movement of ions and water during stimulated secretion. Thus, both in the ducts and in the acini a paracellular transport of calcium appears possible. Whether it occurs at all and the extent to which it contributes to the overall salivary calcium secretion remains, however, to be determined.

  10. Heterogeneity in the expression and subcellular localization of POLYOL/MONOSACCHARIDE TRANSPORTER genes in Lotus japonicus.

    Directory of Open Access Journals (Sweden)

    Lu Tian

    Full Text Available Polyols can serve as a means for the translocation of carbon skeletons and energy between source and sink organs as well as being osmoprotective solutes and antioxidants which may be involved in the resistance of some plants to biotic and abiotic stresses. Polyol/Monosaccharide transporter (PLT proteins previously identified in plants are involved in the loading of polyols into the phloem and are reported to be located in the plasma membrane. The functions of PLT proteins in leguminous plants are not yet clear. In this study, a total of 14 putative PLT genes (LjPLT1-14 were identified in the genome of Lotus japonicus and divided into 4 clades based on phylogenetic analysis. Different patterns of expression of LjPLT genes in various tissues were validated by qRT-PCR analysis. Four genes (LjPLT3, 4, 11, and 14 from clade II were expressed at much higher levels in nodule than in other tissues. Moreover, three of these genes (LjPLT3, 4, and 14 showed significantly increased expression in roots after inoculation with Mesorhizobium loti. Three genes (LjPLT1, 3, and 9 responded when salinity and/or osmotic stresses were applied to L. japonicus. Transient expression of GFP-LjPLT fusion constructs in Arabidopsis and Nicotiana benthamiana protoplasts indicated that the LjPLT1, LjPLT6 and LjPLT7 proteins are localized to the plasma membrane, but LjPLT2 (clade IV, LjPLT3, 4, 5 (clade II and LjPLT8 (clade III proteins possibly reside in the Golgi apparatus. The results suggest that members of the LjPLT gene family may be involved in different biological processes, several of which may potentially play roles in nodulation in this nitrogen-fixing legume.

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

    Science.gov (United States)

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

    2015-10-01

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

  12. Expression and subcellular localization of mammalian formin Fhod3 in the embryonic and adult heart.

    Directory of Open Access Journals (Sweden)

    Meikun Kan-o

    Full Text Available The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from non-muscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain.

  13. Characterization, subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

    DEFF Research Database (Denmark)

    Peracchia, G; Jensen, A B; Culiáñez-Macià, F A

    1999-01-01

    by using in-frame fusions of the maize CK2alpha subunit to the reporter gene encoding beta-glucuronidase (GUS) which were assayed in transiently transformed onion epidermal cells. Analysis of chimeric constructs identified one region containing a nuclear localization signal (NLS) that is highly conserved...

  14. Subcellular Localization and Biochemical Comparison of Cytosolic and Secreted Cytokinin Dehydrogenase Enzymes from Maize

    Science.gov (United States)

    Cytokinin dehydrogenase (CKX, EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in cells of each plant. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a t...

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

    Energy Technology Data Exchange (ETDEWEB)

    Carmo, Lívia A.S.; Dias, Felipe F.; Malta, Kássia K.; Amaral, Kátia B. [Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG (Brazil); Shamri, Revital; Weller, Peter F. [Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States); Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br [Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG (Brazil); Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States)

    2015-10-01

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

  16. The leukemogenic CALM/AF10 fusion protein alters the subcellular localization of the lymphoid regulator Ikaros.

    Science.gov (United States)

    Greif, P A; Tizazu, B; Krause, A; Kremmer, E; Bohlander, S K

    2008-05-01

    The t(10;11)(p13;q14) translocation leads to the fusion of the CALM and AF10 genes. This translocation can be found as the sole cytogenetic abnormality in acute lymphoblastic leukemia, acute myeloid leukemia and in malignant lymphomas. The expression of CALM/AF10 in primary murine bone marrow cells results in the development of an aggressive leukemia in a murine bone marrow transplantation model. Using a yeast two-hybrid screen, we identified the lymphoid regulator Ikaros as an AF10 interacting protein. Interestingly, Ikaros is required for normal development of lymphocytes, and aberrant expression of Ikaros has been found in leukemia. In a murine model, the expression of a dominant negative isoform of Ikaros causes leukemias and lymphomas. The Ikaros interaction domain of AF10 was mapped to the leucine zipper domain of AF10, which is required for malignant transformation both by the CALM/AF10 and the MLL/AF10 fusion proteins. The interaction between AF10 and Ikaros was confirmed by GST pull down and co-immunoprecipitation. Coexpression of CALM/AF10 but not of AF10 alters the subcellular localization of Ikaros in murine fibroblasts. The transcriptional repressor activity of Ikaros is reduced by AF10. These results suggest that CALM/AF10 might interfere with normal Ikaros function, and thereby block lymphoid differentiation in CALM/AF10 positive leukemias.

  17. ClubSub-P: Cluster-Based Subcellular Localization Prediction for Gram-Negative Bacteria and Archaea

    Science.gov (United States)

    Paramasivam, Nagarajan; Linke, Dirk

    2011-01-01

    The subcellular localization (SCL) of proteins provides important clues to their function in a cell. In our efforts to predict useful vaccine targets against Gram-negative bacteria, we noticed that misannotated start codons frequently lead to wrongly assigned SCLs. This and other problems in SCL prediction, such as the relatively high false-positive and false-negative rates of some tools, can be avoided by applying multiple prediction tools to groups of homologous proteins. Here we present ClubSub-P, an online database that combines existing SCL prediction tools into a consensus pipeline from more than 600 proteomes of fully sequenced microorganisms. On top of the consensus prediction at the level of single sequences, the tool uses clusters of homologous proteins from Gram-negative bacteria and from Archaea to eliminate false-positive and false-negative predictions. ClubSub-P can assign the SCL of proteins from Gram-negative bacteria and Archaea with high precision. The database is searchable, and can easily be expanded using either new bacterial genomes or new prediction tools as they become available. This will further improve the performance of the SCL prediction, as well as the detection of misannotated start codons and other annotation errors. ClubSub-P is available online at http://toolkit.tuebingen.mpg.de/clubsubp/ PMID:22073040

  18. A new method for predicting the subcellular localization of eukaryotic proteins with both single and multiple sites: Euk-mPLoc 2.0.

    Directory of Open Access Journals (Sweden)

    Kuo-Chen Chou

    Full Text Available Information of subcellular locations of proteins is important for in-depth studies of cell biology. It is very useful for proteomics, system biology and drug development as well. However, most existing methods for predicting protein subcellular location can only cover 5 to 12 location sites. Also, they are limited to deal with single-location proteins and hence failed to work for multiplex proteins, which can simultaneously exist at, or move between, two or more location sites. Actually, multiplex proteins of this kind usually posses some important biological functions worthy of our special notice. A new predictor called "Euk-mPLoc 2.0" is developed by hybridizing the gene ontology information, functional domain information, and sequential evolutionary information through three different modes of pseudo amino acid composition. It can be used to identify eukaryotic proteins among the following 22 locations: (1 acrosome, (2 cell wall, (3 centriole, (4 chloroplast, (5 cyanelle, (6 cytoplasm, (7 cytoskeleton, (8 endoplasmic reticulum, (9 endosome, (10 extracell, (11 Golgi apparatus, (12 hydrogenosome, (13 lysosome, (14 melanosome, (15 microsome (16 mitochondria, (17 nucleus, (18 peroxisome, (19 plasma membrane, (20 plastid, (21 spindle pole body, and (22 vacuole. Compared with the existing methods for predicting eukaryotic protein subcellular localization, the new predictor is much more powerful and flexible, particularly in dealing with proteins with multiple locations and proteins without available accession numbers. For a newly-constructed stringent benchmark dataset which contains both single- and multiple-location proteins and in which none of proteins has pairwise sequence identity to any other in a same location, the overall jackknife success rate achieved by Euk-mPLoc 2.0 is more than 24% higher than those by any of the existing predictors. As a user-friendly web-server, Euk-mPLoc 2.0 is freely accessible at http

  19. Cellular levels and binding of c-di-GMP control subcellular localization and activity of the Vibrio cholerae transcriptional regulator VpsT.

    Directory of Open Access Journals (Sweden)

    Nicholas J Shikuma

    Full Text Available The second messenger, cyclic diguanylate (c-di-GMP, regulates diverse cellular processes in bacteria. C-di-GMP is produced by diguanylate cyclases (DGCs, degraded by phosphodiesterases (PDEs, and receptors couple c-di-GMP production to cellular responses. In many bacteria, including Vibrio cholerae, multiple DGCs and PDEs contribute to c-di-GMP signaling, and it is currently unclear whether the compartmentalization of c-di-GMP signaling components is required to mediate c-di-GMP signal transduction. In this study we show that the transcriptional regulator, VpsT, requires c-di-GMP binding for subcellular localization and activity. Only the additive deletion of five DGCs markedly decreases the localization of VpsT, while single deletions of each DGC do not impact VpsT localization. Moreover, mutations in residues required for c-di-GMP binding, c-di-GMP-stabilized dimerization and DNA binding of VpsT abrogate wild type localization and activity. VpsT does not co-localize or interact with DGCs suggesting that c-di-GMP from these DGCs diffuses to VpsT, supporting a model in which c-di-GMP acts at a distance. Furthermore, VpsT localization in a heterologous host, Escherichia coli, requires a catalytically active DGC and is enhanced by the presence of VpsT-target sequences. Our data show that c-di-GMP signaling can be executed through an additive cellular c-di-GMP level from multiple DGCs affecting the localization and activity of a c-di-GMP receptor and furthers our understanding of the mechanisms of second messenger signaling.

  20. Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues.

    Science.gov (United States)

    Tarquini, Giulia; Ermacora, Paolo; Bianchi, Gian Luca; De Amicis, Francesca; Pagliari, Laura; Martini, Marta; Loschi, Alberto; Saldarelli, Pasquale; Loi, Nazia; Musetti, Rita

    2017-12-22

    Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.

  1. Anks3 alters the sub-cellular localization of the Nek7 kinase

    Energy Technology Data Exchange (ETDEWEB)

    Ramachandran, Haribaskar; Engel, Christina; Müller, Barbara [Renal Division, Department of Medicine, University Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg (Germany); Dengjel, Jörn [Department of Dermatology, University Freiburg Medical Center and Center of Biological Systems Analysis, Habsburgerstr. 49, 79104 Freiburg (Germany); Walz, Gerd [Renal Division, Department of Medicine, University Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg (Germany); Center for Biological Signaling Studies (BIOSS), Albertstr. 19, 79104 Freiburg (Germany); Yakulov, Toma A., E-mail: toma.antonov.yakulov@uniklinik-freiburg.de [Renal Division, Department of Medicine, University Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg (Germany)

    2015-08-28

    Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease, and a frequent cause of end-stage renal failure in children. To date, 17 NPH-associated gene products (NPHPs) have been identified. Most NPHPs participate in large multi-protein complexes that localize to the cilium and/or basal body; however, the precise composition of these complexes and their biological function remain largely unknown. We recently observed that the ankyrin repeat protein Anks3 interacts with the NPH family member Anks6. Both Anks3 and Anks6 form complexes with multiple other NPHPs, suggesting that both proteins function in similar or overlapping signaling pathways. Here, we show that Anks3, but not Anks6 interacted with the NIMA-related kinase Nek7, and was heavily modified in the presence of Nek7, resulting in an approximately 20 kD increase in molecular weight. Although mass spectrometry revealed increased serine and threonine phosphorylation of Anks3 primarily within the N-terminal ankyrin repeats also required for Nek7 interaction, the molecular weight increase occurred even in the presence of a kinase-dead Nek7 mutant, indicating that this modification was not caused by Nek7-dependent Anks3 phosphorylation. Furthermore, the Anks3 modification was specific for Nek7, and did not occur in the presence of Nek8. Importantly, Anks3 retained Nek7 in the cytoplasm, suggesting that, Nek7 triggers the modification of Anks3, which in turn prevents the nuclear localization of Nek7. - Highlights: • Anks3 interacted with Nek7 kinase, and was heavily modified in the presence of Nek7. • Anks3 N-terminal ankyrin repeats, but not SAM domain required for Nek7 interaction. • Nek7 increased Ser/Thr phosphorylation of Anks3 primarily within ankyrin domain. • Interaction with Anks3 led to cytoplasmic retention and nuclear exclusion of Nek7.

  2. Study of acetowhitening mechanisms in live mammalian cells with label-free subcellular-level multimodal nonlinear optical microscopy

    Science.gov (United States)

    Lin, Jian; Teh, Sengkhoon; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2015-03-01

    The tissue acetowhitening effect in acetic acid instillation procedure is a simple and economic method for neoplasia detection and has been clinically utilized since 1925. It is suspected that the optical property (e.g. scattering) change in acetowhitening is due to coagulation of intracellular proteins, but no experimental proof has been reported yet. In this work, we use third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) to investigate the acetowhitening phenomenon induced by acidic acid in live mammalian cells without labeling. We studied the acetowhitening effect with different acetic acid concentrations and the co-localized TPEF and THG imaging on tryptophan and NADH at subcellular-level reveals that the acetowhitening phenomenon is highly related with proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.

  3. Expression of a rice glutaredoxin in aleurone layers of developing and mature seeds: subcellular localization and possible functions in antioxidant defense.

    Science.gov (United States)

    Morita, Shigeto; Yamashita, Yuki; Fujiki, Masayoshi; Todaka, Rie; Nishikawa, Yuri; Hosoki, Ayaka; Yabe, Chisato; Nakamura, Jun'ichi; Kawamura, Kazuyoshi; Suwastika, I Nengah; Sato, Masa H; Masumura, Takehiro; Ogihara, Yasunari; Tanaka, Kunisuke; Satoh, Shigeru

    2015-11-01

    A rice glutaredoxin isoform (OsGrxC2;2) with antioxidant capacity is expressed abundantly in seed tissues and is localized to storage vacuoles in aleurone layers in developing and mature seeds. Seed tissues undergo drastic water loss at the late stage of seed development, and thus need to tolerate oxidative injuries associated with desiccation. We previously found a rice glutaredoxin isoform, OsGrxC2;2, as a gene expressed abundantly in developing seeds. Since glutaredoxin is involved in antioxidant defense, in the present study we investigated the subcellular localization and expression profile of OsGrxC2;2 and whether OsGrxC2;2 has a role in the defense against reactive oxygen species. Western blotting and immunohistochemistry revealed that the OsGrxC2;2 protein accumulated at a high level in the embryo and aleurone layers of developing and mature seeds. The OsGrxC2;2 in developing seeds was particularly localized to aleurone grains, which are storage organelles derived from vacuoles. Overexpression of OsGrxC2;2 resulted in an enhanced tolerance to menadione in yeast and methyl viologen in green leaves of transgenic rice plants. These results suggest that OsGrxC2;2 participates in the defense against oxidative stress in developing and mature seeds.

  4. Subcellular localization and Ser-137 phosphorylation regulate tumor-suppressive activity of profilin-1.

    Science.gov (United States)

    Diamond, Marc I; Cai, Shirong; Boudreau, Aaron; Carey, Clifton J; Lyle, Nicholas; Pappu, Rohit V; Swamidass, S Joshua; Bissell, Mina; Piwnica-Worms, Helen; Shao, Jieya

    2015-04-03

    The actin-binding protein profilin-1 (Pfn1) inhibits tumor growth and yet is also required for cell proliferation and survival, an apparent paradox. We previously identified Ser-137 of Pfn1 as a phosphorylation site within the poly-l-proline (PLP) binding pocket. Here we confirm that Ser-137 phosphorylation disrupts Pfn1 binding to its PLP-containing ligands with little effect on actin binding. We find in mouse xenografts of breast cancer cells that mimicking Ser-137 phosphorylation abolishes cell cycle arrest and apoptotic sensitization by Pfn1 and confers a growth advantage to tumors. This indicates a previously unrecognized role of PLP binding in Pfn1 antitumor effects. Spatial restriction of Pfn1 to the nucleus or cytoplasm indicates that inhibition of tumor cell growth by Pfn1 requires its nuclear localization, and this activity is abolished by a phosphomimetic mutation on Ser-137. In contrast, cytoplasmic Pfn1 lacks inhibitory effects on tumor cell growth but rescues morphological and proliferative defects of PFN1 null mouse chondrocytes. These results help reconcile seemingly opposed cellular effects of Pfn1, provide new insights into the antitumor mechanism of Pfn1, and implicate Ser-137 phosphorylation as a potential therapeutic target for breast cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.

    Science.gov (United States)

    Harris, Thurl E; Huffman, Todd A; Chi, An; Shabanowitz, Jeffrey; Hunt, Donald F; Kumar, Anil; Lawrence, John C

    2007-01-05

    Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.

  6. Construction of a tagging system for subcellular localization of proteins encoded by open reading frames.

    Science.gov (United States)

    Chuang, C H; Hsu, S C; Hsu, C L; Hsu, T C; Syu, W J

    2001-01-01

    We have previously characterized a monoclonal antibody (SC1D7) that is directed to maltose-binding protein (MBP) of Escherichia coli and other closely related enteric bacteria. SC1D7 does not cross-react with proteins in eucaryotes and appears to be a highly specific tool in immunochemical analyses. To better map the epitope, we took advantage of an available plasmid, pMAL-c2, that encodes the E. coli MBP-coding sequence and constructed plasmids to express MBP fragments. A construct containing the N-terminal portion of MBP does not react with SC1D7, whereas a second construct expressing glutathione S-transferase fused with the C-terminal half of MBP does react with SC1D7. To precisely define the epitope, random peptides displayed on M13 were used to react with SC1D7. Sequences of reactive peptides were aligned, and a consensus sequence of XDXRIPX was deduced. This sequence matches MBP with an amino acid stretch of KDPRIAA. To consolidate the mapping result, a sequence encoding this epitope was inserted into an expression vector and the resulting recombinant protein did react with SC1D7. Thereafter, this epitope was incorporated into a eucaryotic expression plasmid containing a previously defined hepatitis delta virus epitope for protein tagging. This two-epitope-tagging vector is useful in various molecular analyses. We demonstrate its usage for localization of a bacterial virulence factor in host cells. This vector should be applicable for high-throughput characterization of new open reading frames found in genome sequencing. Copyright 2001 National Science Council, ROC and S. Karger AG, Basel

  7. Mutant analysis, protein-protein interactions and subcellular localization of the Arabidopsis B sister (ABS) protein.

    Science.gov (United States)

    Kaufmann, Kerstin; Anfang, Nicole; Saedler, Heinz; Theissen, Günter

    2005-09-01

    Recently, close relatives of class B floral homeotic genes, termed B(sister) genes, have been identified in both angiosperms and gymnosperms. In contrast to the B genes themselves, B(sister) genes are exclusively expressed in female reproductive organs, especially in the envelopes or integuments surrounding the ovules. This suggests an important ancient function in ovule or seed development for B(sister) genes, which has been conserved for about 300 million years. However, investigation of the first loss-of-function mutant for a B(sister) gene (ABS/TT16 from Arabidopsis) revealed only a weak phenotype affecting endothelium formation. Here, we present an analysis of two additional mutant alleles, which corroborates this weak phenotype. Transgenic plants that ectopically express ABS show changes in the growth and identity of floral organs, suggesting that ABS can interact with floral homeotic proteins. Yeast-two-hybrid and three-hybrid analyses indicated that ABS can form dimers with SEPALLATA (SEP) floral homeotic proteins and multimeric complexes that also include the AGAMOUS-like proteins SEEDSTICK (STK) or SHATTERPROOF1/2 (SHP1, SHP2). These data suggest that the formation of multimeric transcription factor complexes might be a general phenomenon among MIKC-type MADS-domain proteins in angiosperms. Heterodimerization of ABS with SEP3 was confirmed by gel retardation assays. Fusion proteins tagged with CFP (Cyan Fluorescent Protein) and YFP (Yellow Fluorescent Protein) in Arabidopsis protoplasts showed that ABS is localized in the nucleus. Phylogenetic analysis revealed the presence of a structurally deviant, but closely related, paralogue of ABS in the Arabidopsis genome. Thus the evolutionary developmental genetics of B(sister) genes can probably only be understood as part of a complex and redundant gene network that may govern ovule formation in a conserved manner, which has yet to be fully explored.

  8. Subcellular localization of cadmium in the root cells of Allium cepa ...

    Indian Academy of Sciences (India)

    Unknown

    mulated by many cereals, potatoes, vegetables and fruits and that humans take up at least 70% of the Cd which originates from plant food (Wagner 1993). Studies ... which the samples were dehydrated in graded acetone and embedded in ERL epoxy resin (Spurr 1969). Ultrathin sections (80 nm thick) were cut on an ultra-.

  9. Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes

    NARCIS (Netherlands)

    Cormont, M; Bortoluzzi, M N; Gautier, N; Mari, M; van Obberghen, E; Le Marchand-Brustel, Y

    1996-01-01

    A role for Rab4 in the translocation of the glucose transporter Glut4 induced by insulin has been recently proposed. To study more directly the role of this small GTPase, freshly isolated adipocytes were transiently transfected with the cDNAs of both an epitope-tagged Glut4-myc and Rab4, a system

  10. Cannabinoid control of brain bioenergetics: Exploring the subcellular localization of the CB1 receptor

    Science.gov (United States)

    Hebert-Chatelain, Etienne; Reguero, Leire; Puente, Nagore; Lutz, Beat; Chaouloff, Francis; Rossignol, Rodrigue; Piazza, Pier-Vincenzo; Benard, Giovanni; Grandes, Pedro; Marsicano, Giovanni

    2014-01-01

    Brain mitochondrial activity is centrally involved in the central control of energy balance. When studying mitochondrial functions in the brain, however, discrepant results might be obtained, depending on the experimental approaches. For instance, immunostaining experiments and biochemical isolation of organelles expose investigators to risks of false positive and/or false negative results. As an example, the functional presence of cannabinoid type 1 (CB1) receptors on brain mitochondrial membranes (mtCB1) was recently reported and rapidly challenged, claiming that the original observation was likely due to artifact results. Here, we addressed this issue by directly comparing the procedures used in the two studies. Our results show that the use of appropriate controls and quantifications allows detecting mtCB1 receptor with CB1 receptor antibodies, and that, if mitochondrial fractions are enriched and purified, CB1 receptor agonists reliably decrease respiration in brain mitochondria. These data further underline the importance of adapted experimental procedures to study brain mitochondrial functions. PMID:24944910

  11. STUDY OF SUBCELLULAR DISTRIBUTION OF CRYSTALLINE MESO-TETRA(3-PYRIDYLBACTERIOCHLORIN NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Yu. S. Maklygina

    2016-01-01

    Full Text Available The results of the study of subcellular distribution of molecular meso-tetra(3-pyridylbacteriochlorin nanocrystals proposed as therapeutic agents for photodynamic therapy are represented in the article. Investigations and measurement of spectroscopic properties of molecular crystals of near-infrared photosensitizer were conducted using special device complex based on fiber-optic spectrometer. Investigation and analysis of the pattern of subcellular accumulation of meso-tetra(3-pyridylbacteriochlorin in molecular (dimethyl sulfoxide (DMSO as solvent and nanocrystalline forms on different cell lines: human monocytes (THP-1, human cervical cancer cells (HeLa and mouse malignant brain tumor cells (glioma C6. The dynamics of subcellylar accumulation of the agent at concentration of 5 and 10 mg/l was assessed with laser microscope-spectrum analyzer and by confocal microscopy. The study showed that in the course of interaction with cell lines molecular nanocrystals of the agent developed ability to fluorescence. Hence, in the cellular environment meso-tetra(3-pyridyl bacteriochlorin nanoparticles became phototoxic giving opportunities for their use for fluorescence diagnosis and photodynamic therapy. Specific role of meso-tetra(3-pyridylbacteriochlorin in the range of photosensitizers is determined by its spectral characteristics, i.e. absorption and fluorescence in near-infrared band, which allows measuring and affecting on deeper layers of biotissue. Thus, the use of meso-tetra(3-pyridylbacteriochlorin nanoparticles as nanophotosensitizers may improve the efficacy of diagnosis and treatment of deep-seated tumors.

  12. Potential involvement of kinesin-1 in the regulation of subcellular localization of Girdin

    Energy Technology Data Exchange (ETDEWEB)

    Muramatsu, Aya [Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Enomoto, Atsushi, E-mail: enomoto@iar.nagoya-u.ac.jp [Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Kato, Takuya; Weng, Liang [Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Kuroda, Keisuke [Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Asai, Naoya; Asai, Masato; Mii, Shinji [Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Takahashi, Masahide, E-mail: mtakaha@med.nagoya-u.ac.jp [Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan)

    2015-08-07

    Girdin is an actin-binding protein that has multiple functions in postnatal neural development and cancer progression. We previously showed that Girdin is a regulator of migration for neuroblasts born from neural stem cells in the subventricular zone (SVZ) and the dentate gyrus of the hippocampus in the postnatal brain. Despite a growing list of Girdin-interacting proteins, the mechanism of Girdin-mediated migration has not been fully elucidated. Girdin interacts with Disrupted-In-Schizophrenia 1 and partitioning-defective 3, both of which have been shown to interact with the kinesin microtubule motor proteins. Based on this, we have identified that Girdin also interacts with kinesin-1, a member of neuronal kinesin proteins. Although a direct interaction of Girdin and kinesin-1 has not been determined, it is of interest to find that Girdin loss-of-function mutant mice with the mutation of a basic amino acid residue-rich region (Basic mut mice) exhibit limited interaction with kinesin-1. Furthermore, expression of a kinesin-1 mutant with motor defects, leads to Girdin mislocalization. Finally, consistent with previous studies on the role of kinesin proteins in trafficking a cell–cell adhesion molecule N-cadherin, Basic mut mice showed an aberrant expression pattern of N-cadherin in migrating SVZ neuroblasts. These findings suggest a potential role of Girdin/kinesin-1 interaction in the regulation of neuroblast migration in the postnatal brain. - Highlights: • Girdin is a regulator of migration for neuroblasts in the postnatal brain. • Girdin interacts with kinesin-1, a member of neuronal kinesin proteins. • Girdin mutant mice showed an aberrant expression of N-cadherin in neuroblasts.

  13. Itm2a expression in the developing mouse first lower molar, and the subcellular localization of Itm2a in mouse dental epithelial cells.

    Directory of Open Access Journals (Sweden)

    Makiko Kihara

    Full Text Available Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6 cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the inner enamel epithelium of the enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with enamel and dentin matrices in the secretory pathway.

  14. Itm2a Expression in the Developing Mouse First Lower Molar, and the Subcellular Localization of Itm2a in Mouse Dental Epithelial Cells

    Science.gov (United States)

    Kihara, Makiko; Kiyoshima, Tamotsu; Nagata, Kengo; Wada, Hiroko; Fujiwara, Hiroaki; Hasegawa, Kana; Someya, Hirotaka; Takahashi, Ichiro; Sakai, Hidetaka

    2014-01-01

    Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6) cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the inner enamel epithelium of the enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with enamel and dentin matrices in the secretory pathway. PMID:25079563

  15. Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM, TEM/X-EDS, and NanoSIMS.

    Science.gov (United States)

    Penen, Florent; Malherbe, Julien; Isaure, Marie-Pierre; Dobritzsch, Dirk; Bertalan, Ivo; Gontier, Etienne; Le Coustumer, Philippe; Schaumlöffel, Dirk

    2016-09-01

    Chemical bioimaging offers an important contribution to the investigation of biochemical functions, biosorption and bioaccumulation processes of trace elements via their localization at the cellular and even at the subcellular level. This paper describes the combined use of high contrast transmission electron microscopy (HC-TEM), energy dispersive X-ray spectroscopy (X-EDS), and nano secondary ion mass spectrometry (NanoSIMS) applied to a model organism, the unicellular green algae Chlamydomonas reinhardtii. HC-TEM providing a lateral resolution of 1nm was used for imaging the ultrastructure of algae cells which have diameters of 5-10μm. TEM coupled to X-EDS (TEM/X-EDS) combined textural (morphology and size) analysis with detection of Ca, P, K, Mg, Fe, and Zn in selected subcellular granules using an X-EDS probe size of approx. 1μm. However, instrumental sensitivity was at the limit for trace element detection. NanoSIMS allowed chemical imaging of macro and trace elements with subcellular resolution (element mapping). Ca, Mg, and P as well as the trace elements Fe, Cu, and Zn present at basal levels were detected in pyrenoids, contractile vacuoles, and granules. Some metals were even localized in small vesicles of about 200nm size. Sensitive subcellular localization of trace metals was possible by the application of a recently developed RF plasma oxygen primary ion source on NanoSIMS which has shown good improvements in terms of lateral resolution (below 50nm), sensitivity, and stability. Furthermore correlative single cell imaging was developed combining the advantages of TEM and NanoSIMS. An advanced sample preparation protocol provided adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell. Thus, the C. reinhardtii cellular ultrastructure could be directly related to the spatial distribution of metals in different cell organelles such as vacuoles and chloroplast. Copyright © 2016 Elsevier GmbH. All rights reserved.

  16. Centromere protein W interacts with beta-transducin repeat-containing protein 1 and modulates its subcellular localization.

    Science.gov (United States)

    Cheon, Yeongmi; Jeon, Seyeong; Lee, Soojin

    2016-12-01

    Beta-transducin repeat-containing protein 1 (β-TrCP1) is a substrate-recognition module of SCF(β-Tr)(CP)(1) ubiquitin ligases and its subcellular distribution is known to be critical for target specificity. Heterogeneous nuclear ribonucleoprotein (hnRNP) U, an abundant nuclear protein, is known to be a unique regulator of β-TrCP1 shuttling between the cytoplasm and the nucleus. In this study, we report that centromere protein W (CENP-W), which is frequently overexpressed in a variety of human cancers, may also contribute to β-TrCP1 shuttling. Although hnRNP U and CENP-W can interact with β-TrCP1 and transport it independently, these proteins do not compete for β-TrCP1 binding, but rather cooperate to form a stable shuttling complex. Intriguingly, we found that overexpression of CENP-W leads to accumulation of β-TrCP1 in the nucleus. Thus, we propose that CENP-W may function as a booster of β-TrCP1 nuclear import to increase the oncogenicity of β-TrCP1. © 2016 Federation of European Biochemical Societies.

  17. Subcellular localization of rice acyl-CoA-binding proteins (ACBPs) indicates that OsACBP6::GFP is targeted to the peroxisomes.

    Science.gov (United States)

    Meng, Wei; Hsiao, An-Shan; Gao, Caiji; Jiang, Liwen; Chye, Mee-Len

    2014-07-01

    Acyl-CoA-binding proteins (ACBPs) show conservation at the acyl-CoA-binding (ACB) domain which facilitates binding to acyl-CoA esters. In Arabidopsis thaliana, six ACBPs participate in development and stress responses. Rice (Oryza sativa) also contains six genes encoding ACBPs. We investigated differences in subcellular localization between monocot rice and eudicot A. thaliana ACBPs. The subcellular localization of the six OsACBPs was achieved via transient expression of green fluorescence protein (GFP) fusions in tobacco (Nicotiana tabacum) epidermal cells, and stable transformation of A. thaliana. As plant ACBPs had not been reported in the peroxisomes, OsACBP6::GFP localization was confirmed by transient expression in rice sheath cells. The function of OsACBP6 was investigated by overexpressing 35S::OsACBP6 in the peroxisomal abc transporter1 (pxa1) mutant defective in peroxisomal fatty acid β-oxidation. As predicted, OsACBP1::GFP and OsACBP2::GFP were localized to the cytosol, and OsACBP4::GFP and OsACBP5::GFP to the endoplasmic reticulum (ER). However, OsACBP3::GFP displayed subcellular multi-localization while OsACBP6::GFP was localized to the peroxisomes. 35S::OsACBP6-OE/pxa1 lines showed recovery in indole-3-butyric acid (IBA) peroxisomal β-oxidation, wound-induced VEGETATIVE STORAGE PROTEIN1 (VSP1) expression and jasmonic acid (JA) accumulation. These findings indicate a role for OsACBP6 in peroxisomal β-oxidation, and suggest that rice ACBPs are involved in lipid degradation in addition to lipid biosynthesis. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  18. Subcellular localization of the histidine kinase receptors Sln1p, Nik1p and Chk1p in the yeast CTG clade species Candida guilliermondii.

    Science.gov (United States)

    Foureau, Emilien; Clastre, Marc; Montoya, Erika J Obando; Besseau, Sébastien; Oudin, Audrey; Glévarec, Gaëlle; Simkin, Andrew J; Crèche, Joël; Atehortùa, Lucia; Giglioli-Guivarc'h, Nathalie; Courdavault, Vincent; Papon, Nicolas

    2014-04-01

    Fungal histidine kinase receptors (HKR) sense and transduce many intra- and extracellular signals that regulate a wide range of physiological processes. Candida CTG clade species commonly possess three types of HKR namely Sln1p (type VI), Nik1p (type III) and Chk1p (type X). Although some recent work has demonstrated the potential involvement of HKR in osmoregulation, morphogenesis, sexual development, adaptation to osmotic stresses and drug resistance in distinct Candida species, little data is available in relation to their subcellular distribution within yeast cells. We describe in this work the comparative subcellular localization of class III, VI, and X HKRs in Candida guilliermondii, a yeast CTG clade species of clinical and biotechnological interest. Using a fluorescent protein fusion approach, we showed that C. guilliermondii Sln1p fused to the yellow fluorescent protein (Sln1p-YFP) appeared to be anchored in the plasma membrane. By contrast, both Chk1p-YFP and YFP-Chk1p were localized in the nucleocytosol of C. guilliermondii transformed cells. Furthermore, while Nik1p-YFP fusion protein always displayed a nucleocytosolic localization, we noted that most of the cells expressing YFP-Nik1p fusion protein displayed an aggregated pattern of fluorescence in the cytosol but not in the nucleus. Interestingly, Sln1p-YFP and Nik1p-YFP fusion protein localization changed in response to hyperosmotic stress by rapidly clustering into punctuated structures that could be associated to osmotic stress signaling. To date, this work provides the first insight into the subcellular localization of the three classes of HKR encoded by CTG clade yeast genomes and constitutes original new data concerning this family of receptors. This represents also an essential prerequisite to open a window into the understanding of the global architecture of HKR-mediated signaling pathways in CTG clade species. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Four Novel NR5A1 Mutations in 46,XY Gonadal Dysgenesis Patients Including Frameshift Mutations with Altered Subcellular SF-1 Localization.

    Science.gov (United States)

    Rehkämper, Jan; Tewes, Ann-Christin; Horvath, Judit; Scherer, Gerd; Wieacker, Peter; Ledig, Susanne

    2017-12-01

    46,XY gonadal dysgenesis (46,XY GD) is a disorder of sexual development caused by mutations in genes involved in early gonadal development (bipotential gonads) and testis differentiation. In 46,XY GD individuals, mutations of the SRY gene are detected most frequently, followed by mutations in the NR5A1 (SF-1) gene, but in a lot of cases, the underlying molecular mechanism remains elusive. In this study, we retrospectively performed sequence analyses of the NR5A1 (SF-1) gene in 84 patients with complete, partial, and syndromic forms of 46,XY GD. In total, 7 heterozygous mutations were found in 6 of 84 patients (7.1%). Among these, we identified 4 mutations that, to the best of our knowledge, have not been reported before (c.268G>T, c.369del, c.871-1G>C, and c.893T>C). Transfection of different mutations revealed altered subcellular localization of the mutant SF-1 protein in the case of the frameshift mutations, indicating an impaired protein function. In conclusion, we present 4 novel mutations of the NR5A1 gene associated with 46,XY GD together with in vitro data pointing towards a possible functional impairment of the mutant SF-1 proteins. © 2017 S. Karger AG, Basel.

  20. Subcellular Localization of Chitinase and of Its Potential Substrate in Tomato Root Tissues Infected by Fusarium oxysporum f. sp. radicis-lycopersici1

    Science.gov (United States)

    Benhamou, Nicole; Joosten, Matthieu H. A. J.; De Wit, Pierre J. G. M.

    1990-01-01

    Antiserum raised against a tomato (Lycopersicon esculentum Mill.) chitinase (molecular mass of 26 kilodaltons) was used as a probe to study the subcellular localization of this enzyme in tomato root tissues infected with Fusarium oxysporum f. sp. radicis-lycopersici. A time-course experiment revealed that chitinase accumulated earlier in the incompatible interaction than in the compatible one. However, in both systems, chitinase deposition was largely correlated with pathogen distribution. The enzyme was found to accumulate in areas where host walls were in close contact with fungal cells. In contrast, the enzyme could not be detected in vacuoles and intracellular spaces. The substantial amount of chitinase found at the fungus cell surface supports the view of an antifungal activity. However, the preferential association of the enzyme with altered fungal wall areas indicates that chitinase activity is either preceded by the hydrolytic action of other enzymes such as β-1,3-glucanases or coincides with these enzymes. The possibility that fungal glucans released through the action of β-1,3-glucanases may act as elicitors of chitinase production is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:16667378

  1. Membrane Orientation and Subcellular Localization of Transmembrane Protein 106B (TMEM106B), a Major Risk Factor for Frontotemporal Lobar Degeneration*♦

    Science.gov (United States)

    Lang, Christina M.; Fellerer, Katrin; Schwenk, Benjamin M.; Kuhn, Peer-Hendrik; Kremmer, Elisabeth; Edbauer, Dieter; Capell, Anja; Haass, Christian

    2012-01-01

    TMEM106B was identified as a major risk factor in a genome-wide association study for frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein (TDP)-43 pathology. The most significant association of TMEM106B single nucleotide polymorphisms with risk of FTLD-TDP was observed in patients with progranulin (GRN) mutations. Subsequent studies suggested an inverse correlation between TMEM106B expression and GRN levels in patient serum. However, in this study, this was not confirmed as we failed to detect a significant alteration of GRN levels upon knockdown or exogenous expression of TMEM106B in heterologous cells. To provide a basis for understanding TMEM106B function in health and disease, we investigated the membrane orientation and subcellular localization of this completely uncharacterized protein. By differential membrane extraction and sequential mutagenesis of potential N-glycosylation sites, we identified TMEM106B as a type 2 integral membrane protein with a highly glycosylated luminal domain. Glycosylation is partially required for the transport of TMEM106B beyond the endoplasmic reticulum to late cellular compartments. Endogenous as well as overexpressed TMEM106B localizes to late endosomes and lysosomes. Interestingly, the inhibition of vacuolar H+-ATPases significantly increased the levels of TMEM106B, a finding that may provide an unexpected biochemical link to GRN, because this protein is also strongly increased under the same conditions. Our findings provide a biochemical and cell biological basis for the understanding of the pathological role of TMEM106B in FTLD, an incurable neurodegenerative disorder. PMID:22511793

  2. Role of NH{sub 2}-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Asaka; Asahina, Kota; Okamoto, Takumi; Kawaguchi, Kosuke [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Kostsin, Dzmitry G. [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Academicheskaya Str. 27, Minsk 220072 (Belarus); Kashiwayama, Yoshinori [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Takanashi, Kojiro; Yazaki, Kazufumi [Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoko University, Uji, Kyoto 611-0011 (Japan); Imanaka, Tsuneo, E-mail: imanaka@pha.u-toyama.ac.jp [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Morita, Masashi [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2014-10-24

    Highlights: • ABCD proteins classifies based on with or without NH{sub 2}-terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH{sub 2}-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH{sub 2}-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH{sub 2}-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH{sub 2}-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH{sub 2}-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH{sub 2}-terminal H0 motif in organelle targeting is widely conserved in living organisms.

  3. Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

    Directory of Open Access Journals (Sweden)

    Hao Chen

    Full Text Available Retinol binding proteins (Rbps are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

  4. Efficient Subcellular Targeting to the Cell Nucleus of Quantum Dots Densely Decorated with a Nuclear Localization Sequence Peptide.

    Science.gov (United States)

    Maity, Amit Ranjan; Stepensky, David

    2016-01-27

    Organelle-targeted drug delivery can enhance the efficiency of the intracellularly acting drugs and reduce their toxicity. We generated core-shell type CdSe-ZnS quantum dots (QDs) densely decorated with NLS peptidic targeting residues using a 3-stage decoration approach and investigated their endocytosis and nuclear targeting efficiencies. The diameter of the generated QDs increased following the individual decoration stages (16.3, 18.9, and 21.9 nm), the ζ-potential became less negative (-33.2, -17.5, and -11.9 mV), and characteristic changes appeared in the FTIR spectra following decoration with the linker and NLS peptides. Quantitative analysis of the last decoration stage revealed that 37.9% and 33.2% of the alkyne-modified NLS groups that were added to the reaction mix became covalently attached or adsorbed to the QDs surface, respectively. These numbers correspond to 63.6 and 55.7 peptides conjugated or adsorbed to a single QD (the surface density of 42 and 37 conjugated and adsorbed peptides per 1000 nm(2) of the QDs surface), which is higher than in the majority of previous studies that reported decoration efficiencies of formulations intended for nuclear-targeted drug delivery. QDs decorated with NLS peptides undergo more efficient endocytosis, as compared to other investigated QDs formulations, and accumulated to a higher extent in the cell nucleus or in close vicinity to it (11.9%, 14.6%, and 56.1% of the QDs endocytosed by an average cell for the QD-COOH, QD-azide, and QD-NLS formulations, respectively). We conclude that dense decoration of QDs with NLS residues increased their endocytosis and led to their nuclear targeting (preferential accumulation in the cells nuclei or in close vicinity to them). The experimental system and research tools that were used in this study allow quantitative investigation of the mechanisms that govern the QDs nuclear targeting and their dependence on the formulation properties. These findings will contribute to the

  5. Functional processing of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N): evidence for a critical role of proteolytic processing in the regulation of its catalytic activity, subcellular localization and substrate targeting in vivo.

    Science.gov (United States)

    Sueyoshi, Noriyuki; Nimura, Takaki; Onouchi, Takashi; Baba, Hiromi; Takenaka, Shinobu; Ishida, Atsuhiko; Kameshita, Isamu

    2012-01-01

    Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear homolog CaMKP-N are Ser/Thr protein phosphatases that belong to the PPM family. These phosphatases are highly specific for multifunctional CaM kinases and negatively regulate their activities. CaMKP-N is only expressed in the brain and specifically localized in the nucleus. In this study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing in both the zebrafish brain and Neuro2a cells. In Neuro2a cells, the proteolytic processing was effectively inhibited by the proteasome inhibitors MG-132, Epoxomicin, and Lactacystin, suggesting that the ubiquitin-proteasome pathway was involved in this processing. Using MG-132, we found that the proteolytic processing changed the subcellular localization of zCaMKP-N from the nucleus to the cytosol. Accompanying this change, the cellular targets of zCaMKP-N in Neuro2a cells were significantly altered. Furthermore, we obtained evidence that the zCaMKP-N activity was markedly activated when the C-terminal domain was removed by the processing. Thus, the proteolytic processing of zCaMKP-N at the C-terminal region regulates its catalytic activity, subcellular localization and substrate targeting in vivo. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Subcellular Localization and Calcium and pH Requirements for Proteolytic Processing of the Hendra Virus Fusion Protein

    OpenAIRE

    Pager, Cara Theresia; Wurth, Mark Allen; Dutch, Rebecca Ellis

    2004-01-01

    Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F1 and F2 subunits, with cleavage occurring after residue K109 in the sequence GDVK↓L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero ...

  7. Paracoccidoides brasiliensis 30 kDa adhesin: identification as a 14-3-3 protein, cloning and subcellular localization in infection models.

    Directory of Open Access Journals (Sweden)

    Julhiany de Fatima da Silva

    Full Text Available Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections and 30 days (chronic infection. An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.

  8. Multitask learning for protein subcellular location prediction.

    Science.gov (United States)

    Xu, Qian; Pan, Sinno Jialin; Xue, Hannah Hong; Yang, Qiang

    2011-01-01

    Protein subcellular localization is concerned with predicting the location of a protein within a cell using computational methods. The location information can indicate key functionalities of proteins. Thus, accurate prediction of subcellular localizations of proteins can help the prediction of protein functions and genome annotations, as well as the identification of drug targets. Machine learning methods such as Support Vector Machines (SVMs) have been used in the past for the problem of protein subcellular localization, but have been shown to suffer from a lack of annotated training data in each species under study. To overcome this data sparsity problem, we observe that because some of the organisms may be related to each other, there may be some commonalities across different organisms that can be discovered and used to help boost the data in each localization task. In this paper, we formulate protein subcellular localization problem as one of multitask learning across different organisms. We adapt and compare two specializations of the multitask learning algorithms on 20 different organisms. Our experimental results show that multitask learning performs much better than the traditional single-task methods. Among the different multitask learning methods, we found that the multitask kernels and supertype kernels under multitask learning that share parameters perform slightly better than multitask learning by sharing latent features. The most significant improvement in terms of localization accuracy is about 25 percent. We find that if the organisms are very different or are remotely related from a biological point of view, then jointly training the multiple models cannot lead to significant improvement. However, if they are closely related biologically, the multitask learning can do much better than individual learning.

  9. Snail transcription factor NLS and importin β1 regulate the subcellular localization of Cathepsin L and Cux1.

    Science.gov (United States)

    Burton, Liza J; Henderson, Veronica; Liburd, Latiffa; Odero-Marah, Valerie A

    2017-09-09

    Several recent studies have highlighted an additional unexpected localization and site of action for Cathepsin L (Cat L) protease within the nucleus in breast, colon and prostate cancer, however, its role in the nucleus was unclear. It was proposed to mediate proteolytic processing of the transcription factor CCAAT-displacement protein/cut homeobox transcription factor (Cux1) from the full-length p200 isoform to generate the p110 and p90 isoforms, of which the p110 isoform was shown to act as a cell cycle regulator to accelerate entry into the S phase. The p110 isoform has also been shown to bind to the promoter regions of Snail and E-cadherin to activate Snail and inactivate E-cadherin transcription, thus promoting epithelial mesenchymal transition (EMT). Mechanistic studies on what drives Cat L nuclear localization have not been reported. Our hypothesis is that Snail shuttles into the nucleus with Cat L through binding to importin-β. Snail knockdown with siRNA in MDA-MB-468 breast cancer cells led to nuclear to cytoplasmic shuttling of Cat L and decreased levels of Cux1, while overexpression of Snail in MCF-7 breast cancer cells or HEK-293 human embryonic kidney cells led to increased nuclear expression of both Cat L and Cux1. Additionally, transient transfection of Snail NLS mutants not only abrogated Snail nuclear localization but also nuclear localization of Cat L and Cux1. Interestingly, importin β1 knockdown with siRNA decreased Snail and Cux1 levels, as well as nuclear localization of Cat L. Therefore, we show for the first time that the nuclear localization of Cat L and its substrate Cux1can be positively regulated by Snail NLS and importin β1, suggesting that Snail, Cat L and Cux1 all utilize importin β1 for nuclear import. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Imbalanced multi-modal multi-label learning for subcellular localization prediction of human proteins with both single and multiple sites.

    Directory of Open Access Journals (Sweden)

    Jianjun He

    Full Text Available It is well known that an important step toward understanding the functions of a protein is to determine its subcellular location. Although numerous prediction algorithms have been developed, most of them typically focused on the proteins with only one location. In recent years, researchers have begun to pay attention to the subcellular localization prediction of the proteins with multiple sites. However, almost all the existing approaches have failed to take into account the correlations among the locations caused by the proteins with multiple sites, which may be the important information for improving the prediction accuracy of the proteins with multiple sites. In this paper, a new algorithm which can effectively exploit the correlations among the locations is proposed by using gaussian process model. Besides, the algorithm also can realize optimal linear combination of various feature extraction technologies and could be robust to the imbalanced data set. Experimental results on a human protein data set show that the proposed algorithm is valid and can achieve better performance than the existing approaches.

  11. Paraformaldehyde Fixation May Lead to Misinterpretation of the Subcellular Localization of Plant High Mobility Group Box Proteins.

    Science.gov (United States)

    Li, Man-Wah; Zhou, Liang; Lam, Hon-Ming

    2015-01-01

    Arabidopsis High Mobility Group Box (HMBG) proteins were previously found associated with the interphase chromatin but not the metaphase chromosome. However, these studies are usually based on immunolocalization analysis involving paraformaldehyde fixation. Paraformaldehyde fixation has been widely adapted to preserved cell morphology before immunofluorescence staining. On one hand, the processed cells are no longer living. On the other hand, the processing may lead to misinterpretation of localization. HMGBs from Arabidopsis were fused with enhanced green fluorescence protein (EGFP) and transformed into tobacco BY-2 cells. Basically, the localization of these HMGB proteins detected with EGFP fluorescence in interphase agreed with previous publications. Upon 4% paraformaldehyde fixation, AtHMGB1 was found associated with interphase but not the metaphase chromosomes as previously reported. However, when EGFP fluorescence signal was directly observed under confocal microscope without fixation, association of AtHMGB1 with metaphase chromosomes can be detected. Paraformaldehyde fixation led to dissociation of EGFP tagged AtHMBG1 protein from metaphase chromosomes. This kind of pre-processing of live specimen may lead to dissociation of protein-protein or protein-nucleic acid interaction. Therefore, using of EGFP fusion proteins in live specimen is a better way to determine the correct localization and interaction of proteins.

  12. Monoterpene biosynthesis potential of plant subcellular compartments

    NARCIS (Netherlands)

    Dong, L.; Jongedijk, E.J.; Bouwmeester, H.J.; Krol, van der A.R.

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana

  13. AtHSBP functions in seed development and the motif is required for subcellular localization and interaction with AtHSFs.

    Science.gov (United States)

    Hsu, Shih-Feng; Jinn, Tsung-Luo

    2010-08-01

    In Arabidopsis thaliana, heat shock factor binding protein (AtHSBP) is a negative regulator of the heat shock response (HSR), and defective AtHSBP leads to seed abortion. We found that the wild-type and AtHSBP-knockout plants did not differ in ovule phenotypes at flower position 3, which indicates that the seed abortion occurs after fertilization and during embryogenesis. The conserved residues of the hydrophobic heptad repeat (HR) domains in AtHSBP were mutated and examined for their subcellular localization and interacting ability with heat shock factors (AtHSFs). The HR domains at the C terminus of AtHSBP are important for retaining AtHSBP in the cytoplasm under normal growth conditions and for interacting with AtHSFs, which negatively affects the DNA-binding capacity and transactivation activity of AtHSFs during the HSR.

  14. Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments.

    Science.gov (United States)

    Pedrajas, José R; Porras, Pablo; Martínez-Galisteo, Emilia; Padilla, C Alicia; Miranda-Vizuete, Antonio; Bárcena, J Antonio

    2002-06-15

    Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a 'classical' Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9 kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99 bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product.

  15. Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

    Science.gov (United States)

    Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

    2012-01-01

    Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can

  16. Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

    Directory of Open Access Journals (Sweden)

    Irene L Ibañez

    Full Text Available The Cyclin-dependent kinase inhibitor 1B (p27Kip1 is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2O(2 in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p at serine 10 (S10 and at threonine 198 (T198 because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2O(2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2O(2 (0.1 µM to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2O(2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization

  17. Subcellular localization of the Staphylococcus aureus heme iron transport components IsdA and IsdB.

    Science.gov (United States)

    Pishchany, Gleb; Dickey, Susan E; Skaar, Eric P

    2009-07-01

    Staphylococcus aureus is a human pathogen that represents a tremendous threat to global public health. An important aspect of S. aureus pathogenicity is the ability to acquire iron from its host during infection. In vertebrates, iron is sequestered predominantly within heme, the majority of which is bound by hemoglobin. To acquire iron, S. aureus binds hemoglobin, removes heme, and transports it into the cytoplasm, where heme is degraded. This process is carried out by the iron-regulated surface determinant system (Isd); however, the mechanism by which hemoglobin recognition occurs is not completely understood. Here we report that the surface receptor components of the Isd system, IsdA and IsdB, physically interact with each other and are anchored to a discrete location within the cell wall. This organized localization pattern is dependent upon the iron status of the bacterium. Furthermore, we have found that hemoglobin colocalizes with IsdB at discrete sites within the cell wall. Virulence studies revealed that IsdB is required for the efficient colonization of the heart and that IsdB is differentially expressed within infected organs, suggesting that S. aureus experiences various degrees of iron starvation depending on the site of infection. These findings significantly expand our understanding of hemoglobin iron acquisition and demonstrate an orchestrated pattern of regulation and localization for the S. aureus heme iron acquisition system.

  18. Subcellular localization of proteins in the anaerobic sulfate reducer Desulfovibrio vulgaris via SNAP-tag labeling and photoconversion

    Energy Technology Data Exchange (ETDEWEB)

    Gorur, A.; Leung, C. M.; Jorgens, D.; Tauscher, A.; Remis, J. P.; Ball, D. A.; Chhabra, S.; Fok, V.; Geller, J. T.; Singer, M.; Hazen, T. C.; Juba, T.; Elias, D.; Wall, J.; Biggin, M.; Downing, K. H.; Auer, M.

    2010-06-01

    Systems Biology studies the temporal and spatial 3D distribution of macromolecular complexes with the aim that such knowledge will allow more accurate modeling of biological function and will allow mathematical prediction of cellular behavior. However, in order to accomplish accurate modeling precise knowledge of spatial 3D organization and distribution inside cells is necessary. And while a number of macromolecular complexes may be identified by its 3D structure and molecular characteristics alone, the overwhelming number of proteins will need to be localized using a reporter tag. GFP and its derivatives (XFPs) have been traditionally employed for subcelllar localization using photoconversion approaches, but this approach cannot be taken for obligate anaerobic bacteria, where the intolerance towards oxygen prevents XFP approaches. As part of the GTL-funded PCAP project (now ENIGMA) genetic tools have been developed for the anaerobe sulfate reducer Desulfovibrio vulgaris that allow the high-throughput generation of tagged-protein mutant strains, with a focus on the commercially available SNAP-tag cell system (New England Biolabs, Ipswich, MA), which is based on a modified O6-alkylguanine-DNA alkyltransferase (AGT) tag, that has a dead-end reaction with a modified O6-benzylguanine (BG) derivative and has been shown to function under anaerobic conditions. After initial challenges with respect to variability, robustness and specificity of the labeling signal we have optimized the labeling. Over the last year, as a result of the optimized labeling protocol, we now obtain robust labeling of 20 out of 31 SNAP strains. Labeling for 13 strains were confirmed at least five times. We have also successfully performed photoconversion on 5 of these 13 strains, with distinct labeling patterns for different strains. For example, DsrC robustly localizes to the periplasmic portion of the inner membrane, where as a DNA-binding protein localizes to the center of the cell, where the

  19. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production.

    Directory of Open Access Journals (Sweden)

    Moon Y F Tay

    2016-09-01

    Full Text Available Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18 alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα, allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization.

  20. Studying subcellular detail in fixed astrocytes: Dissociation of morphologically intact glial cells (DIMIGs)

    OpenAIRE

    Julia eHaseleu; Enrico eAnlauf; Enrico eAnlauf; Sandra eBlaess; Elmar eEndl; Amin eDerouiche; Amin eDerouiche; Amin eDerouiche

    2013-01-01

    Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs), since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from a...

  1. Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs)

    OpenAIRE

    Haseleu, Julia; Anlauf, Enrico; Blaess, Sandra; Endl, Elmar; Derouiche, Amin

    2013-01-01

    Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs), since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from a...

  2. Phylogenetic analysis, subcellular localization, and expression patterns of RPD3/HDA1 family histone deacetylases in plants

    Directory of Open Access Journals (Sweden)

    Yu Chun-Wei

    2009-03-01

    Full Text Available Abstract Background Although histone deacetylases from model organisms have been previously identified, there is no clear basis for the classification of histone deacetylases under the RPD3/HDA1 superfamily, particularly on plants. Thus, this study aims to reconstruct a phylogenetic tree to determine evolutionary relationships between RPD3/HDA1 histone deacetylases from six different plants representing dicots with Arabidopsis thaliana, Populus trichocarpa, and Pinus taeda, monocots with Oryza sativa and Zea mays, and the lower plants with Physcomitrella patens. Results Sixty two histone deacetylases of RPD3/HDA1 family from the six plant species were phylogenetically analyzed to determine corresponding orthologues. Three clusters were formed separating Class I, Class II, and Class IV. We have confirmed lower and higher plant orthologues for AtHDA8 and AtHDA14, classifying both genes as Class II histone deacetylases in addition to AtHDA5, AtHDA15, and AtHDA18. Since Class II histone deacetylases in other eukaryotes have been known to undergo nucleocytoplasmic transport, it remains unknown whether such functional regulation also happens in plants. Thus, bioinformatics studies using different programs and databases were conducted to predict their corresponding localization sites, nuclear export signal, nuclear localization signal, as well as expression patterns. We also found new conserved domains in most of the RPD3/HDA1 histone deacetylases which were similarly conserved in its corresponding orthologues. Assessing gene expression patterns using Genevestigator, it appears that RPD3/HDA1 histone deacetylases are expressed all throughout the plant parts and developmental stages of the plant. Conclusion The RPD3/HDA1 histone deacetylase family in plants is divided into three distinct groups namely, Class I, Class II, and Class IV suggesting functional diversification. Class II comprises not only AtHDA5, AtHDA15, and AtHDA18 but also includes AtHDA8

  3. Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells.

    Science.gov (United States)

    De Muynck, Benoit; Navarre, Catherine; Nizet, Yannick; Stadlmann, Johannes; Boutry, Marc

    2009-06-01

    Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment.

  4. In vivo subcellular localization of Mal de Rio Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

    Energy Technology Data Exchange (ETDEWEB)

    Maroniche, Guillermo A.; Mongelli, Vanesa C.; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar [Instituto de Biotecnologia, CICVyA, Instituto Nacional de Tecnologia Agropecuaria (IB-INTA), Las cabanas y Los Reseros s/n. Hurlingham Cp 1686, Buenos Aires (Argentina); Vas, Mariana del, E-mail: mdelvas@cnia.inta.gov.ar [Instituto de Biotecnologia, CICVyA, Instituto Nacional de Tecnologia Agropecuaria (IB-INTA), Las cabanas y Los Reseros s/n. Hurlingham Cp 1686, Buenos Aires (Argentina)

    2012-09-01

    The in vivo subcellular localization of Mal de Rio Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and {alpha}-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

  5. In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells

    NARCIS (Netherlands)

    Boren, Joan; Ramos-Montoya, Antonio; Bosch, Klazien S.; Vreeling, Heleen; Jonker, Ard; Centelles, Josep J.; Cascante, Marta; Frederiks, Wilma M.

    2006-01-01

    Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in

  6. Subcellular localization of a sporulation membrane protein is achieved through a network of interactions along and across the septum.

    Science.gov (United States)

    Doan, Thierry; Marquis, Kathleen A; Rudner, David Z

    2005-03-01

    During the process of spore formation in Bacillus subtilis many membrane proteins localize to the sporulation septum where they play key roles in morphogenesis and cell-cell signalling. However, the mechanism by which these proteins are anchored at this site is not understood. In this report we have defined the localization requirements for the mother-cell membrane protein SpoIVFA, which anchors a signalling complex in the septal membrane on the mother cell side. We have identified five proteins (SpoIID, SpoIIP, SpoIIM, BofA and SpoIIIAH) synthesized in the mother cell under the control of sigma(E) and one protein (SpoIIQ) synthesized in the forespore under the control of sigma(F) that are all required for the proper localization of SpoIVFA. Surprisingly, these proteins appear to have complementary and overlapping anchoring roles suggesting that SpoIVFA is localized in the septal membrane through a web of protein interactions. Furthermore, we demonstrate a direct biochemical interaction between the extracellular domains of two of the proteins required to anchor SpoIVFA: the forespore protein SpoIIQ and the mother-cell protein SpoIIIAH. This result supports the idea that the web of interactions that anchors SpoIVFA is itself held in the septal membrane through a zipper-like interaction across the sporulation septum. Importantly, our results suggest that a second mechanism independent of forespore proteins participates in anchoring SpoIVFA. Finally, we show that the dynamic localization of SpoIIQ in the forespore is impaired in the absence of SpoIVFA but not SpoIIIAH. Thus, a complex web of interactions among mother cell and forespore proteins is responsible for static and dynamic protein localization in both compartments of the sporangium. We envision that this proposed network is involved in anchoring other sporulation proteins in the septum and that protein networks with overlapping anchoring capacity is a feature of protein localization in all bacteria.

  7. The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization

    Directory of Open Access Journals (Sweden)

    Tam Michael WC

    2010-03-01

    Full Text Available Abstract Background Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants. Results The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1 RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus. Conclusions The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to

  8. hnRNPs H, H' and F behave differently with respect to posttranslational cleavage and subcellular localization

    DEFF Research Database (Denmark)

    Honoré, B; Vorum, H; Baandrup, U

    1999-01-01

    hnRNPs H, H' and F belong to a subfamily of the hnRNPs sharing a high degree of sequence identity. Eukaryotic expression and specific C-terminal antibodies were used to demonstrate great variation in the intracellular fate of the proteins. hnRNPs H and H' become posttranslational cleaved into C......-terminal 35 kDa proteins (H(C), H'(C)) and possibly into N-terminal 22 kDa proteins. No detectable cleavage was observed for hnRNP F. hnRNP H/H' is almost exclusively localized to the nucleus of many cell types while hnRNP F varies from a predominant nuclear localization in some cells to a predominant...... cytoplasmic localization in other cells. The different fates may reflect differences in functional roles that so far only have included nuclear functions. The presence of significant quantities of hnRNP F in the cytoplasm of many cells indicates that it also may have a functional role here. Udgivelsesdato...

  9. Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT through the erythrocytic cycle of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Mitchell Sarah L

    2010-12-01

    Full Text Available Abstract Background The folate pathway enzyme serine hydroxymethyltransferase (SHMT converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc and a related, apparently inactive isoform (PfSHMTm are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated. Methods Polyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites. Results As well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards

  10. The novel CALM interactor CATS influences the subcellular localization of the leukemogenic fusion protein CALM/AF10.

    Science.gov (United States)

    Archangelo, L Fröhlich; Gläsner, J; Krause, A; Bohlander, S K

    2006-07-06

    The Clathrin Assembly Lymphoid Myeloid leukemia gene (CALM or PICALM) was first identified as the fusion partner of AF10 in the t(10;11)(p13;q14) translocation, which is observed in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and malignant lymphoma. The CALM/AF10 fusion protein plays a crucial role in t(10;11)(p13;q14) associated leukemogenesis. Using the N-terminal half of CALM as a bait in a yeast two-hybrid screen, a novel protein named CATS (CALM interacting protein expressed in thymus and spleen) was identified. Multiple tissue Northern blot analysis showed predominant expression of CATS in thymus, spleen and colon. CATS codes for two protein isoforms of 238 and 248 amino acids (aa). The interaction between CALM and CATS could be confirmed using pull down assays, co-immunoprecipitation and colocalization experiments. The CATS interaction domain of CALM was mapped to aa 221-335 of CALM. This domain is contained in the CALM/AF10 fusion protein. CATS localizes to the nucleus and shows a preference for nucleoli. Expression of CATS was able to markedly increase the nuclear localization of CALM and of the leukemogenic fusion protein CALM/AF10. The possible implications of these findings for CALM/AF10-mediated leukemogenesis are discussed.

  11. Distinct effects of subcellular glycogen localization on tetanic relaxation time and endurance in mechanically skinned rat skeletal muscle fibres

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Schrøder, H D; Rix, C G

    2009-01-01

    In vitro experiments indicate a non-metabolic role of muscle glycogen in contracting skeletal muscles. Since the sequence of events in excitation\\#8211;contraction (E\\#8211;C) coupling is known to be located close to glycogen granules, at specific sites on the fibre, we hypothesized...... that the distinct compartments of glycogen have specific effects on muscle fibre contractility and fatigability. Single skeletal muscle fibres (n = 19) from fed and fasted rats were mechanically skinned and divided into two segments. In one segment glycogen localization and volume fraction were estimated......, range 22-252 contractions). Initially the total myofibrillar glycogen volume percentage was 0.46 +/- 0.07%, with 72 +/- 3% in the intermyofibrillar space and 28 +/- 3% in the intramyofibrillar space. The intramyofibrillar glycogen content was positively correlated with the fatigue resistance capacity (r...

  12. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

    Directory of Open Access Journals (Sweden)

    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  13. Dipeptidyl peptidase 9 subcellular localization and a role in cell adhesion involving focal adhesion kinase and paxillin.

    Science.gov (United States)

    Zhang, Hui; Chen, Yiqian; Wadham, Carol; McCaughan, Geoffrey W; Keane, Fiona M; Gorrell, Mark D

    2015-02-01

    Dipeptidyl peptidase 9 (DPP9) is a ubiquitously expressed member of the DPP4 gene and protease family. Deciphering the biological functions of DPP9 and its roles in pathogenesis has implicated DPP9 in tumor biology, the immune response, apoptosis, intracellular epidermal growth factor-dependent signaling and cell adhesion and migration. We investigated the intracellular distribution of DPP9 chimeric fluorescent proteins and consequent functions of DPP9. We showed that while some DPP9 is associated with mitochondria, the strongest co-localization was with microtubules. Under steady state conditions, DPP9 was not seen at the plasma membrane, but upon stimulation with either phorbol 12-myristate 13-acetate or epidermal growth factor, some DPP9 re-distributed towards the ruffling membrane. DPP9 was seen at the leading edge of the migrating cell and co-localized with the focal adhesion proteins, integrin-β1 and talin. DPP9 gene silencing and treatment with a DPP8/DPP9 specific inhibitor both reduced cell adhesion and migration. Expression of integrin-β1 and talin was decreased in DPP9-deficient and DPP9-enzyme-inactive cells. There was a concomitant decrease in the phosphorylation of focal adhesion kinase and paxillin, indicating that DPP9 knockdown or enzyme inhibition suppressed the associated adhesion signaling pathway, causing impaired cell movement. These novel findings provide mechanistic insights into the regulatory role of DPP9 in cell movement, and may thus implicate DPP9 in tissue and tumor growth and metastasis. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations.

    Science.gov (United States)

    Hooper, Cornelia M; Castleden, Ian R; Tanz, Sandra K; Aryamanesh, Nader; Millar, A Harvey

    2017-01-04

    The SUBcellular location database for Arabidopsis proteins (SUBA4, http://suba.live) is a comprehensive collection of manually curated published data sets of large-scale subcellular proteomics, fluorescent protein visualization, protein-protein interaction (PPI) as well as subcellular targeting calls from 22 prediction programs. SUBA4 contains an additional 35 568 localizations totalling more than 60 000 experimental protein location claims as well as 37 new suborganellar localization categories. The experimental PPI data has been expanded to 26 327 PPI pairs including 856 PPI localizations from experimental fluorescent visualizations. The new SUBA4 user interface enables users to choose quickly from the filter categories: 'subcellular location', 'protein properties', 'protein-protein interaction' and 'affiliations' to build complex queries. This allows substantial expansion of search parameters into 80 annotation types comprising 1 150 204 new annotations to study metadata associated with subcellular localization. The 'BLAST' tab contains a sequence alignment tool to enable a sequence fragment from any species to find the closest match in Arabidopsis and retrieve data on subcellular location. Using the location consensus SUBAcon, the SUBA4 toolbox delivers three novel data services allowing interactive analysis of user data to provide relative compartmental protein abundances and proximity relationship analysis of PPI and coexpression partners from a submitted list of Arabidopsis gene identifiers. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Immunoelectron-microscopic investigation of the subcellular localization of pinopsin in the pineal organ of the chicken.

    Science.gov (United States)

    Hirunagi, K; Ebihara, S; Okano, T; Takanaka, Y; Fukada, Y

    1997-08-01

    Pinopsin is a photoreceptive molecule cloned from the chicken pineal organ. An antibody highly specific for pinopsin was applied in light- and electron-microscopic immunocytochemical studies of the pineal organ of 1 to 2-month-old chickens. Intense immunoreactivity was found in the follicular lumen at the light-microscopic level. In addition, small immunoreactive spherical or fibrous structures were diffusely distributed at the parafollicular aspect of the pineal organ. To identify immunoreactive elements precisely, we used pre-embedding immunoelectron microscopy. These studies revealed immunoreactive outer segments of pinealocytes arranged closely side by side in the follicular lumina. The thin initial portion of the outer segment arose from a basal body located in the inner segment. Immunoreactive pear-shaped outer segments occupied small lumina. Follicular lumina displayed immunonegative arrays of whorl-like lamellar membranes. Occasionally, these immunonegative structures were surrounded by immunoreactive concentric lamellar complexes. In the parafollicular pineal parenchyma, long slender cilium-like structures or enlarged cilia and concentric lamellar arrays showed intense immunoreactivity. All immunoreactive structures observed in this study were considered to represent outer segments of pinealocytes of the chicken pineal organ.

  16. Protein Subcellular Relocalization of Duplicated Genes in Arabidopsis

    Science.gov (United States)

    Liu, Shao-Lun; Pan, An Qi; Adams, Keith L.

    2014-01-01

    Gene duplications during eukaroytic evolution, by successive rounds of polyploidy and by smaller scale duplications, have provided an enormous reservoir of new genes for the evolution of new functions. Preservation of many duplicated genes can be ascribed to changes in sequences, expression patterns, and functions. Protein subcellular relocalization (protein targeting to a new location within the cell) is another way that duplicated genes can diverge. We studied subcellular relocalization of gene pairs duplicated during the evolution of the Brassicaceae including gene pairs from the alpha whole genome duplication that occurred at the base of the family. We analyzed experimental localization data from green fluorescent protein experiments for 128 duplicate pairs in Arabidopsis thaliana, revealing 19 pairs with subcellular relocalization. Many more of the duplicate pairs with relocalization than with the same localization showed an accelerated rate of amino acid sequence evolution in one duplicate, and one gene showed evidence for positive selection. We studied six duplicate gene pairs in more detail. We used gene family analysis with several pairs to infer which gene shows relocalization. We identified potential sequence mutations through comparative analysis that likely result in relocalization of two duplicated gene products. We show that four cases of relocalization have new expression patterns, compared with orthologs in outgroup species, including two with novel expression in pollen. This study provides insights into subcellular relocalization of evolutionarily recent gene duplicates and features of genes whose products have been relocalized. PMID:25193306

  17. Subcellular Nutrient Element Localization and Enrichment in Ecto- and Arbuscular Mycorrhizas of Field-Grown Beech and Ash Trees Indicate Functional Differences

    Science.gov (United States)

    Seven, Jasmin; Polle, Andrea

    2014-01-01

    Mycorrhizas are the chief organ for plant mineral nutrient acquisition. In temperate, mixed forests, ash roots (Fraxinus excelsior) are colonized by arbuscular mycorrhizal fungi (AM) and beech roots (Fagus sylvatica) by ectomycorrhizal fungi (EcM). Knowledge on the functions of different mycorrhizal species that coexist in the same environment is scarce. The concentrations of nutrient elements in plant and fungal cells can inform on nutrient accessibility and interspecific differences of mycorrhizal life forms. Here, we hypothesized that mycorrhizal fungal species exhibit interspecific differences in mineral nutrient concentrations and that the differences correlate with the mineral nutrient concentrations of their associated root cells. Abundant mycorrhizal fungal species of mature beech and ash trees in a long-term undisturbed forest ecosystem were the EcM Lactarius subdulcis, Clavulina cristata and Cenococcum geophilum and the AM Glomus sp. Mineral nutrient subcellular localization and quantities of the mycorrhizas were analysed after non-aqueous sample preparation by electron dispersive X-ray transmission electron microscopy. Cenococcum geophilum contained the highest sulphur, Clavulina cristata the highest calcium levels, and Glomus, in which cations and P were generally high, exhibited the highest potassium levels. Lactarius subdulcis-associated root cells contained the highest phosphorus levels. The root cell concentrations of K, Mg and P were unrelated to those of the associated fungal structures, whereas S and Ca showed significant correlations between fungal and plant concentrations of those elements. Our results support profound interspecific differences for mineral nutrient acquisition among mycorrhizas formed by different fungal taxa. The lack of correlation between some plant and fungal nutrient element concentrations may reflect different retention of mineral nutrients in the fungal part of the symbiosis. High mineral concentrations, especially of

  18. Subcellular nutrient element localization and enrichment in ecto- and arbuscular mycorrhizas of field-grown beech and ash trees indicate functional differences.

    Directory of Open Access Journals (Sweden)

    Jasmin Seven

    Full Text Available Mycorrhizas are the chief organ for plant mineral nutrient acquisition. In temperate, mixed forests, ash roots (Fraxinus excelsior are colonized by arbuscular mycorrhizal fungi (AM and beech roots (Fagus sylvatica by ectomycorrhizal fungi (EcM. Knowledge on the functions of different mycorrhizal species that coexist in the same environment is scarce. The concentrations of nutrient elements in plant and fungal cells can inform on nutrient accessibility and interspecific differences of mycorrhizal life forms. Here, we hypothesized that mycorrhizal fungal species exhibit interspecific differences in mineral nutrient concentrations and that the differences correlate with the mineral nutrient concentrations of their associated root cells. Abundant mycorrhizal fungal species of mature beech and ash trees in a long-term undisturbed forest ecosystem were the EcM Lactarius subdulcis, Clavulina cristata and Cenococcum geophilum and the AM Glomus sp. Mineral nutrient subcellular localization and quantities of the mycorrhizas were analysed after non-aqueous sample preparation by electron dispersive X-ray transmission electron microscopy. Cenococcum geophilum contained the highest sulphur, Clavulina cristata the highest calcium levels, and Glomus, in which cations and P were generally high, exhibited the highest potassium levels. Lactarius subdulcis-associated root cells contained the highest phosphorus levels. The root cell concentrations of K, Mg and P were unrelated to those of the associated fungal structures, whereas S and Ca showed significant correlations between fungal and plant concentrations of those elements. Our results support profound interspecific differences for mineral nutrient acquisition among mycorrhizas formed by different fungal taxa. The lack of correlation between some plant and fungal nutrient element concentrations may reflect different retention of mineral nutrients in the fungal part of the symbiosis. High mineral concentrations

  19. Expression and subcellular localization of kinetoplast-associated proteins in the different developmental stages of Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Cavalcanti Danielle

    2009-06-01

    Full Text Available Abstract Background The kinetoplast DNA (kDNA of trypanosomatids consists of an unusual arrangement of circular molecules catenated into a single network. The diameter of the isolated kDNA network is similar to that of the entire cell. However, within the kinetoplast matrix, the kDNA is highly condensed. Studies in Crithidia fasciculata showed that kinetoplast-associated proteins (KAPs are capable of condensing the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasitic protozoon that shows distinct patterns of kDNA condensation during their complex morphogenetic development. In epimastigotes and amastigotes (replicating forms the kDNA fibers are tightly packed into a disk-shaped kinetoplast, whereas trypomastigotes (non-replicating present a more relaxed kDNA organization contained within a rounded structure. It is still unclear how the compact kinetoplast disk of epimastigotes is converted into a globular structure in the infective trypomastigotes. Results In this work, we have analyzed KAP coding genes in trypanosomatid genomes and cloned and expressed two kinetoplast-associated proteins in T. cruzi: TcKAP4 and TcKAP6. Such small basic proteins are expressed in all developmental stages of the parasite, although present a differential distribution within the kinetoplasts of epimastigote, amastigote and trypomastigote forms. Conclusion Several features of TcKAPs, such as their small size, basic nature and similarity with KAPs of C. fasciculata, are consistent with a role in DNA charge neutralization and condensation. Additionally, the differential distribution of KAPs in the kinetoplasts of distinct developmental stages of the parasite, indicate that the kDNA rearrangement that takes place during the T. cruzi differentiation process is accompanied by TcKAPs redistribution.

  20. Janus kinase (Jak) subcellular localization revisited: the exclusive membrane localization of endogenous Janus kinase 1 by cytokine receptor interaction uncovers the Jak.receptor complex to be equivalent to a receptor tyrosine kinase.

    Science.gov (United States)

    Behrmann, Iris; Smyczek, Tanja; Heinrich, Peter C; Schmitz-Van de Leur, Hildegard; Komyod, Waraporn; Giese, Bernd; Müller-Newen, Gerhard; Haan, Serge; Haan, Claude

    2004-08-20

    The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal transduction. We investigated Jak1 localization using subcellular fractionation techniques and fluorescence microscopy (immunofluorescence and yellow fluorescent protein-tagged Jaks). In the different experimental approaches we found Jak1 (as well as Jak2 and Tyk2) predominantly located at membranes. In contrast to previous reports we did not observe Jak proteins in significant amounts within the nucleus or in the cytoplasm. The cytoplasmic localization observed for the Jak1 mutant L80A/Y81A, which is unable to associate with cytokine receptors, indicates that Jak1 does not have a strong intrinsic membrane binding potential and that only receptor binding is crucial for the membrane recruitment. Finally we show that Jak1 remains a membrane-localized protein after cytokine stimulation. These data strongly support the hypothesis that cytokine receptor.Janus kinase complexes can be regarded as receptor tyrosine kinases.

  1. Axonal localization of Ca2+-dependent activator protein for secretion 2 is critical for subcellular locality of brain-derived neurotrophic factor and neurotrophin-3 release affecting proper development of postnatal mouse cerebellum.

    Directory of Open Access Journals (Sweden)

    Tetsushi Sadakata

    Full Text Available Ca2+-dependent activator protein for secretion 2 (CAPS2 is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF and neurotrophin-3 (NT-3 from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2Δex3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2Δex3/Δex3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber-Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum.

  2. Two-Photon Irradiation of an Intracellular Singlet Oxygen Photosensitizer: Achieving Localized Sub-Cellular Excitation in Spatially-Resolved Experiments

    DEFF Research Database (Denmark)

    Pedersen, Brian Wett; Breitenbach, Thomas; Redmond, Robert W.

    2010-01-01

    The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defi ned by the dimensions of the laser...

  3. Skeletal muscle glycogen content and particle size of distinct subcellular localizations in the recovery period after a high-level soccer match

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Krustrup, Peter; Nybo, Lars

    2012-01-01

    biopsy collected immediately after and 24, 48, 72 and 120 h after a competitive soccer match. Transmission electron microscopy was used to estimate the subcellular distribution of glycogen and individual particle size. During the first day of recovery, glycogen content increased by ~60% in all...

  4. Sub-cellular Electrical Heterogeneity Revealed by Loose Patch Recording Reflects Differential Localization of Sarcolemmal Ion Channels in Intact Rat Hearts

    Directory of Open Access Journals (Sweden)

    Igor V. Kubasov

    2018-02-01

    Full Text Available The cardiac action potential (AP is commonly recoded as an integral signal from isolated myocytes or ensembles of myocytes (with intracellular microelectrodes and extracellular macroelectrodes, respectively. These signals, however, do not provide a direct measure of activity of ion channels and transporters located in two major compartments of a cardiac myocyte: surface sarcolemma and the T-tubule system, which differentially contribute to impulse propagation and excitation-contraction (EC coupling. In the present study we investigated electrical properties of myocytes within perfused intact rat heart employing loose patch recording with narrow-tip (2 μm diameter extracellular electrodes. Using this approach, we demonstrated two distinct types of electric signals with distinct waveforms (single peak and multi-peak AP; AP1 and AP2, respectively during intrinsic pacemaker activity. These two types of waveforms depend on the position of the electrode tip on the myocyte surface. Such heterogeneity of electrical signals was lost when electrodes of larger pipette diameter were used (5 or 10 μm, which indicates that the electric signal was assessed from a region of <5 μm. Importantly, both pharmacological and mathematical simulation based on transverse (T-tubular distribution suggested that while the AP1 and the initial peak of AP2 are predominantly attributable to the fast, inward Na+ current in myocyte's surface sarcolemma, the late components of AP2 are likely representative of currents associated with L-type Ca2+ channel and Na+/Ca2+ exchanger (NCX currents which are predominantly located in T-tubules. Thus, loose patch recording with narrow-tip pipette provides a valuable tool for studying cardiac electric activity on the subcellular level in the intact heart.

  5. Subcellular compartmentation of glutathione in dicotyledonous plants

    Science.gov (United States)

    Müller, Maria

    2010-01-01

    This study describes the subcellular distribution of glutathione in roots and leaves of different plant species (Arabidopsis, Cucurbita, and Nicotiana). Glutathione is an important antioxidant and redox buffer which is involved in many metabolic processes including plant defense. Thus information on the subcellular distribution in these model plants especially during stress situations provides a deeper insight into compartment specific defense reactions and reflects the occurrence of compartment specific oxidative stress. With immunogold cytochemistry and computer-supported transmission electron microscopy glutathione could be localized in highest contents in mitochondria, followed by nuclei, peroxisomes, the cytosol, and plastids. Within chloroplasts and mitochondria, glutathione was restricted to the stroma and matrix, respectively, and did not occur in the lumen of cristae and thylakoids. Glutathione was also found at the membrane and in the lumen of the endoplasmic reticulum. It was also associated with the trans and cis side of dictyosomes. None or only very little glutathione was detected in vacuoles and the apoplast of mesophyll and root cells. Additionally, glutathione was found in all cell compartments of phloem vessels, vascular parenchyma cells (including vacuoles) but was absent in xylem vessels. The specificity of this method was supported by the reduction of glutathione labeling in all cell compartments (up to 98%) of the glutathione-deficient Arabidopsis thaliana rml1 mutant. Additionally, we found a similar distribution of glutathione in samples after conventional fixation and rapid microwave-supported fixation. Thus, indicating that a redistribution of glutathione does not occur during sample preparation. Summing up, this study gives a detailed insight into the subcellular distribution of glutathione in plants and presents solid evidence for the accuracy and specificity of the applied method. PMID:20186447

  6. Ischemia-related subcellular redistribution of sodium channels enhances the proarrhythmic effect of class I antiarrhythmic drugs: a simulation study.

    Directory of Open Access Journals (Sweden)

    Kunichika Tsumoto

    Full Text Available Cardiomyocytes located at the ischemic border zone of infarcted ventricle are accompanied by redistribution of gap junctions, which mediate electrical transmission between cardiomyocytes. This ischemic border zone provides an arrhythmogenic substrate. It was also shown that sodium (Na+ channels are redistributed within myocytes located in the ischemic border zone. However, the roles of the subcellular redistribution of Na+ channels in the arrhythmogenicity under ischemia remain unclear.Computer simulations of excitation conduction were performed in a myofiber model incorporating both subcellular Na+ channel redistribution and the electric field mechanism, taking into account the intercellular cleft potentials.We found in the myofiber model that the subcellular redistribution of the Na+ channels under myocardial ischemia, decreasing in Na+ channel expression of the lateral cell membrane of each myocyte, decreased the tissue excitability, resulting in conduction slowing even without any ischemia-related electrophysiological change. The conventional model (i.e., without the electric field mechanism did not reproduce the conduction slowing caused by the subcellular Na+ channel redistribution. Furthermore, Na+ channel blockade with the coexistence of a non-ischemic zone with an ischemic border zone expanded the vulnerable period for reentrant tachyarrhythmias compared to the model without the ischemic border zone. Na+ channel blockade tended to cause unidirectional conduction block at sites near the ischemic border zone. Thus, such a unidirectional conduction block induced by a premature stimulus at sites near the ischemic border zone is associated with the initiation of reentrant tachyarrhythmias.Proarrhythmia of Na+ channel blockade in patients with old myocardial infarction might be partly attributable to the ischemia-related subcellular Na+ channel redistribution.

  7. Liver X receptor ligand cytotoxicity in colon cancer cells and not in normal colon epithelial cells depends on LXRβ subcellular localization.

    Science.gov (United States)

    Courtaut, Flavie; Derangère, Valentin; Chevriaux, Angélique; Ladoire, Sylvain; Cotte, Alexia K; Arnould, Laurent; Boidot, Romain; Rialland, Mickaël; Ghiringhelli, François; Rébé, Cédric

    2015-09-29

    Increasing evidence indicates that Liver X Receptors (LXRs) have some anticancer properties. We recently demonstrated that LXR ligands induce colon cancer cell pyroptosis through an LXRβ-dependent pathway. In the present study, we showed that human colon cancer cell lines presented differential cytoplasmic localizations of LXRβ. This localization correlated with caspase-1 activation and cell death induction under treatment with LXR ligand. The association of LXRβ with the truncated form of RXRα (t-RXRα) was responsible for the sequestration of LXRβ in the cytoplasm in colon cancer cells. Moreover t-RXRα was not expressed in normal colon epithelial cells. These cells presented a predominantly nuclear localization of LXRβ and were resistant to LXR ligand cytotoxicity. Our results showed that predominant cytoplasmic localization of LXRβ, which occurs in colon cancer cells but not in normal colon epithelial cells, allowed LXR ligand-induced pyroptosis. This study strengthens the hypothesis that LXRβ could be a promising target in cancer therapy.

  8. A digital local studies collection

    Directory of Open Access Journals (Sweden)

    Simona Resman

    2005-01-01

    Full Text Available Local studies and local studies departments reflect the entire spectrum of knowledge within the local community. Transition to the new digital environment means just continuing the basic functions of local studies collections: acquisition, cataloguing and preservation of materials in different formats on different media. The background of each digital library is the technical architecture of system that enables interaction between the user and the library, saving digital material and building a platform for searching and indexing digital objects. Using advanced ICT requests from librarians, designers of digital local studies collections a reflection about traditional roles. A digital local studies collection with new technology enlarges local boarders, local contents become more and more interesting for a wider sphere of people. In collecting of local materials a collaboration with archives and museums, with academic community, with community groups and with individuals comes in forefront. Digital local studies collections with their contents support local diversity, lifelong learning and social inclusion. The crucial elements of a digital local studies collection are attractive local contents with fast and simple access from one place – a portal. In the digital age public libraries become managers of knowledge also by establishing digital local studies collections.

  9. Using Femtosecond Laser Subcellular Surgery as a Tool to Study Cell Biology

    Energy Technology Data Exchange (ETDEWEB)

    Shen, N; Colvin, M E; Huser, T

    2007-02-27

    Research on cellular function and regulation would be greatly advanced by new instrumentation using methods to alter cellular processes with spatial discrimination on the nanometer-scale. We present a novel technique for targeting submicrometer sized organelles or other biologically important regions in living cells using femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we can vaporize cellular material inside the cell through nonlinear optical processes. This technique enables non-invasive manipulation of the physical structure of a cell with sub-micrometer resolution. We propose to study the role mitochondria play in cell proliferation and apoptosis. Our technique provides a unique tool for the study of cell biology.

  10. Optimization of transient expression procedures in Catharanthus roseus and Arabidopsis thaliana for subcellular localization studies

    OpenAIRE

    Ribeiro, Diana Margarida da Costa

    2010-01-01

    Dissertação de mestrado em Biotecnologia e Bio-Empreendedorismo em Plantas Aromáticas e Medicinais Nowadays, the availability of much gene sequence information demands the development of tools for their fast characterization at the protein level, where function actually resides. Here, the interest in the characterization of certain of the known Arabidopsis class III peroxidase (Prx) genes, as well as the interest in the characterization of candidate genes implicated in the meta...

  11. Lipidomics in tissues, cells and subcellular compartments

    National Research Council Canada - National Science Library

    Horn, Patrick J; Chapman, Kent D

    2012-01-01

    ...‐infusion MS, localization of lipids in tissues and cells by laser desorption/ionization MS, and even profiling of lipids in individual subcellular compartments by direct‐organelle MS. Applications of these approaches to achieve improved understanding of plant lipid metabolism, compartmentation and function are discussed.

  12. Phytanoyl-CoA hydroxylase from rat liver. Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation

    NARCIS (Netherlands)

    Jansen, G. A.; Ofman, R.; Denis, S.; Ferdinandusse, S.; Hogenhout, E. M.; Jakobs, C.; Wanders, R. J.

    1999-01-01

    Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway. Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes. In rat, however,

  13. A novel BAT3 sequence generated by alternative RNA splicing of exon 11B displays cell type-specific expression and impacts on subcellular localization.

    Directory of Open Access Journals (Sweden)

    Nadine Kämper

    Full Text Available The human lymphocyte antigen (HLA encoded BAT3/BAG6 recently attracted interest as a regulator of protein targeting and degradation, a function that could be exerted in the cytosol and in the nucleus. The BAT3 gene was described to consist of 25 exons. Diversity of transcripts can be generated by alternative RNA splicing, which may control subcellular distribution of BAT3.By cDNA sequencing we identified a novel alternatively spliced sequence of the BAT3 gene located between exons 11 and 12, which was designated as exon 11B. Using PCR and colony hybridization we identified six cDNA variants, which were produced by RNA splicing of BAT3 exons 5, 11B and 24. In four examined cell types the content of BAT3 splice variants was examined. Most of the cDNA clones from monocyte-derived dendritic cells contain exon 11B, whereas this sequence was almost absent in the B lymphoma Raji. Exon 5 was detected in most and exon 24 in approximately half of the cDNA clones. The subcellular distribution of endogenous BAT3 largely correlates with a cell type specific splicing pattern. In cells transfected with BAT3 variants, full-length and Δ24 BAT3 displayed nearly exclusive nuclear staining, whereas variants deleted of exon 11B showed substantial cytosolic expression. We show here that BAT3 is mainly expressed in the cytosol of Raji cells, while other cell types displayed both cytosolic and nuclear staining. Export of BAT3 from the nucleus to the cytosol is inhibited by treatment with leptomycin B, indicating that the Crm1 pathway is involved. Nuclear expression of BAT3 containing exon 11B suggests that this sequence plays a role for nuclear retention of the protein.Cell type-specific subcellular expression of BAT3 suggests distinct functions in the cytosol and in the nucleus. Differential expression of BAT3 variants may reconcile the multiple roles described for BAT3.

  14. Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alpha-synuclein☆

    Science.gov (United States)

    Chen, Tao; Liao, Xiaoping; Wen, Guoqiang; Deng, Yidong; Guo, Min; Long, Zhigang; Ouyang, Feng

    2012-01-01

    The specific and effective α-synuclein RNA interference (RNAi) plasmids, and the α-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 (HEK293) cells using the lipofectamine method. Using an inverted fluorescence microscope, α-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type α-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies (Lewy body-like inclusions) in the cytoplasm of some cells transfected with α-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type α-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type α-synuclein in mitochondria. PMID:25767480

  15. Different subcellular locations of secretome components of Gram-positive bacteria

    NARCIS (Netherlands)

    Buist, Girbe; Ridder, Anja N. J. A.; Kok, Jan; Kuipers, Oscar P.

    2006-01-01

    Gram-positive bacteria contain different types of secretion systems for the transport of proteins into or across the cytoplasmic membrane. Recent studies on subcellular localization of specific components of these secretion systems and their substrates have shown that they can be present at various

  16. Subcellular localization of the magnetosome protein MamC in the marine magnetotactic bacterium Magnetococcus marinus strain MC-1 using immunoelectron microscopy.

    Science.gov (United States)

    Valverde-Tercedor, C; Abadía-Molina, F; Martinez-Bueno, M; Pineda-Molina, Estela; Chen, Lijun; Oestreicher, Zachery; Lower, Brian H; Lower, Steven K; Bazylinski, Dennis A; Jimenez-Lopez, C

    2014-07-01

    Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe3O4) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus. Results show that MamC is located only in the magnetosome membrane of Mc. marinus. Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.

  17. Subcellular localization of the magnetosome protein MamC in the marine magnetotactic bacterium Magnetococcus marinus strain MC-1 using immunoelectron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Valverde-Tercedor, C [Universidad de Granada; Abada-Molina, F [Universidad de Granada; Martinez-Bueno, M [Universidad de Granada; Pineda-Molina, Estela [Laboratorio de Estudios Cristalograficos; Chen, Lijun [Ohio State University; Oestreicher, Zachery [Ohio State University; Lower, Brian H [Ohio State University; Lower, Steven K [Ohio State University; Bazylinski, Dennis A [Ames Laboratory; Jimenez-Lopez, C [Universidad de Granada

    2014-04-24

    Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe3O4) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus. Results show that MamC is located only in the magnetosome membrane of Mc. marinus. Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.

  18. Dynamics of the subcellular localization of RalBP1/RLIP through the cell cycle: the role of targeting signals and of protein-protein interactions.

    Science.gov (United States)

    Fillatre, Jonathan; Delacour, Delphine; Van Hove, Lucie; Bagarre, Thomas; Houssin, Nathalie; Soulika, Marina; Veitia, Reiner A; Moreau, Jacques

    2012-05-01

    The small G protein Ras regulates many cell processes, such as gene expression, proliferation, apoptosis, and cell differentiation. Its mutations are associated with one-third of all cancers. Ras functions are mediated, at least in part, by Ral proteins and their downstream effector the Ral-binding protein 1 (RalBP1). RalBP1 is involved in endocytosis and in regulating the dynamics of the actin cytoskeleton. It also regulates early development since it is required for the completion of gastrulation in Xenopus laevis. RalBP1 has also been reported to be the main transporter of glutathione electrophiles, and it is involved in multidrug resistance. Such a variety of functions could be explained by a differential regulation of RalBP1 localization. In this study, we have detected endogenous RalBP1 in the nucleus of interphasic cells. This nuclear targeting is mediated by nuclear localization sequences that map to the N-terminal third of the protein. Moreover, in X. laevis embryos, a C-terminal coiled-coil sequence mediates RalBP1 retention in the nucleus. We have also observed RalBP1 at the level of the actin cytoskeleton, a localization that depends on interaction of the protein with active Ral. During mitosis RalBP1 also associates with the mitotic spindle and the centrosome, a localization that could be negatively regulated by active Ral. Finally, we demonstrate the presence of post-transcriptional and post-translational isoforms of RalBP1 lacking the Ral-binding domain, which opens new possibilities for the existence of Ral-independent functions.

  19. Polyamine metabolism in prostate cancer : studies on localization, cell growth and apoptosis

    OpenAIRE

    Schipper, Raymond Godfried

    2000-01-01

    The involvement of polyamines in cellular growth and differentiation has prompted many studies of their possible role in cellular neoplasia, including prostatic carcinoma and benign hyperplasia, which represent increasingly important pathologies in men. The precise role of the polyamine system in cellular physiology remains to be clarified. Insight in the exact (sub)cellular localization of polyamine metabolic enzymes might greatly contribute to the elucidation of the specific functions of po...

  20. A Parameter Study of Localization

    Directory of Open Access Journals (Sweden)

    Sandor Stephen Mester

    1996-01-01

    Full Text Available Extensive work has been done on the vibration characteristics of perfectly periodic structures. Disorder in the periodic pattern has been found to lead to localization in one-dimensional periodic structures. It is important to understand localization because it causes energy to be concentrated near the disorder and may cause an overestimation of structural damping. A numerical study is conducted to obtain a better understanding of localization. It is found that any mode, even the first, can localize due to the presence of small imperfections.

  1. Sub-cellular localisation studies may spuriously detect the Yes-associated protein, YAP, in nucleoli leading to potentially invalid conclusions of its function.

    Directory of Open Access Journals (Sweden)

    Megan L Finch

    Full Text Available The Yes-associated protein (YAP is a potent transcriptional co-activator that functions as a nuclear effector of the Hippo signaling pathway. YAP is oncogenic and its activity is linked to its cellular abundance and nuclear localisation. Activation of the Hippo pathway restricts YAP nuclear entry via its phosphorylation by Lats kinases and consequent cytoplasmic retention bound to 14-3-3 proteins. We examined YAP expression in liver progenitor cells (LPCs and surprisingly found that transformed LPCs did not show an increase in YAP abundance compared to the non-transformed LPCs from which they were derived. We then sought to ascertain whether nuclear YAP was more abundant in transformed LPCs. We used an antibody that we confirmed was specific for YAP by immunoblotting to determine YAP's sub-cellular localisation by immunofluorescence. This antibody showed diffuse staining for YAP within the cytosol and nuclei, but, noticeably, it showed intense staining of the nucleoli of LPCs. This staining was non-specific, as shRNA treatment of cells abolished YAP expression to undetectable levels by Western blot yet the nucleolar staining remained. Similar spurious YAP nucleolar staining was also seen in mouse embryonic fibroblasts and mouse liver tissue, indicating that this antibody is unsuitable for immunological applications to determine YAP sub-cellular localisation in mouse cells or tissues. Interestingly nucleolar staining was not evident in D645 cells suggesting the antibody may be suitable for use in human cells. Given the large body of published work on YAP in recent years, many of which utilise this antibody, this study raises concerns regarding its use for determining sub-cellular localisation. From a broader perspective, it serves as a timely reminder of the need to perform appropriate controls to ensure the validity of published data.

  2. Bombyx mori DNA/RNA non-specific nuclease: expression of isoforms in insect culture cells, subcellular localization and functional assays.

    Science.gov (United States)

    Liu, Jisheng; Swevers, Luc; Iatrou, Kostas; Huvenne, Hanneke; Smagghe, Guy

    2012-08-01

    A DNA/RNA non-specific alkaline nuclease (BmdsRNase) was isolated from the digestive juice of Bombyx mori. While originally reported to be produced by the midgut only, in this project it was found that the mRNA of this enzyme was also expressed in the epidermis, fat body, gut, thoracic muscles, Malpighian tubules, brain, and silk glands of 5th instar larvae, indicating additional functions to its reported role in nucleic acid digestion in the midgut. In order to study the functional properties of BmdsRNase, three pEA-BmdsRNase expression constructs were generated, characterized by presence or absence of a signal peptide and a propeptide, and used for expression in lepidopteran Hi5 tissue culture cells. Western blot indicated that these different forms of BmdsRNase protein were not secreted into the growth medium, while they were detected in the pellets and supernatants of Hi5 cell extracts. Nucleic acids cleavage experiments indicated that full-length BmdsRNase could digest dsRNA and that the processed form (absence of signal peptide and propeptide) of BmdsRNase could degrade both DNA and dsRNA in Hi5 cell culture. Using a reporter assay targeted by transfected homologous dsRNA, it was shown that the digestive property of the processed form could interfere with the RNAi response. Immunostaining of processed BmdsRNase protein showed asymmetric localization in the cellular cytoplasm and co-localization with Flag-tagged Dicer-2 was also observed. In conclusion, our in vitro studies indicated that intracellular protein isoforms of BmdsRNase can be functional and involved in the regulation of nucleic acid metabolism in the cytoplasm. In particular, because of its propensity to degrade dsRNA, the enzyme might be involved in the innate immune response against invading nucleic acids such as RNA viruses. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Exploitation of eukaryotic subcellular targeting mechanisms by bacterial effectors.

    Science.gov (United States)

    Hicks, Stuart W; Galán, Jorge E

    2013-05-01

    Several bacterial species have evolved specialized secretion systems to deliver bacterial effector proteins into eukaryotic cells. These effectors have the capacity to modulate host cell pathways in order to promote bacterial survival and replication. The spatial and temporal context in which the effectors exert their biochemical activities is crucial for their function. To fully understand effector function in the context of infection, we need to understand the mechanisms that lead to the precise subcellular localization of effectors following their delivery into host cells. Recent studies have shown that bacterial effectors exploit host cell machinery to accurately target their biochemical activities within the host cell.

  4. ERBB2 influences the subcellular localization of the estrogen receptor in tamoxifen-resistant MCF-7 cells leading to the activation of AKT and RPS6KA2.

    Science.gov (United States)

    Pancholi, Sunil; Lykkesfeldt, Anne E; Hilmi, Caroline; Banerjee, Susana; Leary, Alexandra; Drury, Suzanne; Johnston, Stephen; Dowsett, Mitch; Martin, Lesley-Ann

    2008-12-01

    Acquired resistance to endocrine therapies remains a major clinical obstacle in hormone-sensitive breast tumors. We used an MCF-7 breast tumor cell line (Tam(R)-1) resistant to tamoxifen to investigate this mechanism. We demonstrate that Tam(R)-1 express elevated levels of phosphorylated AKT and MAPK3/1-activated RPS6KA2 compared with the parental MCF-7 cell line (MCF-7). There was no change in the level of total ESR between the two cell lines; however, the Tam(R)-1 cells had increased phosphorylation of ESR1 ser(167). SiRNA blockade of AKT or MAPK3/1 had little effect on ESR1 ser(167) phosphorylation, but a combination of the two siRNAs abrogated this. Co-localization studies revealed an association between ERBB2 and ESR1 in the Tam(R)-1 but not MCF-7 cells. ESR1 was redistributed to extranuclear sites in Tam(R)-1 and was less transcriptionally competent compared with MCF-7 suggesting that nuclear ESR1 activity was suppressed in Tam(R)-1. Tamoxifen resistance in the Tam(R)-1 cells could be partially overcome by the ERBB2 inhibitor AG825 in combination with tamoxifen, and this was associated with re-localization of ESR1 to the nucleus. These data demonstrate that tamoxifen-resistant cells have the ability to switch between ERBB2 or ESR1 pathways promoting cell growth and that pharmacological inhibition of ERBB2 may be a therapeutic strategy for overcoming tamoxifen resistance.

  5. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Daugaard, Tina Fuglsang; Holm, Ida E

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapa is the most predominant isoform. The Gfapd isoform is expressed in proliferating......RNA localization patterns were dependent on the different 39-exon sequences included in Gfapd and Gfapa mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential...

  6. Subcellular targeting strategies for drug design and delivery.

    Science.gov (United States)

    Rajendran, Lawrence; Knölker, Hans-Joachim; Simons, Kai

    2010-01-01

    Many drug targets are localized to particular subcellular compartments, yet current drug design strategies are focused on bioavailability and tissue targeting and rarely address drug delivery to specific intracellular compartments. Insights into how the cell traffics its constituents to these different cellular locations could improve drug design. In this Review, we explore the fundamentals of membrane trafficking and subcellular organization, as well as strategies used by pathogens to appropriate these mechanisms and the implications for drug design and delivery.

  7. Calculation of the relative metastabilities of proteins in subcellular compartments of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Dick Jeffrey M

    2009-07-01

    Full Text Available Abstract Background Protein subcellular localization and differences in oxidation state between subcellular compartments are two well-studied features of the the cellular organization of S. cerevisiae (yeast. Theories about the origin of subcellular organization are assisted by computational models that can integrate data from observations of compositional and chemical properties of the system. Presentation and implications of the hypothesis I adopt the hypothesis that the state of yeast subcellular organization is in a local energy minimum. This hypothesis implies that equilibrium thermodynamic models can yield predictions about the interdependence between populations of proteins and their subcellular chemical environments. Testing the hypothesis Three types of tests are proposed. First, there should be correlations between modeled and observed oxidation states for different compartments. Second, there should be a correspondence between the energy requirements of protein formation and the order the appearance of organelles during cellular development. Third, there should be correlations between the predicted and observed relative abundances of interacting proteins within compartments. Results The relative metastability fields of subcellular homologs of glutaredoxin and thioredoxin indicate a trend from less to more oxidizing as mitochondrion – cytoplasm – nucleus. Representing the overall amino acid compositions of proteins in 23 different compartments each with a single reference model protein suggests that the formation reactions for proteins in the vacuole (in relatively oxidizing conditions, ER and early Golgi (in relatively reducing conditions are relatively highly favored, while that for the microtubule is the most costly. The relative abundances of model proteins for each compartment inferred from experimental data were found in some cases to correlate with the predicted abundances, and both positive and negative correlations were

  8. Domains involved in TAF15 subcellular localisation

    DEFF Research Database (Denmark)

    Marko, Marija; Vlassis, Arsenios; Guialis, Apostolia

    2012-01-01

    to play important roles in the onset of specific tumours, certain forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In this study we identified the domains of TAF15 responsible for its subcellular localisation in human (HeLa) cells and experimentally confirmed...

  9. Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA

    Directory of Open Access Journals (Sweden)

    Lacalandra Giovanni M

    2010-06-01

    Full Text Available Abstract Background Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology. Methods We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye. Results We showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations. Conclusions The results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for

  10. The Subcellular Localization of Tubby-Like Proteins and Participation in Stress Signaling and Root Colonization by the Mutualist Piriformospora indica1[W

    Science.gov (United States)

    Reitz, Marco Uwe; Bissue, Jeff Kweku; Zocher, Kathleen; Attard, Agnès; Hückelhoven, Ralph; Becker, Katja; Imani, Jafargholi; Eichmann, Ruth; Schäfer, Patrick

    2012-01-01

    Tubby and Tubby-like proteins (TLPs) were first discovered in mammals, where they are involved in the development and function of neuronal cells. Due to their importance as plasma membrane (PM)-tethered transcription factors or mediators of vesicle trafficking, their lack causes obesity and other disease syndromes. Phosphatidylinositol 4,5-bisphosphate binding of the carboxyl-terminal Tubby domain attaches these proteins to the PM and vesicles and is essential for function. TLPs are conserved across eukaryotic kingdoms including plants, suggesting fundamental biological functions of TLPs. Plant TLPs possess an amino-terminal F-box domain that distinguishes them from other eukaryotic TLPs. Arabidopsis (Arabidopsis thaliana) encodes 11 AtTLPs that fall into six phylogenetic clades. We identified the significance of AtTLPs for root colonization of Arabidopsis by the mutualistic fungus Piriformospora indica. Our results further indicate conserved phosphatidylinositol 4,5-bisphosphate-binding sites in the Tubby domains that are required for PM anchoring of AtTLPs. More detailed studies revealed phospholipase C-triggered release of AtTLP3 from the PM, indicating a conserved mechanism as reported for mammalian Tubby and TLP3. We further show that hydrogen peroxide stimulates the release of AtTLP3 from the PM, presumably for activating downstream events. Different from mammalian homologs, the amino-terminal part of almost all AtTLPs has nucleocytosolic and plastidial localization patterns. Thus, it is tempting to assume that TLPs translate reactive oxygen species currents into signaling not only for transcriptional regulation in the nucleus but also affect plastid-associated functions after release from the PM. PMID:22751378

  11. The subcellular localization of Tubby-like proteins and participation in stress signaling and root colonization by the mutualist Piriformospora indica.

    Science.gov (United States)

    Reitz, Marco Uwe; Bissue, Jeff Kweku; Zocher, Kathleen; Attard, Agnès; Hückelhoven, Ralph; Becker, Katja; Imani, Jafargholi; Eichmann, Ruth; Schäfer, Patrick

    2012-09-01

    Tubby and Tubby-like proteins (TLPs) were first discovered in mammals, where they are involved in the development and function of neuronal cells. Due to their importance as plasma membrane (PM)-tethered transcription factors or mediators of vesicle trafficking, their lack causes obesity and other disease syndromes. Phosphatidylinositol 4,5-bisphosphate binding of the carboxyl-terminal Tubby domain attaches these proteins to the PM and vesicles and is essential for function. TLPs are conserved across eukaryotic kingdoms including plants, suggesting fundamental biological functions of TLPs. Plant TLPs possess an amino-terminal F-box domain that distinguishes them from other eukaryotic TLPs. Arabidopsis (Arabidopsis thaliana) encodes 11 AtTLPs that fall into six phylogenetic clades. We identified the significance of AtTLPs for root colonization of Arabidopsis by the mutualistic fungus Piriformospora indica. Our results further indicate conserved phosphatidylinositol 4,5-bisphosphate-binding sites in the Tubby domains that are required for PM anchoring of AtTLPs. More detailed studies revealed phospholipase C-triggered release of AtTLP3 from the PM, indicating a conserved mechanism as reported for mammalian Tubby and TLP3. We further show that hydrogen peroxide stimulates the release of AtTLP3 from the PM, presumably for activating downstream events. Different from mammalian homologs, the amino-terminal part of almost all AtTLPs has nucleocytosolic and plastidial localization patterns. Thus, it is tempting to assume that TLPs translate reactive oxygen species currents into signaling not only for transcriptional regulation in the nucleus but also affect plastid-associated functions after release from the PM.

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  13. Dynamic changes in subcellular localization of cattle XLF during cell cycle, and focus formation of cattle XLF at DNA damage sites immediately after irradiation.

    Science.gov (United States)

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2015-09-01

    Clinically, many chemotherapeutics and ionizing radiation (IR) have been applied for the treatment of various types of human and animal malignancies. These treatments kill tumor cells by causing DNA double-strand breaks (DSBs). Core factors of classical nonhomologous DNA-end joining (C-NHEJ) play a vital role in DSB repair. Thus, it is indispensable to clarify the mechanisms of C-NHEJ in order to develop next-generation chemotherapeutics for cancer. The XRCC4-like factor (XLF; also called Cernunnos or NHEJ1) is the lastly identified core NHEJ factor. The localization of core NHEJ factors might play a critical role in regulating NHEJ activity. The localization and function of XLF have not been elucidated in animal species other than mice and humans. Domestic cattle (Bos taurus) are the most common and vital domestic animals in many countries. Here, we show that the localization of cattle XLF changes dynamically during the cell cycle. Furthermore, EYFP-cattle XLF accumulates quickly at microirradiated sites and colocalizes with the DSB marker γH2AX. Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288-299) of the C-terminal region of XLF (XLF CTR). Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals. These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.

  14. Histidine Residues in the Na+-coupled Ascorbic Acid Transporter-2 (SVCT2) Are Central Regulators of SVCT2 Function, Modulating pH Sensitivity, Transporter Kinetics, Na+ Cooperativity, Conformational Stability, and Subcellular Localization*

    Science.gov (United States)

    Ormazabal, Valeska; Zuñiga, Felipe A.; Escobar, Elizabeth; Aylwin, Carlos; Salas-Burgos, Alexis; Godoy, Alejandro; Reyes, Alejandro M.; Vera, Juan Carlos; Rivas, Coralia I.

    2010-01-01

    Na+-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His109, His203, His206, His269, and His413, are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na+ cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His413, localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na+ and loss of Na+ cooperativity, which leads to a decreased Vmax without altering the transport Km; (ii) exofacial histidine residues His203, His206, and His413 may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport Km; and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function. PMID:20843809

  15. Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins

    Energy Technology Data Exchange (ETDEWEB)

    Duband-Goulet, Isabelle; Woerner, Stephanie [Laboratoire du Stress et Pathologies du Cytosquelette, Universite Paris Diderot-Paris 7, CNRS, Institut de Biologie Fonctionnelle et Adaptative, 4 rue M.A. Lagroua Weill Halle, 75205 Paris cedex 13 (France); Gasparini, Sylvaine [Laboratoire de Biologie Structurale et Radiobiologie, URA CNRS 2096, Commissariat a l' Energie Atomique Saclay, 91190 Gif-sur-Yvette (France); Attanda, Wikayatou [Laboratoire du Stress et Pathologies du Cytosquelette, Universite Paris Diderot-Paris 7, CNRS, Institut de Biologie Fonctionnelle et Adaptative, 4 rue M.A. Lagroua Weill Halle, 75205 Paris cedex 13 (France); Konde, Emilie; Tellier-Lebegue, Carine [Laboratoire de Biologie Structurale et Radiobiologie, URA CNRS 2096, Commissariat a l' Energie Atomique Saclay, 91190 Gif-sur-Yvette (France); Craescu, Constantin T. [INSERM U759, Institut Curie/Universite de Paris-Sud, 91405 Orsay Cedex (France); Gombault, Aurelie [Laboratoire du Stress et Pathologies du Cytosquelette, Universite Paris Diderot-Paris 7, CNRS, Institut de Biologie Fonctionnelle et Adaptative, 4 rue M.A. Lagroua Weill Halle, 75205 Paris cedex 13 (France); Roussel, Pascal [Institut Jacques Monod, UMR 7592, Universite Paris Diderot-Paris 7, CNRS, 15 rue Helene Brion, 75205 Paris (France); Vadrot, Nathalie; Vicart, Patrick [Laboratoire du Stress et Pathologies du Cytosquelette, Universite Paris Diderot-Paris 7, CNRS, Institut de Biologie Fonctionnelle et Adaptative, 4 rue M.A. Lagroua Weill Halle, 75205 Paris cedex 13 (France); Oestlund, Cecilia; Worman, Howard J. [Department of Medicine and Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); and others

    2011-12-10

    Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome ( Increment 607-656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.

  16. Localization and regulation of mouse pantothenate kinase 2 [The PanK2 Genes of Mouse and Human Specify Proteins with Distinct Subcellular Locations

    Energy Technology Data Exchange (ETDEWEB)

    Leonardi, Roberta [St. Jude Children' s Research Hospital, Memphis, TN (United States); Zhang, Yong-Mei [St. Jude Children' s Research Hospital, Memphis, TN (United States); Lykidis, Athanasios [DOE Joint Genome Inst., Walnut Creek, CA (United States); Rock, Charles O. [St. Jude Children' s Research Hospital, Memphis, TN (United States); Jackowski, Suzanne [St. Jude Children' s Research Hospital, Memphis, TN (United States)

    2007-09-07

    Coenzyme A (CoA) biosynthesis is initiated by pantothenatekinase (PanK) and CoA levels are controlled through differentialexpression and feedback regulation of PanK isoforms. PanK2 is amitochondrial protein in humans, but comparative genomics revealed thatacquisition of a mitochondrial targeting signal was limited to primates.Human and mouse PanK2 possessed similar biochemical properties, withinhibition by acetylCoA and activation by palmitoylcarnitine. Mouse PanK2localized in the cytosol, and the expression of PanK2 was higher in humanbrain compared to mouse brain. Differences in expression and subcellularlocalization should be considered in developing a mouse model for humanPanK2 deficiency.

  17. New insights into the subcellular localization of Tubby-like proteins and their participation in the Arabidopsis-Piriformospora indica interaction.

    Science.gov (United States)

    Reitz, Marco U; Pai, Subhash; Imani, Jafargholi; Schäfer, Patrick

    2013-08-01

    Tubby-like proteins (TLPs) have been associated with hormone signaling and responses to abiotic and biotic stress in plants. Recently, Arabidopsis thaliana TLP3 was found to translocate from the plasma membrane of cells in response to distinct abiotic stresses, thereby activating cellular signaling. In addition, several AtTLPs were demonstrated to be necessary for normal colonization of roots by the mutualistic fungus Piriformospora indica. Here, we present evidence for the involvement of another two AtTLPs in this interaction. Furthermore, we show that plasma membrane targeting of TLPs might be conserved in other plant species, although we did not find it for all members of the protein family. Finally, the position of a GFP-tag influences the localization of AtTLP3, which needs to be considered when working with TLPs.

  18. Yersinia pestis insecticidal-like toxin complex (Tc family proteins: characterization of expression, subcellular localization, and potential role in infection of the flea vector

    Directory of Open Access Journals (Sweden)

    Spinner Justin L

    2012-12-01

    Full Text Available Abstract Background Toxin complex (Tc family proteins were first identified as insecticidal toxins in Photorhabdus luminescens and have since been found in a wide range of bacteria. The genome of Yersinia pestis, the causative agent of bubonic plague, contains a locus that encodes the Tc protein homologues YitA, YitB, YitC, and YipA and YipB. Previous microarray data indicate that the Tc genes are highly upregulated by Y. pestis while in the flea vector; however, their role in the infection of fleas and pathogenesis in the mammalian host is unclear. Results We show that the Tc proteins YitA and YipA are highly produced by Y. pestis while in the flea but not during growth in brain heart infusion (BHI broth at the same temperature. Over-production of the LysR-type regulator YitR from an exogenous plasmid increased YitA and YipA synthesis in broth culture. The increase in production of YitA and YipA correlated with the yitR copy number and was temperature-dependent. Although highly synthesized in fleas, deletion of the Tc proteins did not alter survival of Y. pestis in the flea or prevent blockage of the proventriculus. Furthermore, YipA was found to undergo post-translational processing and YipA and YitA are localized to the outer membrane of Y. pestis. YitA was also detected by immunofluorescence microscopy on the surface of Y. pestis. Both YitA and YipA are produced maximally at low temperature but persist for several hours after transfer to 37°C. Conclusions Y. pestis Tc proteins are highly expressed in the flea but are not essential for Y. pestis to stably infect or produce a transmissible infection in the flea. However, YitA and YipA localize to the outer membrane and YitA is exposed on the surface, indicating that at least YitA is present on the surface when Y. pestis is transmitted into the mammalian host from the flea.

  19. A multiple information fusion method for predicting subcellular locations of two different types of bacterial protein simultaneously.

    Science.gov (United States)

    Chen, Jing; Xu, Huimin; He, Ping-An; Dai, Qi; Yao, Yuhua

    2016-01-01

    Subcellular localization prediction of bacterial protein is an important component of bioinformatics, which has great importance for drug design and other applications. For the prediction of protein subcellular localization, as we all know, lots of computational tools have been developed in the recent decades. In this study, we firstly introduce three kinds of protein sequences encoding schemes: physicochemical-based, evolutionary-based, and GO-based. The original and consensus sequences were combined with physicochemical properties. And elements information of different rows and columns in position-specific scoring matrix were taken into consideration simultaneously for more core and essence information. Computational methods based on gene ontology (GO) have been demonstrated to be superior to methods based on other features. Then principal component analysis (PCA) is applied for feature selection and reduced vectors are input to a support vector machine (SVM) to predict protein subcellular localization. The proposed method can achieve a prediction accuracy of 98.28% and 97.87% on a stringent Gram-positive (Gpos) and Gram-negative (Gneg) dataset with Jackknife test, respectively. At last, we calculate "absolute true overall accuracy (ATOA)", which is stricter than overall accuracy. The ATOA obtained from the proposed method is also up to 97.32% and 93.06% for Gpos and Gneg. From both the rationality of testing procedure and the success rates of test results, the current method can improve the prediction quality of protein subcellular localization. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. ABC Transporter Subfamily D: Distinct Differences in Behavior between ABCD1–3 and ABCD4 in Subcellular Localization, Function, and Human Disease

    Directory of Open Access Journals (Sweden)

    Kosuke Kawaguchi

    2016-01-01

    Full Text Available ATP-binding cassette (ABC transporters are one of the largest families of membrane-bound proteins and transport a wide variety of substrates across both extra- and intracellular membranes. They play a critical role in maintaining cellular homeostasis. To date, four ABC transporters belonging to subfamily D have been identified. ABCD1–3 and ABCD4 are localized to peroxisomes and lysosomes, respectively. ABCD1 and ABCD2 are involved in the transport of long and very long chain fatty acids (VLCFA or their CoA-derivatives into peroxisomes with different substrate specificities, while ABCD3 is involved in the transport of branched chain acyl-CoA into peroxisomes. On the other hand, ABCD4 is deduced to take part in the transport of vitamin B12 from lysosomes into the cytosol. It is well known that the dysfunction of ABCD1 results in X-linked adrenoleukodystrophy, a severe neurodegenerative disease. Recently, it is reported that ABCD3 and ABCD4 are responsible for hepatosplenomegaly and vitamin B12 deficiency, respectively. In this review, the targeting mechanism and physiological functions of the ABCD transporters are summarized along with the related disease.

  1. ABC Transporter Subfamily D: Distinct Differences in Behavior between ABCD1–3 and ABCD4 in Subcellular Localization, Function, and Human Disease

    Science.gov (United States)

    2016-01-01

    ATP-binding cassette (ABC) transporters are one of the largest families of membrane-bound proteins and transport a wide variety of substrates across both extra- and intracellular membranes. They play a critical role in maintaining cellular homeostasis. To date, four ABC transporters belonging to subfamily D have been identified. ABCD1–3 and ABCD4 are localized to peroxisomes and lysosomes, respectively. ABCD1 and ABCD2 are involved in the transport of long and very long chain fatty acids (VLCFA) or their CoA-derivatives into peroxisomes with different substrate specificities, while ABCD3 is involved in the transport of branched chain acyl-CoA into peroxisomes. On the other hand, ABCD4 is deduced to take part in the transport of vitamin B12 from lysosomes into the cytosol. It is well known that the dysfunction of ABCD1 results in X-linked adrenoleukodystrophy, a severe neurodegenerative disease. Recently, it is reported that ABCD3 and ABCD4 are responsible for hepatosplenomegaly and vitamin B12 deficiency, respectively. In this review, the targeting mechanism and physiological functions of the ABCD transporters are summarized along with the related disease. PMID:27766264

  2. LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization

    Energy Technology Data Exchange (ETDEWEB)

    Rapali, Peter [Dept. Biochemistry, Eoetvoes Lorand University, Budapest (Hungary); Garcia-Mayoral, Maria Flor [Dept. Biological Physical Chemistry, IQFR, CSIC, Madrid (Spain); Martinez-Moreno, Monica [Dept. Biochemistry and Molecular Biology I, Universidad Complutense, Madrid (Spain); Tarnok, Krisztian; Schlett, Katalin [Dept. Physiology and Neurobiology, Eoetvoes Lorand University, Budapest (Hungary); Albar, Juan Pablo [Proteomics Facility, CNB, CSIC, Madrid (Spain); Bruix, Marta [Dept. Biological Physical Chemistry, IQFR, CSIC, Madrid (Spain); Nyitray, Laszlo, E-mail: nyitray@elte.hu [Dept. Biochemistry, Eoetvoes Lorand University, Budapest (Hungary); Rodriguez-Crespo, Ignacio, E-mail: nacho@bbm1.ucm.es [Dept. Biochemistry and Molecular Biology I, Universidad Complutense, Madrid (Spain)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer We have screened a human library with dynein light chain DYNLL1 (DLC8) as bait. Black-Right-Pointing-Pointer Dynein light chain DYNLL1 binds to ATM-kinase interacting protein (ATMIN). Black-Right-Pointing-Pointer ATMIN has 17 SQ/TQ motifs, a motif frequently found in DYNLL1-binding partners. Black-Right-Pointing-Pointer The two proteins interact in vitro, with ATMIN displaying at least five binding sites. Black-Right-Pointing-Pointer The interaction of ATMIN and DYNNL1 in transfected cells can also be observed. -- Abstract: LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1

  3. In vivo localization studies in the stramenopile alga Nannochloropsis oceanica.

    Science.gov (United States)

    Moog, Daniel; Stork, Simone; Reislöhner, Sven; Grosche, Christopher; Maier, Uwe-G

    2015-02-01

    The tiny eustigmatophyte Nannochloropsis sp. recently emerged as a promising model organism for biotechnology as it possesses a considerably high cellular oil content interesting for biodiesel production. Furthermore, the alga was shown to be genetically well accessible providing powerful tools for biotechnological engineering as well as basic research. Nannochloropsis sp. can be transformed very efficiently taking advantage of homologous recombination, however, so far it remained unclear whether these organisms are also suitable model systems for in vivo protein localization studies due to their small cell size. Here we present, to our knowledge, the first protein localization studies based on the expression of chimeric fluorescent fusion proteins in the genus Nannochloropsis using N. oceanica CCMP1779 as a model organism. Besides expressing a cytosolic green fluorescent protein (GFP), the reporter could be directed into subcellular structures such as the mitochondria, the endoplasmic reticulum and secretory pathway as well as the complex plastid including the periplastidal compartment and the stroma via fusion of specific N-terminal targeting sequences. These results expand the potential of N. oceanica as a model system in biotechnology as well as cellular biology for which now an almost complete molecular tool set exists. Copyright © 2015 Elsevier GmbH. All rights reserved.

  4. Structural and functional plasticity of subcellular tethering, targeting and processing of RPGRIP1 by RPGR isoforms

    Directory of Open Access Journals (Sweden)

    Hemangi Patil

    2012-02-01

    Mutations affecting the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1 interactome cause syndromic retinal dystrophies. RPGRIP1 interacts with the retinitis pigmentosa GTPase regulator (RPGR through a domain homologous to RCC1 (RHD, a nucleotide exchange factor of Ran GTPase. However, functional relationships between RPGR and RPGRIP1 and their subcellular roles are lacking. We show by molecular modeling and analyses of RPGR disease-mutations that the RPGR-interacting domain (RID of RPGRIP1 embraces multivalently the shared RHD of RPGR1–19 and RPGRORF15 isoforms and the mutations are non-overlapping with the interface found between RCC1 and Ran GTPase. RPGR disease-mutations grouped into six classes based on their structural locations and differential impairment with RPGRIP1 interaction. RPGRIP1α1 expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1α1 self-aggregation ensue. RPGR1–19 localizes to the endoplasmic reticulum, whereas RPGRORF15 presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1α1. Disease mutations in RPGR1–19, RPGRORF15, or RID of RPGRIP1α1, singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1α1 with RPGR1–19 or RPGRORF15 in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGRORF15, but not RPGR1–19, protects the RID of RPGRIP1α1 from limited proteolysis. These studies define RPGR- and cell-type-dependent targeting pathways with structural and functional plasticity modulating the expression of mutations in RPGR and RPGRIP1. Further, RPGR isoforms distinctively determine the subcellular targeting of RPGRIP1α1, with deficits in RPGRORF15-dependent intracellular localization of RPGRIP1α1 contributing to pathomechanisms shared by etiologically distinct syndromic retinal dystrophies.

  5. Subcellular localization of casein kinase I

    DEFF Research Database (Denmark)

    Grankowski, N; Issinger, O G

    1990-01-01

    An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus....

  6. Subcellular localization of cadmium in hyperaccumulator Populus ...

    African Journals Online (AJOL)

    Yomi

    2012-02-01

    Feb 1, 2012 ... 3College of Mechanical and Electric Engineering, Northwest A&F University, 712100 Yangling, China. 4College of Science ... processes, such as respiration, transpiration, photo- synthesis .... with a uniform shape in all parts.

  7. Subcellular localization of cadmium in hyperaccumulator Populus ...

    African Journals Online (AJOL)

    ... of damage to organs of grey poplar was as follows: root > stem> leaves. It was suggested that the Populus × canescens as a renewable resource has the potential to decontaminate cadmium stress development, accumulation and distribution. Key words: Cadmium, phytoremediation, hyperaccumulator, grey poplar, organ.

  8. Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity.

    Science.gov (United States)

    Staudt, Emanuel; Ramasamy, Pathmanaban; Plattner, Helmut; Simon, Martin

    2016-12-01

    Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP3. In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Pre-embedding staining of single muscle fibers for light and electron microscopy studies of subcellular organization

    DEFF Research Database (Denmark)

    Ralston, E; Ploug, Thorkil

    1996-01-01

    Skeletal muscle fibers are large, multinucleated cells which pose a challenge to the morphologist. In the course of studies of the distribution of the glucose transporter GLUT4, in muscle, we have compared different preparative procedures, for both light (LM) and electron microscopy (EM......) immunocytochemistry. Here we show that pre-embedding staining of single teased fibers, or of single enzymatically dissociated fibers, has several advantages over the use of sections for observing discrete patterns that extend over long distances in the cells. We report on an optimization study carried out...... to establish fixation and permeabilization conditions for EM immunogold labeling of the fibers. We find that a simple fixation with depolymerized paraformaldehyde alone, followed by permeabilization with 0.01% saponin, offers the best compromise between the conflicting demands of unhindered tissue penetration...

  10. Identification of a classic nuclear localization signal at the N terminus that regulates the subcellular localization of Rbfox2 isoforms during differentiation of NMuMG and P19 cells.

    Science.gov (United States)

    Wenzel, Manuel; Schüle, Martin; Casanovas, Sonia; Strand, Dennis; Strand, Susanne; Winter, Jennifer

    2016-12-01

    Nuclear localization of the alternative splicing factor Rbfox2 is achieved by a C-terminal nuclear localization signal (NLS) which can be excluded from some Rbfox2 isoforms by alternative splicing. While this predicts nuclear and cytoplasmic localization, Rbfox2 is exclusively nuclear in some cell types. Here, we identify a second NLS in the N terminus of Rbfox2 isoform 1A that is not included in Rbfox2 isoform 1F. Rbfox2 1A isoforms lacking the C-terminal NLS are nuclear, whereas equivalent 1F isoforms are cytoplasmic. A shift in Rbfox2 expression toward cytoplasmic 1F isoforms occurs during epithelial to mesenchymal transition (EMT) and could be important in regulating the activity and function of Rbfox2. © 2016 Federation of European Biochemical Societies.

  11. Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

    Science.gov (United States)

    Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

    2016-10-07

    Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

  12. A functional dissection of PTEN N-terminus : Implications in PTEN subcellular targeting and tumor suppressor activity

    NARCIS (Netherlands)

    Gil, Anabel; Rodríguez-Escudero, Isabel; Stumpf, Miriam; Molina, María; Cid, Víctor J.; Pulido, Rafael

    2015-01-01

    Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization

  13. Rice DB: an Oryza Information Portal linking annotation, subcellular location, function, expression, regulation, and evolutionary information for rice and Arabidopsis.

    Science.gov (United States)

    Narsai, Reena; Devenish, James; Castleden, Ian; Narsai, Kabir; Xu, Lin; Shou, Huixia; Whelan, James

    2013-12-01

    Omics research in Oryza sativa (rice) relies on the use of multiple databases to obtain different types of information to define gene function. We present Rice DB, an Oryza information portal that is a functional genomics database, linking gene loci to comprehensive annotations, expression data and the subcellular location of encoded proteins. Rice DB has been designed to integrate the direct comparison of rice with Arabidopsis (Arabidopsis thaliana), based on orthology or 'expressology', thus using and combining available information from two pre-eminent plant models. To establish Rice DB, gene identifiers (more than 40 types) and annotations from a variety of sources were compiled, functional information based on large-scale and individual studies was manually collated, hundreds of microarrays were analysed to generate expression annotations, and the occurrences of potential functional regulatory motifs in promoter regions were calculated. A range of computational subcellular localization predictions were also run for all putative proteins encoded in the rice genome, and experimentally confirmed protein localizations have been collated, curated and linked to functional studies in rice. A single search box allows anything from gene identifiers (for rice and/or Arabidopsis), motif sequences, subcellular location, to keyword searches to be entered, with the capability of Boolean searches (such as AND/OR). To demonstrate the utility of Rice DB, several examples are presented including a rice mitochondrial proteome, which draws on a variety of sources for subcellular location data within Rice DB. Comparisons of subcellular location, functional annotations, as well as transcript expression in parallel with Arabidopsis reveals examples of conservation between rice and Arabidopsis, using Rice DB (http://ricedb.plantenergy.uwa.edu.au). © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  14. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation......, regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  15. Internalization and subcellular fate of aptamer and peptide dual-functioned nanoparticles.

    Science.gov (United States)

    Gao, Huile; Yang, Zhi; Zhang, Shuang; Pang, Zhiqing; Jiang, Xinguo

    2014-06-01

    To evaluate the internalization and subcellular fate of AS1411 aptamer (for glioma targeting) and TGN peptide (for blood-brain barrier targeting)-modified nanoparticles (AsTNPs), which was important for optimizing targeted delivery systems and realizing the potential toxicity to cells. Organelles were labelled with specific markers. Several uptake inhibitors were used to determine the endocytosis pathways. Transmission electron microscopy (TEM) was utilized to directly observe the endocytosis procedure and subcellular fate of AsTNPs. Subcellular localization demonstrated that endosomes and mitochondria were involved in the uptake of AsTNPs by both C6 and bEnd.3 cells, however, lysosomes and Golgi apparatus were only involved in the internalization by C6 cells rather than bEnd.3 cells. Uptake mechanism study demonstrated the clathrin- and caveolae-mediated endocytosis were the main pathways in the uptake of AsTNPs by C6 and bEnd.3 cells. However, other pathways, including clathrin- and caveolae-independent endocytosis and macropinocytosis are also involved in the uptake by C6 cells and not by bEnd.3 cells. TEM directly demonstrated the involvement of these pathways. Particles could be found mostly in endosomes. Compared to unmodified nanoparticles, AsTNPs displayed different internalization pathways involved in several cell organelles.

  16. Subcellular distribution of non-muscle myosin IIb is controlled by FILIP through Hsc70.

    Directory of Open Access Journals (Sweden)

    Hideshi Yagi

    Full Text Available The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP. However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology.

  17. Laserspritzer: a simple method for optogenetic investigation with subcellular resolutions.

    Science.gov (United States)

    Sun, Qian-Quan; Wang, Xinjun; Yang, Weiguo

    2014-01-01

    To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2) is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites). We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research.

  18. Laserspritzer: a simple method for optogenetic investigation with subcellular resolutions.

    Directory of Open Access Journals (Sweden)

    Qian-Quan Sun

    Full Text Available To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2 is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites. We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research.

  19. Subcellular Organization of GPCR Signaling.

    Science.gov (United States)

    Eichel, Kelsie; von Zastrow, Mark

    2018-02-01

    G protein-coupled receptors (GPCRs) comprise a large and diverse class of signal-transducing receptors that undergo dynamic and isoform-specific membrane trafficking. GPCRs thus have an inherent potential to initiate or regulate signaling reactions from multiple membrane locations. This review discusses emerging insights into the subcellular organization of GPCR function in mammalian cells, focusing on signaling transduced by heterotrimeric G proteins and β-arrestins. We summarize recent evidence indicating that GPCR-mediated activation of G proteins occurs not only from the plasma membrane (PM) but also from endosomes and Golgi membranes and that β-arrestin-dependent signaling can be transduced from the PM by β-arrestin trafficking to clathrin-coated pits (CCPs) after dissociation from a ligand-activated GPCR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Structural and functional plasticity of subcellular tethering, targeting and processing of RPGRIP1 by RPGR isoforms.

    Science.gov (United States)

    Patil, Hemangi; Guruju, Mallikarjuna R; Cho, Kyoung-In; Yi, Haiqing; Orry, Andrew; Kim, Hyesung; Ferreira, Paulo A

    2012-02-15

    Mutations affecting the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) interactome cause syndromic retinal dystrophies. RPGRIP1 interacts with the retinitis pigmentosa GTPase regulator (RPGR) through a domain homologous to RCC1 (RHD), a nucleotide exchange factor of Ran GTPase. However, functional relationships between RPGR and RPGRIP1 and their subcellular roles are lacking. We show by molecular modeling and analyses of RPGR disease-mutations that the RPGR-interacting domain (RID) of RPGRIP1 embraces multivalently the shared RHD of RPGR(1-19) and RPGR(ORF15) isoforms and the mutations are non-overlapping with the interface found between RCC1 and Ran GTPase. RPGR disease-mutations grouped into six classes based on their structural locations and differential impairment with RPGRIP1 interaction. RPGRIP1α(1) expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1α(1) self-aggregation ensue. RPGR(1-19) localizes to the endoplasmic reticulum, whereas RPGR(ORF15) presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1α(1). Disease mutations in RPGR(1) (-19), RPGR(ORF15), or RID of RPGRIP1α(1), singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1α(1) with RPGR(1-19) or RPGR(ORF15) in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGR(ORF15), but not RPGR(1-19), protects the RID of RPGRIP1α(1) from limited proteolysis. These studies define RPGR- and cell-type-dependent targeting pathways with structural and functional plasticity modulating the expression of mutations in RPGR and RPGRIP1. Further, RPGR isoforms distinctively determine the subcellular targeting of RPGRIP1α(1,) with deficits in RPGR(ORF15)-dependent intracellular localization of RPGRIP1α(1) contributing to pathomechanisms shared by etiologically distinct syndromic

  1. Internalization and Subcellular Trafficking of Poly-l-lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation.

    Science.gov (United States)

    Avaritt, Brittany R; Swaan, Peter W

    2015-06-01

    Internalization and intracellular trafficking of dendrimer-drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-l-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer-drug conjugates requires careful consideration to achieve successful drug delivery.

  2. cAMP signaling in subcellular compartments.

    Science.gov (United States)

    Lefkimmiatis, Konstantinos; Zaccolo, Manuela

    2014-09-01

    In the complex microcosm of a cell, information security and its faithful transmission are critical for maintaining internal stability. To achieve a coordinated response of all its parts to any stimulus the cell must protect the information received from potentially confounding signals. Physical segregation of the information transmission chain ensures that only the entities able to perform the encoded task have access to the relevant information. The cAMP intracellular signaling pathway is an important system for signal transmission responsible for the ancestral 'flight or fight' response and involved in the control of critical functions including frequency and strength of heart contraction, energy metabolism and gene transcription. It is becoming increasingly apparent that the cAMP signaling pathway uses compartmentalization as a strategy for coordinating the large number of key cellular functions under its control. Spatial confinement allows the formation of cAMP signaling "hot spots" at discrete subcellular domains in response to specific stimuli, bringing the information in proximity to the relevant effectors and their recipients, thus achieving specificity of action. In this report we discuss how the different constituents of the cAMP pathway are targeted and participate in the formation of cAMP compartmentalized signaling events. We illustrate a few examples of localized cAMP signaling, with a particular focus on the nucleus, the sarcoplasmic reticulum and the mitochondria. Finally, we discuss the therapeutic potential of interventions designed to perturb specific cAMP cascades locally. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Objective Clustering of Proteins Based on Subcellular Location Patterns

    Directory of Open Access Journals (Sweden)

    Xiang Chen

    2005-01-01

    Full Text Available The goal of proteomics is the complete characterization of all proteins. Efforts to characterize subcellular location have been limited to assigning proteins to general categories of organelles. We have previously designed numerical features to describe location patterns in microscope images and developed automated classifiers that distinguish major subcellular patterns with high accuracy (including patterns not distinguishable by visual examination. The results suggest the feasibility of automatically determining which proteins share a single location pattern in a given cell type. We describe an automated method that selects the best feature set to describe images for a given collection of proteins and constructs an effective partitioning of the proteins by location. An example for a limited protein set is presented. As additional data become available, this approach can produce for the first time an objective systematics for protein location and provide an important starting point for discovering sequence motifs that determine localization.

  4. Palmitoylation of stathmin family proteins domain A controls Golgi versus mitochondrial subcellular targeting.

    Science.gov (United States)

    Chauvin, Stéphanie; Poulain, Fabienne E; Ozon, Sylvie; Sobel, André

    2008-10-01

    Precise localization of proteins to specialized subcellular domains is fundamental for proper neuronal development and function. The neural microtubule-regulatory phosphoproteins of the stathmin family are such proteins whose specific functions are controlled by subcellular localization. Whereas stathmin is cytosolic, SCG10, SCLIP and RB3/RB3'/RB3'' are localized to the Golgi and vesicle-like structures along neurites and at growth cones. We examined the molecular determinants involved in the regulation of this specific subcellular localization in hippocampal neurons in culture. We show that their conserved N-terminal domain A carrying two palmitoylation sites is dominant over the others for Golgi and vesicle-like localization. Using palmitoylation-deficient GFP (green fluorescent protein) fusion mutants, we demonstrate that domains A of stathmin proteins have the particular ability to control protein targeting to either Golgi or mitochondria, depending on their palmitoylation. This regulation involves the co-operation of two subdomains within domain A, and seems also to be under the control of its SLD (stathmin-like domain) extension. Our results unravel that, in specific biological conditions, palmitoylation of stathmin proteins might be able to control their targeting to express their functional activities at appropriate subcellular sites. They, more generally, open new perspectives regarding the role of palmitoylation as a signalling mechanism orienting proteins to their functional subcellular compartments.

  5. Subcellular and cellular locations of nitric-oxide synthase isoforms as determinants of health and disease

    Science.gov (United States)

    Villanueva, Cleva; Giulivi, Cecilia

    2010-01-01

    The effects of nitric oxide in biological systems depend on its steady-state concentration and where it is being produced. The organ where nitric oxide is produced is relevant, and within the organ, which types of cells are actually contributing to this production seem to play a major determinant of its effect. Subcellular compartmentalization of specific nitric-oxide synthase enzymes has been shown to play a major role in health and disease. Pathophysiological conditions affect the cellular expression and localization of nitric oxide synthases, which in turn alter organ cross talk. In this study, we described the compartmentalization of nitric oxide in organs, cells and subcellular organelles, and how its localization relates to several relevant clinical conditions. Understanding the complexity of the compartmentalization of nitric oxide production and the implications of this compartmentalization in terms of cellular targets and downstream effects will eventually contribute toward the development of better strategies for treating or preventing pathological events associated with the increase, inhibition or mislocalization of nitric oxide production. PMID:20388537

  6. A Monte Carlo study of energy deposition at the sub-cellular level for application to targeted radionuclide therapy with low-energy electron emitters

    Science.gov (United States)

    Emfietzoglou, D.; Bousis, C.; Hindorf, C.; Fotopoulos, A.; Pathak, A.; Kostarelos, K.

    2007-03-01

    Optimizing targeted radionuclide therapy for patients with circulating malignant cells (e.g. blood-related cancers) or a micrometastatic spread requires quantification of various dosimetric parameters at the single-cell level. We present results on the energy deposition of monoenergetic electrons of initial energy from 100 eV to 20 keV - relevant to Auger emitting radionuclides - distributed either uniformly or at the surface of spherical volumes of radii from 10 nm to 1 μm which correspond to critical sub-cellular targets. Calculations have been carried out by our detailed-history Monte Carlo (MC) code which simulates event-by-event the complete slowing down (to 1 Ry) of both the primary and all subsequent generations of electrons, as well as, by the continuous-slowing-down-approximation (CSDA) using analytic range-energy relationships. The latter method has been adopted by the MIRD committee of the Society of Nuclear Medicine for dosimetry at the cellular level (>1 μm). Differences between the MC and CSDA results are up to ∼50% and are expected to be even larger at higher energies and/or smaller volumes. They are attributed to the deficiencies of the CSDA method associated with the neglect of straggling and δ-ray transport. The results are particularly relevant to targeted radiotherapy at the genome level by Auger emitters.

  7. Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

    Science.gov (United States)

    Chandra, Subhash

    2008-12-01

    Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells

  8. Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, Subhash [Cornell SIMS Laboratory, Department of Earth and Atmospheric Sciences, Snee Hall, Cornell University, Ithaca, NY 14853 (United States)], E-mail: sc40@cornell.edu

    2008-12-15

    Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O{sub 2}{sup +}) revealed local secondary ion signal enhancements correlated with the water image signals of {sup 19}(H{sub 3}O){sup +}. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K{sup +} and Na{sup +} in physiologically relevant concentrations. The high K

  9. Subcellular targeting domains of sphingomyelin synthase 1 and 2.

    Science.gov (United States)

    Yeang, Calvin; Ding, Tingbo; Chirico, William J; Jiang, Xian-Cheng

    2011-12-14

    Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. Furthermore, its product SM has been implicated in atherogenic processes such as retention of lipoproteins in the blood vessel intima. There are two mammalian sphingomyelin synthases: SMS1 and SMS2. SMS1 is found exclusively in the Golgi at steady state, whereas SMS2 exists in the Golgi and plasma membrane. Conventional motifs responsible for protein targeting to the plasma membrane or Golgi are either not present in, or unique to, SMS1 and SMS2. In this study, we examined how SMS1 and SMS2 achieve their respective subcellular localization patterns. Brefeldin A treatment prevented SMS1 and SMS2 from exiting the ER, demonstrating that they transit through the classical secretory pathway. We created truncations and chimeras of SMS1 and SMS2 to define their targeting signals. We found that SMS1 contains a C-terminal Golgi targeting signal and that SMS2 contains a C-terminal plasma membrane targeting signal.

  10. Mathematical modeling of sub-cellular asymmetry of fat-dachsous heterodimer for generation of planar cell polarity.

    Directory of Open Access Journals (Sweden)

    Mohit Kumar Jolly

    Full Text Available Planar Cell Polarity (PCP is an evolutionarily conserved characteristic of animal tissues marked by coordinated polarization of cells or structures in the plane of a tissue. In insect wing epithelium, for instance, PCP is characterized by en masse orientation of hairs orthogonal to its apical-basal axis and pointing along the proximal-distal axis of the organ. Directional cue for PCP has been proposed to be generated by complex sets of interactions amongst three proteins - Fat (Ft, Dachsous (Ds and Four-jointed (Fj. Ft and Ds are two atypical cadherins, which are phosphorylated by Fj, a Golgi kinase. Ft and Ds from adjacent cells bind heterophilically via their tandem cadherin repeats, and their binding affinities are regulated by Fj. Further, in the wing epithelium, sub-cellular levels of Ft-Ds heterodimers are seen to be elevated at the distal edges of individual cells, prefiguring their PCP. Mechanisms generating this sub-cellular asymmetry of Ft-Ds heterodimer in proximal and distal edges of cells, however, have not been resolved yet. Using a mathematical modeling approach, here we provide a framework for generation of this sub-cellular asymmetry of Ft-Ds heterodimer. First, we explain how the known interactions within Ft-Ds-Fj system translate into sub-cellular asymmetry of Ft-Ds heterodimer. Second, we show that this asymmetric localization of Ft-Ds heterodimer is lost when tissue-level gradient of Fj is flattened, or when phosphorylation of Ft by Fj is abolished, but not when tissue-level gradient of Ds is flattened or when phosphorylation of Ds is abrogated. Finally, we show that distal enrichment of Ds also amplifies Ft-Ds asymmetry. These observations reveal that gradient of Fj expression, phosphorylation of Ft by Fj and sub-cellular distal accumulation of Ds are three critical elements required for generating sub-cellular asymmetry of Ft-Ds heterodimer. Our model integrates the known experimental data and presents testable predictions

  11. Subcellular fractionation associated to radionuclide analysis in various tissues: validation of the technique by using light and electron observations applied on gills bivalves and uranium

    Energy Technology Data Exchange (ETDEWEB)

    Camilleri, V.; Simon, O.; Grasset, G. [CEA Cadarache (DEI/SECRE/LRE), Laboratory of Radioecology and Ecotoxicology, Institute for Radioprotection and Nuclear Safety, 13 - Saint-Paul-lez-Durance (France)

    2004-07-01

    The metal bioaccumulation levels in target-organs associated with micro-localization approaches at the subcellular level provide information for the understanding of the metabolic metal cycle. These findings could be used to select relevant bio-markers of exposure and to focus on specific contaminated organelles to study potential biological effects. Moreover, the metal accumulated in the cytosol fraction can be bound to macromolecules in order to be eliminated and/or to induce a potential cellular effect. Tissular distribution, transfer efficiency from water and subcellular fractionation were investigated on the freshwater bivalve, Corbicula fluminea after uranium aqueous exposure. The subcellular fractionation was performed while measuring associated uranium to each cellular different fraction as follows: cellular debris and nuclei, mitochondria and lysosomes, membranes, microsomes and cytosol. In our experimental conditions, the accumulation in the cytosol fraction was low and more than 80 % of the total uranium in gills and visceral mass was accumulated in the insoluble fraction. Main results presented in this poster come from light and electron microscope observations of subcellular fractions (nuclei/debris and lysosomes/mitochondria) in order to validate the efficiency of the fractionation technique. An adaptation of the fractionation technique is proposed. This set of data confirms high differences of fractionation efficiency as a function of fractionation technique and organs/biological model used (gills of bivalves, digestive gland of crayfish). (author)

  12. Subcellular distribution of calcium during spermatogenesis of zebrafish, Danio rerio.

    Science.gov (United States)

    Golpour, Amin; Pšenička, Martin; Niksirat, Hamid

    2017-08-01

    Calcium plays a variety of vital regulatory functions in many physiological and biochemical events in the cell. The aim of this study was to describe the ultrastructural distribution of calcium during different developmental stages of spermatogenesis in a model organism, the zebrafish (Danio rerio), using a combined oxalate-pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly detectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron-dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The notable changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the

  13. Visualization of Multicolored in vivo Organelle Markers for Co-Localization Studies in Oryza sativa.

    Science.gov (United States)

    Dangol, Sarmina; Singh, Raksha; Chen, Yafei; Jwa, Nam-Soo

    2017-11-06

    Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organellespecific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into dicot plants, but its use is still limited in monocot plants. Here, we generated a set of multicolored organelle markers (fluorescent-tagged proteins) based on wellestablished targeting sequences. We used a series of pGWBs binary vectors to ameliorate localization and co-localization experiments using monocot plants. We constructed different fluorescent-tagged markers to visualize rice cell organelles, i.e., nucleus, plastids, mitochondria, peroxisomes, golgi body, endoplasmic reticulum, plasma membrane, and tonoplast, with four different fluorescent proteins (FPs) (G3GFP, mRFP, YFP, and CFP). Visualization of FP-tagged markers in their respective compartments has been reported for dicot and monocot plants. The comparative localization of the nucleus marker with a nucleus localizing sequence, and the similar, characteristic morphology of mCherry-tagged Arabidopsis organelle markers and our generated organelle markers in onion cells, provide further evidence for the correct subcellular localization of the Oryza sativa (rice) organelle marker. The set of eight different rice organelle markers with four different FPs provides a valuable resource for determining the subcellular localization of newly identified proteins, conducting co-localization assays, and generating stable transgenic localization in monocot plants.

  14. Imaging trace element distributions in single organelles and subcellular features

    Science.gov (United States)

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

  15. Intracellular And Subcellular Partitioning Of Nickel In Aureococcus Anophagefferens

    Science.gov (United States)

    Wang, B.; Axe, L.; Wei, L.; Bagheri, S.; Michalopoulou, Z.

    2008-12-01

    Brown tides are caused by Aureococcus anophagefferens, a species of Pelagophyceae, and have been observed in NY/NJ waterways effecting ecosystems by attenuating light, changing water color, reducing eelgrass beds, decreasing shellfisheries, and further impacting the food web by reducing phytoplankton. Although the impact of macronutrients and iron on A. anophagefferens has been well studied, contaminants, and specifically trace metals have not. In long-term experiments designed to investigate the growth and toxicity, Cd, Cu, Ni, and Zn exposure was evaluated over 10-13 to 10-7 M for the free metal ion. While growth was inhibited or terminated from exposure to Cd and Cu, nickel addition ([Ni2+]: 10-11.23 to 10-10.23 M) promoted A. anophagefferens growth. Short-term experiments are being conducted to better understand mechanistically nickel speciation and distribution. Both total intracellular and subcellular metal concentrations are being assessed with radio-labeled 63Ni. Subcellular fractions are defined as metal-sensitive fractions (MSF) constituting organelles, cell debris, and heat-denatured protein [HDP] and biologically detoxified metal comprising heat-stabilized protein [HSP] and metal-rich granules [MRG]. Based on subcellular distribution, aqueous [Ni2+] concentrations, and A. anophagefferens growth rates, potential reaction pathways promoting A. anophagefferens growth can be addressed.

  16. The Subcellular Distribution of Small Molecules: from Pharmacokinetics to Synthetic Biology

    Science.gov (United States)

    Zheng, Nan; Tsai, Hobart Ng; Zhang, Xinyuan; Rosania, Gus R.

    2011-01-01

    The systemic pharmacokinetics and pharmacodynamics of small molecules are determined by subcellular transport phenomena. Although approaches used to study the subcellular distribution of small molecules have gradually evolved over the past several decades, experimental analysis and prediction of cellular pharmacokinetics remains a challenge. In this article, we surveyed the progress of subcellular distribution research since the 1960s, with a focus on the advantages, disadvantages and limitations of the various experimental techniques. Critical review of the existing body of knowledge pointed to many opportunities to advance the rational design of organelle-targeted chemical agents. These opportunities include: 1) development of quantitative, nonfluorescence-based, whole cell methods and techniques to measure the subcellular distribution of chemical agents in multiple compartments; 2) exploratory experimentation with nonspecific transport probes that have not been enriched with putative, organelle-targeting features; 3) elaboration of hypothesis-driven, mechanistic and modeling-based approaches to guide experiments aimed at elucidating subcellular distribution and transport; and 4) introduction of revolutionary conceptual approaches borrowed from the field of synthetic biology combined with cutting edge experimental strategies. In our laboratory, state-of-the-art subcellular transport studies are now being aimed at understanding the formation of new intracellular membrane structures in response to drug therapy, exploring the function of drug-membrane complexes as intracellular drug depots, and synthesizing new organelles with extraordinary physical and chemical properties. PMID:21805990

  17. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells.

    Science.gov (United States)

    Riskin, Arieh; Mond, Yehudit

    2015-10-26

    Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC) in culture. Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC). To study GLUT1 targeting and recycling in living mouse MEC (MMEC) in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP) and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin. GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90-110 minutes. Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation.

  18. Linking Subcellular Disturbance to Physiological Behavior and Toxicity Induced by Quantum Dots in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Qin; Zhou, Yanfeng; Song, Bin; Zhong, Yiling; Wu, Sicong; Cui, Rongrong; Cong, Haixia; Su, Yuanyuan; Zhang, Huimin; He, Yao

    2016-06-01

    The wide-ranging applications of fluorescent semiconductor quantum dots (QDs) have triggered increasing concerns about their biosafety. Most QD-related toxicity studies focus on the subcellular processes in cultured cells or global physiological effects on whole animals. However, it is unclear how QDs affect subcellular processes in living organisms, or how the subcellular disturbance contributes to the overall toxicity. Here the behavior and toxicity of QDs of three different sizes in Caenorhabditis elegans (C. elegans) are systematically investigated at both the systemic and the subcellular level. Specifically, clear size-dependent distribution and toxicity of the QDs in the digestive tract are observed. Short-term exposure of QDs leads to acute toxicity on C. elegans, yet incurring no lasting, irreversible damage. In contrast, chronic exposure of QDs severely inhibits development and shortens lifespan. Subcellular analysis reveals that endocytosis and nutrition storage are disrupted by QDs, which likely accounts for the severe deterioration in growth and longevity. This work reveals that QDs invasion disrupts key subcellular processes in living organisms, and may cause permanent damage to the tissues and organs over long-term retention. The findings provide invaluable information for safety evaluations of QD-based applications and offer new opportunities for design of novel nontoxic nanoprobes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Local Community Studies In Social Studies Course: An Action Research

    Directory of Open Access Journals (Sweden)

    Tuğba Selanik Ay

    2011-05-01

    Full Text Available Social Studies can be defined as “bonding process based on verification with social reality and dynamic information obtained as a result of this process”. In this context, it is essential to unify the Social Studies course with the real life and to benefit from the society in teaching-learning process in order to enable the learned information to be applied in the real life. In the Social Studies course, students should encounter with the real life itself. Thus, students can produce multidimensional alternative solutions for the cases they encounter and they can explain the best solution with justifications. Considering these arguments, it can be claimed that involving the subjects and studies related to the society and near environment in the Social Studies courses increases the effectiveness of Social Studies teaching. Local community studies, which are associated with the Social Studies program by means of a detailed and good plan, can draw students’ attention and thus permanent learning can occur. In this sense, teachers should benefit from the local community studies in the Social Studies course which reflects the real life.The aim of this study is to determine how local community studies will be applied in Social Studies course in the primary education schools. In line with this aim, the following research questions were addressed:1. How can the activities ofa. benefitting from institutions and organizations in local communitiesb. benefitting from people in local communitiesc. using Internet and library sourcesd. benefitting from special days and current eventswhich are carried out in the Social Studies course taught with local community studies, be arranged?2. Do the local community studies help the students determine the problems in their environments and find out the solutions for these problems?3. What are the students’ opinions about the Social Studies course taught with local community studies ?4. Does the Social Studies

  20. Distinct MicroRNA Subcellular Size and Expression Patterns in Human Cancer Cells

    Directory of Open Access Journals (Sweden)

    Beibei Chen

    2012-01-01

    Full Text Available Introduction. Small noncoding RNAs have important regulatory functions in different cell pathways. It is believed that most of them mainly play role in gene post-transcriptional regulation in the cytoplasm. Recent evidence suggests miRNA and siRNA activity in the nucleus. Here, we show distinct genome-wide sub-cellular localization distribution profiles of small noncoding RNAs in human breast cancer cells. Methods. We separated breast cancer cell nuclei from cytoplasm, and identified small RNA sequences using a high-throughput sequencing platform. To determine the relationship between miRNA sub-cellular distribution and cancer progression, we used microarray analysis to examine the miRNA expression levels in nucleus and cytoplasm of three human cell lines, one normal breast cell line and two breast cancer cell lines. Logistic regression and SVM were used for further analysis. Results. The sub-cellular distribution of small noncoding RNAs shows that numerous miRNAs and their isoforms (isomiR not only locate to the cytoplasm but also appeare in the nucleus. Subsequent microarray analyses indicated that the miRNA nuclear-cytoplasmic-ratio is a significant characteristic of different cancer cell lines. Conclusions. Our results indicate that the sub-cellular distribution is important for miRNA function, and that the characterization of the small RNAs sub-cellular localizome may contribute to cancer research and diagnosis.

  1. In situ spatiotemporal mapping of flow fields around seeded stem cells at the subcellular length scale.

    Directory of Open Access Journals (Sweden)

    Min Jae Song

    2010-09-01

    Full Text Available A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms.

  2. In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

    Science.gov (United States)

    Song, Min Jae; Dean, David; Knothe Tate, Melissa L.

    2010-01-01

    A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms. PMID:20862249

  3. Subcellular targeting of Salmonella virulence proteins by host-mediated S-palmitoylation.

    Science.gov (United States)

    Hicks, Stuart W; Charron, Guillaume; Hang, Howard C; Galán, Jorge E

    2011-07-21

    Several pathogenic bacteria utilize type III secretion systems (TTSS) to deliver into host cells bacterial virulence proteins with the capacity to modulate a variety of cellular pathways. Once delivered into host cells, the accurate targeting of bacterial effectors to specific locations is critical for their proper function. However, little is known about the mechanisms these virulence effectors use to reach their subcellular destination. Here we show that the Salmonella TTSS effector proteins SspH2 and SseI are localized to the plasma membrane of host cells, a process dependent on S-palmitoylation of a conserved cysteine residue within their N-terminal domains. We also show that effector protein lipidation is mediated by a specific subset of host-cell palmitoyltransferases and that lipidation is critical for effector function. This study describes a remarkable mechanism by which a pathogen exploits host-cell machinery to properly target its virulence factors. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model.

    Science.gov (United States)

    Spencer, Netanya Y; Yan, Ziying; Cong, Le; Zhang, Yulong; Engelhardt, John F; Stanton, Robert C

    2016-02-01

    Studies to determine subcellular localization and translocation of proteins are important because subcellular localization of proteins affects every aspect of cellular function. Such studies frequently utilize mutagenesis to alter amino acid sequences hypothesized to constitute subcellular localization signals. These studies often utilize fluorescent protein tags to facilitate live cell imaging. These methods are excellent for studies of monomeric proteins, but for multimeric proteins, they are unable to rule out artifacts from native protein subunits already present in the cells. That is, native monomers might direct the localization of fluorescent proteins with their localization signals obliterated. We have developed a method for ruling out such artifacts, and we use glucose 6-phosphate dehydrogenase (G6PD) as a model to demonstrate the method's utility. Because G6PD is capable of homodimerization, we employed a novel approach to remove interference from native G6PD. We produced a G6PD knockout somatic (hepatic) cell line using CRISPR-Cas9 mediated genome engineering. Transfection of G6PD knockout cells with G6PD fluorescent mutant proteins demonstrated that the major subcellular localization sequences of G6PD are within the N-terminal portion of the protein. This approach sets a new gold standard for similar studies of subcellular localization signals in all homodimerization-capable proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions.

    Directory of Open Access Journals (Sweden)

    Joachim Nielsen

    Full Text Available Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I and low (type II mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h, intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber's oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric

  6. Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation.

    Science.gov (United States)

    Magenau, Andrew J D; Saurabh, Saumya; Andreko, Susan K; Telmer, Cheryl A; Schmidt, Brigitte F; Waggoner, Alan S; Bruchez, Marcel P

    2015-10-01

    The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. AFSC/REFM: Pacific cod Localized Depletion Study

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Data from Localized Depletion study for Pacific cod 2001-2005. Study was conducted using cod pot gear to measure localized abundance of Pacific cod inside and...

  8. Protein localization as a principal feature of the etiology and comorbidity of genetic diseases

    Science.gov (United States)

    Park, Solip; Yang, Jae-Seong; Shin, Young-Eun; Park, Juyong; Jang, Sung Key; Kim, Sanguk

    2011-01-01

    Proteins targeting the same subcellular localization tend to participate in mutual protein–protein interactions (PPIs) and are often functionally associated. Here, we investigated the relationship between disease-associated proteins and their subcellular localizations, based on the assumption that protein pairs associated with phenotypically similar diseases are more likely to be connected via subcellular localization. The spatial constraints from subcellular localization significantly strengthened the disease associations of the proteins connected by subcellular localizations. In particular, certain disease types were more prevalent in specific subcellular localizations. We analyzed the enrichment of disease phenotypes within subcellular localizations, and found that there exists a significant correlation between disease classes and subcellular localizations. Furthermore, we found that two diseases displayed high comorbidity when disease-associated proteins were connected via subcellular localization. We newly explained 7584 disease pairs by using the context of protein subcellular localization, which had not been identified using shared genes or PPIs only. Our result establishes a direct correlation between protein subcellular localization and disease association, and helps to understand the mechanism of human disease progression. PMID:21613983

  9. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    Science.gov (United States)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  10. Sub-cellular force microscopy in single normal and cancer cells.

    Science.gov (United States)

    Babahosseini, H; Carmichael, B; Strobl, J S; Mahmoodi, S N; Agah, M

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Subcellular localization of vanillyl-alcohol oxidase in Penicillium simplicissimum

    NARCIS (Netherlands)

    Fraaije, MW; Sjollema, KA; Veenhuis, M; van Berkel, WJH; Berkel, Willem J.H. van

    1998-01-01

    Growth of Penicillium simplicissimum on anisyl alcohol, veratryl alcohol or 3-(methoxymethyl)phenol, is associated with the synthesis of relatively large amounts of the hydrogen peroxide producing flavoprotein vanillyl-alcohol oxidase (VAO), Immunocytochemistry revealed that the enzyme has a dual

  12. Subcellular localization and expression analysis of the BmDSCLP ...

    African Journals Online (AJOL)

    FJ602779) contained a 642 bp open reading frame (ORF) encoding 213 amino acid residues. The ORF of this gene was inserted into the prokaryotic expression vector pET-28a(+) to construct a recombinant expression plasmid and the fusion protein was expressed in Escherichia coli BL21(DE3) cells. The fusion protein ...

  13. Convolutional LSTM Networks for Subcellular Localization of Proteins

    DEFF Research Database (Denmark)

    Sønderby, Søren Kaae; Sønderby, Casper Kaae; Nielsen, Henrik

    2015-01-01

    Machine learning is widely used to analyze biological sequence data. Non-sequential models such as SVMs or feed-forward neural networks are often used although they have no natural way of handling sequences of varying length. Recurrent neural networks such as the long short term memory (LSTM) model...

  14. Convolutional LSTM Networks for Subcellular Localization of Proteins

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Sønderby, Søren Kaae; Sønderby, Casper Kaae

    Machine learning is widely used to analyze biological sequence data. Non-sequential models such as SVMs or feed-forward neural networks are often used although they have no natural way of handling sequences of varying length. Recurrent neural networks such as the long short term memory (LSTM) model...

  15. Subcellular localization and expression analysis of the BmDSCLP ...

    African Journals Online (AJOL)

    user

    2011-04-04

    Apr 4, 2011 ... Escherichia coli BL21(DE3) cells. The fusion protein was purified by Ni-affinity chromatography and fast protein liquid chromatography (FPLC) and its size was then, determined by liquid chromatography-mass .... collected 10 days after the fourth injection was purified on HiTrap Protein A HP (Amersham), in.

  16. Subcellular localization and expression analysis of the BmDSCLP ...

    African Journals Online (AJOL)

    user

    2011-04-04

    Apr 4, 2011 ... total RNA. The first-strand cDNA was used as the template to amplify a fragment from the coding region by using polymerase chain reaction (PCR). PCR primers were ... into E. coli TG1 competent cells. A positive colony .... various stages of B. mori, total silkworm RNAs from four different stages (egg, larva ...

  17. Protein subcellular location pattern classification in cellular images using latent discriminative models.

    Science.gov (United States)

    Li, Jieyue; Xiong, Liang; Schneider, Jeff; Murphy, Robert F

    2012-06-15

    Knowledge of the subcellular location of a protein is crucial for understanding its functions. The subcellular pattern of a protein is typically represented as the set of cellular components in which it is located, and an important task is to determine this set from microscope images. In this article, we address this classification problem using confocal immunofluorescence images from the Human Protein Atlas (HPA) project. The HPA contains images of cells stained for many proteins; each is also stained for three reference components, but there are many other components that are invisible. Given one such cell, the task is to classify the pattern type of the stained protein. We first randomly select local image regions within the cells, and then extract various carefully designed features from these regions. This region-based approach enables us to explicitly study the relationship between proteins and different cell components, as well as the interactions between these components. To achieve these two goals, we propose two discriminative models that extend logistic regression with structured latent variables. The first model allows the same protein pattern class to be expressed differently according to the underlying components in different regions. The second model further captures the spatial dependencies between the components within the same cell so that we can better infer these components. To learn these models, we propose a fast approximate algorithm for inference, and then use gradient-based methods to maximize the data likelihood. In the experiments, we show that the proposed models help improve the classification accuracies on synthetic data and real cellular images. The best overall accuracy we report in this article for classifying 942 proteins into 13 classes of patterns is about 84.6%, which to our knowledge is the best so far. In addition, the dependencies learned are consistent with prior knowledge of cell organization. http://murphylab.web.cmu.edu/software/.

  18. High-Resolution Secondary Ion Mass Spectrometry Reveals the Contrasting Subcellular Distribution of Arsenic and Silicon in Rice Roots

    National Research Council Canada - National Science Library

    Katie L. Moore; Markus Schröder; Zhongchang Wu; Barry G.H. Martin; Chris R. Hawes; Steve P. McGrath; Malcolm J. Hawkesford; Jian Feng Ma; Fang-Jie Zhao; Chris R.M. Grovenor

    2011-01-01

    .... In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy...

  19. Organelle-targeting surface-enhanced Raman scattering (SERS) nanosensors for subcellular pH sensing.

    Science.gov (United States)

    Shen, Yanting; Liang, Lijia; Zhang, Shuqin; Huang, Dianshuai; Zhang, Jing; Xu, Shuping; Liang, Chongyang; Xu, Weiqing

    2018-01-25

    The pH value of subcellular organelles in living cells is a significant parameter in the physiological activities of cells. Its abnormal fluctuations are commonly believed to be associated with cancers and other diseases. Herein, a series of surface-enhanced Raman scattering (SERS) nanosensors with high sensitivity and targeting function was prepared for the quantification and monitoring of pH values in mitochondria, nucleus, and lysosome. The nanosensors were composed of gold nanorods (AuNRs) functionalized with a pH-responsive molecule (4-mercaptopyridine, MPy) and peptides that could specifically deliver the AuNRs to the targeting subcellular organelles. The localization of our prepared nanoprobes in specific organelles was confirmed by super-high resolution fluorescence imaging and bio-transmission electron microscopy (TEM) methods. By the targeting ability, the pH values of the specific organelles can be determined by monitoring the vibrational spectral changes of MPy with different pH values. Compared to the cases of reported lysosome and cytoplasm SERS pH sensors, more accurate pH values of mitochondria and nucleus, which could be two additional intracellular tracers for subcellular microenvironments, were disclosed by this SERS approach, further improving the accuracy of discrimination of related diseases. Our sensitive SERS strategy can also be employed to explore crucial physiological and biological processes that are related to subcellular pH fluctuations.

  20. Pathogenicity study of local bluetongue virus isolates in local and imported sheep

    Directory of Open Access Journals (Sweden)

    Indrawati Sendow

    2005-03-01

    Full Text Available Bluetongue is one of arboviruses that caused economical impact to sheep farmers. Six local bluetongue virus (BT serotypes isolates were obtained from sentinel cattle blood in West Java and Irian Jaya (Papua, but its pathogenicity has not been identified. Propagation of viraemic blood inoculum from 3 local BT serotypes such as serotypes 1,9 and 21, that had been conducted in Merino sheep, will be used for pathogenicity study. The study was devided into 3 groups, each group contained local and imported sheep as control and infected sheep. All sheep had been tested as negative BT antibodies. Observation on clinical signs had been conducted twice daily for 28 days. Heparinised blood and sera were collected everyday to obtain the viraemia period by Ag-C-ELISA test and antibody respons by C-ELISA test. The clinical signs produced were varied from normal to very mild in local sheep and very mild to mild-moderate in Merino sheep.The lowest severe degree of clinical signs was BT 9 followed by BT 1 and BT 21. No dead, neither local and Merino sheep occurred. Viraemia in Merino sheep occurred between 3-5 days and in local sheep between 4-7 days post inoculation (DPI. Antibody respons occurred as quick as 10 DPI in Merino sheep and 9 DPI in local sheep, and stayed until the end of experiment. This study showed that local BT isolates were not pathogen and not producing clasical BT infection.

  1. Use of correspondence discriminant analysis to predict the subcellular location of bacterial proteins.

    Science.gov (United States)

    Perrière, Guy; Thioulouse, Jean

    2003-02-01

    Correspondence discriminant analysis (CDA) is a multivariate statistical method derived from discriminant analysis which can be used on contingency tables. We have used CDA to separate Gram negative bacteria proteins according to their subcellular location. The high resolution of the discrimination obtained makes this method a good tool to predict subcellular location when this information is not known. The main advantage of this technique is its simplicity. Indeed, by computing two linear formulae on amino acid composition, it is possible to classify a protein into one of the three classes of subcellular location we have defined. The CDA itself can be computed with the ADE-4 software package that can be downloaded, as well as the data set used in this study, from the Pôle Bio-Informatique Lyonnais (PBIL) server at http://pbil.univ-lyon1.fr.

  2. Subcellular Distribution of Glutathione Precursors in Arabidopsis thaliana

    Science.gov (United States)

    Koffler, Barbara Eva; Maier, Romana; Zechmann, Bernd

    2011-01-01

    Abstract Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) – the first enzyme of glutathione synthesis – causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells. PMID:22050910

  3. AAV exploits subcellular stress associated with inflammation, endoplasmic reticulum expansion, and misfolded proteins in models of cystic fibrosis.

    Directory of Open Access Journals (Sweden)

    Jarrod S Johnson

    2011-05-01

    Full Text Available Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR, we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus

  4. Cell type-dependent localization of MLO proteins.

    Science.gov (United States)

    Jones, Daniel S; Kessler, Sharon A

    2017-11-02

    Mildew resistance locus O (MLO) proteins are transmembrane proteins that mediate cell-cell communication in plants. We recently demonstrated the importance of subcellular localization to MLO function during pollen tube reception. NORTIA (NTA), the MLO protein involved in this process, localizes to the Golgi of the synergid cell before interaction with the pollen tube. MLO proteins that can substitute for NTA's function in this pathway all partially localize with the same Golgi marker in the synergid cell. In this study, we report that MLO subcellular localization is cell type-dependent, with different distributions of some MLOs observed when ectopically expressed in the epidermal cells of tobacco and Arabidopsis compared to synergids. This dependency may be due to co-factors that influence MLO function within a given cell type, providing an intriguing new target for understanding MLO distribution and subsequent function in their respective processes.

  5. The GILL study: glycerin-induced local reactions in immunotherapy.

    Science.gov (United States)

    Calabria, Christopher W; Coop, Christopher A; Tankersley, Michael S

    2008-01-01

    The mechanism of local reactions is not well defined. Glycerin, an excellent preservative used commonly in immunotherapy extracts, is a recognized irritant. This study was undertaken to examine whether higher glycerin concentration in immunotherapy extracts is associated with an increase in local reaction rates during immunotherapy. A retrospective analysis of electronic immunotherapy records over a 12-month period was performed from a single site. A small local reaction was defined as induration and/or erythema at the injection site smaller than or equal to the size of the patient's palm. A large local reaction was defined as a reaction larger than the patient's palm. Over the 12-month period, 360 patients received a total of 9678 immunotherapy injections. For all injections, the total local reaction rate was 16.3% (1574/9678), the small local reaction rate was 15.9% (1536/9678), and the large local reaction rate was 0.4% (38/9678). For aeroallergens, small local reaction rates increased significantly with increasing allergen concentrations, from 7.3% (1:1000 vol/vol) to 23.0% (1:1 vol/vol; P glycerin concentration. Large local reactions were infrequent and did not significantly increase with allergen or glycerin concentration. Small local, but not large local, reaction rates increase with higher allergen concentration, number, and volume. Higher glycerin concentrations (even 50%) are not associated with significantly higher small or large local reaction rates.

  6. Subcellular Redox Targeting: Bridging in Vitro and in Vivo Chemical Biology.

    Science.gov (United States)

    Long, Marcus J C; Poganik, Jesse R; Ghosh, Souradyuti; Aye, Yimon

    2017-03-17

    Networks of redox sensor proteins within discrete microdomains regulate the flow of redox signaling. Yet, the inherent reactivity of redox signals complicates the study of specific redox events and pathways by traditional methods. Herein, we review designer chemistries capable of measuring flux and/or mimicking subcellular redox signaling at the cellular and organismal level. Such efforts have begun to decipher the logic underlying organelle-, site-, and target-specific redox signaling in vitro and in vivo. These data highlight chemical biology as a perfect gateway to interrogate how nature choreographs subcellular redox chemistry to drive precision redox biology.

  7. Reinforcement of Science Learning through Local Culture: A Delphi Study

    Science.gov (United States)

    Nuangchalerm, Prasart

    2008-01-01

    This study aims to explore the ways to reinforce science learning through local culture by using Delphi technique. Twenty four participants in various fields of study were selected. The result of study provides a framework for reinforcement of science learning through local culture on the theme life and environment. (Contains 1 table.)

  8. Parasites modify sub-cellular partitioning of metals in the gut of fish

    NARCIS (Netherlands)

    Oyoo-Okoth, E.; Admiraal, W.; Osano, O.; Kraak, M.H.S.; Gichuki, J.; Ogwai, C.

    2012-01-01

    Infestation of fish by parasites may influence metal accumulation patterns in the host. However, the subcellular mechanisms of these processes have rarely been studied. Therefore, this study determined how a cyprinid fish (Rastrineobola argentea) partitioned four metals (Cd, Cr, Zn and Cu) in the

  9. KCC2-dependent subcellular ECl difference of ON-OFF retinal ganglion cells in larval zebrafish

    Directory of Open Access Journals (Sweden)

    Rongwei eZhang

    2013-05-01

    Full Text Available Subcellular difference in the reversal potential of Cl- (ECl has been found in many types of neurons. As local ECl largely determines the action of nearby GABAergic/glycinergic synapses, subcellular ECl difference can effectively regulate neuronal computation. The ON-OFF retinal ganglion cell (RGC processes both ON and OFF visual signals via its ON and OFF dendrites, respectively. It is thus interesting to investigate whether the ON and OFF dendrites of single RGCs exhibit different local ECl. Here, using in vivo gramicidin-perforated patch recording in larval zebrafish ON-OFF RGCs, we examine local ECl at the ON and OFF dendrites, and soma through measuring light-evoked ON and OFF inhibitory responses, and GABA-induced response at the soma, respectively. We find there are subcellular ECl differences between the soma and dendrite, as well as between the ON and OFF dendrites of single RGCs. These somato-dendritic and inter-dendritic ECl differences are dependent on the Cl- extruder, K+/Cl- co-transporter (KCC2, because they are largely diminished by down-regulating kcc2 expression with morpholino oligonucleotides or by blocking KCC2 function with furosemide. Thus, our findings indicate that there exists KCC2-dependent ECl difference between the ON and OFF dendrites of individual ON-OFF RGCs that may differentially affect visual processing in the ON and OFF pathways.

  10. Local Governance and ICTs in Africa : Case Studies and Guidelines ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    1 août 2011 ... Local Governance and ICTs in Africa : Case Studies and Guidelines for Implementation and Evaluation. Couverture du livre Local Governance and ICTs in Africa : Case Studies and Guidelines for Implementation. Directeur(s) : Timothy Mwolo Waema et Edith Ofwona Adera. Maison(s) d'édition : Pambazuka ...

  11. Localization of optical excitations on random surfaces: SNOM studies

    DEFF Research Database (Denmark)

    Bozhevolnyi, Sergey I.

    1999-01-01

    Localization of optical excitations on nanostructured metal surfaces and fractal colloid silver aggregates are studied by using a scanning near-field optical microscope (SNOM). The SNOM images obtained in both configurations exhibit spatially localized (within 150 to 250 nm) light intensity...... enhancement by up to two orders of magnitude. The observed phenomena are related to strong (Anderson) localization of quasi-two-dimensional light waves....

  12. Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy.

    Science.gov (United States)

    Gazi, E; Dwyer, J; Lockyer, N P; Miyan, J; Gardner, P; Hart, C; Brown, M; Clarke, N W

    2005-01-01

    Synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy is a powerful bioanalytical technique for the simultaneous analysis of lipids, proteins, carbohydrates, and a variety of phosphorylated molecules within intact cells. SR-FTIR microspectroscopy can be used in the imaging mode to generate biospectroscopic maps of the distribution and intensity profiles of subcellular biomolecular domains at diffraction-limited spatial resolution. However, the acquisition of highly spatially resolved IR images of cells is not only a function of instrumental parameters (source brightness, sampling aperture size) but also the cell preparation method employed. Additionally, for the IR data to be biochemically relevant the cells must be preserved in a life-like state without introducing artefacts. In the present study we demonstrate, for the first time, the differences in biomolecular localizations observed in SR-FTIR images of cells fixed by formalin, formalin-critical point drying (CPD), and glutaraldehyde-osmium tetroxide-CPD, using the PC-3 prostate cancer cell line. We compare these SR-FTIR images of fixed cells to unfixed cells. The influence of chemical fixatives on the IR spectrum is discussed in addition to the biological significance of the observed localizations. Our experiments reveal that formalin fixation at low concentration preserves lipid, phosphate, and protein components without significantly influencing the IR spectrum of the cell. (c) 2004 Wiley Periodicals, Inc.

  13. Localization in Self-Healing Autonomous Sensor Networks (SASNet): Studies on Cooperative Localization of Sensor Nodes using Distributed Maps

    National Research Council Canada - National Science Library

    Li, Li

    2008-01-01

    .... The cooperative localization scheme adopted in this study computes a local map for each sensor node using all the available link metric constraints, and then merges the local maps into a global...

  14. Intracellular delivery of nanocarriers and targeting to subcellular organelles.

    Science.gov (United States)

    Jhaveri, Aditi; Torchilin, Vladimir

    2016-01-01

    Recent trends in drug delivery indicate a steady increase in the use of targeted therapeutics to enhance the specific delivery of biologically active payloads to diseased tissues while avoiding their off-target effects. However, in most cases, the distribution of therapeutics inside cells and their targeting to intracellular targets still presents a formidable challenge. The main barrier to intracellular delivery is the translocation of therapeutic molecules across the cell membrane, and ultimately through the membrane of their intracellular target organelles. Another prerequisite for an efficient intracellular localization of active molecules is their escape from the endocytic pathway. Pharmaceutical nanocarriers have demonstrated substantial advantages for the delivery of therapeutics and offer elegant platforms for intracellular delivery. They can be engineered with both intracellular and organelle-specific targeting moieties to deliver encapsulated or conjugated cargoes to specific sub-cellular targets. In this review, we discuss important aspects of intracellular drug targeting and delivery with a focus on nanocarriers modified with various ligands to specifically target intracellular organelles. Intracellular delivery affords selective localization of molecules to their target site, thus maximizing their efficacy and safety. The advent of novel nanocarriers and targeting ligands as well as exploration of alternate routes for the intracellular delivery and targeting has prompted extensive research, and promises an exciting future for this field.

  15. A STUDY OF LOCALLY ADVANCED CARCINOMA OF BREAST

    Directory of Open Access Journals (Sweden)

    Prabhakar Jenna

    2017-08-01

    Full Text Available BACKGROUND Worldwide, breast cancer is the most frequent cancer in women and represents the second leading cause of cancer death among women. Locally advanced breast cancer constitutes more than 50-70% of the patients presenting for treatment has two common problems in treatment. Achieving local control and prolonging survival by preventing or delaying distant metastasis. Today, treatment of LABC requires a combination of systemic and local/regional therapies. The aim of the study is to study the clinicopathological presentation, age distribution and various modes of management of locally advanced breast carcinoma. Worldwide breast cancer is the most frequent cancer in women and represents the second leading cause of cancer death among women. Locally advanced breast cancer constitutes more than 50-70% of the patients presenting treatment. MATERIALS AND METHODS The present study includes 50 patients who attended Department of General Surgery for a period of three years. RESULTS The patients were regularly followed up and at the end of the study 35 (70% of the patients were doing well. 4(8% of the patients developed distant metastasis and 3 (6% of the patients developing local recurrence. 8 (16% of the patients were lost follow up. CONCLUSION About half of the cases presenting with breast cancer are in locally advanced stages. Multimodality therapy is the effective treatment of locally advanced carcinoma of breast. Breast cancer management is a challenge and improvement in therapies are needed for disease-free interval and overall survival period.

  16. The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

    Science.gov (United States)

    Domingues, Lia; Ismail, Ahmad; Charro, Nuno; Rodríguez-Escudero, Isabel; Holden, David W; Molina, María; Cid, Víctor J; Mota, Luís Jaime

    2016-07-01

    Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells. © 2015 John Wiley & Sons Ltd.

  17. Vacuoles in mammals: a subcellular structure indispensable for early embryogenesis.

    Science.gov (United States)

    Wada, Yoh

    2013-01-01

    A vacuole is a membrane-bound subcellular structure involved in intracellular digestion. Instead of the large "vacuolar" organelles that are found in plants and fungi, animal cells possess lysosomes that are smaller in size and are enriched with hydrolytic enzymes similar to those found in the vacuoles. Large vacuolar structures are often observed in highly differentiated mammalian tissues such as embryonic visceral endoderm and absorbing epithelium. Vacuoles/lysosomes share a conserved mechanism of biogenesis, and they are at the terminal of the endocytic pathways, Recent genetic studies of the mammalian orthologs of Vam/Vps genes, which have essential functions for vacuole assembly, revealed that the dynamics of vacuoles/lysosomes are important for tissue differentiation and patterning through regulation of various molecular signaling events in mammals.

  18. Differential subcellular distribution of ion channels and the diversity of neuronal function.

    Science.gov (United States)

    Nusser, Zoltan

    2012-06-01

    Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Subcellular distribution and chemical forms of antimony in Ficus tikoua.

    Science.gov (United States)

    Wang, Yong; Chai, Liyuan; Yang, Zhihui; Mubarak, Hussani; Xiao, Ruiyang; Tang, Chongjian

    2017-02-01

    Ficus tikoua (F. tikoua) was a potential species for antimony (Sb) phytoremediation due to its wide growth in the mining area. However, little was known about its tolerance mechanisms toward Sb. The determination of the distribution and chemical speciation of Sb in F. tikoua is essential for understanding the mechanisms involved in Sb accumulation, transportation, and detoxification. The present study investigated the subcellular distribution and chemical forms of Sb in F. tikoua. The plant was exposed to different Sb concentrations (0, 30, 90, and 180 μmol/L) for 30 days. The results showed that F. tikoua possessed a marked ability to tolerate and accumulate Sb. The proportional Sb increased with increasing Sb concentration in the solution, and the highest Sb concentration occurred in roots (1274.5-1580.9 mg/kg), followed by stems (133.5-498.9 mg/kg) and leaves (4.1-15.7 mg/kg). In the subcellular sequestration of Sb in F. tikoua, the largest accumulation of Sb occurred in cell walls (72.4-87.5%) followed by cytoplasmic organelles (8.2-18.6%) and cytoplasmic supernatant. The results suggested that cell walls act as important protective barriers against Sb toxicity in F. tikoua. Although Sb in all plant tissues found primarily in the fractions extracted by ethanol and distilled water, the current study found that the Sb amounts in the HAc-extractable fraction, HCl-extractable fraction, and residue fraction increased at the highest Sb level (180 μmol/L) compared to that under lower Sb levels. These results indicate that excessive Sb accumulated in F. tikoua under Sb stress is bound to non-dissolved or low-bioavailable compounds, a biochemical mechanism that benefits F. tikoua because it helps alleviate Sb toxicity.

  20. Subcellular distribution of glutathione and cysteine in cyanobacteria

    Science.gov (United States)

    Tomašić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

    2010-01-01

    Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria. PMID:20349253

  1. Proteome-wide Subcellular Topologies of E. coli Polypeptides Database (STEPdb)*

    Science.gov (United States)

    Orfanoudaki, Georgia; Economou, Anastassios

    2014-01-01

    Cell compartmentalization serves both the isolation and the specialization of cell functions. After synthesis in the cytoplasm, over a third of all proteins are targeted to other subcellular compartments. Knowing how proteins are distributed within the cell and how they interact is a prerequisite for understanding it as a whole. Surface and secreted proteins are important pathogenicity determinants. Here we present the STEP database (STEPdb) that contains a comprehensive characterization of subcellular localization and topology of the complete proteome of Escherichia coli. Two widely used E. coli proteomes (K-12 and BL21) are presented organized into thirteen subcellular classes. STEPdb exploits the wealth of genetic, proteomic, biochemical, and functional information on protein localization, secretion, and targeting in E. coli, one of the best understood model organisms. Subcellular annotations were derived from a combination of bioinformatics prediction, proteomic, biochemical, functional, topological data and extensive literature re-examination that were refined through manual curation. Strong experimental support for the location of 1553 out of 4303 proteins was based on 426 articles and some experimental indications for another 526. Annotations were provided for another 320 proteins based on firm bioinformatic predictions. STEPdb is the first database that contains an extensive set of peripheral IM proteins (PIM proteins) and includes their graphical visualization into complexes, cellular functions, and interactions. It also summarizes all currently known protein export machineries of E. coli K-12 and pairs them, where available, with the secretory proteins that use them. It catalogs the Sec- and TAT-utilizing secretomes and summarizes their topological features such as signal peptides and transmembrane regions, transmembrane topologies and orientations. It also catalogs physicochemical and structural features that influence topology such as abundance

  2. Multivalency Effect of TAT-Peptide-Functionalized Nanoparticle in Cellular Endocytosis and Subcellular Trafficking.

    Science.gov (United States)

    Dalal, Chumki; Jana, Nikhil R

    2017-04-13

    Although trans-activating transcription (TAT) peptide-functionalized nanoparticle/polymer/liposome is widely used for cellular transfection applications, the multivalency (number of attached peptide per particle) effect on cell uptake mechanism and subcellular targeting performance is largely unexplored. Here we show that multivalency of nanoparticle controls the cellular interaction, cellular entry/exit mechanism, and subcellular targeting performance. We have synthesized TAT-peptide functionalized quantum dot (QD) of 30-35 nm hydrodynamic diameter with varied multivalency from 10 to 75 (e.g., QD(TAT)10, QD(TAT)20, QD(TAT)40, QD(TAT)75) and studied the role of multivalency in endocytosis and subcellular trafficking. We found that both low and high multivalent nanoparticles enter into cell predominantly via lipid-raft mediated endocytosis but the higher multivalency of 40 and 75 induces vesicular trapping followed by exocytosis within 12 h. In contrast, lower multivalency of 10 and 20 offers efficient trafficking toward perinuclear region and Golgi apparatus. This work shows the functional role of nanoparticle multivalency in cellular uptake mechanism and importance of lower multivalency for efficient subcellular targeting.

  3. Subcellular interactions of dietary cadmium, copper and zinc in rainbow trout (Oncorhynchus mykiss)

    Energy Technology Data Exchange (ETDEWEB)

    Kamunde, Collins, E-mail: ckamunde@upei.ca [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3 (Canada); MacPhail, Ruth [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3 (Canada)

    2011-10-15

    Highlights: Interactions of Cu, Cd and Zn were studied at the subcellular level in rainbow trout. Metals accumulated in the liver were predominantly metabolically active. Cd, Cu and Zn exhibited both competitive and cooperative interactions. The metal-metal interactions altered subcellular metals partitioning. - Abstract: Interactions of Cu, Cd and Zn were studied at the subcellular level in juvenile rainbow trout (Oncorhynchus mykiss) fed diets containing ({mu}g/g) 500 Cu, 1000 Zn and 500 Cd singly and as a ternary mixture for 28 days. Livers were harvested and submitted to differential centrifugation to isolate components of metabolically active metal pool (MAP: heat-denaturable proteins (HDP), organelles, nuclei) and metabolically detoxified metal pool (MDP: heat stable proteins (HSP), NaOH-resistant granules). Results indicated that Cd accumulation was enhanced in all the subcellular compartments, albeit at different time points, in fish exposed to the metals mixture relative to those exposed to Cd alone, whereas Cu alone exposure increased Cd partitioning. Exposure to the metals mixture reduced (HDP) and enhanced (HSP, nuclei and granules) Cu accumulation while exposure to Zn alone enhanced Cu concentration in all the fractions analyzed without altering proportional distribution in MAP and MDP. Although subcellular Zn accumulation was less pronounced than that of either Cu or Cd, concentrations of Zn were enhanced in HDP, nuclei and granules from fish exposed to the metals mixture relative to those exposed to Zn alone. Cadmium alone exposure mobilized Zn and Cu from the nuclei and increased Zn accumulation in organelles and Cu in granules, while Cu alone exposure stimulated Zn accumulation in HSP, HDP and organelles. Interestingly, Cd alone exposure increased the partitioning of the three metals in MDP indicative of enhanced detoxification. Generally the accumulated metals were predominantly metabolically active: Cd, 67-83%; Cu, 68-79% and Zn, 60-76%. Taken

  4. A formal ontology of subcellular neuroanatomy

    Directory of Open Access Journals (Sweden)

    Stephen D Larson

    2007-11-01

    Full Text Available The complexity of the nervous system requires high-resolution microscopy to resolve the detailed 3D structure of nerve cells and supracellular domains. The analysis of such imaging data to extract cellular surfaces and cell components often requires the combination of expert human knowledge with carefully engineered software tools. In an effort to make better tools to assist humans in this endeavor, create a more accessible and permanent record of their data, and to aid the process of constructing complex and detailed computational models, we have created a core of formalized knowledge about the structure of the nervous system and have integrated that core into several software applications. In this paper, we describe the structure and content of a formal ontology whose scope is the subcellular anatomy of the nervous system (SAO, covering nerve cells, their parts, and interactions between these parts. Many applications of this ontology to image annotation, content-based retrieval of structural data, and integration of shared data across scales and researchers are also described.

  5. Subcellular drug targeting, pharmacokinetics and bioavailability.

    Science.gov (United States)

    Leucuta, Sorin Emilian

    2014-02-01

    Effective treatment of diseases at the molecular level is possible by directing the drug substance (micromolecular, protein or peptide drugs, DNA, oligonucleotides, siRNA) with the aid of a specialized nanoparticulate carrier, for safe and effective transport to the specific site of action in the cytosol and its organelles including nuclear targeting. To achieve efficient cytosolic delivery of therapeutics or nuclear targeting, different drug delivery systems (DDS) have been developed (macromolecular drug conjugates, chemically or genetically modified proteins, and particulate drug carriers) capable of subcellular internalization overcoming the biological barriers, by passive targeting and especially by active targeting (receptor-targeted delivery). The success depends on the physicochemical nature of DDS, intracellular barriers that these systems need to overcome, the bioavailability of the bioactive drug, biodistribution, the intracellular pharmacokinetics and its influence on the pharmacodynamic effect. Models necessary for this purpose exist but they need to be more developed especially with quantitative treatments, after the development of the means of highlighting the evolution of the drug substance in biophase or at the level of the target cellular organelle by quantitative assays. It is expected that intracellularly targeted drug delivery approaches will be clinically useful using specialized DDSs belonging to the pharmaceutical nanotechnologies.

  6. Multimodal subcellular imaging with microcavity photoacoustic transducer.

    Science.gov (United States)

    Tan, Zhiliang; Tang, Zhilie; Wu, Yongbo; Liao, Yanfei; Dong, Wei; Guo, Lina

    2011-01-31

    Photoacoustic microscopy (PAM) is dominantly sensitive to the endogenous optical absorption compared with the confocal microscopy which images with scattering photons. PAM has similar structure such as optical transportation system, the optical scanning, and light source with the laser scanning confocal microscopy (LSCM). In order to match the PAM with LSCM, a special design microcavity photoacoustic (PA) transducer with high sensitivity is developed to detect the photoacoustic signals induced by modulated continuous wave (CW) laser. By employing a microcavity PA transducer, a PAM can be integrated with LSCM. Thus a simultaneous multimodal imaging can be obtained with the same laser source and optical system. The lateral resolutions of the PAM and the LSCM are both tested to be better than 1.25 μm. Then subcellular multimodal imaging can be achieved. Images from the two modes are corresponding with each other but functionally complementary. Combining PAM and LSCM provides more comprehensive information for the cytological test. This technique is demonstrated for imaging red-blood cells and meristematic cells.

  7. HECTAR: a method to predict subcellular targeting in heterokonts

    National Research Council Canada - National Science Library

    Gschloessl, Bernhard; Guermeur, Yann; Cock, J Mark

    2008-01-01

    .... To understand the biology of these organisms, it is necessary to be able to predict the subcellular localisation of their proteins but this is not straightforward, particularly in photosynthetic...

  8. The Comparative Study Of Local Governance: Towards A Global Approach The Comparative Study Of Local Governance: Towards A Global Approach

    Directory of Open Access Journals (Sweden)

    Gerry Stoker

    2010-12-01

    Full Text Available The comparative study of local governance has been too focused on the institutional arrangements of the Systems of different nation states rather than the more fundamental issue of the societal functions performed by local government. This article focuses attention on four societal roles that local government systems undertake. They can support political identity, underwrite economic development, facilitate social welfare provision or act as a lifestyle co-ordinator through the practice of community governance. Linking our investigation to the embedded societal roles of local government in different systems opens up the opportunity for a more genuinely global comparative perspective. It also helps us to understand the likely forms of politics associated with different systems of local governance. It also enables us to explore the sustainability of different systems of local governance. It is suggested that a strong system of local government is likely to be one that is able to combine societal roles to a substantial degree. A vulnerable local government system is one trapped with one function that in changing societal and economic circumstances could find itself under threat.El estudio comparado de la gobernanza local se ha focalizado excesivamente en los arreglos institucionales de los sistemas de los diferentes Estados-nación en lugar de centrarse en el tema esencial de las funciones sociales que desempeñan los gobiernos locales. Este artículo centra su atención en cuatro roles sociales que desempeñan los sistemas de gobierno local. Pueden proporcionar identidad política, garantizar el desarrollo económico, facilitar la provisión de servicios sociales o actuar como coordinador de la forma de vida mediante la práctica de la gobernanza comunitaria. La vinculación de la investigación a los roles sociales asumidos por los gobiernos locales en los diferentes sistemas proporciona la posibilidad de adoptar una perspectiva global comparada

  9. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Directory of Open Access Journals (Sweden)

    Pei-Chi Yang

    2016-07-01

    Full Text Available Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and

  10. Sub-cellular force microscopy in single normal and cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Babahosseini, H. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Carmichael, B. [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Strobl, J.S. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Mahmoodi, S.N., E-mail: nmahmoodi@eng.ua.edu [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Agah, M., E-mail: agah@vt.edu [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States)

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain.

  11. Towards studies of organizational behaviour with greater local relevance

    OpenAIRE

    Peter B. Smith

    2007-01-01

    Theories of organizational behaviour mostly originated in North America. In testing their applicability elsewhere, attention must be given to differences in local environments and inthe values of local employees. Within Latin America, the prevalence of high collectivism and power distance are particularly important. Employees’ attachment to their organization has been shown to differ within collectivistic cultures. The argument is illustrated by two Latin American studies. Firstly, the ways i...

  12. Towards studies of organizational behaviour with greater local relevance

    OpenAIRE

    Smith, Peter B.

    2012-01-01

    Theories of organizational behaviour mostly originated in North America. In testing their applicability elsewhere, attention must be given to differences in local environments and in the values of local employees. Within Latin America, the prevalence of high collectivism and power distance are particularly important. Employees’ attachment to their organization has been shown to differ within collectivistic cultures. The argument is illustrated by two Latin American studies. Firstly, the ways ...

  13. Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype1

    Science.gov (United States)

    Di Fiore, Stefano; Li, Qiurong; Leech, Mark James; Schuster, Flora; Emans, Neil; Fischer, Rainer; Schillberg, Stefan

    2002-01-01

    Tryptophan decarboxylase (TDC) is a cytosolic enzyme that catalyzes an early step of the terpenoid indole alkaloid biosynthetic pathway by decarboxylation of l-tryptophan to produce the protoalkaloid tryptamine. In the present study, recombinant TDC was targeted to the chloroplast, cytosol, and endoplasmic reticulum (ER) of tobacco (Nicotiana tabacum) plants to evaluate the effects of subcellular compartmentation on the accumulation of functional enzyme and its corresponding enzymatic product. TDC accumulation and in vivo function was significantly affected by the subcellular localization. Immunoblot analysis demonstrated that chloroplast-targeted TDC had improved accumulation and/or stability when compared with the cytosolic enzyme. Because ER-targeted TDC was not detectable by immunoblot analysis and tryptamine levels found in transient expression studies and in transgenic plants were low, it was concluded that the recombinant TDC was most likely unstable if ER retained. Targeting TDC to the chloroplast stroma resulted in the highest accumulation level of tryptamine so far reported in the literature for studies on heterologous TDC expression in tobacco. However, plants accumulating high levels of functional TDC in the chloroplast developed a lesion-mimic phenotype that was probably triggered by the relatively high accumulation of tryptamine in this compartment. We demonstrate that subcellular targeting may provide a useful strategy for enhancing accumulation and/or stability of enzymes involved in secondary metabolism and to divert metabolic flux toward desired end products. However, metabolic engineering of plants is a very demanding task because unexpected, and possibly unwanted, effects may be observed on plant metabolism and/or phenotype. PMID:12114570

  14. Subcellular analysis by laser ablation electrospray ionization mass spectrometry

    Science.gov (United States)

    Vertes, Akos; Stolee, Jessica A; Shrestha, Bindesh

    2014-12-02

    In various embodiments, a method of laser ablation electrospray ionization mass spectrometry (LAESI-MS) may generally comprise micro-dissecting a cell comprising at least one of a cell wall and a cell membrane to expose at least one subcellular component therein, ablating the at least one subcellular component by an infrared laser pulse to form an ablation plume, intercepting the ablation plume by an electrospray plume to form ions, and detecting the ions by mass spectrometry.

  15. Site locality identification study: Hanford Site. Volume II. Data cataloging

    Energy Technology Data Exchange (ETDEWEB)

    1980-07-01

    Data compilation and cataloging for the candidate site locality identification study were conducted in order to provide a retrievable data cataloging system for the present siting study and future site evaluation and licensng processes. This task occurred concurrently with and also independently of other tasks of the candidate site locality identification study. Work in this task provided the data utilized primarily in the development and application of screening and ranking processes to identify candidate site localities on the Hanford Site. The overall approach included two steps: (1) data acquisition and screening; and (2) data compilation and cataloging. Data acquisition and screening formed the basis for preliminary review of data sources with respect to their probable utilization in the candidate site locality identification study and review with respect to the level of completeness and detail of the data. The important working assumption was that the data to be used in the study be based on existing and available published and unpublished literature. The data compilation and cataloging provided the basic product of the Task; a retrievable data cataloging system in the form of an annotated reference list and key word index and an index of compiled data. The annotated reference list and key word index are cross referenced and can be used to trace and retrieve the data sources utilized in the candidate site locality identification study.

  16. Optically-controlled platforms for transfection and single- and sub-cellular surgery

    DEFF Research Database (Denmark)

    Villangca, Mark Jayson; Casey, Duncan; Glückstad, Jesper

    2015-01-01

    Improving the resolution of biological research to the single- or sub-cellular level is of critical importance in a wide variety of processes and disease conditions. Most obvious are those linked to aging and cancer, many of which are dependent upon stochastic processes where individual, unpredic......Improving the resolution of biological research to the single- or sub-cellular level is of critical importance in a wide variety of processes and disease conditions. Most obvious are those linked to aging and cancer, many of which are dependent upon stochastic processes where individual...... and specificity of optical trapping in conjunction with other modalities to perform single and sub-cellular surgery. These tools form highly tuneable platforms for the delivery or removal of material from cells of interest, but can simultaneously excite fluorescent probes for imaging purposes or plasmonic...... structures for very local heating. We discuss both the history and recent applications of the field, highlighting the key findings and developments over the last 40 years of biophotonics research....

  17. Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting

    Directory of Open Access Journals (Sweden)

    Salih Maysoon

    2009-06-01

    Full Text Available Abstract Background Tail anchored (TA membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP possesses two alternatively spliced tail anchors (TA1 or TA2 but their specificity of subcellular targeting remains unknown. Results TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER, whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1. Conclusion Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

  18. Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting.

    Science.gov (United States)

    Byers, Joseph T; Guzzo, Rosa M; Salih, Maysoon; Tuana, Balwant S

    2009-06-19

    Tail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown. TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1. Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

  19. Analysis of subcellular metabolite distributions within Arabidopsis thaliana leaf tissue: a primer for subcellular metabolomics.

    Science.gov (United States)

    Krueger, Stephan; Steinhauser, Dirk; Lisec, Jan; Giavalisco, Patrick

    2014-01-01

    Every biological organism relies for its proper function on interactions between a multitude of molecular entities like RNA, proteins, and metabolites. The comprehensive measurement and the analysis of all these entities would therefore provide the basis for our functional and mechanistic understanding of most biological processes. Next to their amount and identity, it is most crucial to also gain information about the subcellular distribution and the flux of the measured compounds between the cellular compartments. That is, we want to understand not only the individual functions of cellular components but also their functional implications within the whole organism. While the analysis of macromolecules like DNA, RNA, and proteins is quite established and robust, analytical techniques for small metabolites, which are prone to diffusion and degradation processes, provide a host of unsolved challenges. The major limitations here are the metabolite conversion and relocation processes. In this protocol we describe a methodological workflow which includes a nonaqueous fractionation method, a fractionated two-phase liquid/liquid extraction protocol, and a software package, which together allow extracting and analyzing starch, proteins, and especially polar and lipophilic metabolites from a single sample towards the estimation of their subcellular distributions.

  20. a case study of esan west local government area

    African Journals Online (AJOL)

    2013-01-31

    Jan 31, 2013 ... This study assesses the information needs of farmers in Edo State using Esan West Local Government Area as a case study. .... community development and economic respectively. (see table 3). Section 1: Table of socio demographic characteristic. Demographic characteristic. Variables. Frequency (%).

  1. Motion and time study analysis of wooden locally manufactured ...

    African Journals Online (AJOL)

    Studies were carried out on time-and-motion-economy of wooden locally manufactured duplicating machines. Two versions of the machine were used for the study, viz: standard version and semi-mechanized version. Working with both auxiliary and routine operations, the standard duplicator produced printed paper at an ...

  2. Subcellular boron and fluorine distributions with SIMS ion microscopy in BNCT and cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Subhash Chandra

    2008-05-30

    The development of a secondary ion mass spectrometry (SIMS) based technique of Ion Microscopy in boron neutron capture therapy (BNCT) was the main goal of this project, so that one can study the subcellular location of boron-10 atoms and their partitioning between the normal and cancerous tissue. This information is fundamental for the screening of boronated drugs appropriate for neutron capture therapy of cancer. Our studies at Cornell concentrated mainly on studies of glioblastoma multiforme (GBM). The early years of the grant were dedicated to the development of cryogenic methods and correlative microscopic approaches so that a reliable subcellular analysis of boron-10 atoms can be made with SIMS. In later years SIMS was applied to animal models and human tissues of GBM for studying the efficacy of potential boronated agents in BNCT. Under this grant the SIMS program at Cornell attained a new level of excellence and collaborative SIMS studies were published with leading BNCT researchers in the U.S.

  3. Subcellular targeting and dynamic regulation of PTEN: Implications for neuronal cells and neurological disorders

    Directory of Open Access Journals (Sweden)

    Patricia eKreis

    2014-04-01

    Full Text Available PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum, the mitochondria or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein-protein interactions or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.

  4. Subcellular targeting and dynamic regulation of PTEN: implications for neuronal cells and neurological disorders.

    Science.gov (United States)

    Kreis, Patricia; Leondaritis, George; Lieberam, Ivo; Eickholt, Britta J

    2014-01-01

    PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5)P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum (ER), the mitochondria, or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein-protein interactions, or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i) recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii) current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.

  5. Distribution, isomerization and enantiomer selectivity of hexabromocyclododecane (HBCD) diastereoisomers in different tissue and subcellular fractions of earthworms.

    Science.gov (United States)

    Li, Bing; Chen, Hao; Sun, Hongwen; Lan, Zhonghui

    2017-05-01

    In this study, earthworms Eisenia fetida (E. fetida) were exposed to a soil artificially contaminated with individual hexabromocyclododecane (HBCD) diastereoisomers (α-, β- and γ-HBCDs) to investigate the distribution, isomerization and enantiomer selectivity of HBCDs at tissue and subcellular levels. At the tissue level, the concentrations of HBCDs all followed the order of gut>bodyfluid>body wall, which suggested that earthworms accumulated HBCDs mainly via ingesting soil particles. At the subcellular level, the concentrations of HBCDs in an extracellular fraction consisting of granules, tissue fragment, cell membrane and intact cells (fraction A) were higher than those in an intracellular fractions consisting of the microsomal and cytosol (fraction B+C). This confirmed the passive diffusion during the distribution of HBCDs into the intracellular compartment. The distribution proportions of HBCDs varied among different tissue and subcellular fractions, and all changed over time within 14 days. The variable distributions of HBCDs in different fractions were a result of the comprehensive effects of dynamics and thermodynamics processes. The β- and γ-HBCDs were isomerized to α-HBCD in all tissue and subcellular fractions except for fraction C, and the isomerization ratios varied a lot, which seemed to be related to HBCDs residence time. The selective enrichment of (-) α-, (-) β and (-) γ-HBCDs was found in all fractions and this is consistent with that in the whole earthworm. Besides, the extents of enantio-selectivity did not change significantly among different tissue and subcellular fractions. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Studying plastic shear localization in aluminum alloys under dynamic loading

    Science.gov (United States)

    Bilalov, D. A.; Sokovikov, M. A.; Chudinov, V. V.; Oborin, V. A.; Bayandin, Yu. V.; Terekhina, A. I.; Naimark, O. B.

    2016-12-01

    An experimental and theoretical study of plastic shear localization mechanisms observed under dynamic deformation using the shear-compression scheme on a Hopkinson-Kolsky bar has been carried out using specimens of AMg6 alloy. The mechanisms of plastic shear instability are associated with collective effects in the microshear ensemble in spatially localized areas. The lateral surface of the specimens was photographed in the real-time mode using a CEDIP Silver 450M high-speed infrared camera. The temperature distribution obtained at different times allowed us to trace the evolution of the localization of the plastic strain. Based on the equations that describe the effect of nonequilibrium transitions on the mechanisms of structural relaxation and plastic flow, numerical simulation of plastic shear localization has been performed. A numerical experiment relevant to the specimen-loading scheme was carried out using a system of constitutive equations that reflect the part of the structural relaxation mechanisms caused by the collective behavior of microshears with the autowave modes of the evolution of the localized plastic flow. Upon completion of the experiment, the specimens were subjected to microstructure analysis using a New View-5010 optical microscope-interferometer. After the dynamic deformation, the constancy of the Hurst exponent, which reflects the relationship between the behavior of defects and roughness induced by the defects on the surfaces of the specimens is observed in a wider range of spatial scales. These investigations revealed the distinctive features in the localization of the deformation followed by destruction to the script of the adiabatic shear. These features may be caused by the collective multiscale behavior of defects, which leads to a sharp decrease in the stress-relaxation time and, consequently, a localized plastic flow and generation of fracture nuclei in the form of adiabatic shear. Infrared scanning of the localization zone of the

  7. Regional and subcellular distribution of the receptor-targeting protein PIST in the rat central nervous system.

    Science.gov (United States)

    Chen, Annie; Gössling, Enno K; Witkowski, Leora; Bhindi, Amar; Bauch, Carola; Roussy, Geneviève; Sarret, Philippe; Kreienkamp, Hans-Jürgen; Stroh, Thomas

    2012-04-01

    Protein interacting specifically with Tc10, PIST, is a Golgi-associated sorting protein involved in regulating cell-surface targeting of plasma membrane receptors. The present study provides the first comprehensive description of PIST distribution in the mammalian central nervous system and of its subcellular localization by immunocytochemistry. PIST is distributed widely throughout the neuraxis, predominantly associated with neuronal cell bodies and dendrites. In hippocampal neurons, in vitro and in situ, PIST displayed a patchy subcellular distribution in an area surrounding the nucleus and extending into one of the major dendrites. By colocalization with the trans-Golgi marker TGN38, we were able to show that PIST is associated largely but not exclusively with the trans-Golgi network in central neurons. High or moderate to high levels of PIST-like immunoreactivity were found in cortical areas, in particular in layer V of the neocortex. The motor cortex was most strongly labeled. Also, the piriform and insular cortices displayed strong PIST labeling. In the hippocampus, CA2 but not CA1 or CA3 pyramidal cells displayed strong PIST-labeling, extending into their apical dendrites. In the thalamus, ventrolateral and laterodorsal nuclei were most strongly stained, whereas in the hypothalamus the supraoptic nucleus stood out with strong immunoreactivity. Strikingly, in the brainstem all cranial nerve motor nuclei were PIST-positive at varying levels, which is in keeping with the prominent expression of PIST in forebrain motor areas. This selective distribution of PIST suggests that the protein serves distinctive roles in specific neuronal populations, establishing functionally distinct zones, for instance, in the hippocampus. Copyright © 2011 Wiley-Liss, Inc.

  8. Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

    Directory of Open Access Journals (Sweden)

    Takemoto Daigo

    2008-06-01

    Full Text Available Abstract Background Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle. Results Within 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed. Conclusion Our results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the

  9. The study of haematological parameters in the Nigerian local chicken

    African Journals Online (AJOL)

    The study of haematological parameters in the Nigerian local chicken. FUC Mmereole. Abstract. No Abstract. Full Text: EMAIL FULL TEXT EMAIL FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT · AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's · More ...

  10. Acute toxicity studies and characterisation of local dietary salts in ...

    African Journals Online (AJOL)

    Kanwa and Shem are food additives and also local dietary salts which are commonly used in Nigeria for various purposes both in rural and urban communities. Kanwa and Shem were subjected to acute toxicological studies using the modified Lorke method while the elemental, qualitative analysis and pH were determined ...

  11. Numerical study of effect of oxygen fraction on local entropy ...

    Indian Academy of Sciences (India)

    This study considers numerical simulation of the combustion of methane with air, including oxygen and nitrogen, in a burner and the numerical solution of local entropy generation rate due to high temperature and velocity gradients in the combustion chamber. The effects of equivalence ratio () and oxygen percentage () ...

  12. Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Michael I Koukourakis

    Full Text Available LC3s (MAP1-LC3A, B and C are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli, where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

  13. Study of the local structure of materials with novel properties

    Science.gov (United States)

    Cao, Daliang

    The Local structure of a material is important information needed in order to fully understand its macroscopic properties. The X-ray Absorption Fine Structure (XAFS) technique is a local probe that is very useful when studying a system without long range order or a system with significant local distortions. The XAFS technique is also very useful for studying some defects or very lightly doped elements in a system to determine their local physical and chemical environment. A brief introduction to the XAFS theory and experimental setup will be presented in the first part of this thesis. The second part of this thesis is the experimental study of the local structure of several colossal magnetoresistive (CMR) materials, which include La1-xCaxMnO 3 (LCMO) systems as well as Ti and Ga doped LCMO materials. The local distortions of the Mn atoms were studied and compared with the transport properties and magnetization data for each sample. We found that the Mn local distortions are strongly correlated with the magnetization of the material. Although the electron transport in a CMR sample was proposed to have a direct coupling with the sample's magnetization in the Double Exchange (DE) mechanism, we found that was not always the case. The XAFS results for the negative thermal expansion (NTE) material, ZrW 2O8, and thermoelectric materials, filled skutterudites, are presented in the third part. The measurements of the local structure for ZrW2O8 shows that the originally proposed model with a large transverse vibration of the middle O atom in the WO-Zr linkage cannot be the primary origin of NTE in ZrW2O8. A rigid-tentpole model is presented to explain both the XAFS results as well as the absence of a soft-mode displacive phase transition in this material. As to the filled skutterudite, which has a general form of RT4X12 (R = Lanthanide, actinide, or alkali earth; T = Fe, Ru, Os; X = P, As, Sb), both X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption

  14. Subcellular Targeting of Proteins Involved in Modification of Plant N- and O-Glycosylation.

    Science.gov (United States)

    Dicker, Martina; Schoberer, Jennifer; Vavra, Ulrike; Strasser, Richard

    2015-01-01

    Plants are attractive expression hosts for the production of recombinant glycoprotein therapeutics. The quality and efficiency of these biopharmaceuticals are very often influenced by the glycosylation profile. Consequently, approaches are needed that enable the production of recombinant glycoproteins with customized and homogenous N- and O-glycan structures. Here, we describe convenient tools that allow targeting and retention of glycan-modifying enzymes in the early secretory pathway of plants. These protocols can be used to fine-tune the subcellular localization of glycosyltransferases and glycosidases in plants and consequently to increase the homogeneity of glycosylation on recombinant glycoproteins.

  15. Dielectric elastomer actuator for the measurement of cell traction forces with sub-cellular resolution

    Science.gov (United States)

    Rosset, Samuel; Poulin, Alexandre; Zollinger, Alicia; Smith, Michael; Shea, Herbert

    2017-04-01

    We report on the use of dielectric elastomer actuators (DEAs) to measure the traction force field of cells with subcellular resolution. The study of cellular electrochemical and mechanical response to deformation is an important area of research, as mechanotransduction has been shown to be linked with fundamental cell functions, or the progression of diseases such as cancer or atherosclerosis. Experimental cell mechanics is based on two fundamental concepts: the ability to measure cell stiffness, and to apply controlled strains to small clusters of cells. However, there is a lack of tools capable of applying precise deformation to a small cell population while being compatible with an inverted microscope (stable focal plane, transparency, compactness, etc.). Here, we use an anisotropically prestretched silicone-based DEA to deform a soft (7.6kPa) polyacrylamide gel on which the cells are cultured. An array of micro-dots of fluorescent fibronectin is transferred on the gel by micro-contact printing and serves as attachment points for the cells. In addition, the fluorescent dots (which have a diameter of 2 μm with a spacing of 6 μm) are used during the experiment to monitor the traction forces of a single cell (or small cluster of cells). The cell locally exerts traction on the gel, thus deforming the matrix of dots. The position of dots versus time is monitored live when the cells are submitted to a uniaxial strain step. Our deformable bioreactor enables the measurement of the local stiffness of cells submitted to mechanical strain, and is fully compatible with an inverted microscope set-up.

  16. Biorecognition and Subcellular Trafficking of HPMA Copolymer - Anti-PMSA Antibody Conjugates by Prostate Cancer Cells

    Science.gov (United States)

    Liu, Jihua; Kopečková, Pavla; Bühler, Patrick; Wolf, Philipp; Pan, Huaizhong; Bauer, Hillevi; Elsässer-Beile, Ursula

    2009-01-01

    A new generation of antibodies against the prostate specific membrane antigen (PSMA) has been proven to bind specifically to PSMA molecules on the surface of living prostate cancer cells. To explore the potential of anti-PSMA antibodies as targeting moieties for macromolecular therapeutics for prostate cancer, fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymer - anti- PSMA antibody conjugates (P-anti-PSMA) were synthesized and the mechanisms of their endocytosis and subcellular trafficking in C4-2 prostate cancer cells were studied. Radioimmunoassays showed the dissociation constants of P-anti-PSMA for C4-2 prostate cancer cells in the low nanomolar range, close to values for free anti-PSMA. It indicated that conjugation of anti-PSMA to HPMA copolymers did not compromise their binding affinity. The rate of endocytosis of P-anti-PSMA was much faster than that of control HPMA copolymer conjugates containing non-specific IgG. Selective pathway inhibitors of clathrin-mediated endocytosis and of macropinocytosis inhibited the internalization of P-anti-PMSA. Inhibition of clathrin-mediated endocytosis was further evidenced by down-regulation of clathrin heavy chain expression by siRNA. Using a dominant-negative mutant of dynamin (Dyn K44A) to abolish the clathrin-, caveolae-independent endocytic pathway, we found that some of P-anti-PSMA adopted this pathway to be endocytosed into C4-2 cells. Thus multiple receptor-mediated endocytic pathways, including clathrin-mediated endocytosis, macropinocytosis, and dynamin-independent endocytosis, were involved in the internalization of P-anti-PSMA. The extent of the participation of each pathway in P-anti-PSMA endocytosis was estimated. Membrane vesicles containing P-anti-PSMA rapidly co-localized with membrane vesicles overexpressing Rab7, a late endosome localized protein, demonstrating that a part of P-anti-PSMA was transported to late endosomes. PMID:19344119

  17. Water solvent and local anesthetics: A computational study

    Science.gov (United States)

    Bernardi, R. C.; Gomes, D. E. B.; Pascutti, P. G.; Ito, A. S.; Taft, C. A.; Ota, A. T.

    There are various experimental studies regarding the toxicity and the time of action of local anesthetics, which contain general insights about their pharmacological and physicochemical properties. Although a detailed microscopic analysis of the local anesthetics would contribute to understanding these properties, there are relatively few theoretical studies about these molecules. In this article, we present the results from calculations performed for three local anesthetics: tetracaine, procaine, and lidocaine, both in their charged and uncharged forms, in aqueous environment. We have used the density functional theory and molecular dynamics simulations to study the structural characteristics of these compounds. The radial distribution function g(r) was used to examine the structure of water molecules surrounding different regions of the local anesthetics. We demonstrated the nonhomogeneous character of the anesthetics with respect to their affinity to water solvent molecules as well as the modifications in their affinity to water caused by changes in their charge state. We also observed that the biological potency of the anesthetics is more related to the behavior of specific groups within the molecule, which are responsible for the interaction with the lipid phase of membranes, rather than the general properties of the molecule as a whole.

  18. Comparative study of modelling efficiency regarding localization development

    Science.gov (United States)

    Tabourot, Laurent; Sène, Ndèye Awa; Balland, Pascale; Ksiksi, Nesrine; Charleux, Ludovic; Issack, Moustapha

    2017-10-01

    The determination of limit loadings that induce strain localization during material forming operation is always an important industrial concern, especially when it appears during the forming operation during which the material is highly stressed. Several recent studies have demonstrated that the intrinsic structural heterogeneity of metallic materials plays an important part in their mechanical behavior. Nevertheless, the role of heterogeneities on strain localization is not yet really broached. The objective of this study is to determine if the structural heterogeneity of materials has an impact on the localization of ductile materials. A corollary action is then to validate the use of models that integrate this heterogeneity to achieve better results. The study is composed of two parts: experimental and numerical. Concerning the experimental part, in order to obtain different degrees of heterogeneities, titanium samples were prepared to have materials with structure hexagonal and cubic centered which present very different possibilities of plastic accommodation. tensile and equibiaxial tests have been thus realized on Titanium α + β thick sheet, Titanium α thick sheet and Titanium α thin sheet. The simulation of experimental tests is realized using two models firstly a conventional phenomenological model and secondly a modelling in which spatial heterogeneities are introduced through a distribution of hardening parameters. the recourse to a model which integrates a physical dimension, heterogeneity, showed that this last has a notable incidence and makes it possible to improve the prediction of the phenomenon of localization.

  19. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    Directory of Open Access Journals (Sweden)

    Evgeny Bychkov

    Full Text Available G protein-coupled receptor kinases (GRKs and arrestins mediate desensitization of G protein-coupled receptors (GPCR. Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  20. Evolution and comparative genomics of subcellular specializations: EST sequencing of Torpedo electric organ.

    Science.gov (United States)

    Nazarian, Javad; Berry, Deborah L; Sanjari, Salar; Razvi, Mohammed; Brown, Kristy; Hathout, Yetrib; Vertes, Akos; Dadgar, Sherry; Hoffman, Eric P

    2011-03-01

    Uncharacterized open reading frames (ORFs) in human genomic sequence often show a high degree of evolutionary conservation, yet have little or no tissue EST or protein data suggestive of protein product function. The encoded proteins may have highly restricted expression in specialized cells, subcellular specializations, and/or narrow windows during development. One such highly specialized and minute subcellular compartment is the neuromuscular junction (NMJ), where motorneurons contact muscle fibers. The electric Torpedo ray has evolved to expand the NMJ structure to the size of a large organ (electroplax organ), and we hypothesized that Torpedo electroplax proteins would be candidates for human ESTs expressed at the human NMJ. A total of 9719 primary electroplax cDNA clones were sequenced. We identified 44 human ORFs showing high (>63%) amino acid identity to Torpedo electroplax transcripts with enrichment for mRNA splicing motifs (SH2 and pre-mRNA splicing domains), an observation potentially important for the strict nuclear domains maintained by myonuclei underlying the NMJ. We generated antibodies against two uncharacterized human genes (C19orf29 [Drosophila cactin] and C15orf24) and showed that these were indeed expressed at the murine NMJ. Cactin, a member of the Rel transcription factor family in Drosophila, localized to the postsynaptic cytosol of the NMJ and nuclear membrane. C15orf24 protein localized to the murine postsynaptic sarcolemma. We show a novel approach towards identifying proteins expressed at a subcellular specialization using evolutionary diversity of organ function and cross-species mapping. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. HECTAR: a method to predict subcellular targeting in heterokonts.

    Science.gov (United States)

    Gschloessl, Bernhard; Guermeur, Yann; Cock, J Mark

    2008-09-23

    The heterokonts are a particularly interesting group of eukaryotic organisms; they include many key species of planktonic and coastal algae and several important pathogens. To understand the biology of these organisms, it is necessary to be able to predict the subcellular localisation of their proteins but this is not straightforward, particularly in photosynthetic heterokonts which possess a complex chloroplast, acquired as the result of a secondary endosymbiosis. This is because the bipartite target peptides that deliver proteins to these chloroplasts can be easily confused with the signal peptides of secreted proteins, causing currently available algorithms to make erroneous predictions. HECTAR, a subcellular targeting prediction method which takes into account the specific properties of heterokont proteins, has been developed to address this problem. HECTAR is a statistical prediction method designed to assign proteins to five different categories of subcellular targeting: Signal peptides, type II signal anchors, chloroplast transit peptides, mitochondrion transit peptides and proteins which do not possess any N-terminal target peptide. The recognition rate of HECTAR is 96.3%, with Matthews correlation coefficients ranging from 0.67 to 0.95. The method is based on a hierarchical architecture which implements the divide and conquer approach to identify the different possible target peptides one at a time. At each node of the hierarchy, the most relevant outputs of various existing subcellular prediction methods are combined by a Support Vector Machine. The HECTAR method is able to predict the subcellular localisation of heterokont proteins with high accuracy. It also efficiently predicts the subcellular localisation of proteins from cryptophytes, a group that is phylogenetically close to the heterokonts. A variant of HECTAR, called HECTARSEC, can be used to identify signal peptide and type II signal anchor sequences in proteins from any eukaryotic organism. Both

  2. HECTAR: A method to predict subcellular targeting in heterokonts

    Directory of Open Access Journals (Sweden)

    Guermeur Yann

    2008-09-01

    Full Text Available Abstract Background The heterokonts are a particularly interesting group of eukaryotic organisms; they include many key species of planktonic and coastal algae and several important pathogens. To understand the biology of these organisms, it is necessary to be able to predict the subcellular localisation of their proteins but this is not straightforward, particularly in photosynthetic heterokonts which possess a complex chloroplast, acquired as the result of a secondary endosymbiosis. This is because the bipartite target peptides that deliver proteins to these chloroplasts can be easily confused with the signal peptides of secreted proteins, causing currently available algorithms to make erroneous predictions. HECTAR, a subcellular targeting prediction method which takes into account the specific properties of heterokont proteins, has been developed to address this problem. Results HECTAR is a statistical prediction method designed to assign proteins to five different categories of subcellular targeting: Signal peptides, type II signal anchors, chloroplast transit peptides, mitochondrion transit peptides and proteins which do not possess any N-terminal target peptide. The recognition rate of HECTAR is 96.3%, with Matthews correlation coefficients ranging from 0.67 to 0.95. The method is based on a hierarchical architecture which implements the divide and conquer approach to identify the different possible target peptides one at a time. At each node of the hierarchy, the most relevant outputs of various existing subcellular prediction methods are combined by a Support Vector Machine. Conclusion The HECTAR method is able to predict the subcellular localisation of heterokont proteins with high accuracy. It also efficiently predicts the subcellular localisation of proteins from cryptophytes, a group that is phylogenetically close to the heterokonts. A variant of HECTAR, called HECTARSEC, can be used to identify signal peptide and type II signal

  3. Studies of technetium-99m nitridobisdithiocarboxylate leucocyte specific radiopharmaceutical: [{sup 99m}TcN(DTCX){sub 2}], DTCX=CH{sub 3}(CH{sub 2}){sub 8}CS{sub 2}. the cellular and subcellular distribution in human blood cells, and chemical behaviour. synthesis of the analogous rhenium-188 radiopharmaceutical

    Energy Technology Data Exchange (ETDEWEB)

    Demaimay, Florian; Roucoux, Alain E-mail: Alain.Roucoux@ensc-rennes.fr; Dazord, Leontine; Noiret, Nicolas; Moisan, Annick; Patin, Henri

    1999-02-01

    The distribution of the radiopharmaceutical ([{sup 99m}TcN(DTCX){sub 2}], DTCX=CH{sub 3}(CH{sub 2}){sub 8}CS{sub 2}) in the leucocyte population determinated by a density separation with double gradient Polymorphprep{sup TM} was studied. Microautoradiographic analysis showed a subcellular distribution of the radiomarker in human blood cells. This technique confirmed the observed lymphocyte selectivity (69%) and revealed that the uptake was predominantly cytoplasmic around the nucleus. A labeling mechanism by passive endocytosis could be proposed involving a required lipophilicity of the radiopharmaceutical for lymphocyte targeting. Finally, we describe the new synthesis with an efficient yield and radiochemical purity of the analogous radiopharmaceutical [{sup 188}ReN(DTCX){sub 2}].

  4. Biomechanics of subcellular structures by non-invasive Brillouin microscopy

    Science.gov (United States)

    Antonacci, Giuseppe; Braakman, Sietse

    2016-11-01

    Cellular biomechanics play a pivotal role in the pathophysiology of several diseases. Unfortunately, current methods to measure biomechanical properties are invasive and mostly limited to the surface of a cell. As a result, the mechanical behaviour of subcellular structures and organelles remains poorly characterised. Here, we show three-dimensional biomechanical images of single cells obtained with non-invasive, non-destructive Brillouin microscopy with an unprecedented spatial resolution. Our results quantify the longitudinal elastic modulus of subcellular structures. In particular, we found the nucleoli to be stiffer than both the nuclear envelope (p biomechanics and its role in pathophysiology.

  5. Study of local correlations of magnetic and multiferroic compounds

    CERN Multimedia

    Alves, E J

    We propose to study magnetic and multiferroic strongly correlated electron materials using radioactive nuclear probe techniques, at ISOLDE . Following the strategy of a previous project, IS390, our aim is to provide local and element selective information on some of the mechanisms that rule structural, charge and orbital correlations, electronic and magnetic interactions and the coupling of the associated degrees of freedom. The main technique used is Perturbed Angular Correlations (PAC), which allows combined magnetic and electric hyperfine studies. This study is complemented by the use of conventional characterisation techniques, and the investigation of relevant macroscopic properties.

  6. Subcellular targeting and interactions among the Potato virus X TGB proteins.

    Science.gov (United States)

    Samuels, Timmy D; Ju, Ho-Jong; Ye, Chang-Ming; Motes, Christy M; Blancaflor, Elison B; Verchot-Lubicz, Jeanmarie

    2007-10-25

    Potato virus X (PVX) encodes three proteins named TGBp1, TGBp2, and TGBp3 which are required for virus cell-to-cell movement. To determine whether PVX TGB proteins interact during virus cell-cell movement, GFP was fused to each TGB coding sequence within the viral genome. Confocal microscopy was used to study subcellular accumulation of each protein in virus-infected plants and protoplasts. GFP:TGBp2 and TGBp3:GFP were both seen in the ER, ER-associated granular vesicles, and perinuclear X-bodies suggesting that these proteins interact in the same subdomains of the endomembrane network. When plasmids expressing CFP:TGBp2 and TGBp3:GFP were co-delivered to tobacco leaf epidermal cells, the fluorescent signals overlapped in ER-associated granular vesicles indicating that these proteins colocalize in this subcellular compartment. GFP:TGBp1 was seen in the nucleus, cytoplasm, rod-like inclusion bodies, and in punctate sites embedded in the cell wall. The puncta were reminiscent of previous reports showing viral proteins in plasmodesmata. Experiments using CFP:TGBp1 and YFP:TGBp2 or TGBp3:GFP showed CFP:TGBp1 remained in the cytoplasm surrounding the endomembrane network. There was no evidence that the granular vesicles contained TGBp1. Yeast two hybrid experiments showed TGBp1 self associates but failed to detect interactions between TGBp1 and TGBp2 or TGBp3. These experiments indicate that the PVX TGB proteins have complex subcellular accumulation patterns and likely cooperate across subcellular compartments to promote virus infection.

  7. Pendrin gene ablation alters ENaC subcellular distribution and open probability.

    Science.gov (United States)

    Pech, Vladimir; Wall, Susan M; Nanami, Masayoshi; Bao, Hui-Fang; Kim, Young Hee; Lazo-Fernandez, Yoskaly; Yue, Qiang; Pham, Truyen D; Eaton, Douglas C; Verlander, Jill W

    2015-07-15

    The present study explored whether the intercalated cell Cl(-)/HCO3(-) exchanger pendrin modulates epithelial Na(+) channel (ENaC) function by changing channel open probability and/or channel density. To do so, we measured ENaC subunit subcellular distribution by immunohistochemistry, single channel recordings in split open cortical collecting ducts (CCDs), as well as transepithelial voltage and Na(+) absorption in CCDs from aldosterone-treated wild-type and pendrin-null mice. Because pendrin gene ablation reduced 70-kDa more than 85-kDa γ-ENaC band density, we asked if pendrin gene ablation interferes with ENaC cleavage. We observed that ENaC-cleaving protease application (trypsin) increased the lumen-negative transepithelial voltage in pendrin-null mice but not in wild-type mice, which raised the possibility that pendrin gene ablation blunts ENaC cleavage, thereby reducing open probability. In mice harboring wild-type ENaC, pendrin gene ablation reduced ENaC-mediated Na(+) absorption by reducing channel open probability as well as by reducing channel density through changes in subunit total protein abundance and subcellular distribution. Further experiments used mice with blunted ENaC endocytosis and degradation (Liddle's syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddle's syndrome, pendrin gene ablation did not change ENaC subunit total protein abundance, subcellular distribution, or channel density, but markedly reduced channel open probability. We conclude that in mice harboring wild-type ENaC, pendrin modulates ENaC function through changes in subunit abundance, subcellular distribution, and channel open probability. In a mouse model of Liddle's syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability. Copyright © 2015 the American Physiological Society.

  8. Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

    Science.gov (United States)

    da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

    2014-01-01

    The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

  9. A workflow for mathematical modeling of subcellular metabolic pathways in leaf metabolism of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Thomas eNägele

    2013-12-01

    Full Text Available During the last decade genome sequencing has experienced a rapid technological development resulting in numerous sequencing projects and applications in life science. In plant molecular biology, the availability of sequence data on whole genomes has enabled the reconstruction of metabolic networks. Enzymatic reactions are predicted by the sequence information. Pathways arise due to the participation of chemical compounds as substrates and products in these reactions. Although several of these comprehensive networks have been reconstructed for the genetic model plant Arabidopsis thaliana, the integration of experimental data is still challenging. Particularly the analysis of subcellular organization of plant cells limits the understanding of regulatory instances in these metabolic networks in vivo. In this study, we develop an approach for the functional integration of experimental high-throughput data into such large-scale networks. We present a subcellular metabolic network model comprising 524 metabolic intermediates and 548 metabolic interactions derived from a total of 2769 reactions. We demonstrate how to link the metabolite covariance matrix of different Arabidopsis thaliana accessions with the subcellular metabolic network model for the inverse calculation of the biochemical Jacobian, finally resulting in the calculation of a matrix which satisfies a Lyaponov equation involving a covariance matrix. In this way, differential strategies of metabolite compartmentation and involved reactions were identified in the accessions when exposed to low temperature.

  10. Local Magnetic Fields in Ferromagnetics Studied by Positive Muon Precession

    CERN Multimedia

    2002-01-01

    Positive muons are used to study local magnetic fields in different materials. A polarized muon beam is employed with energies of 30-50 MeV, and the muons are stopped in the target being studied. During its lifetime the muon will precess in the magnetic fields present, and after the decay of the muon the emitted positron is detected in plastic scintillators. The time and angle of the detected positron is used to calculate the magnetic field at the position of the muon in the sample. \\\\ \\\\ The detector system consists of plastic scintillators. Most of the measurements are made in an applied magnetic field. A dilution cryostat is used to produce temperatures down to well below $ 1 ^0 $ K. \\\\ \\\\ The present line of experiments concern mainly: \\item a)~~~~Local magnetism in the paramagnetic state of the Lave's phase type REAl$_{2} $ and RENi$_{2} $ systems ~~~where RE is a rare-earth ion. \\item b)~~~~Local magnetic fields and critical behaviour of the magnetism in Gd metal. \\item c)~~~~Investigation of flux exclu...

  11. Measurement of endogenous subcellular concentration of steroids in tissue

    NARCIS (Netherlands)

    Poortman, J.; Landeghem, A.A.J. van; Helmond-Agema, A.; Thussen, J.H.H.

    1984-01-01

    A reliable method for the extraction of steroid hormones from human uterine tissue and the subsequent measurement of these hormones in the subcellular compartments by radioimmunoassay is described. Extraction of radioactive steroid hormones from in vivo labelled human uterine tissue by different

  12. Towards studies of organizational behaviour with greater local relevance

    Directory of Open Access Journals (Sweden)

    Peter B. Smith

    2007-12-01

    Full Text Available Theories of organizational behaviour mostly originated in North America. In testing their applicability elsewhere, attention must be given to differences in local environments and inthe values of local employees. Within Latin America, the prevalence of high collectivism and power distance are particularly important. Employees’ attachment to their organization has been shown to differ within collectivistic cultures. The argument is illustrated by two Latin American studies. Firstly, the ways in which managers handle work events within Mexico, Colombia, Brazil, Chile and Argentina are compared. Secondly, cross-national work problems of employees from these countries are surveyed. The results emphasize the need to take account of variability within the region, and the need to use measures that capture locally important issues. -- Las teorías del comportamiento organizacional se han originado principalmente en Américadel Norte. Al probar su aplicabilidad en otras regiones culturales, debe prestarse atención a las diferencias en los ambientes y en los valores de los empleados locales. Dentro de Latinoamérica, la prevalencia de altos niveles de colectivismo y distancia al poder es particularmenteimportante. En ese sentido, se ha demostrado que el apego de los empleados hacia su organización difiere dentro de las culturas colectivistas. El argumento se ilustra por dosestudios latinoamericanos. En el primero, se compara la forma en que los gerentes manejan los eventos laborales dentro de México, Colombia, Brasil, Chile y Argentina. En el segundo,se examinan los problemas laborales transnacionales de los empleados de estos países. Los resultados enfatizan la necesidad de utilizar medidas que capturen aspectos localmente importantes.

  13. Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5'AMP-activated protein kinase (AMPK). Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique.

    Science.gov (United States)

    Sambandam, Nandakumar; Steinmetz, Michael; Chu, Angel; Altarejos, Judith Y; Dyck, Jason R B; Lopaschuk, Gary D

    2004-07-01

    Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria.

  14. Synthesis and subcellular location of peroxisomal membrane proteins in a peroxisome-deficient mutant of the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Sulter, G.J.; Vrieling, E.G.; Harder, W.; Veenhuis, M.

    We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous

  15. Brain glycogen – new perspectives on its metabolic function and regulation at the subcellular level

    Directory of Open Access Journals (Sweden)

    Linea Frimodt Obel

    2012-03-01

    Full Text Available Glycogen is a complex glucose polymer found in a variety of tissues, including brain, where it is localized primarily in astrocytes. The small quantity found in brain compared to e.g. liver has led to the understanding that brain glycogen is merely used during hypoglycemia or ischemia. In this review evidence is brought forward highlighting what has been an emerging understanding in brain energy metabolism: that glycogen is more than just a convenient way to store energy for use in emergencies – it is a highly dynamic molecule with versatile implications in brain function, i.e. synaptic activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms underlying glycogen metabolism. Based on i the compartmentation of the interconnected second messenger pathways controlling glycogen metabolism (calcium and cAMP, ii alterations in the subcellular location of glycogen-associated enzymes and proteins induced by the metabolic status and iii a sequential component in the intermolecular mechanisms of glycogen metabolism, we suggest that glycogen metabolism in astrocytes is compartmentalized at the subcellular level. As a consequence, the meaning and importance of conventional terms used to describe glycogen metabolism (e.g. turnover is challenged. Overall, this review represents an overview of contemporary knowledge about brain glycogen and its metabolism and function. However, it also has a sharp focus on what we do not know, which is perhaps even more important for the future quest of uncovering the roles of glycogen in brain physiology and pathology.

  16. Biorecognition and subcellular trafficking of HPMA copolymer-anti-PSMA antibody conjugates by prostate cancer cells.

    Science.gov (United States)

    Liu, Jihua; Kopecková, Pavla; Bühler, Patrick; Wolf, Philipp; Pan, Huaizhong; Bauer, Hillevi; Elsässer-Beile, Ursula; Kopecek, Jindrich

    2009-01-01

    A new generation of antibodies against the prostate specific membrane antigen (PSMA) has been proven to bind specifically to PSMA molecules on the surface of living prostate cancer cells. To explore the potential of anti-PSMA antibodies as targeting moieties for macromolecular therapeutics for prostate cancer, fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymer-anti-PSMA antibody conjugates (P-anti-PSMA) were synthesized and the mechanisms of their endocytosis and subcellular trafficking in C4-2 prostate cancer cells were studied. Radioimmunoassays showed the dissociation constants of P-anti-PSMA for C4-2 prostate cancer cells in the low nanomolar range, close to values for free anti-PSMA. It indicated that conjugation of anti-PSMA to HPMA copolymers did not compromise their binding affinity. The rate of endocytosis of P-anti-PSMA was much faster than that of control HPMA copolymer conjugates containing nonspecific IgG. Selective pathway inhibitors of clathrin-mediated endocytosis and of macropinocytosis inhibited the internalization of P-anti-PSMA. Inhibition of clathrin-mediated endocytosis was further evidenced by down-regulation of clathrin heavy chain expression by siRNA. Using a dominant-negative mutant of dynamin (Dyn K44A) to abolish the clathrin-, caveolae-independent endocytic pathway, we found that some of P-anti-PSMA adopted this pathway to be endocytosed into C4-2 cells. Thus multiple receptor-mediated endocytic pathways, including clathrin-mediated endocytosis, macropinocytosis, and clathrin-, caveolae-independent endocytosis, were involved in the internalization of P-anti-PSMA. The extent of the participation of each pathway in P-anti-PSMA endocytosis was estimated. Membrane vesicles containing P-anti-PSMA rapidly colocalized with membrane vesicles overexpressing Rab7, a late endosome localized protein, demonstrating that a part of P-anti-PSMA was transported to late endosomes.

  17. Zn subcellular distribution in liver of goldfish (carassius auratus) with exposure to zinc oxide nanoparticles and mechanism of hepatic detoxification.

    Science.gov (United States)

    Fan, Wenhong; Li, Qian; Yang, Xiuping; Zhang, Li

    2013-01-01

    Zinc Oxide Nanoparticles (ZnO NPs) have attracted increasing concerns because of their widespread use and toxic potential. In this study, Zn accumulations in different tissues (gills, liver, muscle, and gut) of goldfish (Carassius auratus) after exposure to ZnO NPs were studied in comparison with bulk ZnO and Zn(2+). And the technique of subcellular partitioning was firstly used on the liver of goldfish to study the hepatic accumulation of ZnO NPs. The results showed that at sublethal Zn concentration (2 mg/L), bioaccumulation in goldfish was tissue-specific and dependent on the exposure materials. Compared with Zn(2+), the particles of bulk ZnO and the ZnO NPs appeared to aggregate in the environmentally contacted tissues (gills and gut), rather than transport to the internal tissues (liver and muscle). The subcellular distributions of liver differed for the three exposure treatments. After ZnO NPs exposure, Zn percentage in metal-rich granule (MRG) increased significantly, and after Zn(2+) exposure, it increased significantly in the organelles. Metallothionein-like proteins (MTLP) were the main target for Zn(2+), while MRG played dominant role for ZnO NPs. The different results of subcellular distributions revealed that metal detoxification mechanisms of liver for ZnO NPs, bulk ZnO, and Zn(2+) were different. Overall, subcellular partitioning provided an interesting start to better understanding of the toxicity of nano- and conventional materials.

  18. Correlation profiling of brain sub-cellular proteomes reveals co-assembly of synaptic proteins and subcellular distribution

    NARCIS (Netherlands)

    Pandya, N.J. (Nikhil J.); Koopmans, F. (Frank); J.A. Slotman (Johan A.); Paliukhovich, I. (Iryna); A.B. Houtsmuller (Adriaan); A.B. Smit (August); Li, K.W. (Ka Wan)

    2017-01-01

    textabstractProtein correlation profiling might assist in defining co-assembled proteins and subcellular distribution. Here, we quantified the proteomes of five biochemically isolated mouse brain cellular sub-fractions, with emphasis on synaptic compartments, from three brain regions, hippocampus,

  19. GAP Activity, but Not Subcellular Targeting, Is Required for Arabidopsis RanGAP Cellular and Developmental Functions.

    Science.gov (United States)

    Boruc, Joanna; Griffis, Anna H N; Rodrigo-Peiris, Thushani; Zhou, Xiao; Tilford, Bailey; Van Damme, Daniël; Meier, Iris

    2015-07-01

    The Ran GTPase activating protein (RanGAP) is important to Ran signaling involved in nucleocytoplasmic transport, spindle organization, and postmitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis thaliana RanGAP1. A double null mutant of the two Arabidopsis RanGAP homologs is gametophyte lethal. Here, we created a series of mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, the severity of developmental phenotypes increases, but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development is differentially affected by RanGAP mutant allele combinations of increasing severity and requires the GAP activity of RanGAP, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP from the nuclear envelope. © 2015 American Society of Plant Biologists. All rights reserved.

  20. Subcellular distribution and expression of cofilin and ezrin in human colon adenocarcinoma cell lines with different metastatic potential

    Directory of Open Access Journals (Sweden)

    D. Nowak

    2010-04-01

    Full Text Available The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs. Four human colon adenocarcinoma cell lines – parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells. In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.

  1. Localization in random bipartite graphs: Numerical and empirical study

    Science.gov (United States)

    Slanina, František

    2017-05-01

    We investigate adjacency matrices of bipartite graphs with a power-law degree distribution. Motivation for this study is twofold: first, vibrational states in granular matter and jammed sphere packings; second, graphs encoding social interaction, especially electronic commerce. We establish the position of the mobility edge and show that it strongly depends on the power in the degree distribution and on the ratio of the sizes of the two parts of the bipartite graph. At the jamming threshold, where the two parts have the same size, localization vanishes. We found that the multifractal spectrum is nontrivial in the delocalized phase, but still near the mobility edge. We also study an empirical bipartite graph, namely, the Amazon reviewer-item network. We found that in this specific graph the mobility edge disappears, and we draw a conclusion from this fact regarding earlier empirical studies of the Amazon network.

  2. Localization microscopy for the study of amyloid fibril formation

    Science.gov (United States)

    Pinotsi, Dorothea; Kaminski Schierle, Gabriele S.; Rees, Eric; Kaminski, Clemens F.

    2013-09-01

    Super-resolution microscopy has emerged as a powerful and non-invasive tool for the study of molecular processes both in vitro, but also as they occur in live cells. Here we present the application of direct stochastic optical reconstruction microscopy (dSTORM), a super-resolution technique based on single molecule localization, to determine the morphology of protein aggregates and of small extra- and intracellular structures. The technique reveals details down to 20 nm providing information on scales much smaller than the wavelength of the probing light. We use dSTORM in the study of amyloid fibril self-assembly processes associated with neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We show that the aggregation process can be followed kinetically and observe the emergence of amyloid structures in time as they occur in vitro. As an all optical technique, there is translation potential from studies in vitro to in vivo applications.

  3. Endoplasmic reticulum export, subcellular distribution, and fibril formation by Pmel17 require an intact N-terminal domain junction.

    Science.gov (United States)

    Leonhardt, Ralf M; Vigneron, Nathalie; Rahner, Christoph; Van den Eynde, Benoît J; Cresswell, Peter

    2010-05-21

    Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.

  4. Endoplasmic Reticulum Export, Subcellular Distribution, and Fibril Formation by Pmel17 Require an Intact N-terminal Domain Junction*

    Science.gov (United States)

    Leonhardt, Ralf M.; Vigneron, Nathalie; Rahner, Christoph; Van den Eynde, Benoît J.; Cresswell, Peter

    2010-01-01

    Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes. PMID:20231267

  5. Towards studies of organizational behaviour with greater local relevance

    National Research Council Canada - National Science Library

    Smith, Peter B

    2007-01-01

    Theories of organizational behaviour mostly originated in North America. In testing their applicability elsewhere, attention must be given to differences in local environments and in the values of local employees...

  6. Study on local vacuum electron beam welding of flange rim

    CERN Document Server

    He Cheng Dan; Ying Lei; Xu Qi Jin

    2002-01-01

    Local vacuum electron beam welding and its application prospect in military and civil industry are introduced. A home made local vacuum electron beam welding is completed. Its main technical parameters and key techniques are also presented

  7. Local heat preconditioning in skin sparing mastectomy: a pilot study.

    Science.gov (United States)

    Mehta, Saahil; Rolph, Rachel; Cornelius, Victoria; Harder, Yves; Farhadi, Jian

    2013-12-01

    Experimental data has shown an association with a reduction of flap necrosis after local heat-application to a supraphysiological level resulting from the up-regulation of heat shock proteins, such as HSP-32. The proteins maintained capillary perfusion and increased tissue tolerance to ischaemia. The purpose of this translational study was to evaluate the effect of local heat preconditioning before skin sparing mastectomy and immediate breast reconstruction. A prospective non-randomised trial was performed from July 2009-April 2010. 50 consecutive patients at risk of skin flap necrosis (BMI >30, sternal-to-nipple distance>26 cm or breast size>C-cup) were included. Twenty-five patients were asked to heat-precondition their breast 24-h prior to surgery using a hot water bottle with a water temperature of 43 °C (thermometers provided), in three 30-min cycles interrupted by spontaneous cooling to room temperature. Skin flap necrosis was defined by the need for surgical debridement. LDI images were taken pre- and post-mastectomy to demonstrate an increase in tissue vascularity. 36% of women (n=25) without local heat-treatment developed skin flap necrosis, 12% developed skin flap necrosis in the treatment group, resulting in a 24% difference (n=25; p=0.047 (95%CI 1%-47%)). LDI scanning of the heated breast demonstrated an increase in vascularity compared to the contralateral non-heated breast. Median length of inpatient stay for treatment group was 4 days (95%CI(4, 7)), controls 8 days (95%CI(8, 9) (p=mastectomy. ACTRN12612001197820. II. Copyright © 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

  8. Sub-cellular distribution and translocation of TRP channels.

    Science.gov (United States)

    Toro, Carlos A; Arias, Luis A; Brauchi, Sebastian

    2011-01-01

    Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

  9. Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus.

    Science.gov (United States)

    Martínez-Turiño, Sandra; Hernández, Carmen

    2012-02-01

    Replication of all positive RNA viruses occurs in association with intracellular membranes. In many cases, the mechanism of membrane targeting is unknown and there appears to be no correlation between virus phylogeny and the membrane systems recruited for replication. Pelargonium flower break virus (PFBV, genus Carmovirus, family Tombusviridae) encodes two proteins, p27 and its read-through product p86 (the viral RNA dependent-RNA polymerase), that are essential for replication. Recent reports with other members of the family Tombusviridae have shown that the smaller replicase protein is targeted to specific intracellular membranes and it is assumed to determine the subcellular localization of the replication complex. Using in vivo expression of green fluorescent protein (GFP) fusions in plant and yeast cells, we show here that PFBV p27 localizes in mitochondria. The same localization pattern was found for p86 that contains the p27 sequence at its N-terminus. Cellular fractionation of p27GFP-expressing cells confirmed the confocal microscopy observations and biochemical treatments suggested a tight association of the protein to membranes. Analysis of deletion mutants allowed identification of two regions required for targeting of p27 to mitochondria. These regions mapped toward the N- and C-terminus of the protein, respectively, and could function independently though with distinct efficiency. In an attempt to search for putative cellular factors involved in p27 localization, the subcellular distribution of the protein was checked in a selected series of knockout yeast strains and the outcome of this approach is discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Evaluation on subcellular partitioning and biodynamics of pulse copper toxicity in tilapia reveals impacts of a major environmental disturbance.

    Science.gov (United States)

    Ju, Yun-Ru; Yang, Ying-Fei; Tsai, Jeng-Wei; Cheng, Yi-Hsien; Chen, Wei-Yu; Liao, Chung-Min

    2017-07-01

    Fluctuation exposure of trace metal copper (Cu) is ubiquitous in aquatic environments. The purpose of this study was to investigate the impacts of chronically pulsed exposure on biodynamics and subcellular partitioning of Cu in freshwater tilapia (Oreochromis mossambicus). Long-term 28-day pulsed Cu exposure experiments were performed to explore subcellular partitioning and toxicokinetics/toxicodynamics of Cu in tilapia. Subcellular partitioning linking with a metal influx scheme was used to estimate detoxification and elimination rates. A biotic ligand model-based damage assessment model was used to take into account environmental effects and biological mechanisms of Cu toxicity. We demonstrated that the probability causing 50% of susceptibility risk in response to pulse Cu exposure in generic Taiwan aquaculture ponds was ~33% of Cu in adverse physiologically associated, metabolically active pool, implicating no significant susceptibility risk for tilapia. We suggest that our integrated ecotoxicological models linking chronic exposure measurements with subcellular partitioning can facilitate a risk assessment framework that provides a predictive tool for preventive susceptibility reduction strategies for freshwater fish exposed to pulse metal stressors.

  11. Subcellular Targeting of VIP Boutons in Mouse Barrel Cortex is Layer-Dependent and not Restricted to Interneurons.

    Science.gov (United States)

    Zhou, Xiaojuan; Rickmann, Michael; Hafner, Georg; Staiger, Jochen F

    2017-11-01

    Neocortical vasoactive intestinal polypeptide (VIP) expressing cells are a diverse subpopulation of GABAergic interneurons issuing distinct axonal projections. They are known to inhibit other types of interneurons as well as excitatory principal neurons and possess a disinhibitory net effect in cortical circuits. In order to elucidate their targeting specificity, the output connectivity of VIP interneurons was studied at the subcellular level in barrel cortex of interneuron-specific Cre-driver mice, using pre- and postembedding electron microscopy. Systematically sampling VIP boutons across all layers, we found a substantial proportion of the innervated subcellular structures were dendrites (80%), with somata (13%), and spines (7%) being much less targeted. In layer VI, a high proportion of axosomatic synapses was found (39%). GABA-immunopositive ratio was quantified among the targets using statistically validated thresholds: only 37% of the dendrites, 7% of the spines, and 26% of the somata showed above-threshold immunogold labeling. For the main target structure "dendrite", a higher proportion of GABAergic subcellular profiles existed in deep than in superficial layers. In conclusion, VIP interneurons innervate non-GABAergic excitatory neurons and interneurons at their subcellular domains with layer-dependent specificity. This suggests a diverse output of VIP interneurons, which predicts multiple functionality in cortical circuitry beyond disinhibition. © The Author 2017. Published by Oxford University Press.

  12. Studies on locally available three anti-diabetic herbal medicines

    Directory of Open Access Journals (Sweden)

    Mohammad Shoeb

    2011-06-01

    Full Text Available Herbal medicines are being used for the treatment of different diseases for centuries. Sustainable development of herbal medicines need the study of their safety, efficacy and standardization are essential. Two commercially available herbal medicines i.e., Ziabetes (dolabi and Jambadayrist, and a folkloric medicine prepared from four plant materials by a local practitioners were investigated for their chemical compositions. Four compounds were isolated from the extracts of these medicines by silica gel column chromatography. Oleic acid and p-hydroxycinnamic acid were isolated from the aqueous 80%ethanol extract of the folkloric medicine whereas benzoic acid was found to be present in Ziabetes and Jambadayrist. The present investigation revealed that excessive amount of benzoic acid (or sodium benzoate is being added as preservative in commercial herbal medicines.

  13. Intraperitoneal local anesthetics have predominant local analgesic effect: a randomized, double-blind study.

    Science.gov (United States)

    Perniola, Andrea; Magnuson, Anders; Axelsson, Kjell; Gupta, Anil

    2014-08-01

    It remains unclear whether analgesia from intraperitoneal local anesthetics is via local or central mechanisms. This double-blind clinical trial tests the hypothesis that intraperitoneal local anesthetic is superior to continuous IV infusion for pain management. Primary outcome was morphine consumption during 0 to 24 h. Informed consent was obtained from 60 patients, age 30 to 75 yr, American Society of Anesthesiologists physical status I to II, undergoing abdominal hysterectomy. A computer-generated program randomized patients in parallel arms to group IV: continuous infusion of lidocaine 50 mg/h (10 ml) IV and saline 10 ml/h intermittently intraperitoneal; group IP: injection of lidocaine 50 mg/h (10 ml) once every hour intraperitoneally and continuous infusion of saline 10 ml/h intravenously; and group P (placebo): saline 10 ml/h both intravenously and intermittent intraperitoneal injection. Postoperative morphine consumption, pain intensity, recovery, home discharge, and lidocaine concentrations were measured. Morphine consumption during 0 to 24 h was lower in group IP versus group IV, mean difference -22.6 mg (95% CI, 11.4 to 33.8; P lidocaine in group IP was significantly lower than group IV, 0 to 4.5 h postoperatively (P = 0.03) with no evidence of systemic toxicity. Pain intensity and other recovery parameters were similar between the groups. The lower supplemental morphine consumption and plasma lidocaine concentration in group IP would confirm that the effects of local anesthetics are likely to be predominant via local intraperitoneal receptors or anti-inflammatory effects and not via central mechanisms alone.

  14. A CLINICAL STUDY OF LOCALLY ADVANCED CARCINOMA OF BREAST

    Directory of Open Access Journals (Sweden)

    Mrinalini

    2015-06-01

    Full Text Available BACKGROUND : In India it is observed that most of the patients of breast cancer clinically present in late stage due to their ignorance of disease despite so much advancement in its detection and management. Locally advanced breast cancer (LABC accounts for 30 - 35% of all cases of breast cancers in India. This study aims to evaluate C linical features, Investigations, various Treatment modalities and the Clinico - pathological correlation & outcome of various treatment modalities of LABC, with special emphasis on Neo - adjuvant chemotherapy (NACT in Indian setting. MATERIAL AND METHOD : This was a non - randomised prospective observational study. We analyzed 57 patients of LABC Stage IIIB & IIIC presenting at Government Medical College, Nagpur, Maharashtra, a tertiary care C entre from September 2012 to November 2014. RESULTS : Stage IIIB comprised 84.21% patients while remaining 15.79% were having Stage IIIC disease. Skin involvement was observed in 91.23% patients. 15.79% showed supraclavicular lymph node involvement. 32 patients received NACT (2 to 6 cycles. Out of these 32, complete clinical response (cCR was 12.5%, partial response (cPR was 68.75% and pathological CR (pCR was 6.25% with Total Objective response (cCR+cPR 81.25%. Feasibility of Breast Conserving Surgery (BCS was observed in 12.5% patients. 25 patients underwent primary surgery followed by adjuvant chemotherapy. Modified Radical Mastectomy was performed in 89.48% patients. CONCLUSIONS : With overall clinical response of 81.25%, n eoadjuvant chemotherapy is the best treatment option for patients with Locally Advanced Breast Cancer with added advantage of in vivo testing the sensitivity of chemotherapeutic agents, early management of micrometastasis and down staging the primary tumour with feasibility of BCS. Patients presenting LABC constitute a diverse group for whic h a variety of treatment modalities should be instituted with co o rdinated treatment planning among surgeons

  15. Measurement of endogenous subcellular concentration of steroids in tissue

    OpenAIRE

    Poortman, J.; Landeghem, A.A.J. van; Helmond-Agema, A.; Thussen, J.H.H.

    1984-01-01

    A reliable method for the extraction of steroid hormones from human uterine tissue and the subsequent measurement of these hormones in the subcellular compartments by radioimmunoassay is described. Extraction of radioactive steroid hormones from in vivo labelled human uterine tissue by different methods reveals that an almost quantitative extraction of steroid hormones from the nuclear fraction is obtained by sonication in ethanol-acetone. Extraction of steroid hormones with diethylether from...

  16. The punctate localization of rat Eag1 K+ channels is conferred by the proximal post-CNBHD region.

    Science.gov (United States)

    Chuang, Chao-Chin; Jow, Guey-Mei; Lin, Huei-Min; Weng, Yu-Han; Hu, Jui-Hsiang; Peng, Yi-Jheng; Chiu, Yi-Chih; Chiu, Mei-Miao; Jeng, Chung-Jiuan

    2014-02-04

    In mammals, Eag K+ channels (KV10) are exclusively expressed in the brain and comprise two isoforms: Eag1 (KV10.1) and Eag2 (KV10.2). Despite their wide presence in various regions of the brain, the functional role of Eag K+ channels remains obscure. Here we address this question by characterizing the subcellular localization of rat Eag1 (rEag1) and rat Eag2 (rEag2) in hippocampal neurons, as well as determining the structural basis underlying their different localization patterns. Immunofluorescence analysis of young and mature hippocampal neurons in culture revealed that endogenous rEag1 and rEag2 K+ channels were present in both the dendrosomatic and the axonal compartments. Only rEag1 channels displayed a punctate immunostaining pattern and showed significant co-localization with PSD-95. Subcellular fractionation analysis further demonstrated a distinct enrichment of rEag1 in the synaptosomal fraction. Over-expression of recombinant GFP-tagged Eag constructs in hippocampal neurons also showed a significant punctate localization of rEag1 channels. To identify the protein region dictating the Eag channel subcellular localization pattern, we generated a variety of different chimeric constructs between rEag1 and rEag2. Quantitative studies of neurons over-expressing these GFP-tagged chimeras indicated that punctate localization was conferred by a segment (A723-R807) within the proximal post-cyclic nucleotide-binding homology domain (post-CNBHD) region in the rEag1 carboxyl terminus. Our findings suggest that Eag1 and Eag2 K+ channels may modulate membrane excitability in both the dendrosomatic and the axonal compartments and that Eag1 may additionally regulate neurotransmitter release and postsynaptic signaling. Furthermore, we present the first evidence showing that the proximal post-CNBHD region seems to govern the Eag K+ channel subcellular localization pattern.

  17. Using the concept of Chou's pseudo amino acid composition to predict apoptosis proteins subcellular location: an approach by approximate entropy.

    Science.gov (United States)

    Jiang, Xiaoying; Wei, Rong; Zhang, Tongliang; Gu, Quan

    2008-01-01

    The function of protein is closely correlated with it subcellular location. Prediction of subcellular location of apoptosis proteins is an important research area in post-genetic era because the knowledge of apoptosis proteins is useful to understand the mechanism of programmed cell death. Compared with the conventional amino acid composition (AAC), the Pseudo Amino Acid composition (PseAA) as originally introduced by Chou can incorporate much more information of a protein sequence so as to remarkably enhance the power of using a discrete model to predict various attributes of a protein. In this study, a novel approach is presented to predict apoptosis protein solely from sequence based on the concept of Chou's PseAA composition. The concept of approximate entropy (ApEn), which is a parameter denoting complexity of time series, is used to construct PseAA composition as additional features. Fuzzy K-nearest neighbor (FKNN) classifier is selected as prediction engine. Particle swarm optimization (PSO) algorithm is adopted for optimizing the weight factors which are important in PseAA composition. Two datasets are used to validate the performance of the proposed approach, which incorporate six subcellular location and four subcellular locations, respectively. The results obtained by jackknife test are quite encouraging. It indicates that the ApEn of protein sequence could represent effectively the information of apoptosis proteins subcellular locations. It can at least play a complimentary role to many of the existing methods, and might become potentially useful tool for protein function prediction. The software in Matlab is available freely by contacting the corresponding author.

  18. Predicting subcellular location of proteins using integrated-algorithm method.

    Science.gov (United States)

    Cai, Yu-Dong; Lu, Lin; Chen, Lei; He, Jian-Feng

    2010-08-01

    Protein's subcellular location, which indicates where a protein resides in a cell, is an important characteristic of protein. Correctly assigning proteins to their subcellular locations would be of great help to the prediction of proteins' function, genome annotation, and drug design. Yet, in spite of great technical advance in the past decades, it is still time-consuming and laborious to experimentally determine protein subcellular locations on a high throughput scale. Hence, four integrated-algorithm methods were developed to fulfill such high throughput prediction in this article. Two data sets taken from the literature (Chou and Elrod, Protein Eng 12:107-118, 1999) were used as training set and test set, which consisted of 2,391 and 2,598 proteins, respectively. Amino acid composition was applied to represent the protein sequences. The jackknife cross-validation was used to test the training set. The final best integrated-algorithm predictor was constructed by integrating 10 algorithms in Weka (a software tool for tackling data mining tasks, http://www.cs.waikato.ac.nz/ml/weka/ ) based on an mRMR (Minimum Redundancy Maximum Relevance, http://research.janelia.org/peng/proj/mRMR/ ) method. It can achieve correct rate of 77.83 and 80.56% for the training set and test set, respectively, which is better than all of the 60 algorithms collected in Weka. This predicting software is available upon request.

  19. Enhanced Soundings for Local Coupling Studies Field Campaign Report

    Energy Technology Data Exchange (ETDEWEB)

    Ferguson, Craig R [University at Albany, State University of New York; Santanello, Joseph A [NASA Goddard Space Flight Center (GSFC), Greenbelt, MD (United States); Gentine, Pierre [Columbia Univ., New York, NY (United States)

    2016-04-01

    This document presents initial analyses of the enhanced radiosonde observations obtained during the U.S. Department of Energy (DOE) Atmospheric Radiation Measurement (ARM) Climate Research Facility Enhanced Soundings for Local Coupling Studies Field Campaign (ESLCS), which took place at the ARM Southern Great Plains (SGP) Central Facility (CF) from June 15 to August 31, 2015. During ESLCS, routine 4-times-daily radiosonde measurements at the ARM-SGP CF were augmented on 12 days (June 18 and 29; July 11, 14, 19, and 26; August 15, 16, 21, 25, 26, and 27) with daytime 1-hourly radiosondes and 10-minute ‘trailer’ radiosondes every 3 hours. These 12 intensive operational period (IOP) days were selected on the basis of prior-day qualitative forecasts of potential land-atmosphere coupling strength. The campaign captured 2 dry soil convection advantage days (June 29 and July 14) and 10 atmospherically controlled days. Other noteworthy IOP events include: 2 soil dry-down sequences (July 11-14-19 and August 21-25-26), a 2-day clear-sky case (August 15-16), and the passing of Tropical Storm Bill (June 18). To date, the ESLCS data set constitutes the highest-temporal-resolution sampling of the evolution of the daytime planetary boundary layer (PBL) using radiosondes at the ARM-SGP. The data set is expected to contribute to: 1) improved understanding and modeling of the diurnal evolution of the PBL, particularly with regard to the role of local soil wetness, and (2) new insights into the appropriateness of current ARM-SGP CF thermodynamic sampling strategies.

  20. Local History of Jakarta and MulticulturalAttitude (Historical Local Study of Betawi Ethnic

    Directory of Open Access Journals (Sweden)

    Suswandari Suswandari

    2017-03-01

    Full Text Available This is a literature review about local history of Jakarta and multicultural attitude. In the context of local history of Jakarta, ethnic Betawi as ethnic origin Jakarta is formed from the process of inter-ethnic assimilation imported by the Dutch colonial government in its political and economic interests. In its development, the Betawi ethnic group continued to strengthen and succeeded in establishing their own distinctive identity as well as disturbing with other ethnic groups in Indonesia, although in their present development their existence is decreasing due to development interest which is not able to open wide room for Betawi ethnicity itself because various causes. The Betawi ethnic group has a strong identity concerning Betawi cultural heritage such as strong religious soul, respect for diversity, friendly, homoris, helpful, open, tolerant to differences and so on. As a part of Jakarta's local history, ethnic Betawi history can be explored as a source of inspiration and a source of awareness in instilling multicultural souls in Jakarta, as a metropolitan city with increasingly diverse ethnicity towards social life within the framework of peace and harmony.

  1. Using time-of-flight for WLAN localization: feasibility study

    NARCIS (Netherlands)

    Kavitha Muthukrishnan, K.; Koprinkov, G.T.; Meratnia, Nirvana; Lijding, M.E.M.

    Although signal strength based techniques are widely employed for WLAN localization, they generally suffer from providing highly accurate location information. In this paper, we first present the general shortcomings of the signal strength based approaches used for WLANbased localization and then

  2. Grounding local peace organisations: a case study of Southern Sudan

    NARCIS (Netherlands)

    Hilhorst, D.J.M.; Leeuwen, van M.

    2005-01-01

    Since the early 1990s, building peace during and after conflict has been moving away from the conference tables of diplomats to informal settings created by local NGOs. The vast majority, if not all, of the peacebuilding policy and literature argues for strengthening local organisations as vehicles

  3. Epidemiological study of snake bite in some local government areas ...

    African Journals Online (AJOL)

    Envenomation is a common public health problem in many local government areas of Plateau state in Nigeria. Its incidence has increased over the years as reported by the outpatient departments of the General Hospitals in Shendam and Langtang local government areas. In Shendam alone, the increase was from 192 in ...

  4. Local Boundaries of “Illegality”: A Case Study of Local and Federal Police Collaborations on Immigration Enforcement

    OpenAIRE

    Tarrant, Courtney

    2017-01-01

    This research explores Operation Joint Effort (OJE) as a case study of local and federal collaborations on immigration enforcement. OJE is a collective partnership between Escondido, California’s local police department and Immigration and Customs Enforcement (ICE). As individual states continue to opt into such collaborative efforts, the imperative need for a critical review is bellowing. The central concern of this thesis is of how such collaborations impact the daily routine for undocument...

  5. Multiple luteinizing hormone receptor (LHR) protein variants, interspecies reactivity of anti-LHR mAb clone 3B5, subcellular localization of LHR in human placenta, pelvic floor and brain, and possible role for LHR in the development of abnormal pregnancy, pelvic floor disorders and Alzheimer's disease.

    Science.gov (United States)

    Bukovsky, Antonin; Indrapichate, Korakod; Fujiwara, Hiroshi; Cekanova, Maria; Ayala, Maria E; Dominguez, Roberto; Caudle, Michael R; Wimalsena, Jay; Elder, Robert F; Copas, Pleas; Foster, James S; Fernando, Romaine I; Henley, Donald C; Upadhyaya, Nirmala B

    2003-06-03

    Distinct luteinizing hormone receptor (LHR) protein variants exist due to the posttranslational modifications. Besides ovaries, LHR immunoreactivity (LHRI) was also found in other tissues, such as the brain, fallopian tube, endometrium, trophoblast and resident tissue macrophages. The 3B5 mouse monoclonal antibody was raised against purified rat LHR. In rat, porcine and human ovaries, the 3B5 identified six distinct LHR bands migrating at approximately 92, 80, 68, 59, 52 and 48 kDa. Characteristic LHRI was detected in rat, human and porcine corpora lutea. During cellular differentiation, subcellular LHR distribution changed from none to granular cytoplasmic, perinuclear, surface, nuclear and no staining. There were also differences in vascular LHR expression--lack of LHRI in ovarian vessels and strong staining of vessels in other tissues investigated. In normal human term placentae, villous LHRI was associated with blood sinusoids and cytotrophoblast cells, and rarely detected in trophoblastic syncytium. In all abnormal placentae, the LHRI of sinusoids was absent, and syncytium showed either enhanced (immature placental phenotypes) or no LHRI (aged placental phenotype). LHRI in human brain was identified in microglial cells (CD68+ resident macrophages). Protein extracts from human vaginal wall and levator ani muscle and fascia showed strong approximately 92 and 68 kDa species, and LHRI was detected in smooth muscle cells, fibroblasts, resident macrophages and nuclei of skeletal muscle fibers. Our observations indicate that, in contrast to the theory on the role of vascular hormone receptors in preferential pick up of circulating hormones, there is no need to enhance selective pick up rather only prevent LH/CG transport to inappropriate sites. Abnormal placental LHR expression may play a role in the development of abnormal pregnancy. Expression of LHR in the pelvic floor compartments suggests that high LH levels in postmenopausal women may contribute to the pelvic

  6. Multiple luteinizing hormone receptor (LHR protein variants, interspecies reactivity of anti-LHR mAb clone 3B5, subcellular localization of LHR in human placenta, pelvic floor and brain, and possible role for LHR in the development of abnormal pregnancy, pelvic floor disorders and Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Fernando Romaine I

    2003-06-01

    Full Text Available Abstract Distinct luteinizing hormone receptor (LHR protein variants exist due to the posttranslational modifications. Besides ovaries, LHR immunoreactivity (LHRI was also found in other tissues, such as the brain, fallopian tube, endometrium, trophoblast and resident tissue macrophages. The 3B5 mouse monoclonal antibody was raised against purified rat LHR. In rat, porcine and human ovaries, the 3B5 identified six distinct LHR bands migrating at ~92, 80, 68, 59, 52 and 48 kDa. Characteristic LHRI was detected in rat, human and porcine corpora lutea. During cellular differentiation, subcellular LHR distribution changed from none to granular cytoplasmic, perinuclear, surface, nuclear and no staining. There were also differences in vascular LHR expression – lack of LHRI in ovarian vessels and strong staining of vessels in other tissues investigated. In normal human term placentae, villous LHRI was associated with blood sinusoids and cytotrophoblast cells, and rarely detected in trophoblastic syncytium. In all abnormal placentae, the LHRI of sinusoids was absent, and syncytium showed either enhanced (immature placental phenotypes or no LHRI (aged placental phenotype. LHRI in human brain was identified in microglial cells (CD68+ resident macrophages. Protein extracts from human vaginal wall and levator ani muscle and fascia showed strong ~92 and 68 kDa species, and LHRI was detected in smooth muscle cells, fibroblasts, resident macrophages and nuclei of skeletal muscle fibers. Our observations indicate that, in contrast to the theory on the role of vascular hormone receptors in preferential pick up of circulating hormones, there is no need to enhance selective pick up rather only prevent LH/CG transport to inappropriate sites. Abnormal placental LHR expression may play a role in the development of abnormal pregnancy. Expression of LHR in the pelvic floor compartments suggests that high LH levels in postmenopausal women may contribute to the pelvic

  7. Subcellular distribution of molybdenum, ultrastructural and antioxidative responses in soybean seedlings under excess molybdenum stress.

    Science.gov (United States)

    Xu, Shoujun; Hu, Chengxiao; Tan, Qiling; Qin, Shiyu; Sun, Xuecheng

    2017-12-05

    Some studies have shown that excess molybdenum (Mo) could produce toxic effects on plants. However, little is known about the subcellular distribution of Mo and cell ultrastructure within plants under excess Mo stress. Here, we comprehensively analyzed the changes of Mo distribution in subcellular fractions, cell ultrastructure and antioxidant enzymes in leaves and roots of soybean seedlings in response to excess Mo stress. The results showed that roots exhibited higher Mo accumulation than leaves at the 100 mg L-1 Mo level, about 38.58-, 171.48- and 52.99-fold higher in cell walls, cell organelles and soluble fractions, respectively. Subcellular fractionations of Mo-containing tissues indicated that approximately 90% of Mo was accumulated in the soluble fractions and cell walls of the roots and leaves, and soluble fractions (accumulated 66.3-72.2% Mo) might serve as an effective storage site for excess Mo. Furthermore, excess Mo caused ultrastructural alterations in roots and leaves of soybean seedlings, leading to structural abnormality of chloroplast in leaf cells, plasmolysis, cellular deformity, vacuole enlargement and the swelling of cell wall and cytoplasm in root cells. Meanwhile, under excess Mo stress, the activity of POD, CAT and APX enzymes in roots was 1.43, 2.35 and 1.23 times that under standard Mo condition, while that of SOD and CAT enzymes in leaves was 1.23 and 1.94 times, respectively. This study provided novel insights into the mechanisms of excess Mo toxicity in soybean seedlings. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

    Directory of Open Access Journals (Sweden)

    Bo Fu

    2015-12-01

    Full Text Available The in vitro maturation (IVM efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+ oocytes with low glucose-6-phosphate dehydrogenase (G6PDH activity have shown superior quality than BCB negative (− oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9 and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

  9. Proteomics: a subcellular look at spermatozoa

    Directory of Open Access Journals (Sweden)

    Yadav Satya P

    2011-03-01

    Full Text Available Abstract Background Male-factor infertility presents a vexing problem for many reproductively active couples. Many studies have focused on abnormal sperm parameters. Recent advances in proteomic techniques, especially in mass spectrometry, have aided in the study of sperm and more specifically, sperm proteins. The aim of this study was to review the current literature on the various proteomic techniques, and their usefulness in diagnosing sperm dysfunction and potential applications in the clinical setting. Methods Review of PubMed database. Key words: spermatozoa, proteomics, protein, proteome, 2D-PAGE, mass spectrometry. Results Recently employed proteomic methods, such as two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential in gel electrophoresis, have identified numerous sperm-specific proteins. They also have provided a further understanding of protein function involved in sperm processes and for the differentiation between normal and abnormal states. In addition, studies on the sperm proteome have demonstrated the importance of post-translational modifications, and their ability to bring about physiological changes in sperm function. No longer do researchers believe that in order for them to elucidate the biochemical functions of genes, mere knowledge of the human genome sequence is sufficient. Moreover, a greater understanding of the physiological function of every protein in the tissue-specific proteome is essential in order to unravel the biological display of the human genome. Conclusion Recent advances in proteomic techniques have provided insight into sperm function and dysfunction. Several multidimensional separation techniques can be utilized to identify and characterize spermatozoa. Future developments in bioinformatics can further assist researchers in understanding the vast amount of data collected in proteomic studies. Moreover, such advances in proteomics may help to decipher metabolites

  10. Proteomics: a subcellular look at spermatozoa.

    Science.gov (United States)

    du Plessis, Stefan S; Kashou, Anthony H; Benjamin, David J; Yadav, Satya P; Agarwal, Ashok

    2011-03-22

    Male-factor infertility presents a vexing problem for many reproductively active couples. Many studies have focused on abnormal sperm parameters. Recent advances in proteomic techniques, especially in mass spectrometry, have aided in the study of sperm and more specifically, sperm proteins. The aim of this study was to review the current literature on the various proteomic techniques, and their usefulness in diagnosing sperm dysfunction and potential applications in the clinical setting. Review of PubMed database. Key words: spermatozoa, proteomics, protein, proteome, 2D-PAGE, mass spectrometry. Recently employed proteomic methods, such as two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential in gel electrophoresis, have identified numerous sperm-specific proteins. They also have provided a further understanding of protein function involved in sperm processes and for the differentiation between normal and abnormal states. In addition, studies on the sperm proteome have demonstrated the importance of post-translational modifications, and their ability to bring about physiological changes in sperm function. No longer do researchers believe that in order for them to elucidate the biochemical functions of genes, mere knowledge of the human genome sequence is sufficient. Moreover, a greater understanding of the physiological function of every protein in the tissue-specific proteome is essential in order to unravel the biological display of the human genome. Recent advances in proteomic techniques have provided insight into sperm function and dysfunction. Several multidimensional separation techniques can be utilized to identify and characterize spermatozoa. Future developments in bioinformatics can further assist researchers in understanding the vast amount of data collected in proteomic studies. Moreover, such advances in proteomics may help to decipher metabolites which can act as biomarkers in the detection of sperm impairments and

  11. Subcellular targeting of RGS9-2 is controlled by multiple molecular determinants on its membrane anchor, R7BP.

    Science.gov (United States)

    Song, Joseph H; Waataja, Jonathan J; Martemyanov, Kirill A

    2006-06-02

    RGS9-2, a member of the R7 regulators of G protein signaling (RGS) protein family of neuronal RGS, is a critical regulator of G protein signaling. In striatal neurons, RGS9-2 is tightly associated with a novel palmitoylated protein, R7BP (R7 family binding protein). Here we report that R7BP acts to target the localization of RGS9-2 to the plasma membrane. Examination of the subcellular distribution in native striatal neurons revealed that both R7BP and RGS9-2 are almost entirely associated with the neuronal membranes. In addition to the plasma membrane, a large portion of RGS9-2 was found in the neuronal specializations, the postsynaptic densities, where it forms complexes with R7BP and its constitutive partner Gbeta5. Using site-directed mutagenesis we found that the molecular determinants that specify the subcellular targeting of RGS9-2.Gbeta5.R7BP complex are contained within the 21 C-terminal amino acids of R7BP. This function of the C terminus was found to require the synergistic contributions of its two distinct elements, a polybasic motif and palmitoylated cysteines, which when combined are sufficient for directing the intracellular localization of the constituent protein. In differentiated neurons, the C-terminal targeting motif of R7BP was found to be essential for mediating its postsynaptic localization. In addition to the plasma membrane targeting elements, we identified two functional nuclear localization sequences that can mediate the import of R7BP into the nucleus upon depalmitoylation. These findings provide a mechanism for the subcellular targeting of RGS9-2 in neurons.

  12. Triple subcellular targeting of isopentenyl diphosphate isomerases encoded by a single gene.

    Science.gov (United States)

    Guirimand, Grégory; Guihur, Anthony; Phillips, Michael A; Oudin, Audrey; Glévarec, Gaëlle; Mahroug, Samira; Melin, Céline; Papon, Nicolas; Clastre, Marc; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Rodríguez-Concepción, Manuel; Burlat, Vincent; Courdavault, Vincent

    2012-11-01

    Isopentenyl diphosphate isomerase (IDI) is a key enzyme of the isoprenoid pathway, catalyzing the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors of all isoprenoids. In plants, several subcellular compartments, including cytosol/ER, peroxisomes, mitochondria and plastids, are involved in isoprenoid biosynthesis. Here, we report on the unique triple targeting of two Catharanthus roseus IDI isoforms encoded by a single gene (CrIDI1). The triple localization of CrIDI1 in mitochondria, plastids and peroxisomes is explained by alternative transcription initiation of CrIDI1, by the specificity of a bifunctional N-terminal mitochondria/plastid transit peptide and by the presence of a C-terminal peroxisomal targeting signal. Moreover, bimolecular fluorescence complementation assays revealed self-interactions suggesting that the IDI likely acts as a multimer in vivo.

  13. Unified Model for The Study of Diffusion Localization and Dissipation

    CERN Document Server

    Cohen, D

    1997-01-01

    A new model that generalizes the study of quantum Brownian motion (BM) is constructed. We consider disordered environment that may be either static (quenched), noisy or dynamical. The Zwanzig-Caldeira-Leggett BM-model constitutes formally a special case where the disorder auto-correlation length is taken to be infinite. Alternatively, localization problem is obtained if the noise auto-correlation time is taken to be infinite. Also the general case of weak nonlinear coupling to thermal, possibly chaotic bath is handled by the same formalism. A general, Feynman-Vernon type path-integral expression for the propagator is