WorldWideScience

Sample records for similar intracellular localization

  1. Intracellular Localization and Cellular Factors Interaction of HTLV-1 and HTLV-2 Tax Proteins: Similarities and Functional Differences

    Directory of Open Access Journals (Sweden)

    Maria Grazia Romanelli

    2011-05-01

    Full Text Available Human T-lymphotropic viruses type 1 (HTLV-1 and type 2 (HTLV-2 present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity.

  2. Intracellular localization of Arabidopsis sulfurtransferases.

    Science.gov (United States)

    Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D; Papenbrock, Jutta

    2004-06-01

    Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.

  3. Intracellular Localization of Arabidopsis Sulfurtransferases1

    Science.gov (United States)

    Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D.; Papenbrock, Jutta

    2004-01-01

    Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism. PMID:15181206

  4. Intracellular localization of Na + /H + antiporter from Malus zumi ...

    African Journals Online (AJOL)

    In this study, we examined the intracellular localization of the product of Na+/H+ antiporter gene (MzNHX1) cloned from Malus zumi. Analysis using yeast cells expressing a fusion protein of MzNHX1 and green fluorescent protein confirmed the localization of MzNHX1 on the tonoplast.

  5. Bullous pemphigoid antigen localization suggests an intracellular association with hemidesmosomes

    DEFF Research Database (Denmark)

    Westgate, G E; Weaver, A C; Couchman, J R

    1985-01-01

    immunofluorescent staining for BPA is linear at the basement membrane zone (BMZ) of skin and many other epithelial tissues. At higher magnification however, we observed a punctate staining pattern for BPA which was regular in appearance and suggested localization of BPA to discrete structures at the BMZ. Subsequent...... intracellularly both in vivo and in vitro. We suggest that BPA is not normally a lamina lucida component, but that it may form part of a linkage between the cytoskeleton and the basement membrane....

  6. Expanding the boundaries of local similarity analysis.

    Science.gov (United States)

    Durno, W Evan; Hanson, Niels W; Konwar, Kishori M; Hallam, Steven J

    2013-01-01

    Pairwise comparison of time series data for both local and time-lagged relationships is a computationally challenging problem relevant to many fields of inquiry. The Local Similarity Analysis (LSA) statistic identifies the existence of local and lagged relationships, but determining significance through a p-value has been algorithmically cumbersome due to an intensive permutation test, shuffling rows and columns and repeatedly calculating the statistic. Furthermore, this p-value is calculated with the assumption of normality -- a statistical luxury dissociated from most real world datasets. To improve the performance of LSA on big datasets, an asymptotic upper bound on the p-value calculation was derived without the assumption of normality. This change in the bound calculation markedly improved computational speed from O(pm²n) to O(m²n), where p is the number of permutations in a permutation test, m is the number of time series, and n is the length of each time series. The bounding process is implemented as a computationally efficient software package, FASTLSA, written in C and optimized for threading on multi-core computers, improving its practical computation time. We computationally compare our approach to previous implementations of LSA, demonstrate broad applicability by analyzing time series data from public health, microbial ecology, and social media, and visualize resulting networks using the Cytoscape software. The FASTLSA software package expands the boundaries of LSA allowing analysis on datasets with millions of co-varying time series. Mapping metadata onto force-directed graphs derived from FASTLSA allows investigators to view correlated cliques and explore previously unrecognized network relationships. The software is freely available for download at: http://www.cmde.science.ubc.ca/hallam/fastLSA/.

  7. Measuring transferring similarity via local information

    Science.gov (United States)

    Yin, Likang; Deng, Yong

    2018-05-01

    Recommender systems have developed along with the web science, and how to measure the similarity between users is crucial for processing collaborative filtering recommendation. Many efficient models have been proposed (i.g., the Pearson coefficient) to measure the direct correlation. However, the direct correlation measures are greatly affected by the sparsity of dataset. In other words, the direct correlation measures would present an inauthentic similarity if two users have a very few commonly selected objects. Transferring similarity overcomes this drawback by considering their common neighbors (i.e., the intermediates). Yet, the transferring similarity also has its drawback since it can only provide the interval of similarity. To break the limitations, we propose the Belief Transferring Similarity (BTS) model. The contributions of BTS model are: (1) BTS model addresses the issue of the sparsity of dataset by considering the high-order similarity. (2) BTS model transforms uncertain interval to a certain state based on fuzzy systems theory. (3) BTS model is able to combine the transferring similarity of different intermediates using information fusion method. Finally, we compare BTS models with nine different link prediction methods in nine different networks, and we also illustrate the convergence property and efficiency of the BTS model.

  8. Dihydroceramide biology - Structure-specific metabolism and intracellular localization

    NARCIS (Netherlands)

    Kok, JW; NikolovaKarakashian, M; Klappe, K; Alexander, C; Merrill, AH

    1997-01-01

    This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DN-Cer), an intermediate in de novo sphingolipid biosynthesis, When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl-DH-Cer (C-6-NBD-DH-Cer) was incubated with HT29, NRK, BHK,

  9. Conformational determinants of the intracellular localization of midkine

    International Nuclear Information System (INIS)

    Dai Lichen; Xu, Diyong; Yao Xing; Lu Yongliang; Xu Zhengping

    2005-01-01

    Midkine (MK) is a multifunctional growth factor and has been discovered to play important roles in carcinogenesis. MK has been reported to localize to the nucleus and nucleolus, however, the data are not consistent and the signals responsible for the localization are unknown. Here we reported that human MK exclusively localized to the nucleus and nucleolus in HepG2 cells by using GFP as a tracking molecule. In order to identify the motifs required for the nuclear localization and nucleolar accumulation, point- and deletion-mutations were introduced and the corresponding subcellular localizations were analyzed. Data revealed that (i) K79R81, K86K87, and the C-terminal tail of MK constitute the nuclear localization determinant of MK, and (ii) the C-terminal tail is the key element controlling MK nucleolar accumulation though the N-terminal tail, K79R81, and K86K87 also contribute to this process. Taken together, our results provide the first documentation about the determinants required for MK nuclear and nucleolar localization

  10. A method for spatially resolved local intracellular mechanochemical sensing and organelle manipulation

    NARCIS (Netherlands)

    Shekhar, S.; Cambi, A.; Figdor, Carl; Subramaniam, Vinod; Kanger, Johannes S.

    2012-01-01

    Because both the chemical and mechanical properties of living cells play crucial functional roles, there is a strong need for biophysical methods to address these properties simultaneously. Here we present a novel (to our knowledge) approach to measure local intracellular micromechanical and

  11. A method for spatially resolved local intracellular mechanochemical sensing and organelle manipulation.

    NARCIS (Netherlands)

    Shekhar, S.; Cambi, A.; Figdor, C.G.; Subramaniam, V.; Kanger, J.S.

    2012-01-01

    Because both the chemical and mechanical properties of living cells play crucial functional roles, there is a strong need for biophysical methods to address these properties simultaneously. Here we present a novel (to our knowledge) approach to measure local intracellular micromechanical and

  12. A space-efficient algorithm for local similarities.

    Science.gov (United States)

    Huang, X Q; Hardison, R C; Miller, W

    1990-10-01

    Existing dynamic-programming algorithms for identifying similar regions of two sequences require time and space proportional to the product of the sequence lengths. Often this space requirement is more limiting than the time requirement. We describe a dynamic-programming local-similarity algorithm that needs only space proportional to the sum of the sequence lengths. The method can also find repeats within a single long sequence. To illustrate the algorithm's potential, we discuss comparison of a 73,360 nucleotide sequence containing the human beta-like globin gene cluster and a corresponding 44,594 nucleotide sequence for rabbit, a problem well beyond the capabilities of other dynamic-programming software.

  13. Tissue- and Cell-Specific Co-localization of Intracellular Gelatinolytic Activity and Matrix Metalloproteinase 2

    Science.gov (United States)

    Solli, Ann Iren; Fadnes, Bodil; Winberg, Jan-Olof; Uhlin-Hansen, Lars

    2013-01-01

    Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions. PMID:23482328

  14. Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome.

    Directory of Open Access Journals (Sweden)

    Claudia Orelio

    Full Text Available Shwachman-Diamond Syndrome (SDS is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

  15. Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

    Science.gov (United States)

    Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

    2015-01-01

    Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

  16. Studies on the intracellular localization of hHR23B

    International Nuclear Information System (INIS)

    Katiyar, Samiksha; Lennarz, William J.

    2005-01-01

    Yeast Rad23, originally identified as a DNA repair protein, has been proposed to participate in other cellular functions, i.e., the proteasome-degradation pathway, the process of spindle pole body duplication and as a component of the anaphase checkpoint. Two human homologs of yeast Rad23, hHR23A and hHR23B, exhibit high sequence homology with yRad23 and also have been shown to be involved in DNA repair and proteasome-dependent degradation. Previous studies on the intracellular localization of hHR23A and hHR23B revealed their predominant localization in the nucleus during interphase and in the cytoplasm during mitosis. We have analyzed the localization of hHR23B during all the phases of the cell cycle using immunofluorescence. Unlike previous studies, our results suggest localization of hHR23B in the nucleus as well as in the cytoplasm during G1 phase. The nuclear levels of hHR23B decrease during S-phase of the cell cycle. When the cell enters mitosis, hHR23B relocalizes in the cytoplasm without association with chromatin. These results indicate that the intracellular distribution hHR23B is cell cycle dependent

  17. Determination of Six Transmembrane Protein of Prostate 2 Gene Expression and Intracellular Localization in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Bora İrer

    2017-12-01

    Full Text Available Objective: In this study, we aimed to determine the relationship between the RNA and protein expression profile of six transmembrane protein of prostate 2 (STAMP2 gene and androgen and the intracellular localization of STAMP2. Materials and Methods: RNA and protein were obtained from androgen treated lymph node carcinoma of the prostate (LNCaP cells, untreated LNCaP cells, DU145 cells with no androgen receptor, and STAMP2 transfected COS-7 cells. The expression profile of STAMP2 gene and the effect of androgenes on the expression was shown in RNA and protein levels by using Northern and Western blotting methods. In addition, intracellular localization of the naturally synthesized STAMP2 protein and the transfected STAMP2 protein in COS-7 cells after androgen administration in both LNCaP cells was determined by immunofluorescence microscopy. Results: We found that the RNA and protein expression of STAMP2 gene in LNCaP cells are regulated by androgenes, the power of expression is increased with the duration of androgen treatment and there is no STAMP2 expression in DU145 cells which has no androgen receptor. As a result of the immunofluorescence microscopy study we observed that STAMP2 protein was localized at golgi complex and cell membrane. Conclusion: In conclusion, we have demonstrated that STAMP2 may play an important role in the pathogenesis of the prostate cancer and in the androgen-dependent androgen-independent staging of prostate cancer. In addition, STAMP2 protein, which is localized in the intracellular golgi complex and cell membrane, may be a new target molecule for prostate cancer diagnosis and treatment.

  18. Immunomicroscopic localization of aminopeptidase N in the pig enterocyte. Implications for the route of intracellular transport

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Sjöström, H; Norén, Ove

    1987-01-01

    The subcellular localization of aminopeptidase N (EC 3.4.11.2) in the pig enterocyte was investigated by immunofluorescence and immunoelectron microscopy (immunogold staining). By indirect immunofluorescence on either frozen or paraffin-embedded sections, a very intense staining in the microvillar....... Labelling was demonstrated in the Golgi apparatus and in a minor fraction of the intracellular smooth vesicles positioned between the Golgi apparatus and the microvillar membrane. These observations are compatible with the view that newly synthesized aminopeptidase N is delivered directly to the microvillar...

  19. Assimilation as Attraction: Computing Distance, Similarity, and Locality in Phonology

    Science.gov (United States)

    Wayment, Adam

    2009-01-01

    This dissertation explores similarity effects in assimilation, proposing an Attraction Framework to analyze cases of parasitic harmony where a trigger-target pair only results in harmony if the trigger and target agree on other features. Attraction provides a natural model of these effects by relating the pressure for assimilation to the…

  20. Molecular features contributing to virus-independent intracellular localization and dynamic behavior of the herpesvirus transport protein US9.

    Directory of Open Access Journals (Sweden)

    Manuela Pedrazzi

    Full Text Available Reaching the right destination is of vital importance for molecules, proteins, organelles, and cargoes. Thus, intracellular traffic is continuously controlled and regulated by several proteins taking part in the process. Viruses exploit this machinery, and viral proteins regulating intracellular transport have been identified as they represent valuable tools to understand and possibly direct molecules targeting and delivery. Deciphering the molecular features of viral proteins contributing to (or determining this dynamic phenotype can eventually lead to a virus-independent approach to control cellular transport and delivery. From this virus-independent perspective we looked at US9, a virion component of Herpes Simplex Virus involved in anterograde transport of the virus inside neurons of the infected host. As the natural cargo of US9-related vesicles is the virus (or its parts, defining its autonomous, virus-independent role in vesicles transport represents a prerequisite to make US9 a valuable molecular tool to study and possibly direct cellular transport. To assess the extent of this autonomous role in vesicles transport, we analyzed US9 behavior in the absence of viral infection. Based on our studies, Us9 behavior appears similar in different cell types; however, as expected, the data we obtained in neurons best represent the virus-independent properties of US9. In these primary cells, transfected US9 mostly recapitulates the behavior of US9 expressed from the viral genome. Additionally, ablation of two major phosphorylation sites (i.e. Y32Y33 and S34ES36 have no effect on protein incorporation on vesicles and on its localization on both proximal and distal regions of the cells. These results support the idea that, while US9 post-translational modification may be important to regulate cargo loading and, consequently, virion export and delivery, no additional viral functions are required for US9 role in intracellular transport.

  1. Intracellular localization of Treponema denticola chymotrypsin-like proteinase in chronic periodontitis

    Directory of Open Access Journals (Sweden)

    Emilia Marttila

    2014-07-01

    Full Text Available Treponema denticola is an important periodontal pathogen capable of tissue invasion. Its chymotrypsin-like proteinase (CTLP can degrade a number of basement membrane components in vitro, thus suggesting a contribution to tissue invasion by the spirochete. The aim of this study was to analyze the localization of CTLP in chronic periodontitis tissues ex vivo. A polyclonal antibody specific to T. denticola cell-bound CTLP was used to detect the spirochetes in the gingival tissues of patients with moderate to severe chronic periodontitis (n=25 by immunohistochemistry and periodic acid-Schiff staining (PAS. The presence of T. denticola in the periodontal tissue samples was analyzed by PCR. Periodontal tissue samples of 12 of the 25 patients were found to be positive for T. denticola by PCR. Moreover, CTLP could be detected in the periodontal tissues of all these patients by immunohistochemistry. In the epithelium, the CTLP was mostly intracellular. Typically, the positive staining could be seen throughout the whole depth of the epithelium. When detected extracellularly, CTLP was localized mainly as granular deposits. The connective tissue stained diffusely positive in four cases. The positive staining co-localized with the PAS stain in nine cases. T. denticola and its CTLP could be detected in diseased human periodontium both intra- and extracellularly. The granular staining pattern was suggestive of the presence of T. denticola bacteria, whereas the more diffused staining pattern was indicative of the recent presence of the bacterium and shedding of the cell-bound proteinase.

  2. Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins

    Directory of Open Access Journals (Sweden)

    Kahlem Pascal

    2006-06-01

    Full Text Available Abstract Background Trisomy of human chromosome 21 (Chr21 results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. Results We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb to MCM3AP (46.6 Mb, with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. Conclusion The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.

  3. Interspecific variation of intracellular localization and postirradiation movement of Ku70-protein in fibroblastic cells

    International Nuclear Information System (INIS)

    Endoh, Daiji; Hayashi, Masanobu; Okui, Toyo; Kawase, Shiro; Kon, Yasushiro

    2003-01-01

    Ku (Ku70 and Ku80) Proteins are known as components of DNA-dependent protein kinase (DNA-PK) and play an important role for DNA repair. We previously reported that more than 70% of Ku proteins were located in cytoplasm of rat cells, the Ku proteins moved into nuclei of normal rat cells after X-irradiation, Ku proteins also moved into nuclei after X-irradiation but were not retained in nucleus of radiosensitive LEC rat cells. While reports have been shown about mechanisms on nuclear localization of Ku proteins, how Ku proteins export from nucleus is poorly understood. Here we show that C-terminal region of Ku70 protein is important for its cytoplasmic localization. When transfected into LEC rat cells, exogenous intact Ku70 (1-609) tagged with enhanced green fluorescent protein (EGFP-Ku70) localized mainly in the cytoplasm, whereas C-terminal-deletion mutant of Ku70 (1-593) tagged with EGFP (EGFP-Ku70D) was mainly localized in the nucleus. After X-irradiation, the endogenous intact EGFP-Ku70 once moved into nucleus, but returned into the cytoplasm. On the other hand, EGFP-Ku70D was retained in nucleus for two hours after X-irradiation. These results suggest that C-terminal region of Ku70 is included in the postirradiation nuclear export. Next, we investigated the intracellular localization of Ku70 proteins and the movement after X-irradiation of fibroblastic cells prepared from some mammalian species. Ku70 proteins were localized in nucleus and the postirradiation-extranuclear transport was not observed in human and African green monkey cells. On the other hand, Ku70 proteins were mainly localized in cytoplasm and moved into nucleus in mouse, Chinese hamster, Golden hamster, cotton rat, squirrel, cat and dog cells. These results may show that alternatively Ku70 protein is localized in the cytoplasm or nucleus depends on species and translocation of cytoplasmic Ku70 into nucleus is a response against low dose irradiation in fibroblasts of rodents, cats and dogs

  4. USP2 Regulates the Intracellular Localization of PER1 and Circadian Gene Expression

    DEFF Research Database (Denmark)

    Yang, Yaoming; Duguay, David; Fahrenkrug, Jan

    2014-01-01

    . Although Per1 mRNA expression rhythm remained intact in the Usp2 KO MEFs, the expression profiles of other core clock genes were altered. This was also true for the expression of clock-controlled genes (e.g., Dbp, Tef, Hlf, E4bp4). A similar phase advance of PER1 nuclear localization rhythm and alteration...

  5. Intracellular localization and dynamics of Hypericin loaded PLLA nanocarriers by image correlation spectroscopy

    OpenAIRE

    Penjweini, Rozhin; Deville, Sarah; D'Olieslaeger, Lien; Berden, Mandy; Ameloot, Marcel; Ethirajan, Anitha

    2015-01-01

    The study of cell-nanoparticle interactions is an important aspect for understanding drug delivery using nanocarriers. In this regard, advances in fluorescence based microscopy are useful for the investigation of temporal and spatial behavior of nanoparticles (NPs) within the intracellular environment. In this work, we focus on the delivery of the naturally-occurring hydrophobic photosensitizer Hypericin in human lung carcinoma A549 cells by using biodegradable poly L-lactic acid NPs. For the...

  6. Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

    Directory of Open Access Journals (Sweden)

    Muriaux Delphine

    2007-08-01

    Full Text Available Abstract Background The HIV-1 nucleocapsid protein (NC is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT, gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

  7. A new similarity index for nonlinear signal analysis based on local extrema patterns

    Science.gov (United States)

    Niknazar, Hamid; Motie Nasrabadi, Ali; Shamsollahi, Mohammad Bagher

    2018-02-01

    Common similarity measures of time domain signals such as cross-correlation and Symbolic Aggregate approximation (SAX) are not appropriate for nonlinear signal analysis. This is because of the high sensitivity of nonlinear systems to initial points. Therefore, a similarity measure for nonlinear signal analysis must be invariant to initial points and quantify the similarity by considering the main dynamics of signals. The statistical behavior of local extrema (SBLE) method was previously proposed to address this problem. The SBLE similarity index uses quantized amplitudes of local extrema to quantify the dynamical similarity of signals by considering patterns of sequential local extrema. By adding time information of local extrema as well as fuzzifying quantized values, this work proposes a new similarity index for nonlinear and long-term signal analysis, which extends the SBLE method. These new features provide more information about signals and reduce noise sensitivity by fuzzifying them. A number of practical tests were performed to demonstrate the ability of the method in nonlinear signal clustering and classification on synthetic data. In addition, epileptic seizure detection based on electroencephalography (EEG) signal processing was done by the proposed similarity to feature the potentials of the method as a real-world application tool.

  8. Control of local intracellular calcium concentration with dynamic-clamp controlled 2-photon uncaging.

    Directory of Open Access Journals (Sweden)

    Erwin Idoux

    Full Text Available The variations of the intracellular concentration of calcium ion ([Ca(2+](i are at the heart of intracellular signaling, and their imaging is therefore of enormous interest. However, passive [Ca(2+](i imaging provides no control over these variations, meaning that a full exploration of the functional consequences of [Ca(2+](i changes is difficult to attain. The tools designed so far to modify [Ca(2+](i, even qualitatively, suffer drawbacks that undermine their widespread use. Here, we describe an electro-optical technique to quantitatively set [Ca(2+](i, in real time and with sub-cellular resolution, using two-photon Ca(2+ uncaging and dynamic-clamp. We experimentally demonstrate, on neurons from acute olfactory bulb slices of Long Evans rats, various capabilities of this technique previously difficult to achieve, such as the independent control of the membrane potential and [Ca(2+](i variations, the functional knocking-in of user-defined virtual voltage-dependent Ca(2+ channels, and the standardization of [Ca(2+](i patterns across different cells. Our goal is to lay the groundwork for this technique and establish it as a new and versatile tool for the study of cell signaling.

  9. Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells.

    Science.gov (United States)

    Khan, Selina; Bijker, Martijn S; Weterings, Jimmy J; Tanke, Hans J; Adema, Gosse J; van Hall, Thorbald; Drijfhout, Jan W; Melief, Cornelis J M; Overkleeft, Hermen S; van der Marel, Gijsbert A; Filippov, Dmitri V; van der Burg, Sjoerd H; Ossendorp, Ferry

    2007-07-20

    Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen

  10. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    Science.gov (United States)

    Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

    2012-01-01

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  11. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    Energy Technology Data Exchange (ETDEWEB)

    Volland, Stefanie, E-mail: Stefanie.Volland@stud.sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria); Luetz, Cornelius, E-mail: cornelius.luetz@uibk.ac.at [Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck (Austria); Michalke, Bernhard, E-mail: bernhard.michalke@helmholtz-muenchen.de [Helmholtz Zentrum Muenchen, German Research Centre for Environmental Health, Institute of Ecological Chemistry, Ingolstaedter Landstrasse 1, 85764 Neuherberg (Germany); Luetz-Meindl, Ursula, E-mail: ursula.luetz-meindl@sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria)

    2012-03-15

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 {mu}M Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron-oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  12. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    International Nuclear Information System (INIS)

    Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

    2012-01-01

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  13. Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

    International Nuclear Information System (INIS)

    Fujita, Akiko; Sato, Chihiro; Kitajima, Ken.

    2007-01-01

    The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences

  14. Intracellular localization and dynamics of Hypericin loaded PLLA nanocarriers by image correlation spectroscopy.

    Science.gov (United States)

    Penjweini, Rozhin; Deville, Sarah; D'Olieslaeger, Lien; Berden, Mandy; Ameloot, Marcel; Ethirajan, Anitha

    2015-11-28

    The study of cell-nanoparticle interactions is an important aspect for understanding drug delivery using nanocarriers. In this regard, advances in fluorescence based microscopy are useful for the investigation of temporal and spatial behavior of nanoparticles (NPs) within the intracellular environment. In this work, we focus on the delivery of the naturally-occurring hydrophobic photosensitizer Hypericin in human lung carcinoma A549 cells by using biodegradable poly L-lactic acid NPs. For the first time, Hypericin containing NPs are prepared by combining the miniemulsion technique with the solvent evaporation method. This approach yields an efficient loading of the NPs with Hypericin and allows for additional cargo molecules. To monitor the release of Hypercin from the NPs, an additional fluorescent lipophilic dye Coumarin-6 is incorporated in the NPs. Temporal and spatiotemporal image correlation spectroscopy is used to determine the fate of the NPs carrying the potential cargo. Both directed and non-directed motions are detected. By using image cross-correlation spectroscopy and specific fluorescent labeling of endosomes, lysosomes and mitochondria, the dynamics of the cargo loaded NPs in association with the organelles is studied. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Quantifying Differences and Similarities in Whole-Brain White Matter Architecture Using Local Connectome Fingerprints.

    Directory of Open Access Journals (Sweden)

    Fang-Cheng Yeh

    2016-11-01

    Full Text Available Quantifying differences or similarities in connectomes has been a challenge due to the immense complexity of global brain networks. Here we introduce a noninvasive method that uses diffusion MRI to characterize whole-brain white matter architecture as a single local connectome fingerprint that allows for a direct comparison between structural connectomes. In four independently acquired data sets with repeated scans (total N = 213, we show that the local connectome fingerprint is highly specific to an individual, allowing for an accurate self-versus-others classification that achieved 100% accuracy across 17,398 identification tests. The estimated classification error was approximately one thousand times smaller than fingerprints derived from diffusivity-based measures or region-to-region connectivity patterns for repeat scans acquired within 3 months. The local connectome fingerprint also revealed neuroplasticity within an individual reflected as a decreasing trend in self-similarity across time, whereas this change was not observed in the diffusivity measures. Moreover, the local connectome fingerprint can be used as a phenotypic marker, revealing 12.51% similarity between monozygotic twins, 5.14% between dizygotic twins, and 4.51% between none-twin siblings, relative to differences between unrelated subjects. This novel approach opens a new door for probing the influence of pathological, genetic, social, or environmental factors on the unique configuration of the human connectome.

  16. 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.

  17. Dependence of the Ce(iii)/Ce(iv) ratio on intracellular localization in ceria nanoparticles internalized by human cells

    KAUST Repository

    Ferraro, Daniela; Tredici, Ilenia G.; Ghigna, Paolo; Castillio-Michel, Hiram; Falqui, Andrea; Di Benedetto, Cristiano; Alberti, Giancarla; Ricci, Vittorio; Anselmi-Tamburini, Umberto; Sommi, Patrizia

    2017-01-01

    CeO2 nanoparticles (CNPs) have been investigated as promising antioxidant agents with significant activity in the therapy of diseases involving free radicals or oxidative stress. However, the exact mechanism responsible for CNP activity has not been completely elucidated. In particular, in situ evidence of modification of the oxidative state of CNPs in human cells and their evolution during cell internalization and subsequent intracellular distribution has never been presented. In this study we investigated modification of the Ce(iii)/Ce(iv) ratio following internalization in human cells by X-ray absorption near edge spectroscopy (XANES). From this analysis on cell pellets, we observed that CNPs incubated for 24 h showed a significant increase in Ce(iii). By coupling on individual cells synchrotron micro-X-ray fluorescence (μXRF) with micro-XANES (μXANES) we demonstrated that the Ce(iii)/Ce(iv) ratio is also dependent on CNP intracellular localization. The regions with the highest CNP concentrations, suggested to be endolysosomes by transmission electron microscopy, were characterized by Ce atoms in the Ce(iv) oxidation state, while a higher Ce(iii) content was observed in regions surrounding these areas. These observations suggest that the interaction of CNPs with cells involves a complex mechanism in which different cellular areas play different roles.

  18. Dependence of the Ce(iii)/Ce(iv) ratio on intracellular localization in ceria nanoparticles internalized by human cells

    KAUST Repository

    Ferraro, Daniela

    2017-01-09

    CeO2 nanoparticles (CNPs) have been investigated as promising antioxidant agents with significant activity in the therapy of diseases involving free radicals or oxidative stress. However, the exact mechanism responsible for CNP activity has not been completely elucidated. In particular, in situ evidence of modification of the oxidative state of CNPs in human cells and their evolution during cell internalization and subsequent intracellular distribution has never been presented. In this study we investigated modification of the Ce(iii)/Ce(iv) ratio following internalization in human cells by X-ray absorption near edge spectroscopy (XANES). From this analysis on cell pellets, we observed that CNPs incubated for 24 h showed a significant increase in Ce(iii). By coupling on individual cells synchrotron micro-X-ray fluorescence (μXRF) with micro-XANES (μXANES) we demonstrated that the Ce(iii)/Ce(iv) ratio is also dependent on CNP intracellular localization. The regions with the highest CNP concentrations, suggested to be endolysosomes by transmission electron microscopy, were characterized by Ce atoms in the Ce(iv) oxidation state, while a higher Ce(iii) content was observed in regions surrounding these areas. These observations suggest that the interaction of CNPs with cells involves a complex mechanism in which different cellular areas play different roles.

  19. Local-global alignment for finding 3D similarities in protein structures

    Science.gov (United States)

    Zemla, Adam T [Brentwood, CA

    2011-09-20

    A method of finding 3D similarities in protein structures of a first molecule and a second molecule. The method comprises providing preselected information regarding the first molecule and the second molecule. Comparing the first molecule and the second molecule using Longest Continuous Segments (LCS) analysis. Comparing the first molecule and the second molecule using Global Distance Test (GDT) analysis. Comparing the first molecule and the second molecule using Local Global Alignment Scoring function (LGA_S) analysis. Verifying constructed alignment and repeating the steps to find the regions of 3D similarities in protein structures.

  20. Intracellular localization of pregnane X receptor in HepG2 cells cultured by the hanging drop method.

    Science.gov (United States)

    Yokobori, Kosuke; Kobayashi, Kaoru; Azuma, Ikuko; Akita, Hidetaka; Chiba, Kan

    2017-10-01

    Pregnane X receptor (PXR) is localized in the cytoplasm of liver cells, whereas it is localized in the nucleus of monolayer-cultured HepG2 cells. Since cultured cells are affected by the microenvironment in which they are grown, we studied the effect of three-dimensional (3D) culture on the localization of PXR in HepG2 cells using the hanging drop method. The results showed that PXR was retained in the cytoplasm of HepG2 cells and other human hepatocarcinoma cell lines (FLC5, FLC7 and Huh7) when they were cultured by the hanging drop method. Treatment with rifampicin, a ligand of PXR, translocated PXR from the cytoplasm to nucleus and increased expression levels of CYP3A4 mRNA in HepG2 cells cultured by the hanging drop method. These findings suggest that 3D culture is a key factor determining the intracellular localization of PXR in human hepatocarcinoma cells and that PXR that becomes retained in the cytoplasm of HepG2 cells with 3D culture has functions of nuclear translocation and regulation of target genes in response to human PXR ligands. Three-dimensionally cultured hepatocarcinoma cells would be a useful tool to evaluate induction potency of drug candidates and also to study mechanisms of nuclear translocation of PXR by human PXR ligands. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  1. Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

    Energy Technology Data Exchange (ETDEWEB)

    Siyam, Arwa [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); Wang, Suzhen; Qin, Chunlin; Mues, Gabriele [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Stevens, Roy [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); D' Souza, Rena N. [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Lu, Yongbo, E-mail: ylu@bcd.tamhsc.edu [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

  2. Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

    International Nuclear Information System (INIS)

    Siyam, Arwa; Wang, Suzhen; Qin, Chunlin; Mues, Gabriele; Stevens, Roy; D’Souza, Rena N.; Lu, Yongbo

    2012-01-01

    Highlights: ► Nuclear localization of DMP1 in various cell lines. ► Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. ► Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

  3. Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2012-01-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

  4. Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2012-04-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. Copyright © 2011 Wiley Periodicals, Inc.

  5. The Intracellular Localization of the Vanillin Biosynthetic Machinery in Pods of Vanilla planifolia.

    Science.gov (United States)

    Gallage, Nethaji J; Jørgensen, Kirsten; Janfelt, Christian; Nielsen, Agnieszka J Z; Naake, Thomas; Dunski, Eryk; Dalsten, Lene; Grisoni, Michel; Møller, Birger Lindberg

    2018-02-01

    Vanillin is the most important flavor compound in the vanilla pod. Vanilla planifolia vanillin synthase (VpVAN) catalyzes the conversion of ferulic acid and ferulic acid glucoside into vanillin and vanillin glucoside, respectively. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of vanilla pod sections demonstrates that vanillin glucoside is preferentially localized within the mesocarp and placental laminae whereas vanillin is preferentially localized within the mesocarp. VpVAN is present as the mature form (25 kDa) but, depending on the tissue and isolation procedure, small amounts of the immature unprocessed form (40 kDa) and putative oligomers (50, 75 and 100 kDa) may be observed by immunoblotting using an antibody specific to the C-terminal sequence of VpVAN. The VpVAN protein is localized within chloroplasts and re-differentiated chloroplasts termed phenyloplasts, as monitored during the process of pod development. Isolated chloroplasts were shown to convert [14C]phenylalanine and [14C]cinnamic acid into [14C]vanillin glucoside, indicating that the entire vanillin de novo biosynthetic machinery converting phenylalanine to vanillin glucoside is present in the chloroplast.

  6. Global similarity and local divergence in human and mouse gene co-expression networks

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2006-09-01

    Full Text Available Abstract Background A genome-wide comparative analysis of human and mouse gene expression patterns was performed in order to evaluate the evolutionary divergence of mammalian gene expression. Tissue-specific expression profiles were analyzed for 9,105 human-mouse orthologous gene pairs across 28 tissues. Expression profiles were resolved into species-specific coexpression networks, and the topological properties of the networks were compared between species. Results At the global level, the topological properties of the human and mouse gene coexpression networks are, essentially, identical. For instance, both networks have topologies with small-world and scale-free properties as well as closely similar average node degrees, clustering coefficients, and path lengths. However, the human and mouse coexpression networks are highly divergent at the local level: only a small fraction ( Conclusion The dissonance between global versus local network divergence suggests that the interspecies similarity of the global network properties is of limited biological significance, at best, and that the biologically relevant aspects of the architectures of gene coexpression are specific and particular, rather than universal. Nevertheless, there is substantial evolutionary conservation of the local network structure which is compatible with the notion that gene coexpression networks are subject to purifying selection.

  7. MAPPING THE SIMILARITIES OF SPECTRA: GLOBAL AND LOCALLY-BIASED APPROACHES TO SDSS GALAXIES

    Energy Technology Data Exchange (ETDEWEB)

    Lawlor, David [Statistical and Applied Mathematical Sciences Institute (United States); Budavári, Tamás [Dept. of Applied Mathematics and Statistics, The Johns Hopkins University (United States); Mahoney, Michael W. [International Computer Science Institute (United States)

    2016-12-10

    We present a novel approach to studying the diversity of galaxies. It is based on a novel spectral graph technique, that of locally-biased semi-supervised eigenvectors . Our method introduces new coordinates that summarize an entire spectrum, similar to but going well beyond the widely used Principal Component Analysis (PCA). Unlike PCA, however, this technique does not assume that the Euclidean distance between galaxy spectra is a good global measure of similarity. Instead, we relax that condition to only the most similar spectra, and we show that doing so yields more reliable results for many astronomical questions of interest. The global variant of our approach can identify very finely numerous astronomical phenomena of interest. The locally-biased variants of our basic approach enable us to explore subtle trends around a set of chosen objects. The power of the method is demonstrated in the Sloan Digital Sky Survey Main Galaxy Sample, by illustrating that the derived spectral coordinates carry an unprecedented amount of information.

  8. Mapping the Similarities of Spectra: Global and Locally-biased Approaches to SDSS Galaxies

    Science.gov (United States)

    Lawlor, David; Budavári, Tamás; Mahoney, Michael W.

    2016-12-01

    We present a novel approach to studying the diversity of galaxies. It is based on a novel spectral graph technique, that of locally-biased semi-supervised eigenvectors. Our method introduces new coordinates that summarize an entire spectrum, similar to but going well beyond the widely used Principal Component Analysis (PCA). Unlike PCA, however, this technique does not assume that the Euclidean distance between galaxy spectra is a good global measure of similarity. Instead, we relax that condition to only the most similar spectra, and we show that doing so yields more reliable results for many astronomical questions of interest. The global variant of our approach can identify very finely numerous astronomical phenomena of interest. The locally-biased variants of our basic approach enable us to explore subtle trends around a set of chosen objects. The power of the method is demonstrated in the Sloan Digital Sky Survey Main Galaxy Sample, by illustrating that the derived spectral coordinates carry an unprecedented amount of information.

  9. MAPPING THE SIMILARITIES OF SPECTRA: GLOBAL AND LOCALLY-BIASED APPROACHES TO SDSS GALAXIES

    International Nuclear Information System (INIS)

    Lawlor, David; Budavári, Tamás; Mahoney, Michael W.

    2016-01-01

    We present a novel approach to studying the diversity of galaxies. It is based on a novel spectral graph technique, that of locally-biased semi-supervised eigenvectors . Our method introduces new coordinates that summarize an entire spectrum, similar to but going well beyond the widely used Principal Component Analysis (PCA). Unlike PCA, however, this technique does not assume that the Euclidean distance between galaxy spectra is a good global measure of similarity. Instead, we relax that condition to only the most similar spectra, and we show that doing so yields more reliable results for many astronomical questions of interest. The global variant of our approach can identify very finely numerous astronomical phenomena of interest. The locally-biased variants of our basic approach enable us to explore subtle trends around a set of chosen objects. The power of the method is demonstrated in the Sloan Digital Sky Survey Main Galaxy Sample, by illustrating that the derived spectral coordinates carry an unprecedented amount of information.

  10. A Framework for Similarity Search with Space-Time Tradeoffs using Locality Sensitive Filtering

    DEFF Research Database (Denmark)

    Christiani, Tobias Lybecker

    2017-01-01

    that satisfies certain locality-sensitivity properties, we can construct a dynamic data structure that solves the approximate near neighbor problem in $d$-dimensional space with query time $dn^{\\rho_q + o(1)}$, update time $dn^{\\rho_u + o(1)}$, and space usage $dn + n^{1 + \\rho_u + o(1)}$ where $n$ denotes......We present a framework for similarity search based on Locality-Sensitive Filtering~(LSF),generalizing the Indyk-Motwani (STOC 1998) Locality-Sensitive Hashing~(LSH) framework to support space-time tradeoffs. Given a family of filters, defined as a distribution over pairs of subsets of space...... the number of points in the data structure.The space-time tradeoff is tied to the tradeoff between query time and update time (insertions/deletions), controlled by the exponents $\\rho_q, \\rho_u$ that are determined by the filter family. \\\\ Locality-sensitive filtering was introduced by Becker et al. (SODA...

  11. Pairwise local structural alignment of RNA sequences with sequence similarity less than 40%

    DEFF Research Database (Denmark)

    Havgaard, Jakob Hull; Lyngsø, Rune B.; Stormo, Gary D.

    2005-01-01

    detect two genes with low sequence similarity, where the genes are part of a larger genomic region. Results: Here we present such an approach for pairwise local alignment which is based on FILDALIGN and the Sankoff algorithm for simultaneous structural alignment of multiple sequences. We include...... the ability to conduct mutual scans of two sequences of arbitrary length while searching for common local structural motifs of some maximum length. This drastically reduces the complexity of the algorithm. The scoring scheme includes structural parameters corresponding to those available for free energy....... The structure prediction performance for a family is typically around 0.7 using Matthews correlation coefficient. In case (2), the algorithm is successful at locating RNA families with an average sensitivity of 0.8 and a positive predictive value of 0.9 using a BLAST-like hit selection scheme. Availability...

  12. Intracellular localization of phosphatidylcholine and phosphatidylethanolamine synthesis in cotyledons of cotton seedlings

    International Nuclear Information System (INIS)

    Chapman, K.D.; Trelease, R.N.

    1991-01-01

    Subfractionation of clarified cotyledon homogenates of cotton (Gossypium hirsutum L.) seedlings on sucrose gradients revealed a single coincident peak of cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) activities, which equilibrated with the main peak of Anti-mycin A-insensitive NADH: cytochrome c reductase (CCR) activity. The small percentage of CPT and EPT activities in glyoxysome-enriched pellets equilibrated with cytochrome c oxidase activity, not with catalase activity. Preincubation of microsomes in 0.2 millimolar MgCl 2 followed by subfractionation on sucrose gradients resulted in peak CPT and EPT activities equilibrating with peak CCR activity at 24% (w/w) sucrose. Preincubation of microsomes with 14 C-CCP choline (or 14 C-CDPethanolamine) resulted in synthesis and incorporation of 14 C-phosphatidylcholine (PC) (or 14 C-phosphatidylethanolamine, PE) into membranes at the same density. Increasing the Mg 2+ concentration to 2.0 millimolar facilitated binding of ribosomes and caused a concomitant shift in density (to 34% w/w sucrose) of peak CPT, EPT, and CCR activities. under these conditions, newly synthesized and incorporated 14 C-PC (or PE) was recovered in these membranes. These results indicate that Er in cotyledons of germinated cotton seedlings is the primary subcellular site of PC and PE synthesis. This is similar to the situation in endosperm tissue but distinctly different from root and hypocotyl tissue where Golgi are a major subcellular site of PC and PE synthesis

  13. Essential role of flotillin-1 palmitoylation in the intracellular localization and signaling function of IGF-1 receptor.

    Science.gov (United States)

    Jang, Donghwan; Kwon, Hayeong; Jeong, Kyuho; Lee, Jaewoong; Pak, Yunbae

    2015-06-01

    Here, we explored flotillin-1-mediated regulation of insulin-like growth factor-1 (IGF-1) signaling. Flotillin-1-deficient cells exhibited a reduction in the activation of IGF-1 receptor (IGF-1R), ERK1/2 and Akt pathways, and the transcriptional activation of Elk-1 and the proliferation in response to IGF-1 were reduced in these cells. We found that IGF-1-independent flotillin-1 palmitoylation at Cys34 in the endoplasmic reticulum (ER) was required for the ER exit and the plasma membrane localization of flotillin-1 and IGF-1R. IGF-1-dependent depalmitoylation and repalmitoylation of flotillin-1 sustained tyrosine kinase activation of the plasma-membrane-targeted IGF-1R. Dysfunction and blocking the turnover of flotillin-1 palmitoylation abrogated cancer cell proliferation after IGF-1R signaling activation. Our data show that flotillin-1 palmitoylation is a new mechanism by which the intracellular localization and activation of IGF-1R are controlled. © 2015. Published by The Company of Biologists Ltd.

  14. Construction of patient specific atlases from locally most similar anatomical pieces

    Science.gov (United States)

    Ramus, Liliane; Commowick, Olivier; Malandain, Grégoire

    2010-01-01

    Radiotherapy planning requires accurate delineations of the critical structures. To avoid manual contouring, atlas-based segmentation can be used to get automatic delineations. However, the results strongly depend on the chosen atlas, especially for the head and neck region where the anatomical variability is high. To address this problem, atlases adapted to the patient’s anatomy may allow for a better registration, and already showed an improvement in segmentation accuracy. However, building such atlases requires the definition of a criterion to select among a database the images that are the most similar to the patient. Moreover, the inter-expert variability of manual contouring may be high, and therefore bias the segmentation if selecting only one image for each region. To tackle these issues, we present an original method to design a piecewise most similar atlas. Given a query image, we propose an efficient criterion to select for each anatomical region the K most similar images among a database by considering local volume variations possibly induced by the tumor. Then, we present a new approach to combine the K images selected for each region into a piecewise most similar template. Our results obtained with 105 CT images of the head and neck show that our method reduces the over-segmentation seen with an average atlas while being robust to inter-expert manual segmentation variability. PMID:20879395

  15. EKF-GPR-Based Fingerprint Renovation for Subset-Based Indoor Localization with Adjusted Cosine Similarity.

    Science.gov (United States)

    Yang, Junhua; Li, Yong; Cheng, Wei; Liu, Yang; Liu, Chenxi

    2018-01-22

    Received Signal Strength Indicator (RSSI) localization using fingerprint has become a prevailing approach for indoor localization. However, the fingerprint-collecting work is repetitive and time-consuming. After the original fingerprint radio map is built, it is laborious to upgrade the radio map. In this paper, we describe a Fingerprint Renovation System (FRS) based on crowdsourcing, which avoids the use of manual labour to obtain the up-to-date fingerprint status. Extended Kalman Filter (EKF) and Gaussian Process Regression (GPR) in FRS are combined to calculate the current state based on the original fingerprinting radio map. In this system, a method of subset acquisition also makes an immediate impression to reduce the huge computation caused by too many reference points (RPs). Meanwhile, adjusted cosine similarity (ACS) is employed in the online phase to solve the issue of outliers produced by cosine similarity. Both experiments and analytical simulation in a real Wireless Fidelity (Wi-Fi) environment indicate the usefulness of our system to significant performance improvements. The results show that FRS improves the accuracy by 19.6% in the surveyed area compared to the radio map un-renovated. Moreover, the proposed subset algorithm can bring less computation.

  16. EKF–GPR-Based Fingerprint Renovation for Subset-Based Indoor Localization with Adjusted Cosine Similarity

    Science.gov (United States)

    Yang, Junhua; Li, Yong; Cheng, Wei; Liu, Yang; Liu, Chenxi

    2018-01-01

    Received Signal Strength Indicator (RSSI) localization using fingerprint has become a prevailing approach for indoor localization. However, the fingerprint-collecting work is repetitive and time-consuming. After the original fingerprint radio map is built, it is laborious to upgrade the radio map. In this paper, we describe a Fingerprint Renovation System (FRS) based on crowdsourcing, which avoids the use of manual labour to obtain the up-to-date fingerprint status. Extended Kalman Filter (EKF) and Gaussian Process Regression (GPR) in FRS are combined to calculate the current state based on the original fingerprinting radio map. In this system, a method of subset acquisition also makes an immediate impression to reduce the huge computation caused by too many reference points (RPs). Meanwhile, adjusted cosine similarity (ACS) is employed in the online phase to solve the issue of outliers produced by cosine similarity. Both experiments and analytical simulation in a real Wireless Fidelity (Wi-Fi) environment indicate the usefulness of our system to significant performance improvements. The results show that FRS improves the accuracy by 19.6% in the surveyed area compared to the radio map un-renovated. Moreover, the proposed subset algorithm can bring less computation. PMID:29361805

  17. Local self-similarity descriptor for point-of-interest reconstruction of real-world scenes

    International Nuclear Information System (INIS)

    Gao, Xianglu; Wan, Weibing; Zhao, Qunfei; Zhang, Xianmin

    2015-01-01

    Scene reconstruction is utilized commonly in close-range photogrammetry, with diverse applications in fields such as industry, biology, and aerospace industries. Presented surfaces or wireframe three-dimensional (3D) model reconstruction applications are either too complex or too inflexible to accommodate various types of real-world scenes, however. This paper proposes an algorithm for acquiring point-of-interest (referred to throughout the study as POI) coordinates in 3D space, based on multi-view geometry and a local self-similarity descriptor. After reconstructing several POIs specified by a user, a concise and flexible target object measurement method, which obtains the distance between POIs, is described in detail. The proposed technique is able to measure targets with high accuracy even in the presence of obstacles and non-Lambertian surfaces. The method is so flexible that target objects can be measured with a handheld digital camera. Experimental results further demonstrate the effectiveness of the algorithm. (paper)

  18. Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking.

    Science.gov (United States)

    Budayeva, Hanna G; Cristea, Ileana M

    2016-10-01

    Human sirtuin 2 (SIRT2) is an NAD + -dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a

  19. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

    Directory of Open Access Journals (Sweden)

    Violeta Calle-Guisado

    2017-01-01

    Full Text Available AMP-activated kinase (AMPK, a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work′s aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS in the presence or absence of the AMPK inhibitor compound C (CC. AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

  20. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

    Science.gov (United States)

    Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

    2017-01-01

    AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%–80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied. PMID:27678462

  1. In vivo experimental stroke and in vitro organ culture induce similar changes in vasoconstrictor receptors and intracellular calcium handling in rat cerebral arteries

    DEFF Research Database (Denmark)

    Povlsen, Gro Klitgaard; Waldsee, Roya; Ahnstedt, Hilda

    2012-01-01

    Cerebral arteries subjected to different types of experimental stroke upregulate their expression of certain G-protein-coupled vasoconstrictor receptors, a phenomenon that worsens the ischemic brain damage. Upregulation of contractile endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B......)) receptors has been demonstrated after subarachnoid hemorrhage and global ischemic stroke, but the situation is less clear after focal ischemic stroke. Changes in smooth muscle calcium handling have been implicated in different vascular diseases but have not hitherto been investigated in cerebral arteries...... and extracellular sources, whereas 5-HT(1B) receptor-mediated contraction was solely dependent on extracellular calcium. Organ culture and stroke increased basal intracellular calcium levels in MCA smooth muscle cells and decreased the expression of inositol triphosphate receptor and transient receptor potential...

  2. Scaling Relations of Local Magnitude versus Moment Magnitude for Sequences of Similar Earthquakes in Switzerland

    KAUST Repository

    Bethmann, F.

    2011-03-22

    Theoretical considerations and empirical regressions show that, in the magnitude range between 3 and 5, local magnitude, ML, and moment magnitude, Mw, scale 1:1. Previous studies suggest that for smaller magnitudes this 1:1 scaling breaks down. However, the scatter between ML and Mw at small magnitudes is usually large and the resulting scaling relations are therefore uncertain. In an attempt to reduce these uncertainties, we first analyze the ML versus Mw relation based on 195 events, induced by the stimulation of a geothermal reservoir below the city of Basel, Switzerland. Values of ML range from 0.7 to 3.4. From these data we derive a scaling of ML ~ 1:5Mw over the given magnitude range. We then compare peak Wood-Anderson amplitudes to the low-frequency plateau of the displacement spectra for six sequences of similar earthquakes in Switzerland in the range of 0:5 ≤ ML ≤ 4:1. Because effects due to the radiation pattern and to the propagation path between source and receiver are nearly identical at a particular station for all events in a given sequence, the scatter in the data is substantially reduced. Again we obtain a scaling equivalent to ML ~ 1:5Mw. Based on simulations using synthetic source time functions for different magnitudes and Q values estimated from spectral ratios between downhole and surface recordings, we conclude that the observed scaling can be explained by attenuation and scattering along the path. Other effects that could explain the observed magnitude scaling, such as a possible systematic increase of stress drop or rupture velocity with moment magnitude, are masked by attenuation along the path.

  3. Relations between perceptual and conceptual scope: how global versus local processing fits a focus on similarity versus dissimilarity

    NARCIS (Netherlands)

    Förster, J.

    2009-01-01

    Nine studies showed a bidirectional link (a) between a global processing style and generation of similarities and (b) between a local processing style and generation of dissimilarities. In Experiments 1-4, participants were primed with global versus local perception styles and then asked to work on

  4. Scaling Relations of Local Magnitude versus Moment Magnitude for Sequences of Similar Earthquakes in Switzerland

    KAUST Repository

    Bethmann, F.; Deichmann, N.; Mai, Paul Martin

    2011-01-01

    Theoretical considerations and empirical regressions show that, in the magnitude range between 3 and 5, local magnitude, ML, and moment magnitude, Mw, scale 1:1. Previous studies suggest that for smaller magnitudes this 1:1 scaling breaks down

  5. Similarity-transformed perturbation theory on top of truncated local coupled cluster solutions: Theory and applications to intermolecular interactions

    Energy Technology Data Exchange (ETDEWEB)

    Azar, Richard Julian, E-mail: julianazar2323@berkeley.edu; Head-Gordon, Martin, E-mail: mhg@cchem.berkeley.edu [Kenneth S. Pitzer Center for Theoretical Chemistry, Department of Chemistry, University of California and Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (United States)

    2015-05-28

    Your correspondents develop and apply fully nonorthogonal, local-reference perturbation theories describing non-covalent interactions. Our formulations are based on a Löwdin partitioning of the similarity-transformed Hamiltonian into a zeroth-order intramonomer piece (taking local CCSD solutions as its zeroth-order eigenfunction) plus a first-order piece coupling the fragments. If considerations are limited to a single molecule, the proposed intermolecular similarity-transformed perturbation theory represents a frozen-orbital variant of the “(2)”-type theories shown to be competitive with CCSD(T) and of similar cost if all terms are retained. Different restrictions on the zeroth- and first-order amplitudes are explored in the context of large-computation tractability and elucidation of non-local effects in the space of singles and doubles. To accurately approximate CCSD intermolecular interaction energies, a quadratically growing number of variables must be included at zeroth-order.

  6. Local non-similarity method through the Crocco's transformation in boundary layer problem

    International Nuclear Information System (INIS)

    Jardim, R.G.M.

    1981-04-01

    The coordinate transformation developed by L. Crocco to obtain the solution of the compressible fluid flows over isotermal flat plates is originally employed in the present work, with the purpose of adding its inherent advantage to the Non-Similarity Method idealized by E.M. Sparrow, in the solution of the incompressible non-similar boundary layers. The Crocco's transformation is applied to the conservation equation for forced convection, laminar, constant properties and two-dimensional flows over solids. Two non-similar problems arisen from freestream velocity distribution, the cylinder in crossflow and the Howarth's retarded flow, are solved with a view to illustrating the new procedure. In those solutions the effect of frictional heat is also considered. The results of hydrodynamic and thermal problems are compared with available published information and good agreement was observed. (Author) [pt

  7. Chemical similarity and local community assembly in the species rich tropical genus Piper.

    Science.gov (United States)

    Salazar, Diego; Jaramillo, M Alejandra; Marquis, Robert J

    2016-11-01

    Community ecologists have strived to find mechanisms that mediate the assembly of natural communities. Recent evidence suggests that natural enemies could play an important role in the assembly of hyper-diverse tropical plant systems. Classic ecological theory predicts that in order for coexistence to occur, species differences must be maximized across biologically important niche dimensions. For plant-herbivore interactions, it has been recently suggested that, within a particular community, plant species that maximize the difference in chemical defense profiles compared to neighboring taxa will have a relative competitive advantage. Here we tested the hypothesis that plant chemical diversity can affect local community composition in the hyper-diverse genus Piper at a lowland wet forest location in Costa Rica. We first characterized the chemical composition of 27 of the most locally abundant species of Piper. We then tested whether species with different chemical compositions were more likely to coexist. Finally, we assessed the degree to which Piper phylogenetic relationships are related to differences in secondary chemical composition and community assembly. We found that, on average, co-occurring species were more likely to differ in chemical composition than expected by chance. Contrary to expectations, there was no phylogenetic signal for overall secondary chemical composition. In addition we found that species in local communities were, on average, more phylogenetically closely related than expected by chance, suggesting that functional traits other than those measured here also influence local assembly. We propose that selection by herbivores for divergent chemistries between closely related species facilitates the coexistence of a high diversity of congeneric taxa via apparent competition. © 2016 by the Ecological Society of America.

  8. Image-based metal artifact reduction in x-ray computed tomography utilizing local anatomical similarity

    Science.gov (United States)

    Dong, Xue; Yang, Xiaofeng; Rosenfield, Jonathan; Elder, Eric; Dhabaan, Anees

    2017-03-01

    X-ray computed tomography (CT) is widely used in radiation therapy treatment planning in recent years. However, metal implants such as dental fillings and hip prostheses can cause severe bright and dark streaking artifacts in reconstructed CT images. These artifacts decrease image contrast and degrade HU accuracy, leading to inaccuracies in target delineation and dose calculation. In this work, a metal artifact reduction method is proposed based on the intrinsic anatomical similarity between neighboring CT slices. Neighboring CT slices from the same patient exhibit similar anatomical features. Exploiting this anatomical similarity, a gamma map is calculated as a weighted summation of relative HU error and distance error for each pixel in an artifact-corrupted CT image relative to a neighboring, artifactfree image. The minimum value in the gamma map for each pixel is used to identify an appropriate pixel from the artifact-free CT slice to replace the corresponding artifact-corrupted pixel. With the proposed method, the mean CT HU error was reduced from 360 HU and 460 HU to 24 HU and 34 HU on head and pelvis CT images, respectively. Dose calculation accuracy also improved, as the dose difference was reduced from greater than 20% to less than 4%. Using 3%/3mm criteria, the gamma analysis failure rate was reduced from 23.25% to 0.02%. An image-based metal artifact reduction method is proposed that replaces corrupted image pixels with pixels from neighboring CT slices free of metal artifacts. This method is shown to be capable of suppressing streaking artifacts, thereby improving HU and dose calculation accuracy.

  9. Similar local and landscape processes affect both a common and a rare newt species.

    Science.gov (United States)

    Denoël, Mathieu; Perez, Amélie; Cornet, Yves; Ficetola, Gentile Francesco

    2013-01-01

    Although rare species are often the focus of conservation measures, more common species may experience similar decline and suffer from the same threatening processes. We tested this hypothesis by examining, through an information-theoretic approach, the importance of ecological processes at multiple scales in the great crested newt Triturus cristatus, regionally endangered and protected in Europe, and the more common smooth newt, Lissotriton vulgaris. Both species were similarly affected by the same processes, i.e. suitability of aquatic and terrestrial components of their habitat at different scales, connectivity among breeding sites, and the presence of introduced fish. T. cristatus depended more on water depth and aquatic vegetation than L. vulgaris. The results show that environmental pressures threaten both common and rare species, and therefore the more widespread species should not be neglected in conservation programs. Because environmental trends are leading to a deterioration of aquatic and terrestrial habitat features required by newt populations, populations of the common species may follow the fate of the rarest species. This could have substantial conservation implications because of the numerical importance of common species in ecosystems and because commonness could be a transient state moving towards rarity. On the other hand, in agreement with the umbrella species concept, targeting conservation efforts on the most demanding species would also protect part of the populations of the most common species.

  10. Generalized method for sorting Shack-Hartmann spot patterns using local similarity

    International Nuclear Information System (INIS)

    Smith, Daniel G.; Greivenkamp, John E.

    2008-01-01

    The sensitivity and dynamic range of a Shack-Hartmann wavefront sensor is enhanced when the spots produced by the lenslet array are allowed to shift more than one lenslet radius from their on-axis positions. However, this presents the problem of accurately and robustly associating the spots with their respective subapertures. This paper describes a method for sorting spots that takes advantage of the local spot position distortions to unwrap the spot pattern. The described algorithm is both simple and robust and also applicable to any lenslet array geometry that can be described as a two-dimensional lattice, including hexagonal arrays, which are shown here to be more efficient than square arrays

  11. Glycogen synthase kinase 3-{beta} phosphorylates novel S/T-P-S/T domains in Notch1 intracellular domain and induces its nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Han, Xiangzi [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Department of Preventive Medicine, Yanbian University College of Medicine, Yanji (China); Ju, Ji-hyun [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer Novel S/T-P-S/T domains were identified in NICD. Black-Right-Pointing-Pointer Phosphorylation of NICD on the S/T-P-S/T domains induced nuclear localization. Black-Right-Pointing-Pointer GSK-3{beta} phosphorylated S and T residues in NICD S/T-P-S/T domains. -- Abstract: We identified two S/T-P-S/T domains (2122-2124, 2126-2128) inducing Notch intracellular domain (NICD) nuclear localization. The GFP-NICD (1963-2145) fusion protein deletion mutant without classical NLS was localized in the nucleus like the full length GFP-NICD. However, quadruple substitution mutant (T2122A T2124A S2126A T2128A) showed increased cytoplasmic localization. GSK-3{beta} enhanced nuclear localization and transcriptional activity of WT NICD but not of quadruple substitution mutant. In vitro kinase assays revealed that GSK-3{beta} phosphorylated S and T residues in NICD S/T-P-S/T domains. These results suggest that the novel S/T-P-S/T domain, phosphorylated by GSK-3{beta} is also involved in the nuclear localization of NICD as well as classical NLS.

  12. Relations between perceptual and conceptual scope: how global versus local processing fits a focus on similarity versus dissimilarity.

    Science.gov (United States)

    Förster, Jens

    2009-02-01

    Nine studies showed a bidirectional link (a) between a global processing style and generation of similarities and (b) between a local processing style and generation of dissimilarities. In Experiments 1-4, participants were primed with global versus local perception styles and then asked to work on an allegedly unrelated generation task. Across materials, participants generated more similarities than dissimilarities after global priming, whereas for participants with local priming, the opposite was true. Experiments 5-6 demonstrated a bidirectional link whereby participants who were first instructed to search for similarities attended more to the gestalt of a stimulus than to its details, whereas the reverse was true for those who were initially instructed to search for dissimilarities. Because important psychological variables are correlated with processing styles, in Experiments 7-9, temporal distance, a promotion focus, and high power were predicted and shown to enhance the search for similarities, whereas temporal proximity, a prevention focus, and low power enhanced the search for dissimilarities. (PsycINFO Database Record (c) 2009 APA, all rights reserved).

  13. A similar local immune and oxidative stress phenotype in vitiligo and halo nevus.

    Science.gov (United States)

    Yang, Yuqi; Li, Shuli; Zhu, Guannan; Zhang, Qian; Wang, Gang; Gao, Tianwen; Li, Chunying; Wang, Lin; Jian, Zhe

    2017-07-01

    lesions of vitiligo and halo nevus. These results demonstrate a H 2 O 2 -involved autoimmune phenotype in vitiligo and halo nevus, characterized by increased level of IFN-γ-inducible chemokine pair CXCL10-CXCR3, as well as a dense CD8 + T infiltration in the skin lesions, thus suggesting a similar pathogenesis of the two diseases. Copyright © 2017. Published by Elsevier B.V.

  14. Similar local, but different systemic, metabolomics responses of closely related pine subspecies to folivory by caterpillars of the processionary moth

    Energy Technology Data Exchange (ETDEWEB)

    Rivas-Ubach, A. [Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland WA USA; CSIC, Global Ecology Unit CREAF-CEAB-CSIC-UAB, Cerdanyola del Vallès, Catalonia Spain; Cerdanyola del Vallès, CREAF, Catalonia Spain; Sardans, J. [CSIC, Global Ecology Unit CREAF-CEAB-CSIC-UAB, Cerdanyola del Vallès, Catalonia Spain; Cerdanyola del Vallès, CREAF, Catalonia Spain; Hódar, J. A. [Grupo de Ecología Terrestre, Departamento de Biología Animal y Ecología, Facultad de Ciencias, Universidad de Granada, Granada Spain; Garcia-Porta, J. [Institute of Evolutionary Biology, CSIC-Universitat Pompeu Fabra, Barcelona Spain; Guenther, A. [Department of Earth System Science, University of California, Irvine CA USA; Global Change Research Centre, Academy of Sciences of the Czech Republic, Brno Czech Republic; Oravec, M. [Global Change Research Centre, Academy of Sciences of the Czech Republic, Brno Czech Republic; Urban, O. [Global Change Research Centre, Academy of Sciences of the Czech Republic, Brno Czech Republic; Peñuelas, J. [CSIC, Global Ecology Unit CREAF-CEAB-CSIC-UAB, Cerdanyola del Vallès, Catalonia Spain; Cerdanyola del Vallès, CREAF, Catalonia Spain; Leiss, K.

    2016-05-16

    Plants respond locally and systemically to herbivore attack. Most of the research conducted on plant-herbivore relationships at elemental and molecular levels have focused on nutrients or/and certain molecular compounds or specific families of defensive metabolites showing that herbivores tend to select plant individuals or species with higher nutrient concentrations and to avoid those with higher levels of phenolics and terpenes. Unfortunately, the defensive role of phenolics in conifers is still unclear. We performed stoichiometric and metabolomics, local and systemic, analyses in two subspecies of Pinus sylvestris under the herbivorous attack by the caterpillars of the pine processionary moth, an important pest in the Mediterranean Basin. Herbivorous attack was not associated with any of the elements analyzed. Both pine subspecies responded locally to folivory mainly by increasing the concentrations of various terpenes and phenolics. Systemic responses differed between subspecies and most of the metabolites presented intermediate concentrations between those of the affected parts and unattacked trees. Contrary as usually thought, foliar nutrient concentrations did not show to be a main factor of an alleged plant selection by adult female processionary moths for oviposition. Local increases in phenolics were more associated with antioxidant function for protection against oxidative damage produced by folivory. On the other hand, terpenes were directly related to defense against herbivores. Herbivory attack produced a general systemic shift in pines, including both primary and secondary metabolisms, that was, however, less intense and chemically different from the local responses. Subspecies responded similarly locally but differently to folivory at systemic level.

  15. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

    Energy Technology Data Exchange (ETDEWEB)

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy); Caligo, Maria Adelaide [Section of Genetic Oncology, University Hospital and University of Pisa, via Roma 57, 56125 Pisa (Italy); Galli, Alvaro, E-mail: alvaro.galli@ifc.cnr.it [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy)

    2015-04-15

    Highlights: • The human poly (ADP-ribose) polymerase 1 (PARP-1) gene affects growth and UV-induced homologous recombination in yeast. • PARP-1 chemical inhibition impacts yeast growth and UV-induced recombination. • A genome-wide screen identifies 99 yeast genes that suppress the growth defect inferred by PARP-1. • Bioinformatics analysis identifies 41 human orthologues that may have a role in PARP-1 intracellular localization. • The findings suggest that PARP-1 nuclear localization may affect the response to PARP inhibitors in cancer therapy. - Abstract: The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the

  16. Changes in lymphocyte subsets due to local irradiation of a portion of the maxilla in mice. A study of intracellular cytokine detection

    International Nuclear Information System (INIS)

    Momoi, Yukiko; Shirakawa, Masayori; Satoh, Daigo; Yosue, Takashi

    2008-01-01

    It is critical for dentists who perform radiation therapy to comprehend immunity, because irradiation therapy may damage lymphocytes. After local irradiation to the maxillary area of mice, naive T cells and memory T cells in the spleen, and Th1/Th2 balance and Tc1/Tc2 balance were investigated by intracellular cytokine detection. Female BALB/c mice at 5 weeks of age were adopted for the experiments. In the irradiation groups, a portion of the maxilla was exposed to X-rays (2.0 Gy/min, 10 Gy). Then lymphocytes were analyzed using flow cytometry (anti-CD4, CD62L-selectin and CD45RB monoclonal antibodies). The percentage of Th1 cells, Th2 cells, Tc1 cells, and Tc2 cells and the ratio of Th1/Th2 and Tc1/Tc2 were analyzed by intracellular cytokine detection, and the findings were compared with those of non-irradiated groups. The observation was performed 1 day before irradiation and 1, 3, 7, and 14 days after irradiation. The absolute number of naive T cells was significantly lower 1 and 3 days after irradiation. However, the absolute number of memory T cells did not change significantly after irradiation. The percentage of Th1, Th2, Tc1, and Tc2 cells did not change significantly after irradiation, either. There were no significant differences in the Th1/Th2 ratio and Tc1/Tc2 ratio were observed after irradiation. It was suggested that after the local irradiation the absolute number of naive T cells decreased, that the effect on memory T cells was minimal, and that irradiation did not affect either the ratio of Th1/Th2 nor that of Tc1/Tc2. (author)

  17. Extended local similarity analysis (eLSA) of microbial community and other time series data with replicates.

    Science.gov (United States)

    Xia, Li C; Steele, Joshua A; Cram, Jacob A; Cardon, Zoe G; Simmons, Sheri L; Vallino, Joseph J; Fuhrman, Jed A; Sun, Fengzhu

    2011-01-01

    The increasing availability of time series microbial community data from metagenomics and other molecular biological studies has enabled the analysis of large-scale microbial co-occurrence and association networks. Among the many analytical techniques available, the Local Similarity Analysis (LSA) method is unique in that it captures local and potentially time-delayed co-occurrence and association patterns in time series data that cannot otherwise be identified by ordinary correlation analysis. However LSA, as originally developed, does not consider time series data with replicates, which hinders the full exploitation of available information. With replicates, it is possible to understand the variability of local similarity (LS) score and to obtain its confidence interval. We extended our LSA technique to time series data with replicates and termed it extended LSA, or eLSA. Simulations showed the capability of eLSA to capture subinterval and time-delayed associations. We implemented the eLSA technique into an easy-to-use analytic software package. The software pipeline integrates data normalization, statistical correlation calculation, statistical significance evaluation, and association network construction steps. We applied the eLSA technique to microbial community and gene expression datasets, where unique time-dependent associations were identified. The extended LSA analysis technique was demonstrated to reveal statistically significant local and potentially time-delayed association patterns in replicated time series data beyond that of ordinary correlation analysis. These statistically significant associations can provide insights to the real dynamics of biological systems. The newly designed eLSA software efficiently streamlines the analysis and is freely available from the eLSA homepage, which can be accessed at http://meta.usc.edu/softs/lsa.

  18. CMASA: an accurate algorithm for detecting local protein structural similarity and its application to enzyme catalytic site annotation

    Directory of Open Access Journals (Sweden)

    Li Gong-Hua

    2010-08-01

    Full Text Available Abstract Background The rapid development of structural genomics has resulted in many "unknown function" proteins being deposited in Protein Data Bank (PDB, thus, the functional prediction of these proteins has become a challenge for structural bioinformatics. Several sequence-based and structure-based methods have been developed to predict protein function, but these methods need to be improved further, such as, enhancing the accuracy, sensitivity, and the computational speed. Here, an accurate algorithm, the CMASA (Contact MAtrix based local Structural Alignment algorithm, has been developed to predict unknown functions of proteins based on the local protein structural similarity. This algorithm has been evaluated by building a test set including 164 enzyme families, and also been compared to other methods. Results The evaluation of CMASA shows that the CMASA is highly accurate (0.96, sensitive (0.86, and fast enough to be used in the large-scale functional annotation. Comparing to both sequence-based and global structure-based methods, not only the CMASA can find remote homologous proteins, but also can find the active site convergence. Comparing to other local structure comparison-based methods, the CMASA can obtain the better performance than both FFF (a method using geometry to predict protein function and SPASM (a local structure alignment method; and the CMASA is more sensitive than PINTS and is more accurate than JESS (both are local structure alignment methods. The CMASA was applied to annotate the enzyme catalytic sites of the non-redundant PDB, and at least 166 putative catalytic sites have been suggested, these sites can not be observed by the Catalytic Site Atlas (CSA. Conclusions The CMASA is an accurate algorithm for detecting local protein structural similarity, and it holds several advantages in predicting enzyme active sites. The CMASA can be used in large-scale enzyme active site annotation. The CMASA can be available by the

  19. ARP2/3 localization in Arabidopsis leaf pavement cells: a diversity of intracellular pools and cytoskeletal interactions.

    Science.gov (United States)

    Zhang, Chunhua; Mallery, Eileen L; Szymanski, Daniel B

    2013-01-01

    In plant cells the actin cytoskeleton adopts many configurations, but is best understood as an unstable, interconnected track that rearranges to define the patterns of long distance transport of organelles during growth. Actin filaments do not form spontaneously; instead filament nucleators, such as the evolutionarily conserved actin-related protein (ARP) 2/3 complex, can efficiently generate new actin filament networks when in a fully activated state. A growing number of genetic experiments have shown that ARP2/3 is necessary for morphogenesis in processes that range from tip growth during root nodule formation to the diffuse polarized growth of leaf trichomes and pavement cells. Although progress has been rapid in the identification of proteins that function in series to positively regulate ARP2/3, less has been learned about the actual function of ARP2/3 in cells. In this paper, we analyze the localization of ARP2/3 in Arabidopsis leaf pavement cells. We detect a pool of ARP2/3 in the nucleus, and also find that ARP2/3 is efficiently and specifically clustered on multiple organelle surfaces and associates with both the actin filament and microtubule cytoskeletons. Our mutant analyses and ARP2/3 and actin double labeling experiments indicate that the clustering of ARP2/3 on organelle surfaces and an association with actin bundles does not necessarily reflect an active pool of ARP2/3, and instead most of the complex appears to exist as a latent organelle-associated pool.

  20. An evaluation of multi-probe locality sensitive hashing for computing similarities over web-scale query logs.

    Directory of Open Access Journals (Sweden)

    Graham Cormode

    Full Text Available Many modern applications of AI such as web search, mobile browsing, image processing, and natural language processing rely on finding similar items from a large database of complex objects. Due to the very large scale of data involved (e.g., users' queries from commercial search engines, computing such near or nearest neighbors is a non-trivial task, as the computational cost grows significantly with the number of items. To address this challenge, we adopt Locality Sensitive Hashing (a.k.a, LSH methods and evaluate four variants in a distributed computing environment (specifically, Hadoop. We identify several optimizations which improve performance, suitable for deployment in very large scale settings. The experimental results demonstrate our variants of LSH achieve the robust performance with better recall compared with "vanilla" LSH, even when using the same amount of space.

  1. Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

    Science.gov (United States)

    Dong, Lemeng; Miettinen, Karel; Goedbloed, Miriam; Verstappen, Francel W A; Voster, Alessandra; Jongsma, Maarten A; Memelink, Johan; van der Krol, Sander; Bouwmeester, Harro J

    2013-11-01

    Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  2. HTLV-2B Tax oncoprotein is modified by ubiquitination and sumoylation and displays intracellular localization similar to its homologue HTLV-1 Tax

    International Nuclear Information System (INIS)

    Turci, Marco; Lodewick, Julie; Righi, Paola; Polania, Angela; Romanelli, Maria Grazia; Bex, Francoise; Bertazzoni, Umberto

    2009-01-01

    HTLV-1 is more pathogenic than HTLV-2B. The difference is generally attributed to the properties of their individual transactivating Tax proteins. By using internal Flag-6His tagged Tax-1 and Tax-2B, which display transcriptional activities comparable to the untagged proteins and can be recognized by a single anti-Flag antibody, we demonstrate that Tax-2B is modified by ubiquitination and sumoylation. In addition, Tax2B is distributed in punctuate nuclear structures that include the RelA subunit of NF-κB, as has been previously demonstrated for Tax-1

  3. Contribution of local thyroxine monodeiodination to intracellular 3,5, 3'-triiodothyronine in several tissues of hyperthyroid rats at isotopic equilibrium

    International Nuclear Information System (INIS)

    van Doorn, J.; van der Heide, D.; Roelfsema, F.

    1984-01-01

    The local conversion of T4 as a source of intracellular T3 in several organs of both hypothyroid and euthyroid rats has recently been recognized to be an important phenomenon. In the present study the source and quantity of T3 in various peripheral tissues of hyperthyroid rats were investigated. Athyreotic rats received a continuous iv infusion of T4 over a prolonged period in order to attain hyperthyroid conditions. At the same time, the animals also received a continuous iv infusion of [ 125 I]T4 and [ 131 I]T3 until isotopic equilibrium was achieved. After the animals were bled and perfused, the source and quantity of T3 in various tissue homogenates and subcellular preparations of liver, kidney, and the anterior pituitary gland were analyzed. In spite of the elevated plasma T3 and T4 levels, the concentration of T3 in the cerebral cortex and cerebellum was within the normal range. The contribution of T3 derived from local T4 to T3 conversion [Lc T3(T4)] was rather low in both parts of the brain when compared with values previously determined for euthyroid rats. Whereas previous studies revealed that Lc T3(T4) contributes significantly to the T3 in the pituitary glands of both hypothyroid and euthyroid rats, this was not the case for the hyperthyroid animals; virtually all T3 was derived from plasma. It was found that the T3 in muscle was derived exclusively from plasma. Both the liver and kidney showed high concentrations of T3. Whereas Lc T3(T4) was the main source of T3 in the liver, it contributed only a minor fraction of the total T3 content in the kidney

  4. Contribution of local thyroxine monodeiodination to intracellular 3,5, 3'-triiodothyronine in several tissues of hyperthyroid rats at isotopic equilibrium

    Energy Technology Data Exchange (ETDEWEB)

    van Doorn, J.; van der Heide, D.; Roelfsema, F.

    1984-07-01

    The local conversion of T4 as a source of intracellular T3 in several organs of both hypothyroid and euthyroid rats has recently been recognized to be an important phenomenon. In the present study the source and quantity of T3 in various peripheral tissues of hyperthyroid rats were investigated. Athyreotic rats received a continuous iv infusion of T4 over a prolonged period in order to attain hyperthyroid conditions. At the same time, the animals also received a continuous iv infusion of (/sup 125/I)T4 and (/sup 131/I)T3 until isotopic equilibrium was achieved. After the animals were bled and perfused, the source and quantity of T3 in various tissue homogenates and subcellular preparations of liver, kidney, and the anterior pituitary gland were analyzed. In spite of the elevated plasma T3 and T4 levels, the concentration of T3 in the cerebral cortex and cerebellum was within the normal range. The contribution of T3 derived from local T4 to T3 conversion (Lc T3(T4)) was rather low in both parts of the brain when compared with values previously determined for euthyroid rats. Whereas previous studies revealed that Lc T3(T4) contributes significantly to the T3 in the pituitary glands of both hypothyroid and euthyroid rats, this was not the case for the hyperthyroid animals; virtually all T3 was derived from plasma. It was found that the T3 in muscle was derived exclusively from plasma. Both the liver and kidney showed high concentrations of T3. Whereas Lc T3(T4) was the main source of T3 in the liver, it contributed only a minor fraction of the total T3 content in the kidney.

  5. Localization-Free Detection of Replica Node Attacks in Wireless Sensor Networks Using Similarity Estimation with Group Deployment Knowledge

    Directory of Open Access Journals (Sweden)

    Chao Ding

    2017-01-01

    Full Text Available Due to the unattended nature and poor security guarantee of the wireless sensor networks (WSNs, adversaries can easily make replicas of compromised nodes, and place them throughout the network to launch various types of attacks. Such an attack is dangerous because it enables the adversaries to control large numbers of nodes and extend the damage of attacks to most of the network with quite limited cost. To stop the node replica attack, we propose a location similarity-based detection scheme using deployment knowledge. Compared with prior solutions, our scheme provides extra functionalities that prevent replicas from generating false location claims without deploying resource-consuming localization techniques on the resource-constraint sensor nodes. We evaluate the security performance of our proposal under different attack strategies through heuristic analysis, and show that our scheme achieves secure and robust replica detection by increasing the cost of node replication. Additionally, we evaluate the impact of network environment on the proposed scheme through theoretic analysis and simulation experiments, and indicate that our scheme achieves effectiveness and efficiency with substantially lower communication, computational, and storage overhead than prior works under different situations and attack strategies.

  6. Accurate protein structure annotation through competitive diffusion of enzymatic functions over a network of local evolutionary similarities.

    Directory of Open Access Journals (Sweden)

    Eric Venner

    Full Text Available High-throughput Structural Genomics yields many new protein structures without known molecular function. This study aims to uncover these missing annotations by globally comparing select functional residues across the structural proteome. First, Evolutionary Trace Annotation, or ETA, identifies which proteins have local evolutionary and structural features in common; next, these proteins are linked together into a proteomic network of ETA similarities; then, starting from proteins with known functions, competing functional labels diffuse link-by-link over the entire network. Every node is thus assigned a likelihood z-score for every function, and the most significant one at each node wins and defines its annotation. In high-throughput controls, this competitive diffusion process recovered enzyme activity annotations with 99% and 97% accuracy at half-coverage for the third and fourth Enzyme Commission (EC levels, respectively. This corresponds to false positive rates 4-fold lower than nearest-neighbor and 5-fold lower than sequence-based annotations. In practice, experimental validation of the predicted carboxylesterase activity in a protein from Staphylococcus aureus illustrated the effectiveness of this approach in the context of an increasingly drug-resistant microbe. This study further links molecular function to a small number of evolutionarily important residues recognizable by Evolutionary Tracing and it points to the specificity and sensitivity of functional annotation by competitive global network diffusion. A web server is at http://mammoth.bcm.tmc.edu/networks.

  7. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization.

    Science.gov (United States)

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca; Caligo, Maria Adelaide; Galli, Alvaro

    2015-04-01

    The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Air content and O2/N2 tuned chronologies on local insolation signatures in the Vostok ice core are similar

    Science.gov (United States)

    Lipenkov, V.; Raynaud, D.; Loutre, M.-F.; Duval, P.; Lemieux-Dudon, B.

    2009-04-01

    An accurate chronology of ice cores is needed for interpreting the paleoclimatic record and understanding the relation between insolation and climate. A new domain of research in this area has been initially stimulated by the work of M. Bender (2002) linking the record of O2/N2 ratio in the air trapped in the Vostok ice with the local insolation. More recently, it has been proposed that the long-term changes in air content, V, recorded in ice from the high Antarctic plateau is also dominantly imprinted by the local summer insolation (Raynaud et al., 2007). The present paper presents a new V record from Vostok, which is compared with the published Vostok O2/N2 record for the same period of time (150-400 ka BP) by using the same spectral analysis methods. The spectral differences between the two properties and the possible mechanisms linking them with insolation through the surface snow structure and the close-off processes are discussed. The main result of our study is that the two experimentally independent local insolation proxies lead to absolute (orbital) time scales, which agree together within a standard deviation of 0.6 ka. This result strongly adds credibility to the air content of ice and the O2 to N2 ratio of the air trapped in ice as equally reliable and complementary tools for accurate dating of existing and future deep ice cores. References: M. Bender, Orbital tuning chronology for the Vostok climate record supported by trapped gas composition, Earth and Planetary Science Letters 204(2002) 275-289. D. Raynaud, V. Lipenkov, B. Lemieux-Dudon, P. Duval, M.F. Loutre, N. Lhomme, The local insolation signature of air content in Antarctic ice: a new step toward an absolute dating of ice records, Earth and Planetary Science Letters 261(2007) 337-349.

  9. SU-C-206-03: Metal Artifact Reduction in X-Ray Computed Tomography Based On Local Anatomical Similarity

    International Nuclear Information System (INIS)

    Dong, X; Yang, X; Rosenfield, J; Elder, E; Dhabaan, A

    2016-01-01

    Purpose: Metal implants such as orthopedic hardware and dental fillings cause severe bright and dark streaking in reconstructed CT images. These artifacts decrease image contrast and degrade HU accuracy, leading to inaccuracies in target delineation and dose calculation. Additionally, such artifacts negatively impact patient set-up in image guided radiation therapy (IGRT). In this work, we propose a novel method for metal artifact reduction which utilizes the anatomical similarity between neighboring CT slices. Methods: Neighboring CT slices show similar anatomy. Based on this anatomical similarity, the proposed method replaces corrupted CT pixels with pixels from adjacent, artifact-free slices. A gamma map, which is the weighted summation of relative HU error and distance error, is calculated for each pixel in the artifact-corrupted CT image. The minimum value in each pixel’s gamma map is used to identify a pixel from the adjacent CT slice to replace the corresponding artifact-corrupted pixel. This replacement only occurs if the minimum value in a particular pixel’s gamma map is larger than a threshold. The proposed method was evaluated with clinical images. Results: Highly attenuating dental fillings and hip implants cause severe streaking artifacts on CT images. The proposed method eliminates the dark and bright streaking and improves the implant delineation and visibility. In particular, the image non-uniformity in the central region of interest was reduced from 1.88 and 1.01 to 0.28 and 0.35, respectively. Further, the mean CT HU error was reduced from 328 HU and 460 HU to 60 HU and 36 HU, respectively. Conclusions: The proposed metal artifact reduction method replaces corrupted image pixels with pixels from neighboring slices that are free of metal artifacts. This method proved capable of suppressing streaking artifacts, improving HU accuracy and image detectability.

  10. SU-C-206-03: Metal Artifact Reduction in X-Ray Computed Tomography Based On Local Anatomical Similarity

    Energy Technology Data Exchange (ETDEWEB)

    Dong, X; Yang, X; Rosenfield, J; Elder, E; Dhabaan, A [Emory University, Winship Cancer Institute, Atlanta, GA (United States)

    2016-06-15

    Purpose: Metal implants such as orthopedic hardware and dental fillings cause severe bright and dark streaking in reconstructed CT images. These artifacts decrease image contrast and degrade HU accuracy, leading to inaccuracies in target delineation and dose calculation. Additionally, such artifacts negatively impact patient set-up in image guided radiation therapy (IGRT). In this work, we propose a novel method for metal artifact reduction which utilizes the anatomical similarity between neighboring CT slices. Methods: Neighboring CT slices show similar anatomy. Based on this anatomical similarity, the proposed method replaces corrupted CT pixels with pixels from adjacent, artifact-free slices. A gamma map, which is the weighted summation of relative HU error and distance error, is calculated for each pixel in the artifact-corrupted CT image. The minimum value in each pixel’s gamma map is used to identify a pixel from the adjacent CT slice to replace the corresponding artifact-corrupted pixel. This replacement only occurs if the minimum value in a particular pixel’s gamma map is larger than a threshold. The proposed method was evaluated with clinical images. Results: Highly attenuating dental fillings and hip implants cause severe streaking artifacts on CT images. The proposed method eliminates the dark and bright streaking and improves the implant delineation and visibility. In particular, the image non-uniformity in the central region of interest was reduced from 1.88 and 1.01 to 0.28 and 0.35, respectively. Further, the mean CT HU error was reduced from 328 HU and 460 HU to 60 HU and 36 HU, respectively. Conclusions: The proposed metal artifact reduction method replaces corrupted image pixels with pixels from neighboring slices that are free of metal artifacts. This method proved capable of suppressing streaking artifacts, improving HU accuracy and image detectability.

  11. Phosphoinositide 3-kinaseγ controls the intracellular localization of CpG to limit DNA-PKcs-dependent IL-10 production in macrophages.

    Directory of Open Access Journals (Sweden)

    Kaoru Hazeki

    Full Text Available Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG stimulate innate immune responses. Phosphoinositide 3-kinase (PI3K has been implicated in CpG-induced immune activation; however, its precise role has not yet been clarified. CpG-induced production of IL-10 was dramatically increased in macrophages deficient in PI3Kγ (p110γ(-/-. By contrast, LPS-induced production of IL-10 was unchanged in the cells. CpG-induced, but not LPS-induced, IL-10 production was almost completely abolished in SCID mice having mutations in DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Furthermore, wortmannin, an inhibitor of DNA-PKcs, completely inhibited CpG-induced IL-10 production, both in wild type and p110γ(-/- cells. Microscopic analyses revealed that CpG preferentially localized with DNA-PKcs in p110γ(-/- cells than in wild type cells. In addition, CpG was preferentially co-localized with the acidic lysosomal marker, LysoTracker, in p110γ(-/- cells, and with an early endosome marker, EEA1, in wild type cells. Over-expression of p110γ in Cos7 cells resulted in decreased acidification of CpG containing endosome. A similar effect was reproduced using kinase-dead mutants, but not with a ras-binding site mutant, of p110γ. Thus, it is likely that p110γ, in a manner independent of its kinase activity, inhibits the acidification of CpG-containing endosomes. It is considered that increased acidification of CpG-containing endosomes in p110γ(-/- cells enforces endosomal escape of CpG, which results in increased association of CpG with DNA-PKcs to up-regulate IL-10 production in macrophages.

  12. SimShiftDB; local conformational restraints derived from chemical shift similarity searches on a large synthetic database

    International Nuclear Information System (INIS)

    Ginzinger, Simon W.; Coles, Murray

    2009-01-01

    We present SimShiftDB, a new program to extract conformational data from protein chemical shifts using structural alignments. The alignments are obtained in searches of a large database containing 13,000 structures and corresponding back-calculated chemical shifts. SimShiftDB makes use of chemical shift data to provide accurate results even in the case of low sequence similarity, and with even coverage of the conformational search space. We compare SimShiftDB to HHSearch, a state-of-the-art sequence-based search tool, and to TALOS, the current standard tool for the task. We show that for a significant fraction of the predicted similarities, SimShiftDB outperforms the other two methods. Particularly, the high coverage afforded by the larger database often allows predictions to be made for residues not involved in canonical secondary structure, where TALOS predictions are both less frequent and more error prone. Thus SimShiftDB can be seen as a complement to currently available methods

  13. SimShiftDB; local conformational restraints derived from chemical shift similarity searches on a large synthetic database

    Energy Technology Data Exchange (ETDEWEB)

    Ginzinger, Simon W. [Center of Applied Molecular Engineering, University of Salzburg, Department of Molecular Biology, Division of Bioinformatics (Austria)], E-mail: simon@came.sbg.ac.at; Coles, Murray [Max-Planck-Institute for Developmental Biology, Department of Protein Evolution (Germany)], E-mail: Murray.Coles@tuebingen.mpg.de

    2009-03-15

    We present SimShiftDB, a new program to extract conformational data from protein chemical shifts using structural alignments. The alignments are obtained in searches of a large database containing 13,000 structures and corresponding back-calculated chemical shifts. SimShiftDB makes use of chemical shift data to provide accurate results even in the case of low sequence similarity, and with even coverage of the conformational search space. We compare SimShiftDB to HHSearch, a state-of-the-art sequence-based search tool, and to TALOS, the current standard tool for the task. We show that for a significant fraction of the predicted similarities, SimShiftDB outperforms the other two methods. Particularly, the high coverage afforded by the larger database often allows predictions to be made for residues not involved in canonical secondary structure, where TALOS predictions are both less frequent and more error prone. Thus SimShiftDB can be seen as a complement to currently available methods.

  14. Phylogeny, Traits, and Biodiversity of a Neotropical Bat Assemblage: Close Relatives Show Similar Responses to Local Deforestation.

    Science.gov (United States)

    Frank, Hannah K; Frishkoff, Luke O; Mendenhall, Chase D; Daily, Gretchen C; Hadly, Elizabeth A

    2017-08-01

    If species' evolutionary pasts predetermine their responses to evolutionarily novel stressors, then phylogeny could predict species survival in an increasingly human-dominated world. To understand the role of phylogenetic relatedness in structuring responses to rapid environmental change, we focused on assemblages of Neotropical bats, an ecologically diverse and functionally important group. We examined how taxonomic and phylogenetic diversity shift between tropical forest and farmland. We then explored the importance of evolutionary history by ascertaining whether close relatives share similar responses to environmental change and which species traits might mediate these trends. We analyzed a 5-year data set (5,011 captures) from 18 sites in a countryside landscape in southern Costa Rica using statistical models that account and correct for imperfect detection of species across sites, spatial autocorrelation, and consideration of spatial scale. Taxonomic and phylogenetic diversity decreased with deforestation, and assemblages became more phylogenetically clustered. Species' responses to deforestation were strongly phylogenetically correlated. Body mass and absolute wing loading explained a substantial portion of species variation in species' habitat preferences, likely related to these traits' influence on maneuverability in cluttered forest environments. Our findings highlight the role that evolutionary history plays in determining which species will survive human impacts and the need to consider diversity metrics, evolutionary history, and traits together when making predictions about species persistence for conservation or ecosystem functioning.

  15. Histopathological confirmation of similar intramucosal distribution of fluorescein in both intravenous administration and local mucosal application for probe-based confocal laser endomicroscopy of the normal stomach.

    Science.gov (United States)

    Nonaka, Kouichi; Ohata, Ken; Ban, Shinichi; Ichihara, Shin; Takasugi, Rumi; Minato, Yohei; Tashima, Tomoaki; Matsuyama, Yasushi; Takita, Maiko; Matsuhashi, Nobuyuki; Neumann, Helmut

    2015-12-16

    Probe-based confocal laser endomicroscopy (pCLE) is capable of acquiring in vivo magnified cross-section images of the gastric mucosa. Intravenous injection of fluorescein sodium is used for confocal imaging. However, it is still under debate if local administration of the dye to the mucosa is also effective for confocal imaging as it is not yet clear if topical application also reveals the intramucosal distribution of fluorescein. The objective of this study was to evaluate the intramucosal distribution of fluorescein sodium after topical application and to compare the distribution to the conventional intravenous injection used for confocal imaging. pCLE of the stomach uninfected with Helicobacter pylori was performed in a healthy male employing intravenous administration and local mucosal application of fluorescein. The mucosa of the lower gastric body was biopsied 1 min and 5 min after intravenous administration or local mucosal application of fluorescein, and the distribution of fluorescein in the biopsy samples was examined histologically. Green fluorescence was already observed in the cytoplasm of fundic glandular cells in the biopsied deep mucosa 1 min after local mucosal application of fluorescein. It was also observed in the foveolar lumen and inter-foveolar lamina propria, although it was noted at only a few sites. In the tissue biopsied 5 min after the local mucosal application of fluorescein, green fluorescence was more frequently noted in the cytoplasm of fundic glandular cells than in that 1 min after the local mucosal application of fluorescein, although obvious green fluorescence was not identified in the foveolar lumen or inter-foveolar lamina propria. The distribution of intravenously administered fluorescein in the cytoplasm of fundic glandular cells was also clearly observed similarly to that after local mucosal application of fluorescein. Green fluorescence in more cells was observed in many cells 5 min after intravenous administration compared

  16. Specific intracellular signal transduction pathways downstream of CSF-1 receptors: their relationship to breast cancer local recurrence and distant relapse in vivo. Potential targets for the development of new, specific anti-breast cancer therapies to improve local control and block metastatic spread?

    International Nuclear Information System (INIS)

    Kacinski, Barry M.; Sapi, Eva; Flick, Maryann B.; Turner, Bruce; Perrotta, Peter; Maher, M. Grey; Carter, Darryl; Haffy, Bruce

    1997-01-01

    analogues such as Matrigel. Agents which pharmacologically mimic a TYR-721 →PHE mutation by interfering with the activation of elements of intracellular signal transduction downstream of this tyrosine (PI-3 kinase. pp70-S6kinase) produced similar effects. In contrast, a TYR-809→PHE mutation was without effect on anchorage independent growth or the generation of pulmonary metastases but did completely abolish protease production and rendered the transfected cells unable to invade basement membrane analogues. In a parallel line of research, we employed antibodies which recognized CSF-1R and a novel antibody we prepared to recognize CSF-1R only when phosphorylated on TYR-721 in a study of 80 T1 and T2 breast cancer patients treated with tylectomy and primary radiation therapy. Strong staining with the generic anti-CSF-1R antibody correlated strongly with local relapse (P values <.03) in this patient cohort. Staining with the antibody specific for CSF-1R phosphorylated at TYR-721 was not associated with local relapse but did correlate with the development of distant metastases in this cohort of patients, particularly in axillary node-negative patients (P < .006). Conclusion: In summary, our observations demonstrate that CSF-1R activation and phosphorylation at specific tyrosines regulates invasiveness, anchorage, independent growth and tumorigenicity in vitro and in animal models and correlates with metastatic relapse in vivo. They also suggest that such intracellular signaling pathways--particularly those triggered by the phosphorylation of TYR-721 of CSF-1R--are logical targets for the development of a new class of anti-cancer agents which specifically block breast cancer cell metastatic potential without perturbing other normal cellular metabolic processes

  17. Two Phase Non-Rigid Multi-Modal Image Registration Using Weber Local Descriptor-Based Similarity Metrics and Normalized Mutual Information

    Directory of Open Access Journals (Sweden)

    Feng Yang

    2013-06-01

    Full Text Available Non-rigid multi-modal image registration plays an important role in medical image processing and analysis. Existing image registration methods based on similarity metrics such as mutual information (MI and sum of squared differences (SSD cannot achieve either high registration accuracy or high registration efficiency. To address this problem, we propose a novel two phase non-rigid multi-modal image registration method by combining Weber local descriptor (WLD based similarity metrics with the normalized mutual information (NMI using the diffeomorphic free-form deformation (FFD model. The first phase aims at recovering the large deformation component using the WLD based non-local SSD (wldNSSD or weighted structural similarity (wldWSSIM. Based on the output of the former phase, the second phase is focused on getting accurate transformation parameters related to the small deformation using the NMI. Extensive experiments on T1, T2 and PD weighted MR images demonstrate that the proposed wldNSSD-NMI or wldWSSIM-NMI method outperforms the registration methods based on the NMI, the conditional mutual information (CMI, the SSD on entropy images (ESSD and the ESSD-NMI in terms of registration accuracy and computation efficiency.

  18. WE-AB-BRA-01: 3D-2D Image Registration for Target Localization in Spine Surgery: Comparison of Similarity Metrics Against Robustness to Content Mismatch

    International Nuclear Information System (INIS)

    De Silva, T; Ketcha, M; Siewerdsen, J H; Uneri, A; Reaungamornrat, S; Vogt, S; Kleinszig, G; Lo, S F; Wolinsky, J P; Gokaslan, Z L; Aygun, N

    2015-01-01

    Purpose: In image-guided spine surgery, mapping 3D preoperative images to 2D intraoperative images via 3D-2D registration can provide valuable assistance in target localization. However, the presence of surgical instrumentation, hardware implants, and soft-tissue resection/displacement causes mismatches in image content, confounding existing registration methods. Manual/semi-automatic methods to mask such extraneous content is time consuming, user-dependent, error prone, and disruptive to clinical workflow. We developed and evaluated 2 novel similarity metrics within a robust registration framework to overcome such challenges in target localization. Methods: An IRB-approved retrospective study in 19 spine surgery patients included 19 preoperative 3D CT images and 50 intraoperative mobile radiographs in cervical, thoracic, and lumbar spine regions. A neuroradiologist provided truth definition of vertebral positions in CT and radiography. 3D-2D registration was performed using the CMA-ES optimizer with 4 gradient-based image similarity metrics: (1) gradient information (GI); (2) gradient correlation (GC); (3) a novel variant referred to as gradient orientation (GO); and (4) a second variant referred to as truncated gradient correlation (TGC). Registration accuracy was evaluated in terms of the projection distance error (PDE) of the vertebral levels. Results: Conventional similarity metrics were susceptible to gross registration error and failure modes associated with the presence of surgical instrumentation: for GI, the median PDE and interquartile range was 33.0±43.6 mm; similarly for GC, PDE = 23.0±92.6 mm respectively. The robust metrics GO and TGC, on the other hand, demonstrated major improvement in PDE (7.6 ±9.4 mm and 8.1± 18.1 mm, respectively) and elimination of gross failure modes. Conclusion: The proposed GO and TGC similarity measures improve registration accuracy and robustness to gross failure in the presence of strong image content mismatch. Such

  19. WE-AB-BRA-01: 3D-2D Image Registration for Target Localization in Spine Surgery: Comparison of Similarity Metrics Against Robustness to Content Mismatch

    Energy Technology Data Exchange (ETDEWEB)

    De Silva, T; Ketcha, M; Siewerdsen, J H [Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD (United States); Uneri, A; Reaungamornrat, S [Department of Computer Science, Johns Hopkins University, Baltimore, MD (United States); Vogt, S; Kleinszig, G [Siemens Healthcare XP Division, Erlangen, DE (Germany); Lo, S F; Wolinsky, J P; Gokaslan, Z L [Department of Neurosurgery, The Johns Hopkins Hospital, Baltimore, MD (United States); Aygun, N [Department of Raiology and Radiological Sciences, The Johns Hopkins Hospital, Baltimore, MD (United States)

    2015-06-15

    Purpose: In image-guided spine surgery, mapping 3D preoperative images to 2D intraoperative images via 3D-2D registration can provide valuable assistance in target localization. However, the presence of surgical instrumentation, hardware implants, and soft-tissue resection/displacement causes mismatches in image content, confounding existing registration methods. Manual/semi-automatic methods to mask such extraneous content is time consuming, user-dependent, error prone, and disruptive to clinical workflow. We developed and evaluated 2 novel similarity metrics within a robust registration framework to overcome such challenges in target localization. Methods: An IRB-approved retrospective study in 19 spine surgery patients included 19 preoperative 3D CT images and 50 intraoperative mobile radiographs in cervical, thoracic, and lumbar spine regions. A neuroradiologist provided truth definition of vertebral positions in CT and radiography. 3D-2D registration was performed using the CMA-ES optimizer with 4 gradient-based image similarity metrics: (1) gradient information (GI); (2) gradient correlation (GC); (3) a novel variant referred to as gradient orientation (GO); and (4) a second variant referred to as truncated gradient correlation (TGC). Registration accuracy was evaluated in terms of the projection distance error (PDE) of the vertebral levels. Results: Conventional similarity metrics were susceptible to gross registration error and failure modes associated with the presence of surgical instrumentation: for GI, the median PDE and interquartile range was 33.0±43.6 mm; similarly for GC, PDE = 23.0±92.6 mm respectively. The robust metrics GO and TGC, on the other hand, demonstrated major improvement in PDE (7.6 ±9.4 mm and 8.1± 18.1 mm, respectively) and elimination of gross failure modes. Conclusion: The proposed GO and TGC similarity measures improve registration accuracy and robustness to gross failure in the presence of strong image content mismatch. Such

  20. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells

    Science.gov (United States)

    Bach, Thomas J

    2013-01-01

    We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL. PMID:24555083

  1. Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes

    Directory of Open Access Journals (Sweden)

    Hrdličková Radmila

    2012-06-01

    Full Text Available Abstract Background The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in eutherian mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (OanTERT ortholog, and provide a comparison with genes of other vertebrates. Results The OanTERT encodes a protein with a high sequence similarity to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, OanTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in ray-finned fish and eutherian mammals. Several alternatively spliced OanTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues like that of cold-blooded animals and murine rodents. OanTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. Synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males. Conclusions OanTERT shares many features with TERT of the reptilian outgroup, suggesting that OanTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.

  2. 3D–2D image registration for target localization in spine surgery: investigation of similarity metrics providing robustness to content mismatch

    International Nuclear Information System (INIS)

    De Silva, T; Ketcha, M D; Siewerdsen, J H; Uneri, A; Reaungamornrat, S; Kleinszig, G; Vogt, S; Aygun, N; Lo, S-F; Wolinsky, J-P

    2016-01-01

    In image-guided spine surgery, robust three-dimensional to two-dimensional (3D–2D) registration of preoperative computed tomography (CT) and intraoperative radiographs can be challenged by the image content mismatch associated with the presence of surgical instrumentation and implants as well as soft-tissue resection or deformation. This work investigates image similarity metrics in 3D–2D registration offering improved robustness against mismatch, thereby improving performance and reducing or eliminating the need for manual masking. The performance of four gradient-based image similarity metrics (gradient information (GI), gradient correlation (GC), gradient information with linear scaling (GS), and gradient orientation (GO)) with a multi-start optimization strategy was evaluated in an institutional review board-approved retrospective clinical study using 51 preoperative CT images and 115 intraoperative mobile radiographs. Registrations were tested with and without polygonal masks as a function of the number of multistarts employed during optimization. Registration accuracy was evaluated in terms of the projection distance error (PDE) and assessment of failure modes (PDE  >  30 mm) that could impede reliable vertebral level localization. With manual polygonal masking and 200 multistarts, the GC and GO metrics exhibited robust performance with 0% gross failures and median PDE  <  6.4 mm (±4.4 mm interquartile range (IQR)) and a median runtime of 84 s (plus upwards of 1–2 min for manual masking). Excluding manual polygonal masks and decreasing the number of multistarts to 50 caused the GC-based registration to fail at a rate of  >14%; however, GO maintained robustness with a 0% gross failure rate. Overall, the GI, GC, and GS metrics were susceptible to registration errors associated with content mismatch, but GO provided robust registration (median PDE  =  5.5 mm, 2.6 mm IQR) without manual masking and with an improved

  3. 3D-2D image registration for target localization in spine surgery: investigation of similarity metrics providing robustness to content mismatch

    Science.gov (United States)

    De Silva, T.; Uneri, A.; Ketcha, M. D.; Reaungamornrat, S.; Kleinszig, G.; Vogt, S.; Aygun, N.; Lo, S.-F.; Wolinsky, J.-P.; Siewerdsen, J. H.

    2016-04-01

    In image-guided spine surgery, robust three-dimensional to two-dimensional (3D-2D) registration of preoperative computed tomography (CT) and intraoperative radiographs can be challenged by the image content mismatch associated with the presence of surgical instrumentation and implants as well as soft-tissue resection or deformation. This work investigates image similarity metrics in 3D-2D registration offering improved robustness against mismatch, thereby improving performance and reducing or eliminating the need for manual masking. The performance of four gradient-based image similarity metrics (gradient information (GI), gradient correlation (GC), gradient information with linear scaling (GS), and gradient orientation (GO)) with a multi-start optimization strategy was evaluated in an institutional review board-approved retrospective clinical study using 51 preoperative CT images and 115 intraoperative mobile radiographs. Registrations were tested with and without polygonal masks as a function of the number of multistarts employed during optimization. Registration accuracy was evaluated in terms of the projection distance error (PDE) and assessment of failure modes (PDE  >  30 mm) that could impede reliable vertebral level localization. With manual polygonal masking and 200 multistarts, the GC and GO metrics exhibited robust performance with 0% gross failures and median PDE  interquartile range (IQR)) and a median runtime of 84 s (plus upwards of 1-2 min for manual masking). Excluding manual polygonal masks and decreasing the number of multistarts to 50 caused the GC-based registration to fail at a rate of  >14% however, GO maintained robustness with a 0% gross failure rate. Overall, the GI, GC, and GS metrics were susceptible to registration errors associated with content mismatch, but GO provided robust registration (median PDE  =  5.5 mm, 2.6 mm IQR) without manual masking and with an improved runtime (29.3 s). The GO metric

  4. Extended local similarity analysis (eLSA) reveals unique associations between bacterial community structure and odor emission during pig carcasses decomposition.

    Science.gov (United States)

    Ki, Bo-Min; Ryu, Hee Wook; Cho, Kyung-Suk

    2018-02-22

    Soil burial and composting methods have been widely used for the disposal of pig carcasses. The relationship between bacterial community structure and odor emission was examined using extended local similarity analysis (eLSA) during the degradation of pig carcasses in soil and compost. In soil, Hyphomicrobium, Niastella, Rhodanobacter, Polaromonas, Dokdonella and Mesorhizobium were associated with the emission of sulfur-containing odors such as hydrogen sulfide, methyl mercaptan and dimethyl disulfide. Sphingomonas, Rhodanobacter, Mesorhizobium, Dokdonella, Leucobacter and Truepera were associated with the emission of nitrogen-containing odors including ammonia and trimetylamine. In compost, however, Carnobacteriaceae, Lachnospiaceae and Clostridiales were highly correlated with the emission of sulfur-containing odors, while Rumincoccaceae was associated with the emission of nitrogen-containing odors. The emission of organic acids was closely related to Massilia, Sphaerobacter and Bradyrhizobiaceae in soil, but to Actinobacteria, Sporacetigenium, Micromonosporaceae and Solirubrobacteriales in compost. This study suggests that network analysis using eLSA is a useful strategy for exploring the mechanisms of odor emission during biodegradation of pig carcasses.

  5. Characterization of Leptin Intracellular Trafficking

    Directory of Open Access Journals (Sweden)

    E Walum

    2009-12-01

    to engage a monensin and bafilomycin sensitive degradation process in lysosomes. Together, our results provide novel data concerning the uptake, intracellular localization and transport of leptin.

  6. Automated geographic atrophy segmentation for SD-OCT images using region-based C-V model via local similarity factor.

    Science.gov (United States)

    Niu, Sijie; de Sisternes, Luis; Chen, Qiang; Leng, Theodore; Rubin, Daniel L

    2016-02-01

    Age-related macular degeneration (AMD) is the leading cause of blindness among elderly individuals. Geographic atrophy (GA) is a phenotypic manifestation of the advanced stages of non-exudative AMD. Determination of GA extent in SD-OCT scans allows the quantification of GA-related features, such as radius or area, which could be of important value to monitor AMD progression and possibly identify regions of future GA involvement. The purpose of this work is to develop an automated algorithm to segment GA regions in SD-OCT images. An en face GA fundus image is generated by averaging the axial intensity within an automatically detected sub-volume of the three dimensional SD-OCT data, where an initial coarse GA region is estimated by an iterative threshold segmentation method and an intensity profile set, and subsequently refined by a region-based Chan-Vese model with a local similarity factor. Two image data sets, consisting on 55 SD-OCT scans from twelve eyes in eight patients with GA and 56 SD-OCT scans from 56 eyes in 56 patients with GA, respectively, were utilized to quantitatively evaluate the automated segmentation algorithm. We compared results obtained by the proposed algorithm, manual segmentation by graders, a previously proposed method, and experimental commercial software. When compared to a manually determined gold standard, our algorithm presented a mean overlap ratio (OR) of 81.86% and 70% for the first and second data sets, respectively, while the previously proposed method OR was 72.60% and 65.88% for the first and second data sets, respectively, and the experimental commercial software OR was 62.40% for the second data set.

  7. Pathogenic mechanisms of intracellular bacteria.

    Science.gov (United States)

    Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

    2017-06-01

    We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

  8. local

    Directory of Open Access Journals (Sweden)

    Abílio Amiguinho

    2005-01-01

    Full Text Available The process of socio-educational territorialisation in rural contexts is the topic of this text. The theme corresponds to a challenge to address it having as main axis of discussion either the problem of social exclusion or that of local development. The reasons to locate the discussion in this last field of analysis are discussed in the first part of the text. Theoretical and political reasons are there articulated because the question is about projects whose intentions and practices call for the political both in the theoretical debate and in the choices that anticipate intervention. From research conducted for several years, I use contributions that aim at discuss and enlighten how school can be a potential locus of local development. Its identification and recognition as local institution (either because of those that work and live in it or because of those that act in the surrounding context are crucial steps to progressively constitute school as a partner for development. The promotion of the local values and roots, the reconstruction of socio-personal and local identities, the production of sociabilities and the equation and solution of shared problems were the dimensions of a socio-educative intervention, markedly globalising. This scenario, as it is argued, was also, intentionally, one of transformation and of deliberate change of school and of the administration of the educative territoires.

  9. Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

    Science.gov (United States)

    Payne, Christine

    2014-03-01

    Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

  10. Similar decreases in local tumor control are calculated for treatment protraction and for interruptions in the radiotherapy of carcinoma of the larynx in four centers

    International Nuclear Information System (INIS)

    Robertson, Chris; Robertson, A. Gerald; Hendry, Jolyon H.; Roberts, Stephen A.; Slevin, Nicholas J.; Duncan, William B.; MacDougall, R. Hugh; Kerr, Gillian R.; O'Sullivan, B.; Keane, Thomas J.

    1998-01-01

    Purpose: Data on patients with cancer of the larynx are analyzed using statistical models to estimate the effect of gaps in the treatment time on the local control of the tumor. Methods and Materials: Patients from four centers, Edinburgh, Glasgow, Manchester, and Toronto, with carcinoma of the larynx and treated by radiotherapy were followed up and the disease-free period recorded. In all centers the end point was control of the primary tumor after irradiation alone. The local control rates at ≥2 years, P c , were analyzed by log linear models, and Cox proportional hazard models were used to model the disease-free period. Results: T stage, nodal involvement, and site of the tumor were important determinants of the disease-free interval, as was the radiation schedule used. Elongation of the treatment time by 1 day, or a gap of 1 day, was associated with a decrease in P c of 0.68% per day for P c = 0.80, with a 95% confidence interval of (0.28, 1.08)%. An increase of 5 days was associated with a 3.5% reduction in P c from 0.80 to 0.77. At P c = 0.60 an increase of 5 days was associated with an 7.9% decrease in P c . The time factor in the Linear Quadratic model, γ/α, was estimated as 0.89 Gy/day, 95% confidence interval (0.35, 1.43) Gy/day. Conclusions: Any gaps (public holidays are the majority) in the treatment schedule have the same deleterious effect on the disease free period as an increase in the prescribed treatment time. For a schedule, where dose and fraction number are specified, any gap in treatment is potentially damaging

  11. The Patterns of Care Survey of radiation therapy in localized prostate cancer: Similarities between the practice nationally and in minority-rich areas

    International Nuclear Information System (INIS)

    Zietman, Anthony; Moughan, Jennifer; Owen, Jean; Hanks, Gerald

    2001-01-01

    Purpose: Over the last two decades, the chance for the cure of localized prostate cancer by radiation has been improved by the widespread use of PSA for early detection and by a number of technical advances in treatment delivery. This study was designed to determine whether the stage of presentation and the quality of radiation treatment delivered are comparable between Caucasian and minority patients nationally and within minority-rich areas. Methods and Materials: A random survey conducted for the Patterns of Care Study in Radiation Oncology of 80 facilities treating patients with radiation in the USA. Of these, 67 comprise the 'National Survey' and 13 a 'Minority-Rich' survey (>40% of treated patients are minorities). Nine hundred twenty-six men with localized prostate cancer were treated in 1994. Five hundred ninety-five were in the national and 331 in the minority-rich survey. The main outcome measures were the clinical features of Caucasian and minority men at presentation and technical characteristics of the treatment delivered to them. Results: African-American men presented with more advanced disease (higher-presenting PSA and T-stage) than Caucasians in both the national and the minority-rich surveys. Hispanics also presented with later disease and could be grouped with African-American men rather than Caucasians. Overall the stage and PSA at presentation was earlier than seen in the previous Patterns of Care Study survey of 1989. The quality of treatment delivered has improved since 1989, with no distinction seen between those facilities sampled nationally and those within minority-rich areas. Conclusion: African-American and Hispanic men with prostate cancer present for therapy at a later stage than Caucasian men, but when they do, the treatment received is of comparable quality

  12. Dynamics of gradient formation by intracellular shuttling

    Energy Technology Data Exchange (ETDEWEB)

    Berezhkovskii, Alexander M. [Mathematical and Statistical Computing Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892 (United States); Shvartsman, Stanislav Y. [Department of Chemical and Biological Engineering and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544 (United States)

    2015-08-21

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  13. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually

  14. Insecticide resistance and intracellular proteases.

    Science.gov (United States)

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  15. Pyridine nucleotide cycling and control of intracellular redox state in relation to poly (ADP-ribose) polymerase activity and nuclear localization of glutathione during exponential growth of Arabidopsis cells in culture.

    Science.gov (United States)

    Pellny, Till K; Locato, Vittoria; Vivancos, Pedro Diaz; Markovic, Jelena; De Gara, Laura; Pallardó, Federico V; Foyer, Christine H

    2009-05-01

    Pyridine nucleotides, ascorbate and glutathione are major redox metabolites in plant cells, with specific roles in cellular redox homeostasis and the regulation of the cell cycle. However, the regulation of these metabolite pools during exponential growth and their precise functions in the cell cycle remain to be characterized. The present analysis of the abundance of ascorbate, glutathione, and pyridine nucleotides during exponential growth of Arabidopsis cells in culture provides evidence for the differential regulation of each of these redox pools. Ascorbate was most abundant early in the growth cycle, but glutathione was low at this point. The cellular ascorbate to dehydroascorbate and reduced glutathione (GSH) to glutathione disulphide ratios were high and constant but the pyridine nucleotide pools were largely oxidized over the period of exponential growth and only became more reduced once growth had ceased. The glutathione pool increased in parallel with poly (ADP-ribose) polymerase (PARP) activities and with increases in the abundance of PARP1 and PARP2 mRNAs at a time of high cell cycle activity as indicated by transcriptome information. Marked changes in the intracellular partitioning of GSH between the cytoplasm and nucleus were observed. Extension of the exponential growth phase by dilution or changing the media led to increases in the glutathione and nicotinamide adenine dinucleotide, oxidized form (NAD)-plus-nicotinamide adenine dinucleotide, reduced form (NADH) pools and to higher NAD/NADH ratios but the nicotinamide adenine dinucleotide phosphate, oxidized form (NADP)-plus-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) pool sizes, and NAPD/NADPH ratios were much less affected. The ascorbate, glutathione, and pyridine nucleotide pools and PARP activity decreased before the exponential growth phase ended. We conclude that there are marked changes in intracellular redox state during the growth cycle but that redox homeostasis is

  16. Intracellular Cadmium Isotope Fractionation

    Science.gov (United States)

    Horner, T. J.; Lee, R. B.; Henderson, G. M.; Rickaby, R. E.

    2011-12-01

    Recent stable isotope studies into the biological utilization of transition metals (e.g. Cu, Fe, Zn, Cd) suggest several stepwise cellular processes can fractionate isotopes in both culture and nature. However, the determination of fractionation factors is often unsatisfactory, as significant variability can exist - even between different organisms with the same cellular functions. Thus, it has not been possible to adequately understand the source and mechanisms of metal isotopic fractionation. In order to address this problem, we investigated the biological fractionation of Cd isotopes within genetically-modified bacteria (E. coli). There is currently only one known biological use or requirement of Cd, a Cd/Zn carbonic anhydrase (CdCA, from the marine diatom T. weissfloggii), which we introduce into the E. coli genome. We have also developed a cleaning procedure that allows for the treating of bacteria so as to study the isotopic composition of different cellular components. We find that whole cells always exhibit a preference for uptake of the lighter isotopes of Cd. Notably, whole cells appear to have a similar Cd isotopic composition regardless of the expression of CdCA within the E. coli. However, isotopic fractionation can occur within the genetically modified E. coli during Cd use, such that Cd bound in CdCA can display a distinct isotopic composition compared to the cell as a whole. Thus, the externally observed fractionation is independent of the internal uses of Cd, with the largest Cd isotope fractionation occurring during cross-membrane transport. A general implication of these experiments is that trace metal isotopic fractionation most likely reflects metal transport into biological cells (either actively or passively), rather than relating to expression of specific physiological function and genetic expression of different metalloenzymes.

  17. pH-sensitive intracellular photoluminescence of carbon nanotube-fluorescein conjugates in human ovarian cancer cells

    International Nuclear Information System (INIS)

    Chen, M T; Ishikawa, F N; Gundersen, M A; Gomez, L M; Vernier, P T; Zhou, C

    2009-01-01

    To add to the understanding of the properties of functionalized carbon nanotubes in biological applications, we report a monotonic pH sensitivity of the intracellular fluorescence emission of single-walled carbon nanotube-fluorescein carbazide (SWCNT-FC) conjugates in human ovarian cancer cells. Light-stimulated intracellular hydrolysis of the amide linkage and localized intracellular pH changes are proposed as mechanisms. SWCNT-FC conjugates may serve as intracellular pH sensors.

  18. Self-similar cosmological models

    Energy Technology Data Exchange (ETDEWEB)

    Chao, W Z [Cambridge Univ. (UK). Dept. of Applied Mathematics and Theoretical Physics

    1981-07-01

    The kinematics and dynamics of self-similar cosmological models are discussed. The degrees of freedom of the solutions of Einstein's equations for different types of models are listed. The relation between kinematic quantities and the classifications of the self-similarity group is examined. All dust local rotational symmetry models have been found.

  19. Cytoplasmic tail of coronavirus spike protein has intracellular

    Indian Academy of Sciences (India)

    https://www.ias.ac.in/article/fulltext/jbsc/042/02/0231-0244. Keywords. Coronavirus spike protein trafficking; cytoplasmic tail signal; endoplasmic reticulum–Golgi intermediate complex; lysosome. Abstract. Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is ...

  20. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells [v1; ref status: indexed, http://f1000r.es/yx

    Directory of Open Access Journals (Sweden)

    Michael Hartmann

    2013-08-01

    Full Text Available We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL. By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1, shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.

  1. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.

    2013-01-09

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

  2. Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

    DEFF Research Database (Denmark)

    Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    of the physical and biochemical conditions in plant cells. As model system, we use a H(2)O(2) signal originating at the plasma membrane (PM) and spreading through the cytosol. We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits......Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...

  3. Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis

    International Nuclear Information System (INIS)

    Hiraiwa, Tetsuya; Nishikawa, Masatoshi; Shibata, Tatsuo; Nagamatsu, Akihiro; Akuzawa, Naohiro

    2014-01-01

    Eukaryotic chemotaxis is usually mediated by intracellular signals that tend to localize at the front or back of the cell. Such intracellular polarities frequently require no extracellular guidance cues, indicating that spontaneous polarization occurs in the signal network. Spontaneous polarization activity is considered relevant to the persistent motions in random cell migrations and chemotaxis. In this study, we propose a theoretical model that connects spontaneous intracellular polarity and motile ability in a chemoattractant solution. We demonstrate that the intracellular polarity can enhance the accuracy of chemotaxis. Chemotactic accuracy should also depend on chemoattractant concentration through the concentration-dependent correlation time in the polarity direction. Both the polarity correlation time and the chemotactic accuracy depend on the degree of responsiveness to the chemical gradient. We show that optimally accurate chemotaxis occurs at an intermediate responsiveness of intracellular polarity. Experimentally, we find that the persistence time of randomly migrating Dictyostelium cells depends on the chemoattractant concentration, as predicted by our theory. At the optimum responsiveness, this ameboid cell can enhance its chemotactic accuracy tenfold. (paper)

  4. Intracellular calcium levels can regulate Importin-dependent nuclear import

    International Nuclear Information System (INIS)

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

    2014-01-01

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca 2+ on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery

  5. Intracellular calcium levels can regulate Importin-dependent nuclear import

    Energy Technology Data Exchange (ETDEWEB)

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A., E-mail: David.Jans@monash.edu

    2014-07-18

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

  6. Self-organization of intracellular gradients during mitosis

    Directory of Open Access Journals (Sweden)

    Fuller Brian G

    2010-01-01

    Full Text Available Abstract Gradients are used in a number of biological systems to transmit spatial information over a range of distances. The best studied are morphogen gradients where information is transmitted over many cell lengths. Smaller mitotic gradients reflect the need to organize several distinct events along the length of the mitotic spindle. The intracellular gradients that characterize mitosis are emerging as important regulatory paradigms. Intracellular gradients utilize intrinsic auto-regulatory feedback loops and diffusion to establish stable regions of activity within the mitotic cytosol. We review three recently described intracellular mitotic gradients. The Ran GTP gradient with its elaborate cascade of nuclear transport receptors and cargoes is the best characterized, yet the dynamics underlying the robust gradient of Ran-GTP have received little attention. Gradients of phosphorylation have been observed on Aurora B kinase substrates both before and after anaphase onset. In both instances the phosphorylation gradient appears to result from a soluble gradient of Aurora B kinase activity. Regulatory properties that support gradient formation are highlighted. Intracellular activity gradients that regulate localized mitotic events bare several hallmarks of self-organizing biologic systems that designate spatial information during pattern formation. Intracellular pattern formation represents a new paradigm in mitotic regulation.

  7. Renewing the Respect for Similarity

    Directory of Open Access Journals (Sweden)

    Shimon eEdelman

    2012-07-01

    Full Text Available In psychology, the concept of similarity has traditionally evoked a mixture of respect, stemmingfrom its ubiquity and intuitive appeal, and concern, due to its dependence on the framing of the problemat hand and on its context. We argue for a renewed focus on similarity as an explanatory concept, bysurveying established results and new developments in the theory and methods of similarity-preservingassociative lookup and dimensionality reduction — critical components of many cognitive functions, aswell as of intelligent data management in computer vision. We focus in particular on the growing familyof algorithms that support associative memory by performing hashing that respects local similarity, andon the uses of similarity in representing structured objects and scenes. Insofar as these similarity-basedideas and methods are useful in cognitive modeling and in AI applications, they should be included inthe core conceptual toolkit of computational neuroscience.

  8. Synthesis of Tumor-avid Photosensitizer-Gd(III)DTPA conjugates: impact of the number of gadolinium units in T1/T2 relaxivity, intracellular localization, and photosensitizing efficacy.

    Science.gov (United States)

    Goswami, Lalit N; White, William H; Spernyak, Joseph A; Ethirajan, Manivannan; Chen, Yihui; Missert, Joseph R; Morgan, Janet; Mazurchuk, Richard; Pandey, Ravindra K

    2010-05-19

    To develop novel bifunctional agents for tumor imaging (MR) and photodynamic therapy (PDT), certain tumor-avid photosensitizers derived from chlorophyll-a were conjugated with variable number of Gd(III)aminobenzyl DTPA moieties. All the conjugates containing three or six gadolinium units showed significant T(1) and T(2) relaxivities. However, as a bifunctional agent, the 3-(1'-hexyloxyethyl)pyropheophorbide-a (HPPH) containing 3Gd(III) aminophenyl DTPA was most promising with possible applications in tumor-imaging and PDT. Compared to HPPH, the corresponding 3- and 6Gd(III)aminobenzyl DTPA conjugates exhibited similar electronic absorption characteristics with a slightly decreased intensity of the absorption band at 660 nm. However, compared to HPPH, the excitation of the broad "Soret" band (near 400 nm) of the corresponding 3Gd(III)aminobenzyl-DTPA analogues showed a significant decrease in the fluorescence intensity at 667 nm.

  9. Electron Microscopy of Intracellular Protozoa

    Science.gov (United States)

    1988-12-20

    Classification) " ELECTRON MICROSCOPY OF INTRACELLULAR PROTOZOA 12. PERSONAL AUTHOR(S) Aikawa, Masamichi 13a. TYPE OF REPORT I13b. TIME COVERED 114...authors suggest that anti-CS protein antibody is important in reducing the prevalence of malaria with increasing age among persons in such areas and... Hygine 33, 220-226. 0Giudice, G.D., Engers, H.D., Tougne, C., Biro, S.S., Weiss, N., Verdini, A.S., Pessi, A., Degremont, A.A., Freyvogel, T.A., Lambert

  10. Intracellular Detection of Viral Transcription and Replication Using RNA FISH

    Science.gov (United States)

    2016-05-26

    Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses...only allow entirely new investigations into the replication of these viruses, but also how this method can be applied to any virus with a known...localization, TurboFISH, hemorrhagic fever virus replication 1. Introduction RNA FISH was developed as a method to visualize cellular RNA by binding a

  11. New Similarity Functions

    DEFF Research Database (Denmark)

    Yazdani, Hossein; Ortiz-Arroyo, Daniel; Kwasnicka, Halina

    2016-01-01

    spaces, in addition to their similarity in the vector space. Prioritized Weighted Feature Distance (PWFD) works similarly as WFD, but provides the ability to give priorities to desirable features. The accuracy of the proposed functions are compared with other similarity functions on several data sets....... Our results show that the proposed functions work better than other methods proposed in the literature....

  12. Phoneme Similarity and Confusability

    Science.gov (United States)

    Bailey, T.M.; Hahn, U.

    2005-01-01

    Similarity between component speech sounds influences language processing in numerous ways. Explanation and detailed prediction of linguistic performance consequently requires an understanding of these basic similarities. The research reported in this paper contrasts two broad classes of approach to the issue of phoneme similarity-theoretically…

  13. MR imaging of intracellular and extracellular deoxyhemoglobin

    International Nuclear Information System (INIS)

    Janick, P.A.; Grossman, R.I.; Asakura, T.

    1989-01-01

    MR imaging was performed on varying concentrations of intracellular and extracellular deoxyhemoglobin as well as varying proportions of deoxyhemoglobin and oxyhemoglobin in vitro at 1.5T with use of standard spin-echo and gradient-refocused spin sequences. This study indicates that susceptibility-induced T2 shortening occurs over a broad range of intracellular deoxyhemoglobin concentrations (maximal at hematocrits between 20% and 45%), reflecting diffusional effects at the cellular level. T2* gradient-echo imaging enhances the observed hypointensity in images of intracellular deoxyhemoglobin. The characteristic MR appearance of acute hemotomas can be modeled by the behavior of intracellular and extracellular deoxyhemoglobin and oxyhemoglobin

  14. Intracellular localization of hydroxyproline-rich glycoprotein biosynthesis

    International Nuclear Information System (INIS)

    Robinson, D.G.; Andreae, M.; Glas, A.R.; Sauer, A.

    1984-01-01

    The structural proteins of plant cell walls are glycoproteins characterized by O-glucosidic linkages to hydroxyproline or serine. Proline, not hydroxyproline, is the translatable amino acid in hydroxyproline-rich glycoproteins (HRGP). Hydroxylation and arabinosylation of proline are sequential, post-translational events. Because of this, there is no a priori reason for expecting HRGP synthesis to follow the well-established route for secretory and plasma membrane (PM) glycoproteins, i.e., from endoplasmic reticulum (ER) via the Golgi apparatus (GA) to the PM. In this paper, two plausible alternatives for HRGO secretion are examined. Because a feature of the majority of dicotyledons is overlapping GA and PM regions in sucrose density gradients, the authors have used two monocotyledonous systems to determine the distribution of HRGP and enzyme activity

  15. Antibody- and TRIM21-dependent intracellular restriction of Salmonella enterica.

    Science.gov (United States)

    Rakebrandt, Nikolas; Lentes, Sabine; Neumann, Heinz; James, Leo C; Neumann-Staubitz, Petra

    2014-11-01

    TRIM21 ('tripartite motif-containing protein 21', Ro52) is a ubiquitously expressed cytosolic Fc receptor, which has a potent role in protective immunity against nonenveloped viruses. TRIM21 mediates intracellular neutralisation of antibody-coated viruses, a process called ADIN (antibody-dependent intracellular neutralisation). Our results reveal a similar mechanism to fight bacterial infections. TRIM21 is recruited to the intracellular pathogen Salmonella enterica in epithelial cells early in infection. TRIM21 does not bind directly to S. enterica, but to antibodies opsonising it. Most importantly, bacterial restriction is dependent on TRIM21 as well as on the opsonisation state of the bacteria. Finally, Salmonella and TRIM21 colocalise with the autophagosomal marker LC3, and intracellular defence is enhanced in starved cells suggesting an involvement of the autophagocytic pathway. Our data extend the protective role of TRIM21 from viruses to bacteria and thereby strengthening the general role of ADIN in cellular immunity. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  16. Growth-dependent catalase localization in Exiguobacterium oxidotolerans T-2-2T reflected by catalase activity of cells.

    Science.gov (United States)

    Hanaoka, Yoshiko; Takebe, Fumihiko; Nodasaka, Yoshinobu; Hara, Isao; Matsuyama, Hidetoshi; Yumoto, Isao

    2013-01-01

    A psychrotolerant and H2O2-resistant bacterium, Exiguobacterium oxidotolerans T-2-2(T), exhibits extraordinary H2O2 resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state.

  17. Growth-Dependent Catalase Localization in Exiguobacterium oxidotolerans T-2-2T Reflected by Catalase Activity of Cells

    Science.gov (United States)

    Hanaoka, Yoshiko; Takebe, Fumihiko; Nodasaka, Yoshinobu; Hara, Isao; Matsuyama, Hidetoshi; Yumoto, Isao

    2013-01-01

    A psychrotolerant and H2O2-resistant bacterium, Exiguobacterium oxidotolerans T-2-2T, exhibits extraordinary H2O2 resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state. PMID:24204687

  18. Molecular similarity measures.

    Science.gov (United States)

    Maggiora, Gerald M; Shanmugasundaram, Veerabahu

    2011-01-01

    Molecular similarity is a pervasive concept in chemistry. It is essential to many aspects of chemical reasoning and analysis and is perhaps the fundamental assumption underlying medicinal chemistry. Dissimilarity, the complement of similarity, also plays a major role in a growing number of applications of molecular diversity in combinatorial chemistry, high-throughput screening, and related fields. How molecular information is represented, called the representation problem, is important to the type of molecular similarity analysis (MSA) that can be carried out in any given situation. In this work, four types of mathematical structure are used to represent molecular information: sets, graphs, vectors, and functions. Molecular similarity is a pairwise relationship that induces structure into sets of molecules, giving rise to the concept of chemical space. Although all three concepts - molecular similarity, molecular representation, and chemical space - are treated in this chapter, the emphasis is on molecular similarity measures. Similarity measures, also called similarity coefficients or indices, are functions that map pairs of compatible molecular representations that are of the same mathematical form into real numbers usually, but not always, lying on the unit interval. This chapter presents a somewhat pedagogical discussion of many types of molecular similarity measures, their strengths and limitations, and their relationship to one another. An expanded account of the material on chemical spaces presented in the first edition of this book is also provided. It includes a discussion of the topography of activity landscapes and the role that activity cliffs in these landscapes play in structure-activity studies.

  19. Dynamics of intracellular information decoding

    International Nuclear Information System (INIS)

    Kobayashi, Tetsuya J; Kamimura, Atsushi

    2011-01-01

    A variety of cellular functions are robust even to substantial intrinsic and extrinsic noise in intracellular reactions and the environment that could be strong enough to impair or limit them. In particular, of substantial importance is cellular decision-making in which a cell chooses a fate or behavior on the basis of information conveyed in noisy external signals. For robust decoding, the crucial step is filtering out the noise inevitably added during information transmission. As a minimal and optimal implementation of such an information decoding process, the autocatalytic phosphorylation and autocatalytic dephosphorylation (aPadP) cycle was recently proposed. Here, we analyze the dynamical properties of the aPadP cycle in detail. We describe the dynamical roles of the stationary and short-term responses in determining the efficiency of information decoding and clarify the optimality of the threshold value of the stationary response and its information-theoretical meaning. Furthermore, we investigate the robustness of the aPadP cycle against the receptor inactivation time and intrinsic noise. Finally, we discuss the relationship among information decoding with information-dependent actions, bet-hedging and network modularity

  20. Dynamics of intracellular information decoding.

    Science.gov (United States)

    Kobayashi, Tetsuya J; Kamimura, Atsushi

    2011-10-01

    A variety of cellular functions are robust even to substantial intrinsic and extrinsic noise in intracellular reactions and the environment that could be strong enough to impair or limit them. In particular, of substantial importance is cellular decision-making in which a cell chooses a fate or behavior on the basis of information conveyed in noisy external signals. For robust decoding, the crucial step is filtering out the noise inevitably added during information transmission. As a minimal and optimal implementation of such an information decoding process, the autocatalytic phosphorylation and autocatalytic dephosphorylation (aPadP) cycle was recently proposed. Here, we analyze the dynamical properties of the aPadP cycle in detail. We describe the dynamical roles of the stationary and short-term responses in determining the efficiency of information decoding and clarify the optimality of the threshold value of the stationary response and its information-theoretical meaning. Furthermore, we investigate the robustness of the aPadP cycle against the receptor inactivation time and intrinsic noise. Finally, we discuss the relationship among information decoding with information-dependent actions, bet-hedging and network modularity.

  1. Secretome of obligate intracellular Rickettsia

    Science.gov (United States)

    Gillespie, Joseph J.; Kaur, Simran J.; Rahman, M. Sayeedur; Rennoll-Bankert, Kristen; Sears, Khandra T.; Beier-Sexton, Magda; Azad, Abdu F.

    2014-01-01

    The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’. PMID:25168200

  2. HYPERTHERMIA, INTRACELLULAR FREE CALCIUM AND CALCIUM IONOPHORES

    NARCIS (Netherlands)

    STEGE, GJJ; WIERENGA, PK; KAMPINGA, HH; KONINGS, AWT

    1993-01-01

    It is shown that heat-induced increase of intracellular calcium does not correlate with hyperthermic cell killing. Six different cell lines were investigated; in four (EAT, HeLa S3, L5178Y-R and L5178Y-S) heat treatments killing 90% of the cells did not affect the levels of intracellular free

  3. Similarity Measure of Graphs

    Directory of Open Access Journals (Sweden)

    Amine Labriji

    2017-07-01

    Full Text Available The topic of identifying the similarity of graphs was considered as highly recommended research field in the Web semantic, artificial intelligence, the shape recognition and information research. One of the fundamental problems of graph databases is finding similar graphs to a graph query. Existing approaches dealing with this problem are usually based on the nodes and arcs of the two graphs, regardless of parental semantic links. For instance, a common connection is not identified as being part of the similarity of two graphs in cases like two graphs without common concepts, the measure of similarity based on the union of two graphs, or the one based on the notion of maximum common sub-graph (SCM, or the distance of edition of graphs. This leads to an inadequate situation in the context of information research. To overcome this problem, we suggest a new measure of similarity between graphs, based on the similarity measure of Wu and Palmer. We have shown that this new measure satisfies the properties of a measure of similarities and we applied this new measure on examples. The results show that our measure provides a run time with a gain of time compared to existing approaches. In addition, we compared the relevance of the similarity values obtained, it appears that this new graphs measure is advantageous and  offers a contribution to solving the problem mentioned above.

  4. Processes of Similarity Judgment

    Science.gov (United States)

    Larkey, Levi B.; Markman, Arthur B.

    2005-01-01

    Similarity underlies fundamental cognitive capabilities such as memory, categorization, decision making, problem solving, and reasoning. Although recent approaches to similarity appreciate the structure of mental representations, they differ in the processes posited to operate over these representations. We present an experiment that…

  5. Judgments of brand similarity

    NARCIS (Netherlands)

    Bijmolt, THA; Wedel, M; Pieters, RGM; DeSarbo, WS

    This paper provides empirical insight into the way consumers make pairwise similarity judgments between brands, and how familiarity with the brands, serial position of the pair in a sequence, and the presentation format affect these judgments. Within the similarity judgment process both the

  6. Endothelial remodelling and intracellular calcium machinery.

    Science.gov (United States)

    Moccia, F; Tanzi, F; Munaron, L

    2014-05-01

    Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca(2+) signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca(2+) response to injury are yet to be fully elucidated. Similarly, the components of the Ca(2+) toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca(2+) signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca(2+) channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca(2+) channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca(2+) entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

  7. Intracellular calcium homeostasis and signaling.

    Science.gov (United States)

    Brini, Marisa; Calì, Tito; Ottolini, Denis; Carafoli, Ernesto

    2013-01-01

    Ca(2+) is a universal carrier of biological information: it controls cell life from its origin at fertilization to its end in the process of programmed cell death. Ca(2+) is a conventional diffusible second messenger released inside cells by the interaction of first messengers with plasma membrane receptors. However, it can also penetrate directly into cells to deliver information without the intermediation of first or second messengers. Even more distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma membrane receptor to set in motion intracellular signaling pathways that involve Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes an agent that mediates cell distress, or even (toxic) cell death, if its concentration and movements inside cells are not carefully tuned. Ca(2+) is controlled by reversible complexation to specific proteins, which could be pure Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its signal to pass it on to targets. The most important actors in the buffering of cell Ca(2+) are proteins that transport it across the plasma membrane and the membrane of the organelles: some have high Ca(2+) affinity and low transport capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+) uptake system of mitochondria). Between the initial event of fertilization, and the terminal event of programmed cell death, the Ca(2+) signal regulates the most important activities of the cell, from the expression of genes, to heart and muscle contraction and other motility processes, to diverse metabolic pathways involved in the generation of cell fuels.

  8. The semantic similarity ensemble

    Directory of Open Access Journals (Sweden)

    Andrea Ballatore

    2013-12-01

    Full Text Available Computational measures of semantic similarity between geographic terms provide valuable support across geographic information retrieval, data mining, and information integration. To date, a wide variety of approaches to geo-semantic similarity have been devised. A judgment of similarity is not intrinsically right or wrong, but obtains a certain degree of cognitive plausibility, depending on how closely it mimics human behavior. Thus selecting the most appropriate measure for a specific task is a significant challenge. To address this issue, we make an analogy between computational similarity measures and soliciting domain expert opinions, which incorporate a subjective set of beliefs, perceptions, hypotheses, and epistemic biases. Following this analogy, we define the semantic similarity ensemble (SSE as a composition of different similarity measures, acting as a panel of experts having to reach a decision on the semantic similarity of a set of geographic terms. The approach is evaluated in comparison to human judgments, and results indicate that an SSE performs better than the average of its parts. Although the best member tends to outperform the ensemble, all ensembles outperform the average performance of each ensemble's member. Hence, in contexts where the best measure is unknown, the ensemble provides a more cognitively plausible approach.

  9. Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport

    Directory of Open Access Journals (Sweden)

    Tjeldhorn Lena

    2010-09-01

    Full Text Available Abstract Background Activated protein C (PC is a serine protease that regulates blood coagulation by inactivating coagulation factors Va and VIIIa. PC deficiency is an autosomally inherited disorder associated with a high risk of recurrent venous thrombosis. The aim of the study was to explore the mechanisms responsible for severe PC deficiency in a patient with the protein C A267T mutation by in-vitro expression studies. Results Huh7 and CHO-K1 cells were transiently transfected with expression vectors containing wild-type (WT PC and mutated PC (A267T PC cDNAs. PC mRNA levels were assessed by qRT-PCR and the PC protein levels were measured by ELISA. The mRNA levels of WT PC and A267T PC were similar, while the intracellular protein level of A267T PC was moderately decreased compared to WT PC. The secretion of A267T PC into the medium was severely impaired. No differences in molecular weights were observed between WT and A267T PC before and after treatment with endo-β-N-acetylglucosaminidase. Proteasomal and lysosomal degradations were examined using lactacystin and bafilomycin, respectively, and revealed that A267T PC was slightly more susceptible for proteasomal degradation than WT PC. Intracellular co-localization analysis indicated that A267T PC was mainly located in the endoplasmic reticulum (ER, whereas WT PC was observed in both ER and Golgi. Conclusions In contrast to what has been reported for other PC mutants, intracellular degradation of A267T PC was not the main/dominant mechanism underlying the reduced intracellular and secretion levels of PC. Our results indicate that the A267T mutation most likely caused misfolding of PC, which might lead to increased retention of the mutated PC in ER.

  10. Gender similarities and differences.

    Science.gov (United States)

    Hyde, Janet Shibley

    2014-01-01

    Whether men and women are fundamentally different or similar has been debated for more than a century. This review summarizes major theories designed to explain gender differences: evolutionary theories, cognitive social learning theory, sociocultural theory, and expectancy-value theory. The gender similarities hypothesis raises the possibility of theorizing gender similarities. Statistical methods for the analysis of gender differences and similarities are reviewed, including effect sizes, meta-analysis, taxometric analysis, and equivalence testing. Then, relying mainly on evidence from meta-analyses, gender differences are reviewed in cognitive performance (e.g., math performance), personality and social behaviors (e.g., temperament, emotions, aggression, and leadership), and psychological well-being. The evidence on gender differences in variance is summarized. The final sections explore applications of intersectionality and directions for future research.

  11. Ortholog-based screening and identification of genes related to intracellular survival.

    Science.gov (United States)

    Yang, Xiaowen; Wang, Jiawei; Bing, Guoxia; Bie, Pengfei; De, Yanyan; Lyu, Yanli; Wu, Qingmin

    2018-04-20

    Bioinformatics and comparative genomics analysis methods were used to predict unknown pathogen genes based on homology with identified or functionally clustered genes. In this study, the genes of common pathogens were analyzed to screen and identify genes associated with intracellular survival through sequence similarity, phylogenetic tree analysis and the λ-Red recombination system test method. The total 38,952 protein-coding genes of common pathogens were divided into 19,775 clusters. As demonstrated through a COG analysis, information storage and processing genes might play an important role intracellular survival. Only 19 clusters were present in facultative intracellular pathogens, and not all were present in extracellular pathogens. Construction of a phylogenetic tree selected 18 of these 19 clusters. Comparisons with the DEG database and previous research revealed that seven other clusters are considered essential gene clusters and that seven other clusters are associated with intracellular survival. Moreover, this study confirmed that clusters screened by orthologs with similar function could be replaced with an approved uvrY gene and its orthologs, and the results revealed that the usg gene is associated with intracellular survival. The study improves the current understanding of intracellular pathogens characteristics and allows further exploration of the intracellular survival-related gene modules in these pathogens. Copyright © 2018. Published by Elsevier B.V.

  12. Pico gauges for minimally invasive intracellular hydrostatic pressure measurements

    DEFF Research Database (Denmark)

    Knoblauch, Jan; Mullendore, Daniel L.; Jensen, Kaare Hartvig

    2014-01-01

    Intracellular pressure has a multitude of functions in cells surrounded by a cell wall or similar matrix in all kingdoms of life. The functions include cell growth, nastic movements, and penetration of tissue by parasites. The precise measurement of intracellular pressure in the majority of cells......, however, remains difficult or impossible due to their small size and/or sensitivity to manipulation. Here, we report on a method that allows precise measurements in basically any cell type over all ranges of pressure. It is based on the compression of nanoliter and picoliter volumes of oil entrapped...... in the tip of microcapillaries, which we call pico gauges. The production of pico gauges can be accomplished with standard laboratory equipment, and measurements are comparably easy to conduct. Example pressure measurements are performed on cells that are difficult or impossible to measure with other methods....

  13. Similarity or difference?

    DEFF Research Database (Denmark)

    Villadsen, Anders Ryom

    2013-01-01

    While the organizational structures and strategies of public organizations have attracted substantial research attention among public management scholars, little research has explored how these organizational core dimensions are interconnected and influenced by pressures for similarity....... In this paper I address this topic by exploring the relation between expenditure strategy isomorphism and structure isomorphism in Danish municipalities. Different literatures suggest that organizations exist in concurrent pressures for being similar to and different from other organizations in their field......-shaped relation exists between expenditure strategy isomorphism and structure isomorphism in a longitudinal quantitative study of Danish municipalities....

  14. Nanoparticles for intracellular-targeted drug delivery

    International Nuclear Information System (INIS)

    Paulo, Cristiana S O; Pires das Neves, Ricardo; Ferreira, Lino S

    2011-01-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  15. Biological synthesis and characterization of intracellular gold ...

    Indian Academy of Sciences (India)

    thods of reduction of metal ions using plants or microorganisms are often ... have several advantages over bacteria, they are often pre- ferred. ... in static condition for a period of 7 days. ... work was focused on the production of intracellular gold.

  16. Mycobacterium intracellulare Infection Mimicking Progression of Scleroderma

    DEFF Research Database (Denmark)

    Krabbe, Simon; Engelhart, Merete; Thybo, Sören

    2017-01-01

    This case report describes a patient with scleroderma who developed Mycobacterium intracellulare infection, which for more than a year mimicked worsening of her connective tissue disorder. The patient was diagnosed with scleroderma based on puffy fingers that developed into sclerodactyly, abnormal......, unfortunately with significant scarring. Immunodeficiency testing was unremarkable. In summary, an infection with Mycobacterium intracellulare was mistaken for an unusually severe progression of scleroderma....

  17. Comparing Harmonic Similarity Measures

    NARCIS (Netherlands)

    de Haas, W.B.; Robine, M.; Hanna, P.; Veltkamp, R.C.; Wiering, F.

    2010-01-01

    We present an overview of the most recent developments in polyphonic music retrieval and an experiment in which we compare two harmonic similarity measures. In contrast to earlier work, in this paper we specifically focus on the symbolic chord description as the primary musical representation and

  18. New intracellular activities of matrix metalloproteinases shine in the moonlight.

    Science.gov (United States)

    Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

    2017-11-01

    Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Intracellular Hg(0) Oxidation in Desulfovibrio desulfuricans ND132.

    Science.gov (United States)

    Wang, Yuwei; Schaefer, Jeffra K; Mishra, Bhoopesh; Yee, Nathan

    2016-10-03

    The disposal of elemental mercury (Hg(0)) wastes in mining and manufacturing areas has caused serious soil and groundwater contamination issues. Under anoxic conditions, certain anaerobic bacteria can oxidize dissolved elemental mercury and convert the oxidized Hg to neurotoxic methylmercury. In this study, we conducted experiments with the Hg-methylating bacterium Desulfovibrio desulfuricans ND132 to elucidate the role of cellular thiols in anaerobic Hg(0) oxidation. The concentrations of cell-surface and intracellular thiols were measured, and specific fractions of D. desulfuricans ND132 were examined for Hg(0) oxidation activity and analyzed with extended X-ray absorption fine structure (EXAFS) spectroscopy. The experimental data indicate that intracellular thiol concentrations are approximately six times higher than those of the cell wall. Cells reacted with a thiol-blocking reagent were severely impaired in Hg(0) oxidation activity. Spheroplasts lacking cell walls rapidly oxidized Hg(0) to Hg(II), while cell wall fragments exhibited low reactivity toward Hg(0). EXAFS analysis of spheroplast samples revealed that multiple different forms of Hg-thiols are produced by the Hg(0) oxidation reaction and that the local coordination environment of the oxidized Hg changes with reaction time. The results of this study indicate that Hg(0) oxidation in D. desulfuricans ND132 is an intracellular process that occurs by reaction with thiol-containing molecules.

  20. Intracellular distribution of 57Bleomycin

    International Nuclear Information System (INIS)

    Konings, A.W.T.; Rasker, J.J.; Posthuma, G.A.

    1978-01-01

    Since the first promising results of Nouel et al. 1972, additional positive experience has been obtained with 57 Co-Bleomycin ( 57 Co-BLM) as a tumour-localizing agent. In this preclinical study, mice with transplanted osteosarcoma and lymphosarcoma were used and rats with transplanted rhabdomyosarcoma. 57 CoCl 2 served as a control substance. 57 Co-BLM had concentrated in the tumours with a factor 2 to 10 as compared to the (normal) liver of the animals. No preferential concentration in the tumours was found when 57 CoCl 2 was used. The highest specific activity of 57 Co-BLM (cpm/mg protein) was found in a fraction containing mitochondria and lysosomes. Evidence for a lysosomal localization of this diagnostic compound was obtained from experiments in which the mitochondrial-lysosomal fraction was treated with hypertonic media of different osmolarities. Conditions could be found in which many lysosomes burst while almost all mitochondria were intact. From these experiments it appeared that the radioactivity in the particles obtained from animals injected with 57 Co-BLM was released very rapidly. (Auth.)

  1. Heavy metals toxicity after acute exposure of cultured renal cells. Intracellular accumulation and repartition

    International Nuclear Information System (INIS)

    Khodja, Hicham; Carriere, Marie; Avoscan, Laure; Gouget, Barbara

    2005-01-01

    Lead (Pb), cadmium (Cd) and uranium (U) present no known biological function but are toxic in various concentration ranges. Pb and Cd lead generally to nephrotoxicity consisting in proximal renal tubular dysfunction and accumulation while U has been reported to induce chemical kidney toxicity, functional and histological damages being as well mainly observed in proximal tubule cells. This work address the question of Cd, Pb, and U cytotoxicity, intracellular accumulation and repartition after acute intoxication of renal proximal tubule epithelial cells. After cells exposure to different concentrations of metals for various times, morphological changes were observed and intracellular concentrations and distributions of toxic metals were specified by PIXE coupled to RBS. Cell viability, measured by biochemical tests, was used as toxicity indicator. A direct correlation between cytotoxicity and intracellular accumulation in renal epithelial cells have been established. Finally, intracellular Pb and U localizations were detected while Cd was found to be uniformly distributed in renal cells. (author)

  2. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

    Directory of Open Access Journals (Sweden)

    Onkar Sharma

    2016-03-01

    Full Text Available A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase. When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO, and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells.

  3. Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization

    International Nuclear Information System (INIS)

    Lawrence, J.B.; Singer, R.H.; Villnave, C.A.; Stein, J.L.; Stein, G.S.

    1988-01-01

    We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs

  4. INTRACELLULAR SYNTHESIS OF CHONDROITIN SULFATE

    Science.gov (United States)

    Dziewiatkowski, Dominic D.

    1962-01-01

    In autoradiograms of slices of costal cartilage, incubated for 4 hours in a salt solution containing S35-sulfate and then washed extensively and dehydrated, about 85 per cent of the radioactivity was assignable to the chondrocytes. From alkaline extracts of similarly prepared slices of cartilage, 64 to 83 per cent of the total sulfur-35 in the slices was isolated as chondroitin sulfate by chromatography on an anion-exchange resin. In view of the estimate that only about 15 per cent of the radioactivity was in the matrix, the isolation of 64 to 83 per cent of the total sulfur-35 as chondroitin sulfate is a strong argument that the chondrocytes are the loci in which chondroitin sulfate(s) is synthesized. PMID:13888910

  5. Efficient intracellular delivery and improved biocompatibility of colloidal silver nanoparticles towards intracellular SERS immuno-sensing.

    Science.gov (United States)

    Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J

    2015-06-21

    High throughput intracellular delivery strategies, electroporation, passive and TATHA2 facilitated diffusion of colloidal silver nanoparticles (AgNPs) are investigated for cellular toxicity and uptake using state-of-art analytical techniques. The TATHA2 facilitated approach efficiently delivered high payload with no toxicity, pre-requisites for intracellular applications of plasmonic metal nanoparticles (PMNPs) in sensing and therapeutics.

  6. Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

    Directory of Open Access Journals (Sweden)

    Birkedal Rikke

    2009-12-01

    Full Text Available Abstract Background Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss, which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine. Results Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. Conclusions The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that

  7. Investigating Internalization and Intracellular Trafficking of GPCRs

    DEFF Research Database (Denmark)

    Foster, Simon R; Bräuner-Osborne, Hans

    2017-01-01

    for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal...... independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies...

  8. Similar or different?

    DEFF Research Database (Denmark)

    Cornér, Solveig; Pyhältö, Kirsi; Peltonen, Jouni

    2018-01-01

    Previous research has identified researcher community and supervisory support as key determinants of the doctoral journey contributing to students’ persistence and robustness. However, we still know little about cross-cultural variation in the researcher community and supervisory support experien...... counter partners, whereas the Finnish students perceived lower levels of instrumental support than the Danish students. The findings imply that seemingly similar contexts hold valid differences in experienced social support and educational strategies at the PhD level....... experienced by PhD students within the same discipline. This study explores the support experiences of 381 PhD students within the humanities and social sciences from three research-intensive universities in Denmark (n=145) and Finland (n=236). The mixed methods design was utilized. The data were collected...... counter partners. The results also indicated that the only form of support in which the students expressed more matched support than mismatched support was informational support. Further investigation showed that the Danish students reported a high level of mismatch in emotional support than their Finnish...

  9. Intracellular calcium signals display an avalanche-like behavior over multiple lengthscales.

    Directory of Open Access Journals (Sweden)

    Lucía eLopez

    2012-09-01

    Full Text Available Many natural phenomena display "self-organized criticality'' (SOC. This refers to spatially extended systems for which patterns of activity characterized by different lengthscales can occur with a probability density that follows a power law with pattern size. Differently from power laws at phase transitions, systems displaying SOC do not need the tuning of an external parameter. Here we analyze intracellular calcium Ca2+ signals, a key component of the signaling toolkit of almost any cell type. Ca2+ signals can either be spatially restricted (local or propagate throughout the cell (global. Different models have suggested that the transition from local to global signals is similar to that of directed percolation. Directed percolation has been associated, in turn, to the appearance of self-organized criticality. In this paper we discuss these issues within the framework of simple models of Ca2+ signal propagation. We also analyze the size distribution of local signals ("puffs'' observed in immature Xenopus Laevis oocytes. The puff amplitude distribution obtained from observed local signals is not Gaussian with a noticeable fraction of large size events. The experimental distribution of puff areas in the spatio-temporal record of the image has a long tail that is approximately log-normal. The distribution can also be fitted with a power law relationship albeit with a smaller goodness of fit. The power law behavior is encountered within a simple model that includes some coupling among individual signals for a wide range of parameter values. An analysis of the model shows that a global elevation of the Ca2+ concentration plays a major role in determining whether the puff size distribution is long-tailed or not. This suggests that Ca2+-clearing from the cytosol is key to determine whether IP3-mediated Ca2+ signals can display a SOC-like behavior or not.

  10. Brucella abortus nicotinamidase (PncA) contributes to its intracellular replication and infectivity in mice.

    Science.gov (United States)

    Kim, Suk; Kurokawa, Daisuke; Watanabe, Kenta; Makino, Sou-Ichi; Shirahata, Toshikazu; Watarai, Masahisa

    2004-05-15

    Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. Nicotinamidase/pyrazinamidase mutant (pncA mutant) of Brucella abortus failed to replicate in HeLa cells, and showed a lower rate of intracellular replication than that of wild-type strain in macrophages. Addition of nicotinic acid, but not nicotinamide, into medium supported intracellular replication of pncA mutant in HeLa cells and macrophages. The pncA mutant was not co-localizing with either late endosomes or lysosomes. The B. abortus virB4 mutant was completely cleared from the spleens of mice after 4 weeks, while the pncA mutant showed a 1.5-log reduction of the number of bacteria isolated from spleens after 10 weeks. Although pncA mutant showed reduced virulence in mice and defective intracellular replication, its ability to confer protection against the virulent B. abortus strain 544 was fully retained. These results suggest that PncA does not contribute to intracellular trafficking of B. abortus, but contributes to utilization of nutrients required for intracellular growth. Our results indicate that detailed characterizations of the pncA mutant may help the improvement of currently available live vaccines. Copyright 2004 Federation of European Microbiological Societies

  11. Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

    Science.gov (United States)

    García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

    2013-01-01

    Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

  12. Cationic polymers for intracellular delivery of proteins

    NARCIS (Netherlands)

    Coué, G.M.J.P.C.; Engbersen, Johannes F.J.; Samal, Sangram; Dubruel, Peter

    2015-01-01

    Many therapeutic proteins exert their pharmaceutical action inside the cytoplasm or onto individual organelles inside the cell. Intracellular protein delivery is considered to be the most direct, fastest and safest approach for curing gene-deficiency diseases, enhancing vaccination and triggering

  13. Molecular detection and characterization of sustainable intracellular ...

    African Journals Online (AJOL)

    3Centre for Biopolymer and Bio-Molecular Research, Athlone College of Technology, Republic of Ireland. ... cells was associated with the elongation of micro-villar extension that ... Keywords: Intracellular contaminants, cell cultures, bacteria culture, pre-clinical studies. ... production work involving culture technology.

  14. Spatial Cytoskeleton Organization Supports Targeted Intracellular Transport

    Science.gov (United States)

    Hafner, Anne E.; Rieger, Heiko

    2018-03-01

    The efficiency of intracellular cargo transport from specific source to target locations is strongly dependent upon molecular motor-assisted motion along the cytoskeleton. Radial transport along microtubules and lateral transport along the filaments of the actin cortex underneath the cell membrane are characteristic for cells with a centrosome. The interplay between the specific cytoskeleton organization and the motor performance realizes a spatially inhomogeneous intermittent search strategy. In order to analyze the efficiency of such intracellular search strategies we formulate a random velocity model with intermittent arrest states. We evaluate efficiency in terms of mean first passage times for three different, frequently encountered intracellular transport tasks: i) the narrow escape problem, which emerges during cargo transport to a synapse or other specific region of the cell membrane, ii) the reaction problem, which considers the binding time of two particles within the cell, and iii) the reaction-escape problem, which arises when cargo must be released at a synapse only after pairing with another particle. Our results indicate that cells are able to realize efficient search strategies for various intracellular transport tasks economically through a spatial cytoskeleton organization that involves only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments.

  15. Biological synthesis and characterization of intracellular gold ...

    Indian Academy of Sciences (India)

    In the present study, Aspergillus fumigatus was used for the intracellular synthesis of gold nanoparticles. Stable nanoparticles were produced when an aqueous solution of chloroauric acid (HAuCl4) was reduced by A. fumigatus biomass as the reducing agent. Production of nanoparticles was confirmed by the colour ...

  16. Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

    Science.gov (United States)

    Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun

    2018-03-20

    Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane

  17. Dysfunction of bovine endogenous retrovirus K2 envelope glycoprotein is related to unsuccessful intracellular trafficking.

    Science.gov (United States)

    Nakaya, Yuki; Miyazawa, Takayuki

    2014-06-01

    Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs. Retroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition of the entry

  18. Intracellular Zn(2+) signaling in the dentate gyrus is required for object recognition memory.

    Science.gov (United States)

    Takeda, Atsushi; Tamano, Haruna; Ogawa, Taisuke; Takada, Shunsuke; Nakamura, Masatoshi; Fujii, Hiroaki; Ando, Masaki

    2014-11-01

    The role of perforant pathway-dentate granule cell synapses in cognitive behavior was examined focusing on synaptic Zn(2+) signaling in the dentate gyrus. Object recognition memory was transiently impaired when extracellular Zn(2+) levels were decreased by injection of clioquinol and N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylendediamine. To pursue the effect of the loss and/or blockade of Zn(2+) signaling in dentate granule cells, ZnAF-2DA (100 pmol, 0.1 mM/1 µl), an intracellular Zn(2+) chelator, was locally injected into the dentate molecular layer of rats. ZnAF-2DA injection, which was estimated to chelate intracellular Zn(2+) signaling only in the dentate gyrus, affected object recognition memory 1 h after training without affecting intracellular Ca(2+) signaling in the dentate molecular layer. In vivo dentate gyrus long-term potentiation (LTP) was affected under the local perfusion of the recording region (the dentate granule cell layer) with 0.1 mM ZnAF-2DA, but not with 1-10 mM CaEDTA, an extracellular Zn(2+) chelator, suggesting that the blockade of intracellular Zn(2+) signaling in dentate granule cells affects dentate gyrus LTP. The present study demonstrates that intracellular Zn(2+) signaling in the dentate gyrus is required for object recognition memory, probably via dentate gyrus LTP expression. Copyright © 2014 Wiley Periodicals, Inc.

  19. Detection of ubiquitinated huntingtin species in intracellular aggregates

    Directory of Open Access Journals (Sweden)

    Katrin eJuenemann

    2015-01-01

    Full Text Available Protein conformation diseases, including polyglutamine diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease is one of nine diseases caused by an expanded polyglutamine repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated huntingtin such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated huntingtin fragments offers an important possibility to understand huntingtin degradation and aggregation processes within the cell. For the identification of aggregated huntingtin and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant huntingtin. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

  20. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    Energy Technology Data Exchange (ETDEWEB)

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Fujita, Norihisa, E-mail: nori@ph.ritsumei.ac.jp [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); School of Pharmacy, Ristumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  1. Leishmania hijacking of the macrophage intracellular compartments.

    Science.gov (United States)

    Liévin-Le Moal, Vanessa; Loiseau, Philippe M

    2016-02-01

    Leishmania spp., transmitted to humans by the bite of the sandfly vector, are responsible for the three major forms of leishmaniasis, cutaneous, diffuse mucocutaneous and visceral. Leishmania spp. interact with membrane receptors of neutrophils and macrophages. In macrophages, the parasite is internalized within a parasitophorous vacuole and engages in a particular intracellular lifestyle in which the flagellated, motile Leishmania promastigote metacyclic form differentiates into non-motile, metacyclic amastigote form. This phenomenon is induced by Leishmania-triggered events leading to the fusion of the parasitophorous vacuole with vesicular members of the host cell endocytic pathway including recycling endosomes, late endosomes and the endoplasmic reticulum. Maturation of the parasitophorous vacuole leads to the intracellular proliferation of the Leishmania amastigote forms by acquisition of host cell nutrients while escaping host defense responses. © 2015 FEBS.

  2. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-05-01

    The intracellular glutathione (GSH) content in HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulfoximine (BSO) or diethyl maleate (DEM). Clonogenicity, single strand DNA breaks (ssb) and double strand DNA breaks (dsb) were used as criteria for radiation induced damage after X- or γ irradiation. In survival experiments DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the OER was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (Auth.)

  3. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-01-01

    The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or γ-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (author)

  4. Intracellular mechanisms of solar water disinfection

    Science.gov (United States)

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  5. Intracellular serpins, firewalls and tissue necrosis.

    Science.gov (United States)

    Marciniak, Stefan J; Lomas, David A

    2008-02-01

    Luke and colleagues have recently attributed a new role to a member of the serpin superfamily of serine proteinase inhibitors. They have used Caenorhabditis elegans to show that an intracellular serpin is crucial for maintaining lysosomal integrity. We examine the role of this firewall in preventing necrosis and attempt to integrate this with current theories of stress-induced protein degradation. We discuss how mutant serpins cause disease either through polymerization or now, perhaps, by unleashing necrosis.

  6. Fluorescent nanoparticles for intracellular sensing: A review

    International Nuclear Information System (INIS)

    Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.

    2012-01-01

    Highlights: ► Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. ► Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. ► Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  7. Fluorescent nanoparticles for intracellular sensing: A review

    Energy Technology Data Exchange (ETDEWEB)

    Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  8. Intracellular Cholesterol Trafficking and Impact in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Fabian Arenas

    2017-11-01

    Full Text Available Cholesterol is a critical component of membrane bilayers where it plays key structural and functional roles by regulating the activity of diverse signaling platforms and pathways. Particularly enriched in brain, cholesterol homeostasis in this organ is singular with respect to other tissues and exhibits a heterogeneous regulation in distinct brain cell populations. Due to the key role of cholesterol in brain physiology and function, alterations in cholesterol homeostasis and levels have been linked to brain diseases and neurodegeneration. In the case of Alzheimer disease (AD, however, this association remains unclear with evidence indicating that either increased or decreased total brain cholesterol levels contribute to this major neurodegenerative disease. Here, rather than analyzing the role of total cholesterol levels in neurodegeneration, we focus on the contribution of intracellular cholesterol pools, particularly in endolysosomes and mitochondria through its trafficking via specialized membrane domains delineated by the contacts between endoplasmic reticulum and mitochondria, in the onset of prevalent neurodegenerative diseases such as AD, Parkinson disease, and Huntington disease as well as in lysosomal disorders like Niemann-Pick type C disease. We dissect molecular events associated with intracellular cholesterol accumulation, especially in mitochondria, an event that results in impaired mitochondrial antioxidant defense and function. A better understanding of the mechanisms involved in the distribution of cholesterol in intracellular compartments may shed light on the role of cholesterol homeostasis disruption in neurodegeneration and may pave the way for specific intervention opportunities.

  9. A bacteriophage endolysin that eliminates intracellular streptococci

    Science.gov (United States)

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-01-01

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001 PMID:26978792

  10. An active matter analysis of intracellular Active Transport

    Science.gov (United States)

    Wang, Bo; Chen, Kejia; Bae, Sung Chul; Granick, Steve

    2012-02-01

    Tens of thousands of fluorescence-based trajectories at nm resolution have been analyzed, regarding active transport along microtubules in living cells. The following picture emerges. Directed motion to pre-determined locations is certainly an attractive idea, but cannot be pre-programmed as to do so would sacrifice adaptability. The polarity of microtubules is inadequate to identify these directions in cells, and no other mechanism is currently known. We conclude that molecular motors carry cargo through disordered intracellular microtubule networks in a statistical way, with loud cellular ``noise'' both in directionality and speed. Programmed random walks describe how local 1D active transport traverses crowded cellular space efficiently, rapidly, minimizing the energy waste that would result from redundant activity. The mechanism of statistical regulation is not yet understood, however.

  11. A Similarity Search Using Molecular Topological Graphs

    Directory of Open Access Journals (Sweden)

    Yoshifumi Fukunishi

    2009-01-01

    Full Text Available A molecular similarity measure has been developed using molecular topological graphs and atomic partial charges. Two kinds of topological graphs were used. One is the ordinary adjacency matrix and the other is a matrix which represents the minimum path length between two atoms of the molecule. The ordinary adjacency matrix is suitable to compare the local structures of molecules such as functional groups, and the other matrix is suitable to compare the global structures of molecules. The combination of these two matrices gave a similarity measure. This method was applied to in silico drug screening, and the results showed that it was effective as a similarity measure.

  12. Plasmalemmal V-H+-ATPases regulate intracellular pH in human lung microvascular endothelial cells

    International Nuclear Information System (INIS)

    Rojas, Jose D.; Sennoune, Souad R.; Maiti, Debasish; Martinez, Gloria M.; Bakunts, Karina; Wesson, Donald E.; Martinez-Zaguilan, Raul

    2004-01-01

    The lung endothelium layer is exposed to continuous CO 2 transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na + /H + exchanger and HCO 3 - -dependent H + -transporting mechanisms regulate intracellular pH (pH cyt ) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H + -ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na + /H + exchanger and HCO 3 - -based H + -transporting mechanisms, to maintain pH cyt homeostasis. Immunocytochemical studies revealed V-H + -ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na + and HCO 3 - that were similar to those observed in the presence of either Na + , or Na + and HCO 3 - . The Na + - and HCO 3 - -independent pH cyt recovery was inhibited by bafilomycin A 1 , a V-H + -ATPase inhibitor. These studies show a Na + - and HCO 3 - -independent pH cyt regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases

  13. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    Wagner, G.J.

    1979-01-01

    The mechanisms underlying heavy metal accumulation, toxicity and tolerance in higher plants are poorly understood. Since subcellular processes are undoubtedly involved in all these phenomena, it is of interest to study the extent of, subcellular site of and nature of intracellularly accumulated cadmium in higher plants. Whole plants supplied 109 CdCl 2 or 112 CdSO 4 accumulated Cd into roots and aerial tissues. Preparation of protoplasts from aerial tissue followed by subcellular fractionation of the protoplasts to obtain intact vacuoles, chloroplasts and cytosol revealed the presence of Cd in the cytosol but not in vacuoles or chloroplasts. Particulate materials containing other cell components were also labeled. Of the 109 Cd supplied to plants, 2 to 10% was recovered in both cytosol preparations and in particulate materials. Cytosol contained proteinaceous--Cd complexes, free metal and low molecular weight Cd complexes. Labeling of protoplasts gave similar results. No evidence was obtained for the production of volatile Cd complexes in tobacco

  14. Local mechanical stimulation induces components of the pathogen defense response in parsley

    Science.gov (United States)

    Gus-Mayer, Sabine; Naton, Beatrix; Hahlbrock, Klaus; Schmelzer, Elmon

    1998-01-01

    Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response to Phytophthora sojae. In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli. Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes. When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected. A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation. By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations. Neither papilla formation nor hypersensitive cell death occurred under the conditions tested. These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes. As yet unidentified signals may be involved in papilla formation and hypersensitive cell death. PMID:9653198

  15. Mechanism of H. pylori intracellular entry: an in vitro study

    Directory of Open Access Journals (Sweden)

    Hui eLiu

    2012-03-01

    Full Text Available The majority of H. pylori reside on gastric epithelial cell surfaces and in the overlying mucus, but a small fraction of H. pylori enter host epithelial and immune cells. To explore the role of the nudA invasin in host cell entry, a ΔnudA deletion derivative of strain J99 was constructed and transformants were verified by PCR and by fluorescence in situ hybridization. AGS cells were inoculated with either wild type (WT strain J99 or its ΔnudA mutant to determine the fraction of bacteria that were bound to the cells and inside these cells using the gentamicin protection assay. We observed no significant difference between either the density of H. pylori bound to AGS cell membranes or the density of intracellular H. pylori. To further explore this finding, separate chambers of each culture were fixed in glutaraldehyde for transmission electron microscopy (TEM and immunogold TEM. This addition to the classical gentamicin assay demonstrated that there were significantly more intracellular, and fewer membrane-bound, H. pylori in WT-infected AGS cells than in ΔnudA allele infected cells. Thus, the sum of intracellular and membrane-bound H. pylori was similar in the two groups. Since no other similar TEM study has been performed, it is at present unknown whether our observations can be reproduced by others Taken together however, our observations suggest that the classical gentamicin protection assay is not sufficiently sensitive to analyze H. pylori cell entry and that the addition of TEM to the test demonstrate that nudA plays a role in H. pylori entry into AGS cells in vitro. In addition, deletion of the invasin gene appears to limit H. pylori to the AGS cell surface, where it may be partly protected against gentamicin. In contrast, this specific environment may render H. pylori more vulnerable to host defense and therapeutic intervention, and less prone to trigger normal immune, carcinogenic, and other developmental response pathways.

  16. Intracellular phase for an extracellular bacterial pathogen: MgtC shows the way

    Directory of Open Access Journals (Sweden)

    Audrey Bernut

    2015-08-01

    Full Text Available Pseudomonas aeruginosa is an extracellular pathogen known to impair host phagocytic functions. However, our recent results identify MgtC as a novel actor in P. aeruginosa virulence, which plays a role in an intramacrophage phase of this pathogen. In agreement with its intracellular function, P. aeruginosa mgtC gene expression is strongly induced when the bacteria reside within macrophages. MgtC was previously known as a horizontally-acquired virulence factor important for multiplication inside macrophages in several intracellular bacterial pathogens. MgtC thus provides a singular example of a virulence determinant that subverts macrophages both in intracellular and extracellular pathogens. Moreover, we demonstrate that P. aeru-ginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosaMgtC prevents biofilm formation. We propose that MgtC has a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to the host in relation to the different bacterial lifestyles. MgtC thus appears as an attractive target for antivirulence strategies and our work provides a natural peptide as MgtC antagonist, which paves the way for the development of MgtC inhibitors.

  17. Azithromycin effectiveness against intracellular infections of Francisella

    Directory of Open Access Journals (Sweden)

    Mann Barbara J

    2010-04-01

    Full Text Available Abstract Background Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F. tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A may have different susceptibilities to Az, a widely used and well-tolerated antibiotic. Results In vitro susceptibility testing of Az confirmed that F. tularensis subsp. holarctica Live Vaccine Strain (LVS (Type B was not sensitive while F. philomiragia, F. novicida, and Type A F. tularensis (NIH B38 and Schu S4 strain were susceptible. In J774A.1 mouse macrophage cells infected with F. philomiragia, F. novicida, and F. tularensis LVS, 5 μg/ml Az applied extracellularly eliminated intracellular Francisella infections. A concentration of 25 μg/ml Az was required for Francisella-infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (tolC and ftlC in F. novicida demonstrated less sensitivity to Az by MIC than the parental strain, but the tolC disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of acrA and acrB mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in F. novicida. In contrast, F. tularensis Schu S4 mutants ΔacrB and ΔacrA were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in F. tularensis Schu S4. F. novicida LPS O-antigen mutants (wbtN, wbtE, wbtQ and wbtA were found to be less sensitive in vitro to Az compared to the wild

  18. Intracellular bacteria: the origin of dinoflagellate toxicity.

    Science.gov (United States)

    Silva, E S

    1990-01-01

    Dinoflagellate blooms of the same species have been registered either as toxic or nontoxic and, in the latter case, toxicity may be of different types. A hypothesis has been formulated according to which the bacteria having in some way taken part in the toxin formation are either inside the dinoflagellate cell or in the nutritive liquid. The presence of intracellular bacteria in those microorganisms has been studied mainly in material from cultures, a few from the sea, and several strains were isolated from different species. Experiments with crossed inoculations have shown that the bacterial strain from Gonyaulax tamarensis caused the cells of some other species to become toxic. From nontoxic clonal cultures of Prorocentrum balticum, Glenodinium foliaceum, and Gyrodinium instriatum, after inoculation of that bacterial strain, cultures were obtained whose cell extracts showed the same kind of toxicity as G. tamarensis. No toxic action could be found in the extracts of the bacterial cells form the assayed strains. The interference of intracellular bacteria in the metabolism of dinoflagellates must be the main cause of their toxicity.

  19. Fluorescent nanoparticles for intracellular sensing: a review.

    Science.gov (United States)

    Ruedas-Rama, Maria J; Walters, Jamie D; Orte, Angel; Hall, Elizabeth A H

    2012-11-02

    Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Mechanisms of cellular invasion by intracellular parasites.

    Science.gov (United States)

    Walker, Dawn M; Oghumu, Steve; Gupta, Gaurav; McGwire, Bradford S; Drew, Mark E; Satoskar, Abhay R

    2014-04-01

    Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

  1. [Intracellular signaling mechanisms in thyroid cancer].

    Science.gov (United States)

    Mondragón-Terán, Paul; López-Hernández, Luz Berenice; Gutiérrez-Salinas, José; Suárez-Cuenca, Juan Antonio; Luna-Ceballos, Rosa Isela; Erazo Valle-Solís, Aura

    2016-01-01

    Thyroid cancer is the most common malignancy of the endocrine system, the papillary variant accounts for 80-90% of all diagnosed cases. In the development of papillary thyroid cancer, BRAF and RAS genes are mainly affected, resulting in a modification of the system of intracellular signaling proteins known as «protein kinase mitogen-activated» (MAPK) which consist of «modules» of internal signaling proteins (Receptor/Ras/Raf/MEK/ERK) from the cell membrane to the nucleus. In thyroid cancer, these signanling proteins regulate diverse cellular processes such as differentiation, growth, development and apoptosis. MAPK play an important role in the pathogenesis of thyroid cancer as they are used as molecular biomarkers for diagnostic, prognostic and as possible therapeutic molecular targets. Mutations in BRAF gene have been correlated with poor response to treatment with traditional chemotherapy and as an indicator of poor prognosis. To review the molecular mechanisms involved in intracellular signaling of BRAF and RAS genes in thyroid cancer. Molecular therapy research is in progress for this type of cancer as new molecules have been developed in order to inhibit any of the components of the signaling pathway (RET/PTC)/Ras/Raf/MEK/ERK; with special emphasis on the (RET/PTC)/Ras/Raf section, which is a major effector of ERK pathway. Copyright © 2016 Academia Mexicana de Cirugía A.C. Publicado por Masson Doyma México S.A. All rights reserved.

  2. Brucella abortus Induces a Warburg Shift in Host Metabolism That Is Linked to Enhanced Intracellular Survival of the Pathogen.

    Science.gov (United States)

    Czyż, Daniel M; Willett, Jonathan W; Crosson, Sean

    2017-08-01

    Intracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected with Brucella abortus undergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well described in cancer cells and also occurs in activated inflammatory cells. B. abortus efficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affect in vitro growth of B. abortus in axenic culture but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, and B. abortus uses this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens. IMPORTANCE Brucella spp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected with Brucella abortus undergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism

  3. Sigma-1 receptor: The novel intracellular target of neuropsychotherapeutic drugs

    Directory of Open Access Journals (Sweden)

    Teruo Hayashi

    2015-01-01

    Full Text Available Sigma-1 receptor ligands have been long expected to serve as drugs for treatment of human diseases such as neurodegenerative disorders, depression, idiopathic pain, drug abuse, and cancer. Recent research exploring the molecular function of the sigma-1 receptor started unveiling underlying mechanisms of the therapeutic activity of those ligands. Via the molecular chaperone activity, the sigma-1 receptor regulates protein folding/degradation, ER/oxidative stress, and cell survival. The chaperone activity is activated or inhibited by synthetic sigma-1 receptor ligands in an agonist-antagonist manner. Sigma-1 receptors are localized at the endoplasmic reticulum (ER membranes that are physically associated with the mitochondria (MAM: mitochondria-associated ER membrane. In specific types of neurons (e.g., those at the spinal cord, sigma-1 receptors are also clustered at ER membranes that juxtapose postsynaptic plasma membranes. Recent studies indicate that sigma-1 receptors, partly in sake of its unique subcellular localization, regulate the mitochondria function that involves bioenergetics and free radical generation. The sigma-1 receptor may thus provide an intracellular drug target that enables controlling ER stress and free radical generation under pathological conditions.

  4. Enlightening intracellular complexity of living cells with quantitative phase microscopy

    Science.gov (United States)

    Martinez Torres, C.; Laperrousaz, B.; Berguiga, L.; Boyer Provera, E.; Elezgaray, J.; Nicolini, F. E.; Maguer-Satta, V.; Arneodo, A.; Argoul, F.

    2016-03-01

    The internal distribution of refractive indices (RIs) of a living cell is much more complex than usually admitted in multi-shell models. The reconstruction of RI maps from single phase images has rarely been achieved for several reasons: (i) we still have very little knowledge of the impact of internal macromolecular complexes on the local RI and (ii) phase changes produced by light propagation through the sample are mixed with diffraction effects by internal cell bodies. We propose the implementation a 2D wavelet-based contour chain detection method to distinguish internal boundaries thanks to their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are morphological indicators for distinguishing cells of different origins and to follow their transformation in pathologic situations. We use this method to compare non adherent blood cells from primary and laboratory culture origins, in healthy and pathological situations (chronic myelogenous leukaemia). In a second part of this presentation, we concentrate on the temporal dynamics of the phase contour chains and we discuss the spectral decomposition of their dynamics in both health and disease.

  5. Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

    Science.gov (United States)

    Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

    2007-08-17

    Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

  6. Intracellular distribution of organic anions (131I-BSP and 3H-bilirubin)

    International Nuclear Information System (INIS)

    Kamisaka, Kazuaki; Iida, Yoshitaka; Azegami, Nobuhisa; Oda, Hiroyuki; Maezawa, Hidenori

    1981-01-01

    About 2 μ Ci of 131 I-BSP were injected intravenously into normal wister rats and the distributions of the isotope were determined in subcellular fractions of rat liver by the method of De Duve et al. Approximately 33% of the total activity was localized in nuclear fraction and cell debris, 28.5% was in supernatant fraction, 16.5% in microsome, 13% in lysosome and 8% in mitochondrial fraction. The subcellular distributions of radioactivity remained unchanged for 1.5 hours. Using autoradiographic method, the intracellular distribution of 3 H-bilirubin was examined by the extracted liver, 5 min, after intravenous injection of 3 H-bilirubin. 3 H-bilirubin was localized mainly in the cytoplasm and small amounts was already distributed on the canalicular membrane. It is suggested that these small molecules are mainly transported through cytoplasm and there is no specific pathway for the hepatic intracellular transport system. (author)

  7. A COMPARISON OF SEMANTIC SIMILARITY MODELS IN EVALUATING CONCEPT SIMILARITY

    Directory of Open Access Journals (Sweden)

    Q. X. Xu

    2012-08-01

    Full Text Available The semantic similarities are important in concept definition, recognition, categorization, interpretation, and integration. Many semantic similarity models have been established to evaluate semantic similarities of objects or/and concepts. To find out the suitability and performance of different models in evaluating concept similarities, we make a comparison of four main types of models in this paper: the geometric model, the feature model, the network model, and the transformational model. Fundamental principles and main characteristics of these models are introduced and compared firstly. Land use and land cover concepts of NLCD92 are employed as examples in the case study. The results demonstrate that correlations between these models are very high for a possible reason that all these models are designed to simulate the similarity judgement of human mind.

  8. Similarity solutions for phase-change problems

    Science.gov (United States)

    Canright, D.; Davis, S. H.

    1989-01-01

    A modification of Ivantsov's (1947) similarity solutions is proposed which can describe phase-change processes which are limited by diffusion. The method has application to systems that have n-components and possess cross-diffusion and Soret and Dufour effects, along with convection driven by density discontinuities at the two-phase interface. Local thermal equilibrium is assumed at the interface. It is shown that analytic solutions are possible when the material properties are constant.

  9. An intracellular interaction network regulates conformational transitions in the dopamine transporter

    DEFF Research Database (Denmark)

    Kniazeff, Julie; Shi, Lei; Løland, Claus Juul

    2008-01-01

    Neurotransmitter:sodium symporters (NSS)(1) mediate sodium-dependent reuptake of neurotransmitters from the synaptic cleft and are targets for many psychoactive drugs. The crystal structure of the prokaryotic NSS protein, LeuT, was recently solved at high resolution; however, the mechanistic...... and the intracellular milieu. The mechanism that emerges from these findings may be unique to the NSS family, where the local disruption of ionic interactions modulates the transition of the transporter between the outward- and inward-facing conformations....

  10. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

    Science.gov (United States)

    Schumacher, Dominik; Helma, Jonas; Schneider, Anselm F. L.; Leonhardt, Heinrich

    2018-01-01

    Abstract Nanobodies can be seen as next‐generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site‐specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens. PMID:28913971

  11. Drosophila VAMP7 regulates Wingless intracellular trafficking.

    Science.gov (United States)

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

    2017-01-01

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

  12. Intracellular pH in sperm physiology.

    Science.gov (United States)

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications.

    Science.gov (United States)

    Schumacher, Dominik; Helma, Jonas; Schneider, Anselm F L; Leonhardt, Heinrich; Hackenberger, Christian P R

    2018-02-23

    Nanobodies can be seen as next-generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site-specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  14. Intracellular Signalling by C-Peptide

    Directory of Open Access Journals (Sweden)

    Claire E. Hills

    2008-01-01

    Full Text Available C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.

  15. Intracellular sphingosine releases calcium from lysosomes.

    Science.gov (United States)

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-11-27

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

  16. [Limbic encephalitis with antibodies against intracellular antigens].

    Science.gov (United States)

    Morita, Akihiko; Kamei, Satoshi

    2010-04-01

    Limbic encephalitis is a paraneoplastic syndrome that is often associated with small cell lung cancer (SCLC), breast cancer, testicular tumors, teratoma, Hodgkin's lymphoma and thymoma. The common clinical manifestations of limbic encephalitis are subacute onset, cognitive dysfunction, seizures and psychiatric symptoms. Paraneoplastic neurological disorders are considered to occur because of cytotoxic T cell responses and antibodies against target neuronal proteins that are usually expressed by an underlying tumor. The main intracellular antigens related to limbic encephalitis are Hu, Ma2, and less frequently CV2/CRMP5 and amphiphysin. The anti-Hu antibody, which is involved in cerebellar degeneration and extensive or multifocal encephalomyelitis such as limbic encephalitis is closely associated with a history of smoking and SCLC. The anti-Ma2 antibody is associated with encephalitis of the limbic system, hypothalamus and brain-stem. For this reason, some patients with limbic encephalitis have sleep disorders (including REM sleep abnormalities), severe hypokinesis and gaze palsy in addition to limbic dysfunction. In men aged less than 50 years, anti-Ma2 antibody encephalitis is almost always associated with testicular germ-cell tumors that are occasionally difficult to detect. In older men and women, the most common tumors are non-SCLC and breast cancer. Limbic encephalitis associated with cell-surface antigens (e.g., voltage-gated potassium channels, NMDA receptors) is mediated by antibodies and often improves after a reduction in the antibody titer and after tumor resection. Patients with antibodies against intracellular antigens, except for those with anti-Ma2 antibodies and testicular tumors, are less responsive. Early diagnosis and treatment with immunotherapy, tumor resection or both are important for improving or stabilizing the condition of limbic encephalitis.

  17. Single-cell intracellular nano-pH probes†

    OpenAIRE

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular p...

  18. Self-similar factor approximants

    International Nuclear Information System (INIS)

    Gluzman, S.; Yukalov, V.I.; Sornette, D.

    2003-01-01

    The problem of reconstructing functions from their asymptotic expansions in powers of a small variable is addressed by deriving an improved type of approximants. The derivation is based on the self-similar approximation theory, which presents the passage from one approximant to another as the motion realized by a dynamical system with the property of group self-similarity. The derived approximants, because of their form, are called self-similar factor approximants. These complement the obtained earlier self-similar exponential approximants and self-similar root approximants. The specific feature of self-similar factor approximants is that their control functions, providing convergence of the computational algorithm, are completely defined from the accuracy-through-order conditions. These approximants contain the Pade approximants as a particular case, and in some limit they can be reduced to the self-similar exponential approximants previously introduced by two of us. It is proved that the self-similar factor approximants are able to reproduce exactly a wide class of functions, which include a variety of nonalgebraic functions. For other functions, not pertaining to this exactly reproducible class, the factor approximants provide very accurate approximations, whose accuracy surpasses significantly that of the most accurate Pade approximants. This is illustrated by a number of examples showing the generality and accuracy of the factor approximants even when conventional techniques meet serious difficulties

  19. Dynamic similarity in erosional processes

    Science.gov (United States)

    Scheidegger, A.E.

    1963-01-01

    A study is made of the dynamic similarity conditions obtaining in a variety of erosional processes. The pertinent equations for each type of process are written in dimensionless form; the similarity conditions can then easily be deduced. The processes treated are: raindrop action, slope evolution and river erosion. ?? 1963 Istituto Geofisico Italiano.

  20. Personalized recommendation with corrected similarity

    International Nuclear Information System (INIS)

    Zhu, Xuzhen; Tian, Hui; Cai, Shimin

    2014-01-01

    Personalized recommendation has attracted a surge of interdisciplinary research. Especially, similarity-based methods in applications of real recommendation systems have achieved great success. However, the computations of similarities are overestimated or underestimated, in particular because of the defective strategy of unidirectional similarity estimation. In this paper, we solve this drawback by leveraging mutual correction of forward and backward similarity estimations, and propose a new personalized recommendation index, i.e., corrected similarity based inference (CSI). Through extensive experiments on four benchmark datasets, the results show a greater improvement of CSI in comparison with these mainstream baselines. And a detailed analysis is presented to unveil and understand the origin of such difference between CSI and mainstream indices. (paper)

  1. Towards Personalized Medicine: Leveraging Patient Similarity and Drug Similarity Analytics

    Science.gov (United States)

    Zhang, Ping; Wang, Fei; Hu, Jianying; Sorrentino, Robert

    2014-01-01

    The rapid adoption of electronic health records (EHR) provides a comprehensive source for exploratory and predictive analytic to support clinical decision-making. In this paper, we investigate how to utilize EHR to tailor treatments to individual patients based on their likelihood to respond to a therapy. We construct a heterogeneous graph which includes two domains (patients and drugs) and encodes three relationships (patient similarity, drug similarity, and patient-drug prior associations). We describe a novel approach for performing a label propagation procedure to spread the label information representing the effectiveness of different drugs for different patients over this heterogeneous graph. The proposed method has been applied on a real-world EHR dataset to help identify personalized treatments for hypercholesterolemia. The experimental results demonstrate the effectiveness of the approach and suggest that the combination of appropriate patient similarity and drug similarity analytics could lead to actionable insights for personalized medicine. Particularly, by leveraging drug similarity in combination with patient similarity, our method could perform well even on new or rarely used drugs for which there are few records of known past performance. PMID:25717413

  2. pH-Dependent Toxicity of High Aspect Ratio ZnO Nanowires in Macrophages Due to Intracellular Dissolution

    KAUST Repository

    H. Müller, Karin

    2010-11-23

    High-aspect ratio ZnO nanowires have become one of the most promising products in the nanosciences within the past few years with a multitude of applications at the interface of optics and electronics. The interaction of zinc with cells and organisms is complex, with both deficiency and excess causing severe effects. The emerging significance of zinc for many cellular processes makes it imperative to investigate the biological safety of ZnO nanowires in order to guarantee their safe economic exploitation. In this study, ZnO nanowires were found to be toxic to human monocyte macrophages (HMMs) at similar concentrations as ZnCl2. Confocal microscopy on live cells confirmed a rise in intracellular Zn2+ concentrations prior to cell death. In vitro, ZnO nanowires dissolved very rapidly in a simulated body fluid of lysosomal pH, whereas they were comparatively stable at extracellular pH. Bright-field transmission electron microscopy (TEM) showed a rapid macrophage uptake of ZnO nanowire aggregates by phagocytosis. Nanowire dissolution occurred within membrane-bound compartments, triggered by the acidic pH of the lysosomes. ZnO nanowire dissolution was confirmed by scanning electron microscopy/energy-dispersive X-ray spectrometry. Deposition of electron-dense material throughout the ZnO nanowire structures observed by TEM could indicate adsorption of cellular components onto the wires or localized zinc-induced protein precipitation. Our study demonstrates that ZnO nanowire toxicity in HMMs is due to pH-triggered, intracellular release of ionic Zn2+ rather than the high-aspect nature of the wires. Cell death had features of necrosis as well as apoptosis, with mitochondria displaying severe structural changes. The implications of these findings for the application of ZnO nanowires are discussed. © 2010 American Chemical Society.

  3. Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

    Science.gov (United States)

    Briers, Yves; Staubli, Titu; Schmid, Markus C; Wagner, Michael; Schuppler, Markus; Loessner, Martin J

    2012-01-01

    Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

  4. Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

    Directory of Open Access Journals (Sweden)

    Yves Briers

    Full Text Available Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

  5. Blockade of intracellular Zn2+ signaling in the basolateral amygdala affects object recognition memory via attenuation of dentate gyrus LTP.

    Science.gov (United States)

    Fujise, Yuki; Kubota, Mitsuyasu; Suzuki, Miki; Tamano, Haruna; Takeda, Atsushi

    2017-09-01

    Hippocampus-dependent memory is modulated by the amygdala. However, it is unknown whether intracellular Zn 2+ signaling in the amygdala is involved in hippocampus-dependent memory. On the basis of the evidence that intracellular Zn 2+ signaling in dentate granule cells (DGC) is necessary for object recognition memory via LTP at medial perforant pathway (PP)-DGC synapses, the present study examined whether intracellular Zn 2+ signaling in the amygdala influences object recognition memory via modulation of LTP at medial PP-DGC synapses. When ZnAF-2DA (100 μM, 2 μl) was injected into the basolateral amygdala (BLA), intracellular ZnAF-2 locally chelated intracellular Zn 2+ in the amygdala. Recognition memory was affected when training of object recognition test was performed 20 min after ZnAF-2DA injection into the BLA. Twenty minutes after injection of ZnAF-2DA into the BLA, LTP induction at medial PP-DGC synapses was attenuated, while LTP induction at PP-BLA synapses was potentiated and LTP induction at BLA-DGC synapses was attenuated. These results suggest that intracellular Zn 2+ signaling in the BLA is involved in BLA-associated LTP and modulates LTP at medial PP-DGC synapses, followed by modulation of object recognition memory. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

    Science.gov (United States)

    Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

    2014-01-01

    Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

  7. Effect of bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration.

    Science.gov (United States)

    Bilgin, M; Burgazli, K M; Rafiq, A; Mericliler, M; Neuhof, C; Oliva, M L; Parahuleva, M; Soydan, N; Doerr, O; Abdallah, Y; Erdogan, A

    2014-01-01

    Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium. HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 µM 12% and 13%. Furthermore, BbKI (100 µM) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control. The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential. BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.

  8. Intracellular calcium mobilization in human lymphocytes in the presence of synthetic IgG Fc peptides

    International Nuclear Information System (INIS)

    Plummer, J.M.; Panahi, Y.P.; McClurg, M.R.; Hahn, G.S.; Naemura, J.R.

    1986-01-01

    Certain synthetic peptides derived from the Fc region of human IgG can suppress the mixed lymphocyte response. These peptides were tested for the ability to induce intracellular calcium mobilization in human lymphocytes using fura-2/calcium fluorescence. T cells were isolated by rosetting and were > 90% OKT3 positive. Lymphocytes were incubated with the acetoxymethyl ester of fura-2 (10 μM) for 60 minutes at 37 0 C. Fluorescence intensity changes at 505 nm were monitored at an excitation lambda of 340 nm. Fura-2 was not cytotoxic compared to quin-2 since fura-2 loaded mononuclear cells incorporated 3 H-thymidine when stimulated by PHA, succinyl Con A, PWM or LPS-STM whereas quin-2 loaded cells showed a dose dependent inhibition of proliferation. Those synthetic peptides (5 to 400 μg/ml) that suppressed the MLR induced a dose dependent increase in intracellular calcium in mononuclear cells, lymphocytes, non-T cells and T cells. The fura-2 calcium fluorescence time course response was similar for peptide, PHA and succinyl Con A. These results suggest that these immunoregulatory peptides suppress 3 H-thymidine incorporation at a point after intracellular calcium mobilization and that fura-2 has advantages over quin-2 in measuring intracellular calcium levels in lymphocytes

  9. Cyclic AMP Pathway Activation and Extracellular Zinc Induce Rapid Intracellular Zinc Mobilization in Candida albicans

    Science.gov (United States)

    Kjellerup, Lasse; Winther, Anne-Marie L.; Wilson, Duncan; Fuglsang, Anja T.

    2018-01-01

    Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this “zinc flux” could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen. PMID:29619016

  10. Polymeric nanoparticles affect the intracellular delivery, antiretroviral activity and cytotoxicity of the microbicide drug candidate dapivirine.

    Science.gov (United States)

    das Neves, José; Michiels, Johan; Ariën, Kevin K; Vanham, Guido; Amiji, Mansoor; Bahia, Maria Fernanda; Sarmento, Bruno

    2012-06-01

    To assess the intracellular delivery, antiretroviral activity and cytotoxicity of poly(ε-caprolactone) (PCL) nanoparticles containing the antiretroviral drug dapivirine. Dapivirine-loaded nanoparticles with different surface properties were produced using three surface modifiers: poloxamer 338 NF (PEO), sodium lauryl sulfate (SLS) and cetyl trimethylammonium bromide (CTAB). The ability of nanoparticles to promote intracellular drug delivery was assessed in different cell types relevant for vaginal HIV transmission/microbicide development. Also, antiretroviral activity of nanoparticles was determined in different cell models, as well as their cytotoxicity. Dapivirine-loaded nanoparticles were readily taken up by different cells, with particular kinetics depending on the cell type and nanoparticles, resulting in enhanced intracellular drug delivery in phagocytic cells. Different nanoparticles showed similar or improved antiviral activity compared to free drug. There was a correlation between increased antiviral activity and increased intracellular drug delivery, particularly when cell models were submitted to a single initial short-course treatment. PEO-PCL and SLS-PCL nanoparticles consistently showed higher selectivity index values than free drug, contrasting with high cytotoxicity of CTAB-PCL. These results provide evidence on the potential of PCL nanoparticles to affect in vitro toxicity and activity of dapivirine, depending on surface engineering. Thus, this formulation approach may be a promising strategy for the development of next generation microbicides.

  11. Synthesis and analysis of separation networks for the recovery of intracellular chemicals generated from microbial-based conversions.

    Science.gov (United States)

    Yenkie, Kirti M; Wu, Wenzhao; Maravelias, Christos T

    2017-01-01

    Bioseparations can contribute to more than 70% in the total production cost of a bio-based chemical, and if the desired chemical is localized intracellularly, there can be additional challenges associated with its recovery. Based on the properties of the desired chemical and other components in the stream, there can be multiple feasible options for product recovery. These options are composed of several alternative technologies, performing similar tasks. The suitability of a technology for a particular chemical depends on (1) its performance parameters, such as separation efficiency; (2) cost or amount of added separating agent; (3) properties of the bioreactor effluent (e.g., biomass titer, product content); and (4) final product specifications. Our goal is to first synthesize alternative separation options and then analyze how technology selection affects the overall process economics. To achieve this, we propose an optimization-based framework that helps in identifying the critical technologies and parameters. We study the separation networks for two representative classes of chemicals based on their properties. The separation network is divided into three stages: cell and product isolation (stage I), product concentration (II), and product purification and refining (III). Each stage exploits differences in specific product properties for achieving the desired product quality. The cost contribution analysis for the two cases (intracellular insoluble and intracellular soluble) reveals that stage I is the key cost contributor (>70% of the overall cost). Further analysis suggests that changes in input conditions and technology performance parameters lead to new designs primarily in stage I. The proposed framework provides significant insights for technology selection and assists in making informed decisions regarding technologies that should be used in combination for a given set of stream/product properties and final output specifications. Additionally, the

  12. LIPID SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION

    Science.gov (United States)

    Stein, Olga; Stein, Yechezkiel

    1967-01-01

    In the mammary glands of lactating albino mice injected intravenously with 9, 10-oleic acid-3H or 9, 10-palmitic acid-3H, it has been shown that the labeled fatty acids are incorporated into mammary gland glycerides. The labeled lipid in the mammary gland 1 min after injection was in esterified form (> 95%), and the radioautographic reaction was seen over the rough endoplasmic reticulum and over lipid droplets, both intracellular and intraluminal. At 10–60 min after injection, the silver grains were concentrated predominantly over lipid droplets. There was no concentration of radioactivity over the granules in the Golgi apparatus, at any time interval studied. These findings were interpreted to indicate that after esterification of the fatty acid into glycerides in the rough endoplasmic reticulum an in situ aggregation of lipid occurs, with acquisition of droplet form. The release of the lipid into the lumen proceeds directly and not through the Golgi apparatus, in contradistinction to the mode of secretion of casein in the mammary gland or of lipoprotein in the liver. The presence of strands of endoplasmic reticulum attached to intraluminal lipid droplets provides a structural counterpart to the milk microsomes described in ruminant milk. PMID:6033535

  13. Intracellular events regulating cross-presentation

    Directory of Open Access Journals (Sweden)

    Peter eCresswell

    2012-06-01

    Full Text Available Cross-presentation plays a fundamental role in the induction of CD8-T cell immunity. However, although more than three decades have passed since its discovery, surprisingly little is known about the exact mechanisms involved. Here we give an overview of the components involved at different stages of this process. First, antigens must be internalized into the cross-presenting cell. The involvement of different receptors, method of antigen uptake, and nature of the antigen can influence intracellular trafficking and access to the cross-presentation pathway. Once antigens access the endocytic system, different requirements for endosomal/phagosomal processing arise, such as proteolysis and reduction of disulfide bonds. The majority of cross-presented peptides are generated by proteasomal degradation. Therefore, antigens must cross a membrane barrier in a manner analogous to the fate of misfolded proteins in the endoplasmic reticulum (ER that are retrotranslocated into the cytosol for degradation. Indeed, some components of the ER-associated degradation (ERAD machinery have been implicated in cross-presentation. Further complicating the matter, endosomal and phagosomal compartments have been suggested as alternative sites to the ER for loading of peptides on MHC class I molecules. Finally, the antigen presenting cells involved, particularly dendritic cell subsets and their state of maturation, influence the efficiency of cross-presentation.

  14. Intracellular recording from a spider vibration receptor.

    Science.gov (United States)

    Gingl, Ewald; Burger, Anna-M; Barth, Friedrich G

    2006-05-01

    The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ's cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.

  15. On the Computing Potential of Intracellular Vesicles.

    Science.gov (United States)

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing.

  16. Domain similarity based orthology detection.

    Science.gov (United States)

    Bitard-Feildel, Tristan; Kemena, Carsten; Greenwood, Jenny M; Bornberg-Bauer, Erich

    2015-05-13

    Orthologous protein detection software mostly uses pairwise comparisons of amino-acid sequences to assert whether two proteins are orthologous or not. Accordingly, when the number of sequences for comparison increases, the number of comparisons to compute grows in a quadratic order. A current challenge of bioinformatic research, especially when taking into account the increasing number of sequenced organisms available, is to make this ever-growing number of comparisons computationally feasible in a reasonable amount of time. We propose to speed up the detection of orthologous proteins by using strings of domains to characterize the proteins. We present two new protein similarity measures, a cosine and a maximal weight matching score based on domain content similarity, and new software, named porthoDom. The qualities of the cosine and the maximal weight matching similarity measures are compared against curated datasets. The measures show that domain content similarities are able to correctly group proteins into their families. Accordingly, the cosine similarity measure is used inside porthoDom, the wrapper developed for proteinortho. porthoDom makes use of domain content similarity measures to group proteins together before searching for orthologs. By using domains instead of amino acid sequences, the reduction of the search space decreases the computational complexity of an all-against-all sequence comparison. We demonstrate that representing and comparing proteins as strings of discrete domains, i.e. as a concatenation of their unique identifiers, allows a drastic simplification of search space. porthoDom has the advantage of speeding up orthology detection while maintaining a degree of accuracy similar to proteinortho. The implementation of porthoDom is released using python and C++ languages and is available under the GNU GPL licence 3 at http://www.bornberglab.org/pages/porthoda .

  17. Modeling HIV-1 intracellular replication: two simulation approaches

    NARCIS (Netherlands)

    Zarrabi, N.; Mancini, E.; Tay, J.; Shahand, S.; Sloot, P.M.A.

    2010-01-01

    Many mathematical and computational models have been developed to investigate the complexity of HIV dynamics, immune response and drug therapy. However, there are not many models which consider the dynamics of virus intracellular replication at a single level. We propose a model of HIV intracellular

  18. Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids.

    Science.gov (United States)

    Treyer, Andrea; Mateus, André; Wiśniewski, Jacek R; Boriss, Hinnerk; Matsson, Pär; Artursson, Per

    2018-06-04

    Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( F ic ) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F ic . The induction of NL did not further increase drug binding but led to altered F ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.

  19. Distributed and dynamic intracellular organization of extracellular information.

    Science.gov (United States)

    Granados, Alejandro A; Pietsch, Julian M J; Cepeda-Humerez, Sarah A; Farquhar, Iseabail L; Tkačik, Gašper; Swain, Peter S

    2018-06-05

    Although cells respond specifically to environments, how environmental identity is encoded intracellularly is not understood. Here, we study this organization of information in budding yeast by estimating the mutual information between environmental transitions and the dynamics of nuclear translocation for 10 transcription factors. Our method of estimation is general, scalable, and based on decoding from single cells. The dynamics of the transcription factors are necessary to encode the highest amounts of extracellular information, and we show that information is transduced through two channels: Generalists (Msn2/4, Tod6 and Dot6, Maf1, and Sfp1) can encode the nature of multiple stresses, but only if stress is high; specialists (Hog1, Yap1, and Mig1/2) encode one particular stress, but do so more quickly and for a wider range of magnitudes. In particular, Dot6 encodes almost as much information as Msn2, the master regulator of the environmental stress response. Each transcription factor reports differently, and it is only their collective behavior that distinguishes between multiple environmental states. Changes in the dynamics of the localization of transcription factors thus constitute a precise, distributed internal representation of extracellular change. We predict that such multidimensional representations are common in cellular decision-making.

  20. Dynamics of inorganic nutrients in intertidal sediments: porewater, exchangeable and intracellular pools

    Directory of Open Access Journals (Sweden)

    Emilio eGarcia-Robledo

    2016-05-01

    Full Text Available The study of inorganic nutrients dynamics in shallow sediments usually focuses on two main pools: the porewater (PW nutrients and the exchangeable (EX ammonium and phosphate. Recently, it has been found that microphytobenthos (MPB and other microorganisms can accumulate large amounts of nutrients intracellularly (IC, highlighting the biogeochemical importance of this nutrient pool. Storing nutrients could support the growth of autotrophs when nutrients are not available, and could also provide alternative electron acceptors for dissimilatory processes such as nitrate reduction. Here, we studied the magnitude and relative importance of these three nutrient pools (PW, IC and EX and their relation to chlorophylls (used as a proxy for MPB abundance and organic matter (OM contents in an intertidal mudflat of Cadiz Bay (Spain. MPB was localized in the first 4 mm of the sediment and showed a clear seasonal pattern; highest chlorophylls content was found during autumn and lowest during spring-summer. The temporal and spatial distribution of nutrients pools and MPB were largely correlated. Ammonium was higher in the IC and EX fractions, representing on average 59 and 37% of the total ammonium pool, respectively. Similarly, phosphate in the IC and EX fractions accounted on average for 40 and 31% of the total phosphate pool, respectively. Nitrate in the PW was low, suggesting low nitrification activity and rapid consumption. Nitrate accumulated in the IC pool during periods of moderate MPB abundance, being up to 66% of the total nitrate pool, whereas it decreased when chlorophyll concentration peaked likely due to a high nitrogen demand. EX-Nitrate accounted for the largest fraction of total sediment nitrate, 66% on average. The distribution of EX-Nitrate was significantly correlated with chlorophyll and OM, which probably indicates a relation of this pool to an increased availability of sites for ionic adsorption. This EX-Nitrate pool could represent an

  1. Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols.

    Science.gov (United States)

    Pinu, Farhana R; Villas-Boas, Silas G; Aggio, Raphael

    2017-10-23

    Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

  2. Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols

    Directory of Open Access Journals (Sweden)

    Farhana R. Pinu

    2017-10-01

    Full Text Available Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

  3. Yersinia pestis intracellular parasitism of macrophages from hosts exhibiting high and low severity of plague.

    Directory of Open Access Journals (Sweden)

    Duraisamy Ponnusamy

    Full Text Available BACKGROUND: Yersinia pestis causes severe disease in natural rodent hosts, but mild to inapparent disease in certain rodent predators such as dogs. Y. pestis initiates infection in susceptible hosts by parasitizing and multiplying intracellularly in local macrophages prior to systemic dissemination. Thus, we hypothesize that Y. pestis disease severity may depend on the degree to which intracellular Y. pestis overcomes the initial host macrophage imposed stress. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, the progression of in vitro infection by Y. pestis KIM62053.1+ of mouse splenic and RAW264.7 tissue culture macrophages and dog peripheral blood-derived and DH82 tissue culture macrophages was studied using microscopy and various parameters of infection. The study showed that during the early stage of infection, intracellular Y. pestis assumed filamentous cellular morphology with multiple copies of the genome per bacterium in both mouse and dog macrophages. Later, in mouse macrophages, the infection elicited spacious vacuolar extension of Yersinia containing vacuoles (YCV, and the filamentous Y. pestis reverted to coccobacillary morphology with genomic equivalents approximately equaling colony forming units. In contrast, Y. pestis infected dog macrophages did not show noticeable extension of YCV, and intracellular Y. pestis retained the filamentous cellular morphology for the entire experiment in DH82 cells or were killed by blood-derived macrophages. In addition, during the later stage of infection, Y. pestis infected mouse macrophages exhibited cell lysis whereas dog macrophages did not. CONCLUSION/SIGNIFICANCE: Overall, these results support our hypothesis that Y. pestis in mouse macrophages can overcome the initial intracellular stress necessary for subsequent systemic infection. However, in dogs, failure of Y. pestis to overcome macrophage imposed stress may result in mild or in apparent disease in dogs.

  4. Development of viral nanoparticles for efficient intracellular delivery

    Science.gov (United States)

    Wu, Zhuojun; Chen, Kevin; Yildiz, Ibrahim; Dirksen, Anouk; Fischer, Rainer; Dawson, Philip E.; Steinmetz, Nicole F.

    2012-05-01

    Viral nanoparticles (VNPs) based on plant viruses such as Cowpea mosaic virus (CPMV) can be used for a broad range of biomedical applications because they present a robust scaffold that allows functionalization by chemical conjugation and genetic modification, thereby offering an efficient drug delivery platform that can target specific cells and tissues. VNPs such as CPMV show natural affinity to cells; however, cellular uptake is inefficient. Here we show that chemical modification of the CPMV surface with a highly reactive, specific and UV-traceable hydrazone linker allows bioconjugation of polyarginine (R5) cell penetrating peptides (CPPs), which can overcome these limitations. The resulting CPMV-R5 particles were taken up into a human cervical cancer cell line (HeLa) more efficiently than native particles. Uptake efficiency was dependent on the density of R5 peptides on the surface of the VNP; particles displaying 40 R5 peptides per CPMV (denoted as CPMV-R5H) interact strongly with the plasma membrane and are taken up into the cells via an energy-dependent mechanism whereas particles displaying 10 R5 peptides per CPMV (CPMV-R5L) are only slowly taken up. The fate of CPMV-R5 versus native CPMV particles within cells was evaluated in a co-localization time course study. It was indicated that the intracellular localization of CPMV-R5 and CPMV differs; CPMV remains trapped in Lamp-1 positive endolysosomes over long time frames; in contrast, 30-50% of the CPMV-R5 particles transitioned from the endosome into other cellular vesicles or compartments. Our data provide the groundwork for the development of efficient drug delivery formulations based on CPMV-R5.Viral nanoparticles (VNPs) based on plant viruses such as Cowpea mosaic virus (CPMV) can be used for a broad range of biomedical applications because they present a robust scaffold that allows functionalization by chemical conjugation and genetic modification, thereby offering an efficient drug delivery platform

  5. Recent advances in intracellular and in vivo ROS sensing: focus on nanoparticle and nanotube applications.

    Science.gov (United States)

    Uusitalo, Larissa M; Hempel, Nadine

    2012-01-01

    Reactive oxygen species (ROS) are increasingly being implicated in the regulation of cellular signaling cascades. Intracellular ROS fluxes are associated with cellular function ranging from proliferation to cell death. Moreover, the importance of subtle, spatio-temporal shifts in ROS during localized cellular signaling events is being realized. Understanding the biochemical nature of the ROS involved will enhance our knowledge of redox-signaling. An ideal intracellular sensor should therefore resolve real-time, localized ROS changes, be highly sensitive to physiologically relevant shifts in ROS and provide specificity towards a particular molecule. For in vivo applications issues such as bioavailability of the probe, tissue penetrance of the signal and signal-to-noise ratio also need to be considered. In the past researchers have heavily relied on the use of ROS-sensitive fluorescent probes and, more recently, genetically engineered ROS sensors. However, there is a great need to improve on current methods to address the above issues. Recently, the field of molecular sensing and imaging has begun to take advantage of the unique physico-chemical properties of nanoparticles and nanotubes. Here we discuss the recent advances in the use of these nanostructures as alternative platforms for ROS sensing, with particular emphasis on intracellular and in vivo ROS detection and quantification.

  6. Similarity measures for face recognition

    CERN Document Server

    Vezzetti, Enrico

    2015-01-01

    Face recognition has several applications, including security, such as (authentication and identification of device users and criminal suspects), and in medicine (corrective surgery and diagnosis). Facial recognition programs rely on algorithms that can compare and compute the similarity between two sets of images. This eBook explains some of the similarity measures used in facial recognition systems in a single volume. Readers will learn about various measures including Minkowski distances, Mahalanobis distances, Hansdorff distances, cosine-based distances, among other methods. The book also summarizes errors that may occur in face recognition methods. Computer scientists "facing face" and looking to select and test different methods of computing similarities will benefit from this book. The book is also useful tool for students undertaking computer vision courses.

  7. Intracellular delivery of poly(I:C) induces apoptosis of fibroblast-like synoviocytes via an unknown dsRNA sensor

    Energy Technology Data Exchange (ETDEWEB)

    Karpus, Olga N.; Hsiao, Cheng-Chih; Kort, Hanneke de; Tak, Paul P.; Hamann, Jörg, E-mail: j.hamann@amc.uva.nl

    2016-08-26

    Fibroblast-like synoviocytes (FLS) express functional membranous and cytoplasmic sensors for double-stranded (ds)RNA. Notably, FLS undergo apoptosis upon transfection with the synthetic dsRNA analog poly(I:C). We here studied the mechanism of intracellular poly(I:C) recognition and subsequent cell death in FLS. FLS responded similarly to poly(I:C) or 3pRNA transfection; however, only intracellular delivery of poly(I:C) induced significant cell death, accompanied by upregulation of pro-apoptotic proteins Puma and Noxa, caspase 3 cleavage, and nuclear segregation. Knockdown of the DExD/H-box helicase MDA5 did not affect the response to intracellular poly(I:C); in contrast, knockdown of RIG-I abrogated the response to 3pRNA. Knockdown of the downstream adaptor proteins IPS, STING, and TRIF or inhibition of TBK1 did not affect the response to intracellular poly(I:C), while knockdown of IFNAR blocked intracellular poly(I:C)-mediated signaling and cell death. We conclude that a so far unknown intracellular sensor recognizes linear dsRNA and induces apoptosis in FLS. - Highlights: • Intracellular poly(I:C) and 3pRNA evoke immune responses in FLS. • Only intracellular delivery of poly(I:C) induces FLS apoptosis. • FLS do not require MDA5 for their response to intracellular poly(I:C). • FLS respond to intracellular poly(I:C) independent of IPS and STING. • An unknown intracellular sensor recognizes linear dsRNA in FLS.

  8. Effects of inhibitors of protein synthesis and intracellular transport on the gamma-aminobutyric acid agonist-induced functional differentiation of cultured cerebellar granule cells

    DEFF Research Database (Denmark)

    Belhage, B; Hansen, Gert Helge; Meier, E

    1990-01-01

    The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological...... an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low-affinity GABA receptors. In contrast, the inhibitors of intracellular transport...

  9. Intracellular magnetophoresis of amyloplasts and induction of root curvature

    Science.gov (United States)

    Kuznetsov, O. A.; Hasenstein, K. H.

    1996-01-01

    High-gradient magnetic fields (HGMFs) were used to induce intracellular magnetophoresis of amyloplasts. The HGMFs were generated by placing a small ferromagnetic wedge into a uniform magnetic field or at the gap edge between two permanent magnets. In the vicinity of the tip of the wedge the dynamic factor of the magnetic field, delta(H2/2), was about 10(9) Oe2.cm-1, which subjected the amyloplasts to a force comparable to that of gravity. When roots of 2-d-old seedlings of flax (Linum usitatissimum L.) were positioned vertically and exposed to an HGMF, curvature away from the wedge was transient and lasted approximately 1 h. Average curvature obtained after placing magnets, wedge and seedlings on a 1-rpm clinostat for 2 h was 33 +/- 5 degrees. Roots of horizontally placed control seedlings without rotation curved about 47 +/- 4 degrees. The time course of curvature and changes in growth rate were similar for gravicurvature and for root curvature induced by HGMFs. Microscopy showed displacement of amyloplasts in vitro and in vivo. Studies with Arabidopsis thaliana (L.) Heynh. showed that the wild type responded to HGMFs but the starchless mutant TC7 did not. The data indicate that a magnetic force can be used to study the gravisensing and response system of roots.

  10. Prion protein modulates glucose homeostasis by altering intracellular iron.

    Science.gov (United States)

    Ashok, Ajay; Singh, Neena

    2018-04-26

    The prion protein (PrP C ), a mainly neuronal protein, is known to modulate glucose homeostasis in mouse models. We explored the underlying mechanism in mouse models and the human pancreatic β-cell line 1.1B4. We report expression of PrP C on mouse pancreatic β-cells, where it promoted uptake of iron through divalent-metal-transporters. Accordingly, pancreatic iron stores in PrP knockout mice (PrP -/- ) were significantly lower than wild type (PrP +/+ ) controls. Silencing of PrP C in 1.1B4 cells resulted in significant depletion of intracellular (IC) iron, and remarkably, upregulation of glucose transporter GLUT2 and insulin. Iron overloading, on the other hand, resulted in downregulation of GLUT2 and insulin in a PrP C -dependent manner. Similar observations were noted in the brain, liver, and neuroretina of iron overloaded PrP +/+ but not PrP -/- mice, indicating PrP C -mediated modulation of insulin and glucose homeostasis through iron. Peripheral challenge with glucose and insulin revealed blunting of the response in iron-overloaded PrP +/+ relative to PrP -/- mice, suggesting that PrP C -mediated modulation of IC iron influences both secretion and sensitivity of peripheral organs to insulin. These observations have implications for Alzheimer's disease and diabetic retinopathy, known complications of type-2-diabetes associated with brain and ocular iron-dyshomeostasis.

  11. Getting Across the Plasma Membrane and Beyond: Intracellular Uses of Colloidal Semiconductor Nanocrystals

    Directory of Open Access Journals (Sweden)

    Camilla Luccardini

    2007-01-01

    Full Text Available Semiconductor nanocrystals (NCs are increasingly being used as photoluminescen markers in biological imaging. Their brightness, large Stokes shift, and high photostability compared to organic fluorophores permit the exploration of biological phenomena at the single-molecule scale with superior temporal resolution and spatial precision. NCs have predominantly been used as extracellular markers for tagging and tracking membrane proteins. Successful internalization and intracellular labelling with NCs have been demonstrated for both fixed immunolabelled and live cells. However, the precise localization and subcellular compartment labelled are less clear. Generally, live cell studies are limited by the requirement of fairly invasive protocols for loading NCs and the relatively large size of NCs compared to the cellular machinery, along with the subsequent sequestration of NCs in endosomal/lysosomal compartments. For long-period observation the potential cytotoxicity of cytoplasmically loaded NCs must be evaluated. This review focuses on the challenges of intracellular uses of NCs.

  12. Accumulation of intra-cellular polyphosphate in Chlorella vulgaris cells is related to indole-3-acetic acid produced by Azospirillum brasilense.

    Science.gov (United States)

    Meza, Beatriz; de-Bashan, Luz E; Hernandez, Juan-Pablo; Bashan, Yoav

    2015-06-01

    Accumulation of intra-cellular phosphate, as polyphosphate, was measured when the microalga Chlorella vulgaris was immobilized in alginate with either of two wild-type strains of the microalgae growth-promoting bacterium Azospirillum brasilense or their corresponding IAA-attenuated mutants. Wild type strains of A. brasilense induced higher amounts of intra-cellular phosphate in Chlorella than their respective mutants. Calculations comparing intra-cellular phosphate accumulation by culture or net accumulation by the cell and the amount of IAA that was produced by each of these strains revealed that higher IAA was linked to higher accumulations of intra-cellular phosphate. Application of four levels of exogenous IAA reported for A. brasilense and their IAA-attenuated mutants to cultures of C. vulgaris enhanced accumulation of intra-cellular phosphate; the higher the content of IAA per culture or per single cell, the higher was the amount of accumulated phosphate. When an IAA-attenuated mutant was complemented with exogenous IAA, accumulation of intra-cellular phosphate at the culture level was even higher than phosphate accumulation with the respective wild type strains. When calculating the net accumulation of intra-cellular phosphate in the complementation experiment, net intra-cellular phosphate induced by the IAA-attenuated mutant was completely restored and was similar to the wild strains. We propose that IAA produced by A. brasilense is linked to polyphosphate accumulation in C. vulgaris. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  13. Revisiting Inter-Genre Similarity

    DEFF Research Database (Denmark)

    Sturm, Bob L.; Gouyon, Fabien

    2013-01-01

    We revisit the idea of ``inter-genre similarity'' (IGS) for machine learning in general, and music genre recognition in particular. We show analytically that the probability of error for IGS is higher than naive Bayes classification with zero-one loss (NB). We show empirically that IGS does...... not perform well, even for data that satisfies all its assumptions....

  14. Fast business process similarity search

    NARCIS (Netherlands)

    Yan, Z.; Dijkman, R.M.; Grefen, P.W.P.J.

    2012-01-01

    Nowadays, it is common for organizations to maintain collections of hundreds or even thousands of business processes. Techniques exist to search through such a collection, for business process models that are similar to a given query model. However, those techniques compare the query model to each

  15. Glove boxes and similar containments

    International Nuclear Information System (INIS)

    Anon.

    1975-01-01

    According to the present invention a glove box or similar containment is provided with an exhaust system including a vortex amplifier venting into the system, the vortex amplifier also having its main inlet in fluid flow connection with the containment and a control inlet in fluid flow connection with the atmosphere outside the containment. (U.S.)

  16. pH-Dependent Toxicity of High Aspect Ratio ZnO Nanowires in Macrophages Due to Intracellular Dissolution

    KAUST Repository

    H. Müller, Karin; Kulkarni, Jaideep; Motskin, Michael; Goode, Angela; Winship, Peter; Skepper, Jeremy N.; Ryan, Mary P.; Porter, Alexandra E.

    2010-01-01

    exploitation. In this study, ZnO nanowires were found to be toxic to human monocyte macrophages (HMMs) at similar concentrations as ZnCl2. Confocal microscopy on live cells confirmed a rise in intracellular Zn2+ concentrations prior to cell death. In vitro, Zn

  17. Popularity versus similarity in growing networks

    Science.gov (United States)

    Krioukov, Dmitri; Papadopoulos, Fragkiskos; Kitsak, Maksim; Serrano, Mariangeles; Boguna, Marian

    2012-02-01

    Preferential attachment is a powerful mechanism explaining the emergence of scaling in growing networks. If new connections are established preferentially to more popular nodes in a network, then the network is scale-free. Here we show that not only popularity but also similarity is a strong force shaping the network structure and dynamics. We develop a framework where new connections, instead of preferring popular nodes, optimize certain trade-offs between popularity and similarity. The framework admits a geometric interpretation, in which preferential attachment emerges from local optimization processes. As opposed to preferential attachment, the optimization framework accurately describes large-scale evolution of technological (Internet), social (web of trust), and biological (E.coli metabolic) networks, predicting the probability of new links in them with a remarkable precision. The developed framework can thus be used for predicting new links in evolving networks, and provides a different perspective on preferential attachment as an emergent phenomenon.

  18. Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

    Science.gov (United States)

    Peng, Tao; Hang, Howard C

    2016-11-02

    Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

  19. Large margin classification with indefinite similarities

    KAUST Repository

    Alabdulmohsin, Ibrahim

    2016-01-07

    Classification with indefinite similarities has attracted attention in the machine learning community. This is partly due to the fact that many similarity functions that arise in practice are not symmetric positive semidefinite, i.e. the Mercer condition is not satisfied, or the Mercer condition is difficult to verify. Examples of such indefinite similarities in machine learning applications are ample including, for instance, the BLAST similarity score between protein sequences, human-judged similarities between concepts and words, and the tangent distance or the shape matching distance in computer vision. Nevertheless, previous works on classification with indefinite similarities are not fully satisfactory. They have either introduced sources of inconsistency in handling past and future examples using kernel approximation, settled for local-minimum solutions using non-convex optimization, or produced non-sparse solutions by learning in Krein spaces. Despite the large volume of research devoted to this subject lately, we demonstrate in this paper how an old idea, namely the 1-norm support vector machine (SVM) proposed more than 15 years ago, has several advantages over more recent work. In particular, the 1-norm SVM method is conceptually simpler, which makes it easier to implement and maintain. It is competitive, if not superior to, all other methods in terms of predictive accuracy. Moreover, it produces solutions that are often sparser than more recent methods by several orders of magnitude. In addition, we provide various theoretical justifications by relating 1-norm SVM to well-established learning algorithms such as neural networks, SVM, and nearest neighbor classifiers. Finally, we conduct a thorough experimental evaluation, which reveals that the evidence in favor of 1-norm SVM is statistically significant.

  20. An Alfven eigenmode similarity experiment

    International Nuclear Information System (INIS)

    Heidbrink, W W; Fredrickson, E; Gorelenkov, N N; Hyatt, A W; Kramer, G; Luo, Y

    2003-01-01

    The major radius dependence of Alfven mode stability is studied by creating plasmas with similar minor radius, shape, magnetic field (0.5 T), density (n e ≅3x10 19 m -3 ), electron temperature (1.0 keV) and beam ion population (near-tangential 80 keV deuterium injection) on both NSTX and DIII-D. The major radius of NSTX is half the major radius of DIII-D. The super-Alfvenic beam ions that drive the modes have overlapping values of v f /v A in the two devices. Observed beam-driven instabilities include toroidicity-induced Alfven eigenmodes (TAE). The stability threshold for the TAE is similar in the two devices. As expected theoretically, the most unstable toroidal mode number n is larger in DIII-D

  1. Compressional Alfven Eigenmode Similarity Study

    Science.gov (United States)

    Heidbrink, W. W.; Fredrickson, E. D.; Gorelenkov, N. N.; Rhodes, T. L.

    2004-11-01

    NSTX and DIII-D are nearly ideal for Alfven eigenmode (AE) similarity experiments, having similar neutral beams, fast-ion to Alfven speed v_f/v_A, fast-ion pressure, and shape of the plasma, but with a factor of 2 difference in the major radius. Toroidicity-induced AE with ˜100 kHz frequencies were compared in an earlier study [1]; this paper focuses on higher frequency AE with f ˜ 1 MHz. Compressional AE (CAE) on NSTX have a polarization, dependence on the fast-ion distribution function, frequency scaling, and low-frequency limit that are qualitatively consistent with CAE theory [2]. Global AE (GAE) are also observed. On DIII-D, coherent modes in this frequency range are observed during low-field (0.6 T) similarity experiments. Experiments will compare the CAE stability limits on DIII-D with the NSTX stability limits, with the aim of determining if CAE will be excited by alphas in a reactor. Predicted differences in the frequency splitting Δ f between excited modes will also be used. \\vspace0.25em [1] W.W. Heidbrink, et al., Plasmas Phys. Control. Fusion 45, 983 (2003). [2] E.D. Fredrickson, et al., Princeton Plasma Physics Laboratory Report PPPL-3955 (2004).

  2. Modulating intracellular acidification by regulating the incubation time of proton caged compounds.

    Science.gov (United States)

    Carbone, Marilena; Sabbatella, Gianfranco; Antonaroli, Simonetta; Orlando, Viviana; Biagioni, Stefano; Nucara, Alessandro

    2016-09-01

    A proton caged compound, the 1-(2-nitrophenyl)- ethylhexadecyl sulfonate (HDNS), was dosed into HEK-293 at different incubation times. Samples were irradiated with filtered UV light for inducing photolysis of the HDNS and then probed by infrared spectroscopy. The intracellular acidification reaction can be followed by monitoring the consequent CO2 peak intensity variation. The total CO2 produced is similar for all the samples, hence it is only a function of the initial HDNS concentration. The way it is achieved, though, is different for the different incubation times and follows kinetics, which results in a combination of a linear CO2 increase and a steep CO2 increase followed by a decay. This is interpreted in terms of confinement of the HDNS into intracellular vesicles of variable average size and sensitive to UV light when they reach critical dimensions.

  3. New perspective in the assessment of total intracellular magnesium

    Directory of Open Access Journals (Sweden)

    Azzurra Sargenti

    2014-01-01

    Full Text Available Magnesium (Mg is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ, that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg.

  4. Plectasin shows intracellular activity against Staphylococcus aureus in human THP-1 monocytes and in a mouse peritonitis model

    DEFF Research Database (Denmark)

    Brinch, Karoline Sidelmann; Sandberg, Anne; Baudoux, Pierre

    2009-01-01

    was maintained (maximal relative efficacy [E(max)], 1.0- to 1.3-log reduction in CFU) even though efficacy was inferior to that of extracellular killing (E(max), >4.5-log CFU reduction). Animal studies included a novel use of the mouse peritonitis model, exploiting extra- and intracellular differentiation assays...... concentration. These findings stress the importance of performing studies of extra- and intracellular activity since these features cannot be predicted from traditional MIC and killing kinetic studies. Application of both the THP-1 and the mouse peritonitis models showed that the in vitro results were similar...

  5. Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection

    Science.gov (United States)

    Damalakiene, Leona; Karabanovas, Vitalijus; Bagdonas, Saulius; Valius, Mindaugas; Rotomskis, Ricardas

    2013-01-01

    Background: The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines. Methods: The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker®. Results: The uptake of quantum dots into cells was dramatically reduced at a low temperature (4°C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37°C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5–8 μm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours. Conclusion: Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison

  6. Vacuolar zinc transporter Zrc1 is required for detoxification of excess intracellular zinc in the human fungal pathogen Cryptococcus neoformans.

    Science.gov (United States)

    Cho, Minsu; Hu, Guanggan; Caza, Mélissa; Horianopoulos, Linda C; Kronstad, James W; Jung, Won Hee

    2018-01-01

    Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular zinc to protect the cell. In this study, we identified an ortholog of the S. cerevisiae ZRC1 gene in the human fungal pathogen Cryptococcus neoformans. Zrc1 was localized in the vacuolar membrane in C. neoformans, and a mutant lacking ZRC1 showed significant growth defects under high-zinc conditions. These results suggested a role for Zrc1 in zinc detoxification. However, contrary to our expectation, the expression of Zrc1 was induced in cells grown in zinc-limited conditions and decreased upon the addition of zinc. These expression patterns were similar to those of Zip1, the high-affinity zinc transporter in the plasma membrane of C. neoformans. Furthermore, we used the zrc1 mutant in a murine model of cryptococcosis to examine whether a mammalian host could inhibit the survival of C. neoformans using zinc toxicity. We found that the mutant showed no difference in virulence compared with the wildtype strain. This result suggests that Zrc1-mediated zinc detoxification is not required for the virulence of C. neoformans, and imply that zinc toxicity may not be an important aspect of the host immune response to the fungus.

  7. Subcellular neuropharmacology: the importance of intracellular targeting.

    Science.gov (United States)

    Miyashiro, Kevin Y; Bell, Thomas J; Sul, Jai-Yoon; Eberwine, James

    2009-04-01

    Few cell types are more adapted for cell-cell signaling than neurons. Their responsiveness lies in the formation of highly specialized compartments composed of unique repertoires of selectively distributed protein complexes generated, in part, by the local translation of mRNAs and regulated by their RNA-binding proteins. Utilizing the selective distribution of these neuronal proteins and the underlying mechanisms that generate the differential patterns of expression as central facets of drug design promises to enhance the therapeutic ratio of a drug. It is in this context that we discuss the unique arrangement of mRNAs, RNA-binding proteins and the protein macromolecular complexes at the dendrite, which is the postsynaptic site of synaptic transmission. Recent advances in identifying the function of dendritic components of the mechanisms of protein and RNA transport, non-nuclear RNA splicing and localized translation underscore their importance as targets of neuropharmacology.

  8. Involvement of intracellular Zn2+ signaling in LTP at perforant pathway-CA1 pyramidal cell synapse.

    Science.gov (United States)

    Tamano, Haruna; Nishio, Ryusuke; Takeda, Atsushi

    2017-07-01

    Physiological significance of synaptic Zn 2+ signaling was examined at perforant pathway-CA1 pyramidal cell synapses. In vivo long-term potentiation (LTP) at perforant pathway-CA1 pyramidal cell synapses was induced using a recording electrode attached to a microdialysis probe and the recording region was locally perfused with artificial cerebrospinal fluid (ACSF) via the microdialysis probe. Perforant pathway LTP was not attenuated under perfusion with CaEDTA (10 mM), an extracellular Zn 2+ chelator, but attenuated under perfusion with ZnAF-2DA (50 μM), an intracellular Zn 2+ chelator, suggesting that intracellular Zn 2+ signaling is required for perforant pathway LTP. Even in rat brain slices bathed in CaEDTA in ACSF, intracellular Zn 2+ level, which was measured with intracellular ZnAF-2, was increased in the stratum lacunosum-moleculare where perforant pathway-CA1 pyramidal cell synapses were contained after tetanic stimulation. These results suggest that intracellular Zn 2+ signaling, which originates in internal stores/proteins, is involved in LTP at perforant pathway-CA1 pyramidal cell synapses. Because the influx of extracellular Zn 2+ , which originates in presynaptic Zn 2+ release, is involved in LTP at Schaffer collateral-CA1 pyramidal cell synapses, synapse-dependent Zn 2+ dynamics may be involved in plasticity of postsynaptic CA1 pyramidal cells. © 2017 Wiley Periodicals, Inc.

  9. Similarity analysis between quantum images

    Science.gov (United States)

    Zhou, Ri-Gui; Liu, XingAo; Zhu, Changming; Wei, Lai; Zhang, Xiafen; Ian, Hou

    2018-06-01

    Similarity analyses between quantum images are so essential in quantum image processing that it provides fundamental research for the other fields, such as quantum image matching, quantum pattern recognition. In this paper, a quantum scheme based on a novel quantum image representation and quantum amplitude amplification algorithm is proposed. At the end of the paper, three examples and simulation experiments show that the measurement result must be 0 when two images are same, and the measurement result has high probability of being 1 when two images are different.

  10. Similarity flows in relativistic hydrodynamics

    International Nuclear Information System (INIS)

    Blaizot, J.P.; Ollitrault, J.Y.

    1986-01-01

    In ultra-relativistic heavy ion collisions, one expects in particular to observe a deconfinement transition leading to a formation of quark gluon plasma. In the framework of the hydrodynamic model, experimental signatures of such a plasma may be looked for as observable consequences of a first order transition on the evolution of the system. In most of the possible scenario, the phase transition is accompanied with discontinuities in the hydrodynamic flow, such as shock waves. The method presented in this paper has been developed to treat without too much numerical effort such discontinuous flow. It relies heavily on the use of similarity solutions of the hydrodynamic equations

  11. SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome.

    Science.gov (United States)

    Perez, Yonatan; Shorer, Zamir; Liani-Leibson, Keren; Chabosseau, Pauline; Kadir, Rotem; Volodarsky, Michael; Halperin, Daniel; Barber-Zucker, Shiran; Shalev, Hanna; Schreiber, Ruth; Gradstein, Libe; Gurevich, Evgenia; Zarivach, Raz; Rutter, Guy A; Landau, Daniel; Birk, Ohad S

    2017-04-01

    A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9's highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through

  12. Self-similar gravitational clustering

    International Nuclear Information System (INIS)

    Efstathiou, G.; Fall, S.M.; Hogan, C.

    1979-01-01

    The evolution of gravitational clustering is considered and several new scaling relations are derived for the multiplicity function. These include generalizations of the Press-Schechter theory to different densities and cosmological parameters. The theory is then tested against multiplicity function and correlation function estimates for a series of 1000-body experiments. The results are consistent with the theory and show some dependence on initial conditions and cosmological density parameter. The statistical significance of the results, however, is fairly low because of several small number effects in the experiments. There is no evidence for a non-linear bootstrap effect or a dependence of the multiplicity function on the internal dynamics of condensed groups. Empirical estimates of the multiplicity function by Gott and Turner have a feature near the characteristic luminosity predicted by the theory. The scaling relations allow the inference from estimates of the galaxy luminosity function that galaxies must have suffered considerable dissipation if they originally formed from a self-similar hierarchy. A method is also developed for relating the multiplicity function to similar measures of clustering, such as those of Bhavsar, for the distribution of galaxies on the sky. These are shown to depend on the luminosity function in a complicated way. (author)

  13. Intracellular trafficking of new anticancer therapeutics: antibody–drug conjugates

    Science.gov (United States)

    Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

    2017-01-01

    Antibody–drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs. PMID:28814834

  14. Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates.

    Science.gov (United States)

    Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

    2017-01-01

    Antibody-drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs.

  15. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    Science.gov (United States)

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  16. Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection

    DEFF Research Database (Denmark)

    Ernstsen, Christina Lundgaard; Login, Frédéric H.; Jensen, Helene Halkjær

    2017-01-01

    Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacteria...

  17. Arylthiazole antibiotics targeting intracellular methicillin-resistant Staphylococcus aureus (MRSA) that interfere with bacterial cell wall synthesis.

    Science.gov (United States)

    Eid, Islam; Elsebaei, Mohamed M; Mohammad, Haroon; Hagras, Mohamed; Peters, Christine E; Hegazy, Youssef A; Cooper, Bruce; Pogliano, Joe; Pogliano, Kit; Abulkhair, Hamada S; Seleem, Mohamed N; Mayhoub, Abdelrahman S

    2017-10-20

    The promising antibacterial potency of arylthiazole antibiotics is offset by their limited activity against intracellular bacteria (namely methicillin-resistant Staphylococcus aureus (MRSA)), similar to many clinically-approved antibiotics. The failure to target these hidden pathogens is due to the compounds' lack of proper characteristics to accumulate intracellularly. Fine tuning of the size and polar-surface-area of the linking heteroaromatic ring provided a new series of 5-thiazolylarylthiazoles with balanced properties that allow them to sufficiently cross and accumulate inside macrophages infected with MRSA. The most promising compound 4i exhibited rapid bactericidal activity, good metabolic stability and produced over 80% reduction of intracellular MRSA in infected macrophages. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. Relationship between genetic similarity and some productive traits ...

    African Journals Online (AJOL)

    Admin

    Random amplified polymorphic DNA (RAPD) technique was applied to detect genetic similarity between five local chicken strains that have been selected for eggs and meat production in Egypt. Based on six oligonucleotide primers, the genetic similarity between the egg-producing strains (Anshas, Silver. Montazah and ...

  19. Intracellular sodium hydrogen exchange inhibition and clinical myocardial protection.

    Science.gov (United States)

    Mentzer, Robert M; Lasley, Robert D; Jessel, Andreas; Karmazyn, Morris

    2003-02-01

    Although the mechanisms underlying ischemia/reperfusion injury remain elusive, evidence supports the etiologic role of intracellular calcium overload and oxidative stress induced by reactive oxygen species. Activation of the sodium hydrogen exchanger (NHE) is associated with intracellular calcium accumulation. Inhibition of the NHE-1 isoform may attenuate the consequences of this injury. Although there is strong preclinical and early clinical evidence that NHE inhibitors may be cardioprotective, definitive proof of this concept in humans awaits the results of ongoing clinical trials.

  20. Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification.

    Directory of Open Access Journals (Sweden)

    Sha Liu

    Full Text Available Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1 in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs were incubated with either growth medium (1.4 mmol/L Pi or calcification medium (CM (3.0 mmol/L Pi. Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and

  1. Uptake and intracellular activity of AM-1155 in phagocytic cells.

    Science.gov (United States)

    Yamamoto, T; Kusajima, H; Hosaka, M; Fukuda, H; Oomori, Y; Shinoda, H

    1996-01-01

    The uptake and intracellular activity of AM-1155 in murine J774.1 macrophages and human polymorphonuclear leukocytes were investigated. AM-1155 penetrated phagocytic cells rapidly and reversibly, although the penetration process was not affected by metabolic inhibitors such as sodium fluoride, cyanide m-chlorophenylhydrazone, or ouabain or by nucleoside transport system inhibitors such as adenosine. The intracellular concentration-to-extracellular concentration ratio of AM-1155 in both cell types of phagocytes ranged from 5 to 7. These ratios were almost equal to those for sparfloxacin. The intracellular activity of AM-1155 in J774.1 macrophages, examined with Staphylococcus aureus 209P as a test bacterium, was dependent on the extracellular concentration. AM-1155 at a concentration of 1 microgram/ml reduced the number of viable cells of S. aureus ingested by more than 90%. The intracellular activity of AM-1155 was more potent than those of sparfloxacin, ofloxacin, ciprofloxacin, flomoxef, and erythromycin. These results suggest that the potent intracellular activity of AM-1155 might mainly be due to the high intracellular concentration and its potent in vitro activity. PMID:9124835

  2. Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Xianglu Li

    2009-01-01

    Full Text Available HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization.

  3. Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting.

    Science.gov (United States)

    Staquicini, Daniela I; Rangel, Roberto; Guzman-Rojas, Liliana; Staquicini, Fernanda I; Dobroff, Andrey S; Tarleton, Christy A; Ozbun, Michelle A; Kolonin, Mikhail G; Gelovani, Juri G; Marchiò, Serena; Sidman, Richard L; Hajjar, Katherine A; Arap, Wadih; Pasqualini, Renata

    2017-06-26

    Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

  4. The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles.

    Directory of Open Access Journals (Sweden)

    Dimitris Missirlis

    Full Text Available BACKGROUND: Peptide amphiphiles (PAs are a class of amphiphilic molecules able to self-assemble into nanomaterials that have shown efficient in vivo targeted delivery. Understanding the interactions of PAs with cells and the mechanisms of their internalization and intracellular trafficking is critical in their further development for therapeutic delivery applications. METHODOLOGY/PRINCIPAL FINDINGS: PAs of a novel, cell- and tissue-penetrating peptide were synthesized possessing two different lipophilic tail architectures and their interactions with prostate cancer cells were studied in vitro. Cell uptake of peptides was greatly enhanced post-modification. Internalization occurred via lipid-raft mediated endocytosis and was common for the two analogs studied. On the contrary, we identified the non-peptidic part as the determining factor of differences between intracellular trafficking and retention of PAs. PAs composed of di-stearyl lipid tails linked through poly(ethylene glycol to the peptide exhibited higher exocytosis rates and employed different recycling pathways compared to ones consisting of di-palmitic-coupled peptides. As a result, cell association of the former PAs decreased with time. CONCLUSIONS/SIGNIFICANCE: Control over peptide intracellular localization and retention is possible by appropriate modification with synthetic hydrophobic tails. We propose this as a strategy to design improved peptide-based delivery systems.

  5. Hypothetical physicochemical mechanisms of some intracellular processes: The hydrate hypothesis of mitosis and DNA replication

    International Nuclear Information System (INIS)

    Kadyshevich, E.A.; Ostrovskii, V.E.

    2007-01-01

    A DNA replication, mitosis, and binary fission hydrate hypothesis (MRH hypothesis) allowing non-trivial explanations for the physicochemical mechanisms of some intracellular processes is proposed. The hypothesis has a thermodynamic basis and is initiated by original experimental calorimetric and kinetic studies of the behavior of functional organic polymer and monomer substances in highly concentrated aqueous solutions. Experimental data demonstrating the occurrence of a short-range ordering in concentrated aqueous solutions of such substances are included. Hypothetical simple non-enzymatic unified mechanisms for the natural processes of DNA local unwinding preceding the start of duplication, DNA replication, formation and disappearance of the protein bonds between sister chromatids in the centromere region of eukaryotic DNA and in the centromere-like region of prokaryotic DNA, moving of daughter chromosomes apart to the opposite sides of cells in late anaphase, and formation of the nuclear envelopes in telophase and intracellular membranes between the newly formed nuclei in cytokinesis are formulated. The nature of a number of other intracellular phenomena is discussed

  6. Seniority bosons from similarity transformations

    International Nuclear Information System (INIS)

    Geyer, H.B.

    1986-01-01

    The requirement of associating in the boson space seniority with twice the number of non-s bosons defines a similarity transformation which re-expresses the Dyson pair boson images in terms of seniority bosons. In particular the fermion S-pair creation operator is mapped onto an operator which, unlike the pair boson image, does not change the number of non-s bosons. The original results of Otsuka, Arima and Iachello are recovered by this procedure while at the same time they are generalized to include g-bosons or even bosons with J>4 as well as any higher order boson terms. Furthermore the seniority boson images are valid for an arbitrary number of d- or g-bosons - a result which is not readily obtainable within the framework of the usual Marumori- or OAI-method

  7. SERS-Active Nanoinjector for Intracellular Spectroscopy

    Science.gov (United States)

    Vitol, Elina; Orynbayeva, Zulfiya; Bouchard, Michael; Azizkhan-Clifford, Jane; Friedman, Gary; Gogotsi, Yury

    2009-03-01

    We developed a multifunctional nanopipette which allows simultaneous cell injection and intacellular surface-enhanced Raman spectroscopy (SERS) analysis. SERS spectra contain the characteristic frequencies of molecular bond vibrations. This is a unique method for studying cell biochemistry and physiology on a single organelle level. Unlike the fluorescence spectroscopy, it does not require any specific staining. The principle of SERS is based on very large electromagnetic field enhancement localized around a nano-rough metallic surface. Gold colloids are widely used SERS substrates. Previously, the colloidal nanoparticles were introduced into a cell by the mechanism of endocytosis. The disadvantage of this method is the uncontrollable aggregation and distribution of gold nanoparticles inside a cell which causes a significant uncertainty in the origin of the acquired data. At the same time, the nanoparticle uptake is irreversible. We present a SERS-active nanoinjector, coated with gold nanoparticles, which enables selective signal acquisition from any point-of-interest inside a cell. The nanoinjector provides a highly localized SERS signal with sub-nanometer resolution in real time.

  8. Localization of SERBP1 in stress granules and nucleoli.

    Science.gov (United States)

    Lee, Yu-Jen; Wei, Hung-Ming; Chen, Ling-Yun; Li, Chuan

    2014-01-01

    SERPINE1 mRNA-binding protein 1 (SERBP1) is an arginine-methylated RNA-binding protein whose modification affects protein interaction and intracellular localization. In the present study, we show that, under normal growth conditions without stress, SERBP1 interacts with arginine-methylated and stress granule-associated proteins such as heterogeneous nuclear ribonucleoprotein A1, fragile X mental retardation protein and fragile X mental retardation syndrome-related protein 1 in an RNA-dependent manner. We also show that, after arsenite treatment, a proportion of full-length SERBP1 protein co-localizes with the typical stress granule marker T-cell intracellular antigen-1 in the cytoplasmic stress granules. Truncated SERBP1 with an N-terminal, central RG or C-terminal deletion, or single-domain segments comprising the N-terminal, central or C-terminal region, were recruited to stress granules upon arsenite treatment but with reduced efficiency. In addition, upon arsenite treatment, the localization of SERBP1 changed from a diffuse cytoplasmic localization to nuclear-dominant (concentrated in the nucleolus) A similar distribution was observed when cells were treated with the methylation inhibitor adenosine periodate, and was also detected for N- or C-terminal domain deletions and all three single-domain fragments even without stress induction. We further demonstrate that adenosine periodate treatment delays the association/dissociation of SERBP1 with stress granules. Hypomethylation retains SERBP1 in the nucleus/nucleolus regardless of arsenite treatment. Our study indicates that arginine methylation is correlated with recruitment of SERBP to stress granules and nucleoli and its retention therein. To our knowledge, this is the first report of an RNA-binding protein that is shifted simultaneously to cytoplasmic stress granules and nucleoli, two ribonucleoprotein-enriched subcellular compartments, upon stress. © 2013 FEBS.

  9. Decreased intracellular [Ca2+ ] coincides with reduced expression of Dhprα1s, RyR1, and diaphragmatic dysfunction in a rat model of sepsis.

    Science.gov (United States)

    Wang, Meng-Meng; Hao, Li-Ying; Guo, Feng; Zhong, Bin; Zhong, Xiao-Mei; Yuan, Jing; Hao, Yi-Fei; Zhao, Shuang; Sun, Xue-Fei; Lei, Ming; Jiao, Guang-Yu

    2017-12-01

    Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca 2+ ]) and the distribution and co-localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca 2+ ] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis. We measured diaphragm contractility and isolated diaphragm muscle cells in a rat model of sepsis. The distribution and co-localization of DHPRα1s and RyR1 were determined using immunohistochemistry and immunofluorescence, whereas intracellular [Ca 2+ ] was measured by confocal microscopy and fluorescence spectrophotometry. Septic rat diaphragm contractility, expression of DHPRα1s and RyR1, and intracellular [Ca 2+ ] were significantly decreased in the rat sepsis model compared with controls. Decreased intracellular [Ca 2+ ] coincides with diaphragmatic contractility and decreased expression of DHPRα1s and RyR1 in sepsis. Muscle Nerve 56: 1128-1136, 2017. © 2017 Wiley Periodicals, Inc.

  10. Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens

    Science.gov (United States)

    Gao, Junyuan; Sun, Xiurong; White, Thomas W.; Delamere, Nicholas A.; Mathias, Richard T.

    2015-01-01

    In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity. PMID:26536260

  11. Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro.

    Directory of Open Access Journals (Sweden)

    Alberto Canfrán-Duque

    Full Text Available First- and second-generation antipsychotics (FGAs and SGAs, respectively, both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [3H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.

  12. Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats.

    Science.gov (United States)

    Stern, Javier E; Potapenko, Evgeniy S

    2013-08-15

    An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca²⁺ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca²⁺ levels (NMDA-ΔCa²⁺) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa²⁺ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa²⁺ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca²⁺ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca²⁺ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa²⁺ signaling during HF are discussed.

  13. Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bjørnar Sporsheim

    2015-12-01

    Full Text Available Volatile allyl isothiocyanate (AITC derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER, vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system.

  14. A general mechanism for intracellular toxicity of metal-containing nanoparticles

    KAUST Repository

    Sabella, Stefania

    2014-04-09

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment-where particles are abundantly internalized-is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. The Royal Society of Chemistry 2014.

  15. A general mechanism for intracellular toxicity of metal-containing nanoparticles

    KAUST Repository

    Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

    2014-01-01

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment-where particles are abundantly internalized-is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. The Royal Society of Chemistry 2014.

  16. Alaska, Gulf spills share similarities

    International Nuclear Information System (INIS)

    Usher, D.

    1991-01-01

    The accidental Exxon Valdez oil spill in Alaska and the deliberate dumping of crude oil into the Persian Gulf as a tactic of war contain both glaring differences and surprising similarities. Public reaction and public response was much greater to the Exxon Valdez spill in pristine Prince William Sound than to the war-related tragedy in the Persian Gulf. More than 12,000 workers helped in the Alaskan cleanup; only 350 have been involved in Kuwait. But in both instances, environmental damages appear to be less than anticipated. Natures highly effective self-cleansing action is primarily responsible for minimizing the damages. One positive action growing out of the two incidents is increased international cooperation and participation in oil-spill clean-up efforts. In 1990, in the aftermath of the Exxon Valdez spill, 94 nations signed an international accord on cooperation in future spills. The spills can be historic environmental landmarks leading to creation of more sophisticated response systems worldwide

  17. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

    Science.gov (United States)

    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

  18. Factors influencing intracellular uptake and radiosensitization by 2-nitroimidazoles in vitro

    International Nuclear Information System (INIS)

    Brown, D.M.; Gonzalez-Mendez, R.; Brown, J.M.

    1983-01-01

    In this study it is shown that the radiosensitization of hypoxic Chinese hamster ovary (HA-1) cells in vitro by misonidazole (MIS) and other 1-substituted 2-nitroimidazoles depends on the rate and extent of intracellular uptake of these radiosensitizers, which in turn is governed by their lipophilicity [expressed as the octanol:water partition coefficient (P)]. As the lipophilicity of the compounds decreased, the rate of drug entry into the cells was slower, and below P values of approximately 0.05, peak intracellular drug concentrations were found to be lower than that of MIS (P=0.43). In addition, the number of hydroxyl groups on the side chain of the nitroimidazole molecule influenced the uptake of drug into the cells. For compounds of similar P, but differing in the number of side-chain hydroxyl groups, the addition of a single hydroxyl group to the molecule decreased the amount of drug entering the cell by a factor of approximately 2. These compounds enter the cell by nonmediated passive diffusion since altering the energy (ATP) capacity of the cell by 2-deoxyglucose did not affect uptake. It is also shown that increases in temperature or decreases in pH can increase the intracellular uptake of MIS. For example, equal intracellular and extracellular concentrations (100% uptake) of MIS were obtained if cells were heated to 44-45 0 C for 15 min compared to 20-40% uptake at 37 0 C. Increases in MIS uptake by factors of 2 to 3 could be demonstrated within 30 min when cells were incubated in Hanks' balanced salt solution at pH between 6.0 and 6.3 without loss of cell viability. In addition, MIS uptake in aerobic cultured cells varied from 15 to 60% depending on the cell line and culure conditions used

  19. Identification of key amino acid residues modulating intracellular and in vitro microcin E492 amyloid formation

    Directory of Open Access Journals (Sweden)

    Paulina eAguilera

    2016-01-01

    with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.

  20. Intracellular disposition of chitosan nanoparticles in macrophages: intracellular uptake, exocytosis, and intercellular transport

    Directory of Open Access Journals (Sweden)

    Jiang LQ

    2017-08-01

    Full Text Available Li Qun Jiang,1 Ting Yu Wang,1 Thomas J Webster,2 Hua-Jian Duan,1 Jing Ying Qiu,1 Zi Ming Zhao,1 Xiao Xing Yin,1,* Chun Li Zheng3,* 1Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, People’s Republic of China; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3School of Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Biodegradable nanomaterials have been widely used in numerous medical fields. To further improve such efforts, this study focused on the intracellular disposition of chitosan nanoparticles (CsNPs in macrophages, a primary cell of the mononuclear phagocyte system (MPS. Such interactions with the MPS determine the nanoparticle retention time in the body and consequently play a significant role in their own clinical safety. In this study, various dye-labeled CsNPs (about 250 nm were prepared, and a murine macrophage cell line (RAW 264.7 was selected as a model macrophage. The results showed two mechanisms of macrophage incorporation of CsNPs, ie, a clathrin-mediated endocytosis pathway (the primary and phagocytosis. Following internalization, the particles partly dissociated in the cells, indicating cellular digestion of the nanoparticles. It was proved that, after intracellular uptake, a large proportion of CsNPs were exocytosed within 24 h; this excretion induced a decrease in fluorescence intensity in cells by 69%, with the remaining particles possessing difficulty being cleared. Exocytosis could be inhibited by both wortmannin and vacuolin-1, indicating that CsNP uptake was mediated by lysosomal and multivesicular body pathways, and after exocytosis, the reuptake of CsNPs by neighboring cells was verified by further experiments. This study, thus, elucidated the fate of CsNPs in macrophages as well as identified cellular disposition

  1. Intracellular chemical gradients: morphing principle in bacteria

    Directory of Open Access Journals (Sweden)

    Endres Robert G

    2012-09-01

    Full Text Available Abstract Advances in computational biology allow systematic investigations to ascertain whether internal chemical gradients can be maintained in bacteria – an open question at the resolution limit of fluorescence microscopy. While it was previously believed that the small bacterial cell size and fast diffusion in the cytoplasm effectively remove any such gradient, a new computational study published in BMC Biophysics supports the emerging view that gradients can exist. The study arose from the recent observation that phosphorylated CtrA forms a gradient prior to cell division in Caulobacter crescentus, a bacterium known for its complicated cell cycle. Tropini et al. (2012 postulate that such gradients can provide an internal chemical compass, directing protein localization, cell division and cell development. More specifically, they describe biochemical and physical constraints on the formation of such gradients and explore a number of existing bacterial cell morphologies. These chemical gradients may limit in vitro analyses, and may ensure timing control and robustness to fluctuations during critical stages in cell development.

  2. Intracellular transport of fat-soluble vitamins A and E.

    Science.gov (United States)

    Kono, Nozomu; Arai, Hiroyuki

    2015-01-01

    Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E. © 2014 The Authors. Traffic published by John Wiley & Sons Ltd.

  3. Exploring anti-bacterial compounds against intracellular Legionella.

    Directory of Open Access Journals (Sweden)

    Christopher F Harrison

    Full Text Available Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.

  4. Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

    Directory of Open Access Journals (Sweden)

    William A. McEwan

    2016-11-01

    Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

  5. Advances in genetic manipulation of obligate intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Paul eBeare

    2011-05-01

    Full Text Available Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

  6. Exploring Anti-Bacterial Compounds against Intracellular Legionella

    Science.gov (United States)

    Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

    2013-01-01

    Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an ‘accidental’ human pathogen and cause a severe pneumonia known as Legionnaires’ disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoeba castellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

  7. Intracellular renin disrupts chemical communication between heart cells. Pathophysiological implications

    Directory of Open Access Journals (Sweden)

    Walmor eDe Mello

    2015-01-01

    Full Text Available The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; 1 under normal conditions, Lucifer Yellow flows from cell-to-cell through gap junctions; 2 the intracellular dialysis of renin (100nM disrupts chemical communication-an effect enhanced by simultaneous administration of angiotensinogen (100nM; 3 enalaprilat (10-9M administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; 4 aliskiren (10-8M inhibited the effect of renin on chemical communication;5 the possible role of intracellular renin independently of angiotensin II (Ang II was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; 6 the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed;7 the present results indicate that intracellular renin due to internalization or in situ synthesis, causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.

  8. Popularity versus similarity in growing networks.

    Science.gov (United States)

    Papadopoulos, Fragkiskos; Kitsak, Maksim; Serrano, M Ángeles; Boguñá, Marián; Krioukov, Dmitri

    2012-09-27

    The principle that 'popularity is attractive' underlies preferential attachment, which is a common explanation for the emergence of scaling in growing networks. If new connections are made preferentially to more popular nodes, then the resulting distribution of the number of connections possessed by nodes follows power laws, as observed in many real networks. Preferential attachment has been directly validated for some real networks (including the Internet), and can be a consequence of different underlying processes based on node fitness, ranking, optimization, random walks or duplication. Here we show that popularity is just one dimension of attractiveness; another dimension is similarity. We develop a framework in which new connections optimize certain trade-offs between popularity and similarity, instead of simply preferring popular nodes. The framework has a geometric interpretation in which popularity preference emerges from local optimization. As opposed to preferential attachment, our optimization framework accurately describes the large-scale evolution of technological (the Internet), social (trust relationships between people) and biological (Escherichia coli metabolic) networks, predicting the probability of new links with high precision. The framework that we have developed can thus be used for predicting new links in evolving networks, and provides a different perspective on preferential attachment as an emergent phenomenon.

  9. Fungal ABC Transporter Deletion and Localization Analysis

    NARCIS (Netherlands)

    Kovalchuk, A.; Weber, S.S.; Nijland, J.G.; Bovenberg, R.A.L.; Driessen, A.J.M.

    2012-01-01

    Fungal cells are highly complex as their metabolism is compartmentalized harboring various types of subcellular organelles that are bordered by one or more membranes. Knowledge about the intracellular localization of transporter proteins is often required for the understanding of their biological

  10. Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

    Directory of Open Access Journals (Sweden)

    Beijing K. Huang

    2014-01-01

    Full Text Available Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies.

  11. Simple Recovery of Intracellular Gold Nanoparticles from Peanut Seedling Roots.

    Science.gov (United States)

    Raju, D; Mehta, Urmil J; Ahmad, Absar

    2015-02-01

    Fabrication of inorganic nanomaterials via a biological route witnesses the formation either extracellularly, intracellulary or both. Whereas extracellular formation of these nanomaterials is cherished owing to their easy and economical extraction and purification processes; the intracellular formation of nanomaterials, due to the lack of a proper recovery protocol has always been dreaded, as the extraction processes used so far were tedious, costly, time consuming and often resulting in very low recovery. The aim of the present study was to overcome the problems related with the extraction and recovery of intracellularly synthesized inorganic nanoparticles, and to devise a method to increasing the output, the shape, size, composition and dispersal of nanoparticles is not altered. Water proved to be much better system as it provided well dispersed, stable gold nanoparticles and higher recovery. This is the first report, where intracellular nanoparticles have been recovered using a very cost-effective and eco-friendly approach.

  12. Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells.

    Science.gov (United States)

    Stitt, A W; Anderson, H R; Gardiner, T A; Bailie, J R; Archer, D B

    1994-08-01

    The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. These results illustrate the internalization and intracellular

  13. Cholera toxin subunit B-mediated intracellular trafficking of mesoporous silica nanoparticles toward the endoplasmic reticulum

    Science.gov (United States)

    Walker, William Andrew

    In recent decades, pharmaceutical research has led to the development of numerous treatments for human disease. Nanoscale delivery systems have the potential to maximize therapeutic outcomes by enabling target specific delivery of these therapeutics. The intracellular localization of many of these materials however, is poorly controlled, leading to sequestration in degradative cellular pathways and limiting the efficacy of their payloads. Numerous proteins, particularly bacterial toxins, have evolved mechanisms to subvert the degradative mechanisms of the cell. Here, we have investigated a possible strategy for shunting intracellular delivery of encapsulated cargoes from these pathways by modifying mesoporous silica nanoparticles (MSNs) with the well-characterized bacterial toxin Cholera toxin subunit B (CTxB). Using established optical imaging methods we investigated the internalization, trafficking, and subcellular localization of our modified MSNs in an in vitro animal cell model. We then attempted to demonstrate the practical utility of this approach by using CTxB-modified mesoporous silica nanoparticles to deliver propidium iodide, a membrane-impermeant fluorophore.

  14. Regulating Intracellular Calcium in Plants: From Molecular Genetics to Physiology

    International Nuclear Information System (INIS)

    Sze, Heven

    2008-01-01

    To grow, develop, adapt, and reproduce, plants have evolved mechanisms to regulate the uptake, translocation and sorting of calcium ions into different cells and subcellular compartments. Yet how plants accomplish this remarkable feat is still poorly understood. The spatial and temporal changes in intracellular (Ca2+) during growth and during responses to hormonal and environmental stimuli indicate that Ca2+ influx and efflux transporters are diverse and tightly regulated in plants. The specific goals were to determine the biological roles of multiple Ca pumps (ECAs) in the model plant Arabidopsis thaliana. We had pioneered the use of K616 yeast strain to functionally express plant Ca pumps, and demonstrated two distinct types of Ca pumps in plants (Sze et al., 2000. Annu Rev Plant Biol. 51,433). ACA2 represented one type that was auto-inhibited by the N-terminal region and stimulated by calmodulin. ECA1 represented another type that was not sensitive to calmodulin and phylogenetically distinct from ACAs. The goal to determine the biological roles of multiple ECA-type Ca pumps in Arabidopsis has been accomplished. Although we demonstrated ECA1 was a Ca pump by functional expression in yeast, the in vivo roles of ECAs was unclear. A few highlights are described. ECA1 and/or ECA4 are Ca/Mn pumps localized to the ER and are highly expressed in all cell types. Using homozygous T-DNA insertional mutants of eca1, we demonstrated that the ER-bound ECA1 supports growth and confers tolerance of plants growing on medium low in Ca or containing toxic levels of Mn. This is the first genetic study to determine the in vivo function of a Ca pump in plants. A phylogenetically distinct ECA3 is also a Ca/Mn pump that is localized to endosome, such as post-Golgi compartments. Although it is expressed at lower levels than ECA1, eca3 mutants are impaired in Ca-dependent root growth and in pollen tube elongation. Increased secretion of wall proteins in mutants suggests that Ca and Mn

  15. Tethering factors as organizers of intracellular vesicular traffic.

    Science.gov (United States)

    Yu, I-Mei; Hughson, Frederick M

    2010-01-01

    Intracellular trafficking entails the budding, transport, tethering, and fusion of transport vesicles and other membrane carriers. Here we review recent progress toward a mechanistic understanding of vesicle tethering. The known tethering factors are large complexes important for one or more intracellular trafficking pathways and are capable of interacting directly with many of the other principal components of the cellular trafficking machinery. Our review emphasizes recent developments in the in vitro reconstitution of vesicle tethering and the structural characterization of multisubunit tethering factors. The combination of these and other approaches has led to exciting progress toward understanding how these essential nanomachines work.

  16. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms.

    Science.gov (United States)

    Pareja, Maria Eugenia Mansilla; Colombo, Maria I

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.

  17. Intracellular trafficking of a pH-responsive drug metal complex.

    Science.gov (United States)

    Kheirolomoom, Azadeh; Ingham, Elizabeth S; Commisso, Joel; Abushaban, Neveen; Ferrara, Katherine W

    2016-12-10

    We previously developed a pH-responsive copper-doxorubicin (CuDox) cargo in lysolipid-based temperature-sensitive liposomes (LTSLs). The CuDox complex is released from the particle by elevated temperature; however, full release of doxorubicin from CuDox requires a reduced pH, such as that expected in lysosomes. The primary goal of this study is to evaluate the cellular uptake and intracellular trafficking of the drug-metal complex in comparison with intact liposomes and free drug. We found that the CuDox complex was efficiently internalized by mammary carcinoma cells after release from LTSLs. Intracellular doxorubicin and copper were 6-fold and 5-fold greater, respectively, after a 0.5h incubation with the released CuDox complex, as compared to incubation with intact liposomes containing the complex. Total cellular doxorubicin fluorescence was similar following CuDox and free doxorubicin incubation. Imaging and mass spectrometry assays indicated that the CuDox complex was initially internalized intact but breaks down over time within cells, with intracellular copper decreasing more rapidly than intracellular doxorubicin. Doxorubicin fluorescence was reduced when complexed with copper, and nuclear fluorescence was reduced when cells were incubated with the CuDox complex as compared with free doxorubicin. Therapeutic efficacy, which typically results from intercalation of doxorubicin with DNA, was equivalent for the CuDox complex and free doxorubicin and was superior to that of liposomal doxorubicin formulations. Taken together, the results suggest that quenched CuDox reaches the nucleus and remains efficacious. In order to design protocols for the use of these temperature-sensitive particles in cancer treatment, the timing of hyperthermia relative to drug administration must be examined. When cells were heated to 42°C prior to the addition of free doxorubicin, nuclear drug accumulation increased by 1.8-fold in cancer cells after 5h, and cytotoxicity increased 1

  18. Structure-Function Correlation Analysis of Connexin50 Missense Mutations Causing Congenital Cataract: Electrostatic Potential Alteration Could Determine Intracellular Trafficking Fate of Mutants

    Directory of Open Access Journals (Sweden)

    Devroop Sarkar

    2014-01-01

    Full Text Available Connexin50 (Cx50 mutations are reported to cause congenital cataract probably through the disruption of intercellular transport in the lens. Cx50 mutants that undergo mistrafficking have generally been associated with failure to form functional gap junction channels; however, sometimes even properly trafficked mutants were found to undergo similar consequences. We hereby wanted to elucidate any structural bases of the varied functional consequences of Cx50 missense mutations through in silico approach. Computational studies have been done based on a Cx50 homology model to assess conservation, solvent accessibility, and 3-dimensional localization of mutated residues as well as mutation-induced changes in surface electrostatic potential, H-bonding, and steric clash. This was supplemented with meta-analysis of published literature on the functional properties of connexin missense mutations. Analyses revealed that the mutation-induced critical alterations of surface electrostatic potential in Cx50 mutants could determine their fate in intracellular trafficking. A similar pattern was observed in case of mutations involving corresponding conserved residues in other connexins also. Based on these results the trafficking fates of 10 uncharacterized Cx50 mutations have been predicted. Further experimental analyses are needed to validate the observed correlation.

  19. Development of similarity theory for control systems

    Science.gov (United States)

    Myshlyaev, L. P.; Evtushenko, V. F.; Ivushkin, K. A.; Makarov, G. V.

    2018-05-01

    The area of effective application of the traditional similarity theory and the need necessity of its development for systems are discussed. The main statements underlying the similarity theory of control systems are given. The conditions for the similarity of control systems and the need for similarity control control are formulated. Methods and algorithms for estimating and similarity control of control systems and the results of research of control systems based on their similarity are presented. The similarity control of systems includes the current evaluation of the degree of similarity of control systems and the development of actions controlling similarity, and the corresponding targeted change in the state of any element of control systems.

  20. The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more.

    Science.gov (United States)

    Johannes, Ludger

    2002-07-01

    Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

  1. Intracellular Methamphetamine Prevents the Dopamine-induced Enhancement of Neuronal Firing*

    Science.gov (United States)

    Saha, Kaustuv; Sambo, Danielle; Richardson, Ben D.; Lin, Landon M.; Butler, Brittany; Villarroel, Laura; Khoshbouei, Habibeh

    2014-01-01

    The dysregulation of the dopaminergic system is implicated in multiple neurological and neuropsychiatric disorders such as Parkinson disease and drug addiction. The primary target of psychostimulants such as amphetamine and methamphetamine is the dopamine transporter (DAT), the major regulator of extracellular dopamine levels in the brain. However, the behavioral and neurophysiological correlates of methamphetamine and amphetamine administration are unique from one another, thereby suggesting these two compounds impact dopaminergic neurotransmission differentially. We further examined the unique mechanisms by which amphetamine and methamphetamine regulate DAT function and dopamine neurotransmission; in the present study we examined the impact of extracellular and intracellular amphetamine and methamphetamine on the spontaneous firing of cultured midbrain dopaminergic neurons and isolated DAT-mediated current. In dopaminergic neurons the spontaneous firing rate was enhanced by extracellular application of amphetamine > dopamine > methamphetamine and was DAT-dependent. Amphetamine > methamphetamine similarly enhanced DAT-mediated inward current, which was sensitive to isosmotic substitution of Na+ or Cl− ion. Although isosmotic substitution of extracellular Na+ ions blocked amphetamine and methamphetamine-induced DAT-mediated inward current similarly, the removal of extracellular Cl− ions preferentially blocked amphetamine-induced inward current. The intracellular application of methamphetamine, but not amphetamine, prevented the dopamine-induced increase in the spontaneous firing of dopaminergic neurons and the corresponding DAT-mediated inward current. The results reveal a new mechanism for methamphetamine-induced dysregulation of dopaminergic neurons. PMID:24962577

  2. Mixed quantization dimensions of self-similar measures

    International Nuclear Information System (INIS)

    Dai Meifeng; Wang Xiaoli; Chen Dandan

    2012-01-01

    Highlights: ► We define the mixed quantization dimension of finitely many measures. ► Formula of mixed quantization dimensions of self-similar measures is given. ► Illustrate the behavior of mixed quantization dimension as a function of order. - Abstract: Classical multifractal analysis studies the local scaling behaviors of a single measure. However recently mixed multifractal has generated interest. The purpose of this paper is some results about the mixed quantization dimensions of self-similar measures.

  3. Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy

    Science.gov (United States)

    There is increasing interest in using live-cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of multip...

  4. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

  5. Intracellular pH gradients in migrating cells

    DEFF Research Database (Denmark)

    Martin, Christine; Pedersen, Stine Helene Falsig; Schwab, Albrecht

    2011-01-01

    might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of p...

  6. Structural rearrangement of the intracellular domains during AMPA receptor activation

    DEFF Research Database (Denmark)

    Zachariassen, Linda Grønborg; Katchan, Ljudmila; Jensen, Anna Guldvang

    2016-01-01

    -clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence...

  7. The interferon response to intracellular DNA: why so many receptors?

    Science.gov (United States)

    Unterholzner, Leonie

    2013-11-01

    The detection of intracellular DNA has emerged to be a key event in the innate immune response to viruses and intracellular bacteria, and during conditions of sterile inflammation and autoimmunity. One of the consequences of the detection of DNA as a 'stranger' and a 'danger' signal is the production of type I interferons and pro-inflammatory cytokines. Much work has been dedicated to the elucidation of the signalling cascades that activate this DNA-induced gene expression programme. However, while many proteins have been proposed to act as sensors for intracellular DNA in recent years, none has been met with universal acceptance, and a theory linking all the recent observations is, as yet, lacking. This review presents the evidence for the various interferon-inducing DNA receptors proposed to date, and examines the hypotheses that might explain why so many different receptors appear to be involved in the innate immune recognition of intracellular DNA. Copyright © 2013 Elsevier GmbH. All rights reserved.

  8. Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

    Directory of Open Access Journals (Sweden)

    Dhritiman Samanta

    2017-05-01

    Full Text Available Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

  9. Engineering of obligate intracellular bacteria: progress, challenges and paradigms

    Science.gov (United States)

    Over twenty years have passed since the first report of genetic manipulation of an obligate intracellular bacterium. Through progress interspersed by bouts of stagnation, microbiologists and geneticists have developed approaches to genetically manipulate obligates. A brief overview of the current ge...

  10. Galectin-3 guides intracellular trafficking of some human serotransferrin glycoforms

    DEFF Research Database (Denmark)

    Carlsson, Carl Michael; Bengtson, Per; Cucak, Helena

    2013-01-01

    these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3 bound glycoform increased in cancer, suggesting...

  11. CONTRIBUTIONS OF INTRACELLULAR IONS TO Kv CHANNEL VOLTAGE SENSOR DYNAMICS.

    Directory of Open Access Journals (Sweden)

    Samuel eGoodchild

    2012-06-01

    Full Text Available Voltage sensing domains of Kv channels control ionic conductance through coupling of the movement of charged residues in the S4 segment to conformational changes at the cytoplasmic region of the pore domain, that allow K+ ions to flow. Conformational transitions within the voltage sensing domain caused by changes in the applied voltage across the membrane field are coupled to the conducting pore region and the gating of ionic conductance. However, several other factors not directly linked to the voltage dependent movement of charged residues within the voltage sensor impact the dynamics of the voltage sensor, such as inactivation, ionic conductance, intracellular ion identity and block of the channel by intracellular ligands. The effect of intracellular ions on voltage sensor dynamics is of importance in the interpretation of gating current measurements and the physiology of pore/voltage sensor coupling. There is a significant amount of variability in the reported kinetics of voltage sensor deactivation kinetics of Kv channels attributed to different mechanisms such as open state stabilization, immobilization and relaxation processes of the voltage sensor. Here we separate these factors and focus on the causal role that intracellular ions can play in allosterically modulating the dynamics of Kv voltage sensor deactivation kinetics. These considerations are of critical importance in understanding the molecular determinants of the complete channel gating cycle from activation to deactivation.

  12. Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses

    Directory of Open Access Journals (Sweden)

    Jinhua Li

    2016-02-01

    Full Text Available The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, ultramicrotomy, and scanning transmission X-ray microscopy (STXM. Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.

  13. Facilitating Intracellular Drug Delivery by Ultrasound-Activated Microbubbles

    NARCIS (Netherlands)

    Lammertink, BHA

    2017-01-01

    The goal of this thesis was to investigate the combination of ultrasound and microbubbles (USMB) for intracellular delivery of (model) drugs in vitro. We have focused on clinically approved drugs, i.e. cisplatin, and microbubbles, i.e. SonoVue™, to facilitate clinical translation. In addition, model

  14. Intracellular localization of Na /H antiporter from Malus zumi (MzNHX1)

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... protein since 1980s. The exogenous protein can be located on different membrane in yeast cells, which is impossible in bacteria. There are ... porter protein which locates on the plasma membrane or the tonoplast, it is wise to use eGFP as fluorescence mark in yeast cells, because that the technology of this.

  15. Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome

    NARCIS (Netherlands)

    Orelio, Claudia; van der Sluis, Renée M.; Verkuijlen, Paul; Nethe, Micha; Hordijk, Peter L.; van den Berg, Timo K.; Kuijpers, Taco W.

    2011-01-01

    Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS

  16. Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants

    NARCIS (Netherlands)

    Huang, M.; Jongejan, L.; Zheng, H.; Zhang, L.; Bol, J. F.

    2001-01-01

    Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In

  17. Induction of a stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased dependence on mitochondrial metabolism.

    Directory of Open Access Journals (Sweden)

    Eleanor C Saunders

    2014-01-01

    Full Text Available Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using (13C-stable isotope resolved metabolomics and (2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.

  18. Intergenotypic replacement of lyssavirus matrix proteins demonstrates the role of lyssavirus M proteins in intracellular virus accumulation.

    Science.gov (United States)

    Finke, Stefan; Granzow, Harald; Hurst, Jose; Pollin, Reiko; Mettenleiter, Thomas C

    2010-02-01

    Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

  19. Sequence relationships between the genome and the intracellular RNA species 1,3,6 and 7 of mouse hepatitis virus strain A59

    NARCIS (Netherlands)

    Horzinek, M.C.; Spaan, W.J.M.; Rottier, P.J.M.; Zeijst, B.A.M. van der

    1982-01-01

    We have shown by T1 oligonucleotide fingerprinting that the genome of mouse hepatitis virus strain A59 and its intracellular RNA 1 have identical fingerprints and that RNA 1 and the subgenomic RNAs 3, 6, and 7 contain common sequences. To localize the homologous region between the RNAs, we compared

  20. Single-cell intracellular nano-pH probes†

    Science.gov (United States)

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2016-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

  1. Single-cell intracellular nano-pH probes.

    Science.gov (United States)

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  2. Legionella pneumophila transcriptome during intracellular multiplication in human macrophages

    Directory of Open Access Journals (Sweden)

    Sebastien P Faucher

    2011-04-01

    Full Text Available Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot Type IVB secretion system (TFBSS, which is used to translocate more than 150 effector proteins to host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called SCOTS to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to L-arginine, L-histidine and L-proline as well as many transport systems involved in amino acid and iron uptake. Gene involved in catabolism of glycerol is also induced during intracellular growth and could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.

  3. Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

    2008-12-09

    For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.

  4. Marriage Matters: Spousal Similarity in Life Satisfaction

    OpenAIRE

    Ulrich Schimmack; Richard Lucas

    2006-01-01

    Examined the concurrent and cross-lagged spousal similarity in life satisfaction over a 21-year period. Analyses were based on married couples (N = 847) in the German Socio-Economic Panel (SOEP). Concurrent spousal similarity was considerably higher than one-year retest similarity, revealing spousal similarity in the variable component of life satisfac-tion. Spousal similarity systematically decreased with length of retest interval, revealing simi-larity in the changing component of life sati...

  5. Similarity queries for temporal toxicogenomic expression profiles.

    Directory of Open Access Journals (Sweden)

    Adam A Smith

    2008-07-01

    Full Text Available We present an approach for answering similarity queries about gene expression time series that is motivated by the task of characterizing the potential toxicity of various chemicals. Our approach involves two key aspects. First, our method employs a novel alignment algorithm based on time warping. Our time warping algorithm has several advantages over previous approaches. It allows the user to impose fairly strong biases on the form that the alignments can take, and it permits a type of local alignment in which the entirety of only one series has to be aligned. Second, our method employs a relaxed spline interpolation to predict expression responses for unmeasured time points, such that the spline does not necessarily exactly fit every observed point. We evaluate our approach using expression time series from the Edge toxicology database. Our experiments show the value of using spline representations for sparse time series. More significantly, they show that our time warping method provides more accurate alignments and classifications than previous standard alignment methods for time series.

  6. Localization Study of Co-Phthalocyanines in Cells by Raman Micro(spectro)scopy

    NARCIS (Netherlands)

    Arzhantsev, S.Y.; Arzhantsev, S.Y.; Chikishev, A.Y.; Chikishev, A.Y.; Koroteev, N.I.; Greve, Jan; Otto, Cornelis; Sijtsema, N.M.

    1999-01-01

    An investigation of intracellular localization of Co-phthalocyanines is reported. The Raman images of K562 cells stained with phthalocyanine were acquired. To understand the peculiarities of the Raman images, measurements were performed at different z-axis positions. The intracellular concentration

  7. Localization study of Co-phthalocyanines in cells by Raman micro(spectro)scopy

    NARCIS (Netherlands)

    Arzhantsev, S Y; Chikishev, A Y; Koroteev, N I; Greve, J; Otto, C; Sijtsema, N M

    An investigation of intracellular localization of Co-phthalocyanines is reported. The Raman images of K562 cells stained with phthalocyanine were acquired. To understand the peculiarities of the Raman images, measurements were performed at different z-axis positions. The intracellular concentration

  8. Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia

    DEFF Research Database (Denmark)

    Alexander, R Todd; Beggs, Megan R; Zamani, Reza

    2015-01-01

    role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestine, while pan-specific Pmca antibodies...... the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In human kidney, a similar pattern of distribution was observed, with highest PMCA4 expression in NCC positive tubules. Electron microscopy demonstrated Pmca4 localization...... in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments, but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing...

  9. Multi-Scale Scattering Transform in Music Similarity Measuring

    Science.gov (United States)

    Wang, Ruobai

    Scattering transform is a Mel-frequency spectrum based, time-deformation stable method, which can be used in evaluating music similarity. Compared with Dynamic time warping, it has better performance in detecting similar audio signals under local time-frequency deformation. Multi-scale scattering means to combine scattering transforms of different window lengths. This paper argues that, multi-scale scattering transform is a good alternative of dynamic time warping in music similarity measuring. We tested the performance of multi-scale scattering transform against other popular methods, with data designed to represent different conditions.

  10. Multielemental accumulation and its intracellular distribution in tissues of some aquatic birds

    International Nuclear Information System (INIS)

    Nam, Dong-Ha; Anan, Yasumi; Ikemoto, Tokutaka; Tanabe, Shinsuke

    2005-01-01

    This study was aimed at determining multielemental concentration and its intracellular distribution in selected tissues of cormorant and waterfowl species. Non-essential elements such as Hg, Tl, Cd, Pb and V in tissues were generally consistent with those in ingested items, indicating the significance of food sources of non-essential metal accumulation in great cormorants and mallards. Great cormorants and four waterfowl species examined reflected natural background levels of toxic metals such as Cd, Hg and Pb as well as some essential elements, indicating no specific metal exposure from local sources. Most of Cu, Zn, Se, Rb, Ag, Cd, Cs, and Hg contents were present in the hepatocytosolic fraction, whereas a large percentage of V and Mo were present in insoluble fraction in great cormorant, mallard, and spot-billed duck. The major role of these subcelluar fractions in elemental regulation accounts for the high percentage contribution of each cellular fraction to the total metal contents. Cadmium and Cu are chiefly sequestered through binding to metallothioneins (MTs) of hepatocytosolic fraction in these three avian species. Both MTs and high-molecular-weight substance (HMWS) for Zn and low-molecular-weight substance (LMWS) for Rb were also involved in their sequestration in cytosolic fractions. Relatively different species-specific cytosolic substances were responsible for varying degrees of Ag, Mn, and Co accumulation. It is worth noting that these intracellular metal levels in birds are closely regulated by metal-associated cellular constituents. Therefore, risk assessment studies of metal accumulation in such wild birds should take intracellular metal distribution and specific cellular constituents into account

  11. Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Zhenghang Zhao

    Full Text Available Recent studies have suggested that mitochondria may play important roles in the Ca(2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca(2+ flux can regulate the generation of Ca(2+ waves (CaWs and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca(2+ (Cai (2+ was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR Ca(2+ release and CaWs were induced in the presence of high (4 mM external Ca(2+ (Cao (2+. The protonophore carbonyl cyanide p-(trifluoromethoxyphenylhydrazone (FCCP reversibly raised basal Cai (2+ levels even after depletion of SR Ca(2+ in the absence of Cao (2+ , suggesting Ca(2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m or Ru360 (a mitochondrial Ca(2+ uniporter inhibitor, but not by oligomycin (an ATP synthase inhibitor or iodoacetic acid (a glycolytic inhibitor, excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca(2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca(2+ release and uptake exquisitely control the local Ca(2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.

  12. Characterization of intracellular palladium nanoparticles synthesized by Desulfovibrio desulfuricans and Bacillus benzeovorans

    Energy Technology Data Exchange (ETDEWEB)

    Omajali, Jacob B., E-mail: JBO037@bham.ac.uk, E-mail: jbomajali@gmail.com; Mikheenko, Iryna P. [University of Birmingham, Unit of Functional Bionanomaterials, School of Biosciences, Institute of Microbiology and Infection (United Kingdom); Merroun, Mohamed L. [University of Granada, Department of Microbiology, Faculty of Sciences (Spain); Wood, Joseph [University of Birmingham, School of Chemical Engineering (United Kingdom); Macaskie, Lynne E. [University of Birmingham, Unit of Functional Bionanomaterials, School of Biosciences, Institute of Microbiology and Infection (United Kingdom)

    2015-06-15

    Early studies have focused on the synthesis of palladium nanoparticles within the periplasmic layer or on the outer membrane of Desulfovibrio desulfuricans and on the S-layer protein of Bacillus sphaericus. However, it has remained unclear whether the synthesis of palladium nanoparticles also takes place in the bacterial cell cytoplasm. This study reports the use of high-resolution scanning transmission electron microscopy with a high-angle annular dark field detector and energy dispersive X-ray spectrometry attachment to investigate the intracellular synthesis of palladium nanoparticles (Pd NPs). We show the intracellular synthesis of Pd NPs within cells of two anaerobic strains of D. desulfuricans and an aerobic strain of B. benzeovorans using hydrogen and formate as electron donors. The Pd nanoparticles were small and largely monodispersed, between 0.2 and 8 nm, occasionally from 9 to 12 nm with occasional larger nanoparticles. With D. desulfuricans NCIMB 8307 (but not D. desulfuricans NCIMB 8326) and with B. benzeovorans NCIMB 12555, the NPs were larger when made at the expense of formate, co-localizing with phosphate in the latter, and were crystalline, but were amorphous when made with H{sub 2,} with no phosphorus association. The intracellular Pd nanoparticles were mainly icosahedrons with surfaces comprising {111} facets and about 5 % distortion when compared with that of bulk palladium. The particles were more concentrated in the cell cytoplasm than the cell wall, outer membrane, or periplasm. We provide new evidence for synthesis of palladium nanoparticles within the cytoplasm of bacteria, which were confirmed to maintain cellular integrity during this synthesis.

  13. Intracellular serotonin modulates insulin secretion from pancreatic beta-cells by protein serotonylation.

    Directory of Open Access Journals (Sweden)

    Nils Paulmann

    2009-10-01

    Full Text Available While serotonin (5-HT co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/- and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.

  14. Role of individual histidines in the pH-dependent global stability of human chloride intracellular channel 1.

    Science.gov (United States)

    Achilonu, Ikechukwu; Fanucchi, Sylvia; Cross, Megan; Fernandes, Manuel; Dirr, Heini W

    2012-02-07

    Chloride intracellular channel proteins exist in both a soluble cytosolic form and a membrane-bound form. The mechanism of conversion between the two forms is not properly understood, although one of the contributing factors is believed to be the variation in pH between the cytosol (~7.4) and the membrane (~5.5). We systematically mutated each of the three histidine residues in CLIC1 to an alanine at position 74 and a phenylalanine at positions 185 and 207. We examined the effect of the histidine-mediated pH dependence on the structure and global stability of CLIC1. None of the mutations were found to alter the global structure of the protein. However, the stability of H74A-CLIC1 and H185F-CLIC1, as calculated from the equilibrium unfolding data, is no longer dependent on pH because similar trends are observed at pH 7.0 and 5.5. The crystal structures show that the mutations result in changes in the local hydrogen bond coordination. Because the mutant total free energy change upon unfolding is not different from that of the wild type at pH 7.0, despite the presence of intermediates that are not seen in the wild type, we propose that it may be the stability of the intermediate state rather than the native state that is dependent on pH. On the basis of the lower stability of the intermediate in the H74A and H185F mutants compared to that of the wild type, we conclude that both His74 and His185 are involved in triggering the pH changes to the conformational stability of wild-type CLIC1 via their protonation, which stabilizes the intermediate state.

  15. A general mechanism for intracellular toxicity of metal-containing nanoparticles

    Science.gov (United States)

    Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman M.; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

    2014-05-01

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where particles are abundantly internalized - is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a ``lysosome-enhanced Trojan horse effect'' since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments.The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where

  16. Enhanced intracellular delivery and antibacterial efficacy of enrofloxacin-loaded docosanoic acid solid lipid nanoparticles against intracellular Salmonella.

    Science.gov (United States)

    Xie, Shuyu; Yang, Fei; Tao, Yanfei; Chen, Dongmei; Qu, Wei; Huang, Lingli; Liu, Zhenli; Pan, Yuanhu; Yuan, Zonghui

    2017-01-23

    Enrofloxacin-loaded docosanoic acid solid lipid nanoparticles (SLNs) with different physicochemical properties were developed to enhance activity against intracellular Salmonella. Their cellular uptake, intracellular elimination and antibacterial activity were studied in RAW 264.7 cells. During the experimental period, SLN-encapsulated enrofloxacin accumulated in the cells approximately 27.06-37.71 times more efficiently than free drugs at the same extracellular concentration. After incubation for 0.5 h, the intracellular enrofloxacin was enhanced from 0.336 to 1.147 μg/mg of protein as the sizes of nanoparticles were increased from 150 to 605 nm, and from 0.960 to 1.147 μg/mg of protein when the charge was improved from -8.1 to -24.9 mv. The cellular uptake was more significantly influenced by the size than it was by the charge, and was not affected by whether the charge was positive or negative. The elimination of optimal SLN-encapsulated enrofloxacin from the cells was significantly slower than that of free enrofloxacin after removing extracellular drug. The inhibition effect against intracellular Salmonella CVCC541 of 0.24 and 0.06 μg/mL encapsulated enrofloxacin was stronger than 0.6 μg/mL free drug after all of the incubation periods and at 48 h, respectively. Docosanoic acid SLNs are thus considered as a promising carrier for intracellular bacterial treatment.

  17. Cultural similarity, cultural competence, and nurse workforce diversity.

    Science.gov (United States)

    McGinnis, Sandra L; Brush, Barbara L; Moore, Jean

    2010-11-01

    Proponents of health workforce diversity argue that increasing the number of minority health care providers will enhance cultural similarity between patients and providers as well as the health system's capacity to provide culturally competent care. Measuring cultural similarity has been difficult, however, given that current benchmarks of workforce diversity categorize health workers by major racial/ethnic classifications rather than by cultural measures. This study examined the use of national racial/ethnic categories in both patient and registered nurse (RN) populations and found them to be a poor indicator of cultural similarity. Rather, we found that cultural similarity between RN and patient populations needs to be established at the level of local labor markets and broadened to include other cultural parameters such as country of origin, primary language, and self-identified ancestry. Only then can the relationship between cultural similarity and cultural competence be accurately determined and its outcomes measured.

  18. Ultrafine particles cause cytoskeletal dysfunctions in macrophages: role of intracellular calcium

    Directory of Open Access Journals (Sweden)

    Brown David M

    2005-10-01

    Full Text Available Abstract Background Particulate air pollution is reported to cause adverse health effects in susceptible individuals. Since most of these particles are derived form combustion processes, the primary composition product is carbon with a very small diameter (ultrafine, less than 100 nm in diameter. Besides the induction of reactive oxygen species and inflammation, ultrafine particles (UFP can cause intracellular calcium transients and suppression of defense mechanisms of alveolar macrophages, such as impaired migration or phagocytosis. Methods In this study the role of intracellular calcium transients caused by UFP was studied on cytoskeleton related functions in J774A.1 macrophages. Different types of fine and ultrafine carbon black particles (CB and ufCB, respectively, such as elemental carbon (EC90, commercial carbon (Printex 90, diesel particulate matter (DEP and urban dust (UD, were investigated. Phagosome transport mechanisms and mechanical cytoskeletal integrity were studied by cytomagnetometry and cell viability was studied by fluorescence microscopy. Macrophages were exposed in vitro with 100 and 320 μg UFP/ml/million cells for 4 hours in serum free medium. Calcium antagonists Verapamil, BAPTA-AM and W-7 were used to block calcium channels in the membrane, to chelate intracellular calcium or to inhibit the calmodulin signaling pathways, respectively. Results Impaired phagosome transport and increased cytoskeletal stiffness occurred at EC90 and P90 concentrations of 100 μg/ml/million cells and above, but not with DEP or UD. Verapamil and W-7, but not BAPTA-AM inhibited the cytoskeletal dysfunctions caused by EC90 or P90. Additionally the presence of 5% serum or 1% bovine serum albumin (BSA suppressed the cytoskeletal dysfunctions. Cell viability showed similar results, where co-culture of ufCB together with Verapamil, W-7, FCS or BSA produced less cell dead compared to the particles only.

  19. Polymeric nanoparticles for the intracellular delivery of paclitaxel in lung and breast cancer

    Science.gov (United States)

    Zubris, Kimberly Ann Veronica

    Nanoparticles are useful for addressing many of the difficulties encountered when administering therapeutic compounds. Nanoparticles are able to increase the solubility of hydrophobic drugs, improve pharmacokinetics through sustained release, alter biodistribution, protect sensitive drugs from low pH environments or enzymatic alteration, and, in some cases, provide targeting of the drug to the desired tissues. The use of functional nanocarriers can also provide controlled intracellular delivery of a drug. To this end, we have developed functional pH-responsive expansile nanoparticles for the intracellular delivery of paclitaxel. The pH-responsiveness of these nanoparticles occurs due to a hydrophobic to hydrophilic transition of the polymer occurring under mildly acidic conditions. These polymeric nanoparticles were systematically evaluated for the delivery of paclitaxel in vitro and in vivo to improve local therapy for lung and breast cancers. Nanoparticles were synthesized using a miniemulsion polymerization process and were subsequently characterized and found to swell when exposed to acidic environments. Paclitaxel was successfully encapsulated within the nanoparticles, and the particles exhibited drug release at pH 5 but not at pH 7.4. In addition, the uptake of nanoparticles was observed using flow cytometry, and the anticancer efficacy of the paclitaxel-loaded nanoparticles was measured using cancer cell lines in vitro. The potency of the paclitaxel-loaded nanoparticles was close to that of free drug, demonstrating that the drug was effectively delivered by the particles and that the particles could act as an intracellular drug depot. Following in vitro characterization, murine in vivo studies demonstrated the ability of the paclitaxel-loaded responsive nanoparticles to delay recurrence of lung cancer and to prevent establishment of breast cancer in the mammary fat pads with higher efficacy than paclitaxel alone. In addition, the ability of nanoparticles to

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

    International Nuclear Information System (INIS)

    Liu Xiaolong; Yuan Zhenglong; Chung, Maureen

    2008-01-01

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

  1. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Science.gov (United States)

    Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

    2012-01-01

    Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

  2. Preparation and characterization of vinculin-targeted polymer-lipid nanoparticle as intracellular delivery vehicle.

    Science.gov (United States)

    Wang, Junping; Ornek-Ballanco, Ceren; Xu, Jiahua; Yang, Weiguo; Yu, Xiaojun

    2013-01-01

    Intracellular delivery vehicles have been extensively investigated as these can serve as an effective tool in studying the cellular mechanism, by delivering functional protein to specific locations of the cells. In the current study, a polymer-lipid nanoparticle (PLN) system was developed as an intracellular delivery vehicle specifically targeting vinculin, a focal adhesion protein associated with cellular adhesive structures, such as focal adhesions and adherens junctions. The PLNs possessed an average size of 106 nm and had a positively charged surface. With a lower encapsulation efficiency 32% compared with poly(lactic-co-glycolic) acid (PLGA) nanoparticles (46%), the PLNs showed the sustained release profile of model drug BSA, while PLGA nanoparticles demonstrated an initial burst-release property. Cell-uptake experiments using mouse embryonic fibroblasts cultured in fibrin-fibronectin gels observed, under confocal microscope, that the anti-vinculin conjugated PLNs could successfully ship the cargo to the cytoplasm of fibroblasts, adhered to fibronectin-fibrin. With the use of cationic lipid, the unconjugated PLNs were shown to have high gene transfection efficiency. Furthermore, the unconjugated PLNs had nuclear-targeting capability in the absence of nuclear-localization signals. Therefore, the PLNs could be manipulated easily via different type of targeting ligands and could potentially be used as a powerful tool for cellular mechanism study, by delivering drugs to specific cellular organelles.

  3. Local food:

    DEFF Research Database (Denmark)

    Sundbo, Donna Isabella Caroline

    2013-01-01

    are identified and then categorised according to whether they pertain to the food product itself or the production methods and facilities and whether they describe physical or social properties of local food. From this a model with four categories is developed. It is found that properties of the product are more......Recently there has been more focus on food in general and local food in particular. But what is local food? And what are the perceptions of this concept according to theory and to providers and consumers of local food? This article first summarises and compares three different theoretical...... perspectives on local food, namely experience economy, local food systems and what is termed pro-industrialism. These have differing and sometimes opposite conceptualisations and aims for the concept of local food. Using the perspective of experience economy as theoretical background, the concept of local food...

  4. On different forms of self similarity

    International Nuclear Information System (INIS)

    Aswathy, R.K.; Mathew, Sunil

    2016-01-01

    Fractal geometry is mainly based on the idea of self-similar forms. To be self-similar, a shape must able to be divided into parts that are smaller copies, which are more or less similar to the whole. There are different forms of self similarity in nature and mathematics. In this paper, some of the topological properties of super self similar sets are discussed. It is proved that in a complete metric space with two or more elements, the set of all non super self similar sets are dense in the set of all non-empty compact sub sets. It is also proved that the product of self similar sets are super self similar in product metric spaces and that the super self similarity is preserved under isometry. A characterization of super self similar sets using contracting sub self similarity is also presented. Some relevant counterexamples are provided. The concepts of exact super and sub self similarity are introduced and a necessary and sufficient condition for a set to be exact super self similar in terms of condensation iterated function systems (Condensation IFS’s) is obtained. A method to generate exact sub self similar sets using condensation IFS’s and the denseness of exact super self similar sets are also discussed.

  5. Local perturbations perturb—exponentially–locally

    International Nuclear Information System (INIS)

    De Roeck, W.; Schütz, M.

    2015-01-01

    We elaborate on the principle that for gapped quantum spin systems with local interaction, “local perturbations [in the Hamiltonian] perturb locally [the groundstate].” This principle was established by Bachmann et al. [Commun. Math. Phys. 309, 835–871 (2012)], relying on the “spectral flow technique” or “quasi-adiabatic continuation” [M. B. Hastings, Phys. Rev. B 69, 104431 (2004)] to obtain locality estimates with sub-exponential decay in the distance to the spatial support of the perturbation. We use ideas of Hamza et al. [J. Math. Phys. 50, 095213 (2009)] to obtain similarly a transformation between gapped eigenvectors and their perturbations that is local with exponential decay. This allows to improve locality bounds on the effect of perturbations on the low lying states in certain gapped models with a unique “bulk ground state” or “topological quantum order.” We also give some estimate on the exponential decay of correlations in models with impurities where some relevant correlations decay faster than one would naively infer from the global gap of the system, as one also expects in disordered systems with a localized groundstate

  6. Local Content

    CSIR Research Space (South Africa)

    Gibberd, Jeremy

    2016-10-01

    Full Text Available Local content refers to materials and products made in a country as opposed those that are imported. There is an increasing interest in the concept of local content as a means of supporting local economies and providing jobs (Belderbos & Sleuwaegen...

  7. Regulation of dopamine transporter trafficking by intracellular amphetamine

    DEFF Research Database (Denmark)

    Kahlig, Kristopher M; Lute, Brandon J; Wei, Yuqiang

    2006-01-01

    -induced cell surface DAT redistribution may result in long-lasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent...... alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wild-type human DAT (WT-hDAT). However, AMPH did not induce cytosolic...... redistribution in an uptake-impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn(2+)) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn(2+) and AMPH consistently reduced WT-hDAT trafficking but stimulated cytosolic redistribution of Y335A...

  8. Intracellular compartmentalization of skeletal muscle glycogen metabolism and insulin signalling

    DEFF Research Database (Denmark)

    Prats Gavalda, Clara; Gomez-Cabello, Alba; Vigelsø Hansen, Andreas

    2011-01-01

    The interest in skeletal muscle metabolism and insulin signalling has increased exponentially in recent years as a consequence of their role in the development of type 2 diabetes mellitus. Despite this, the exact mechanisms involved in the regulation of skeletal muscle glycogen metabolism...... and insulin signalling transduction remain elusive. We believe that one of the reasons is that the role of intracellular compartmentalization as a regulator of metabolic pathways and signalling transduction has been rather ignored. This paper briefly reviews the literature to discuss the role of intracellular...... compartmentalization in the regulation of skeletal muscle glycogen metabolism and insulin signalling. As a result, a hypothetical regulatory mechanism is proposed by which cells could direct glycogen resynthesis towards different pools of glycogen particles depending on the metabolic needs. Furthermore, we discuss...

  9. Intracellular Chemistry: Integrating Molecular Inorganic Catalysts with Living Systems.

    Science.gov (United States)

    Ngo, Anh H; Bose, Sohini; Do, Loi H

    2018-03-23

    This concept article focuses on the rapid growth of intracellular chemistry dedicated to the integration of small-molecule metal catalysts with living cells and organisms. Although biological systems contain a plethora of biomolecules that can deactivate inorganic species, researchers have shown that small-molecule metal catalysts could be engineered to operate in heterogeneous aqueous environments. Synthetic intracellular reactions have recently been reported for olefin hydrogenation, hydrolysis/oxidative cleavage, azide-alkyne cycloaddition, allylcarbamate cleavage, C-C bond cross coupling, and transfer hydrogenation. Other promising targets for new biocompatible reaction discovery will also be discussed, with a special emphasis on how such innovations could lead to the development of novel technologies and chemical tools. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. An intracellular adrenomedullin system reduces IL-6 release via a NF-kB-mediated, cAMP-independent transcriptional mechanism in rat thymic epithelial cells.

    Science.gov (United States)

    Castellani, Giulia; Paliuri, Giovanna; Orso, Genny; Paccagnella, Nicola; D'Amore, Claudio; Facci, Laura; Cima, Francesca; Caicci, Federico; Palatini, Pietro; Bova, Sergio; De Martin, Sara

    2016-12-01

    Thymic epithelial cells (TECs) play a key role in the regulation of central immune tolerance by expressing autoantigens and eliminating self-reactive T cells. In a previous paper we reported that adrenomedullin (ADM) and its co-receptor protein RAMP2 are located intracellularly in newborn human thymic epithelial cells (TECs). This work has two main aims: (1) to examine the cellular localization of ADM and its receptor in TECs of adult Wistar rats to validate this animal model for the study of the ADM system and its function(s) in thymus; (2) to investigate the potential modulating effect of ADM on the NF-kB pathway, which is involved through the production of cytokines such as IL-6, in the maturation of T-lymphocytes and immunological tolerance. Our results show that, similarly to human newborn TECs, ADM is localized to the cytoplasm of adult rat TECs, and RAMP2 is expressed in the nucleus but not in the plasma membrane. Pretreatment of TECs for 4h with ADM significantly reduced lipopolysaccharide (LPS)-induced release of IL-6 (PkB, while doubled the expression of IkBα (PkB nuclear translocation. These effects were not mediated by activation of the cAMP pathway, a signalling cascade that is rapidly activated by ADM in cells that express plasma membrane RAMP2, but were the consequence of a reduction in the transcription of p65 (PkB genes transcription through an interaction with a receptor localized to the nucleus. This may partly explain the protective effects of ADM in autoimmune diseases and points to the ADM system of TECs as a novel potential target for immunomodulating drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Chelation of intracellular calcium blocks insulin action in the adipocyte

    International Nuclear Information System (INIS)

    Pershadsingh, H.A.; Shade, D.L.; Delfert, D.M.; McDonald, J.M.

    1987-01-01

    The hypothesis that intracellular Ca 2+ is an essential component of the intracellular mechanism of insulin action in the adipocyte was evaluated. Cells were loaded with the Ca 2+ chelator quin-2, by preincubating them with quin-2 AM, the tetrakis(acetoxymethyl) ester of quin-2. Quin-2 loading inhibited insulin-stimulated glucose transport without affecting basal activity. The ability of insulin to stimulate glucose uptake in quin-2-loaded cells could be partially restored by preincubating cells with buffer supplemented with 1.2 mM CaCl 2 and the Ca 2+ ionophore A23187. These conditions had no effect on basal activity and omission of CaCl 2 from the buffer prevented the restoration of insulin-stimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation and the ability of insulin to inhibit cAMP-stimulated lipolysis without affecting their basal activities. Incubation of cells with 100 μM quin-2 or quin-2 AM had no effect on intracellular ATP concentration or the specific binding of 125 I=labeled insulin to adipocytes. These findings suggest that intracellular Ca 2+ is an essential component in the coupling of the insulin-activated receptor complex to cellular physiological/metabolic machinery. Furthermore, differing quin-2 AM dose-response profiles suggest the presence of dual Ca 2+ -dependent pathways in the adipocyte. One involves insulin stimulation of glucose transport and oxidation, whereas the other involves the antilipolytic action of insulin

  12. Mycobacterium avium-intracellulare: a rare cause of subacromial bursitis.

    Science.gov (United States)

    Sinha, Raj; Tuckett, John; Hide, Geoff; Dildey, Petra; Karsandas, Alvin

    2015-01-01

    Septic subacromial bursitis is an uncommon disorder with only a few reported cases in the literature. The most common causative organism is Staphylococcus aureus. We report the case of a 61-year-old female with a septic subacromial bursitis where the causative organism was found to be Mycobacterium avium-intracellulare (MAI). The diagnosis was only made following a biopsy, and we use this case to highlight the importance of recognising the need to consider a biopsy and aspiration in atypical situations.

  13. Molecular evolution, intracellular organization, and the quinary structure of proteins.

    OpenAIRE

    McConkey, E H

    1982-01-01

    High-resolution two-dimensional polyacrylamide gel electrophoresis shows that at least half of 370 denatured polypeptides from hamster cells and human cells are indistinguishable in terms of isoelectric points and molecular weights. Molecular evolution may have been more conservative for this set of proteins than sequence studies on soluble proteins have implied. This may be a consequence of complexities of intracellular organization and the numerous macromolecular interactions in which most ...

  14. Control of intracellular heme levels: Heme transporters and heme oxygenases

    OpenAIRE

    Khan, Anwar A.; Quigley, John G.

    2011-01-01

    Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number...

  15. Rising Intracellular Zinc by Membrane Depolarization and Glucose in Insulin-Secreting Clonal HIT-T15 Beta Cells

    Directory of Open Access Journals (Sweden)

    Kira G. Slepchenko

    2012-01-01

    Full Text Available Zinc (Zn2+ appears to be intimately involved in insulin metabolism since insulin secretion is correlated with zinc secretion in response to glucose stimulation, but little is known about the regulation of zinc homeostasis in pancreatic beta-cells. This study set out to identify the intracellular zinc transient by imaging free cytosolic zinc in HIT-T15 beta-cells with fluorescent zinc indicators. We observed that membrane depolarization by KCl (30–60 mM was able to induce a rapid increase in cytosolic concentration of zinc. Multiple zinc transients of similar magnitude were elicited during repeated stimulations. The amplitude of zinc responses was not affected by the removal of extracellular calcium or zinc. However, the half-time of the rising slope was significantly slower after removing extracellular zinc with zinc chelator CaEDTA, suggesting that extracellular zinc affect the initial rising phase of zinc response. Glucose (10 mM induced substantial and progressive increases in intracellular zinc concentration in a similar way as KCl, with variation in the onset and the duration of zinc mobilization. It is known that the depolarization of beta-cell membrane is coupled with the secretion of insulin. Rising intracellular zinc concentration may act as a critical signaling factor in insulin metabolism of pancreatic beta-cells.

  16. Rising intracellular zinc by membrane depolarization and glucose in insulin-secreting clonal HIT-T15 beta cells.

    Science.gov (United States)

    Slepchenko, Kira G; Li, Yang V

    2012-01-01

    Zinc (Zn(2+)) appears to be intimately involved in insulin metabolism since insulin secretion is correlated with zinc secretion in response to glucose stimulation, but little is known about the regulation of zinc homeostasis in pancreatic beta-cells. This study set out to identify the intracellular zinc transient by imaging free cytosolic zinc in HIT-T15 beta-cells with fluorescent zinc indicators. We observed that membrane depolarization by KCl (30-60 mM) was able to induce a rapid increase in cytosolic concentration of zinc. Multiple zinc transients of similar magnitude were elicited during repeated stimulations. The amplitude of zinc responses was not affected by the removal of extracellular calcium or zinc. However, the half-time of the rising slope was significantly slower after removing extracellular zinc with zinc chelator CaEDTA, suggesting that extracellular zinc affect the initial rising phase of zinc response. Glucose (10 mM) induced substantial and progressive increases in intracellular zinc concentration in a similar way as KCl, with variation in the onset and the duration of zinc mobilization. It is known that the depolarization of beta-cell membrane is coupled with the secretion of insulin. Rising intracellular zinc concentration may act as a critical signaling factor in insulin metabolism of pancreatic beta-cells.

  17. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    Directory of Open Access Journals (Sweden)

    Hannah Karlsson

    Full Text Available CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

  18. Fatty Acid Signaling: The New Function of Intracellular Lipases

    Directory of Open Access Journals (Sweden)

    Zuzana Papackova

    2015-02-01

    Full Text Available Until recently, intracellular triacylglycerols (TAG stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed.

  19. Estimating the biophysical properties of neurons with intracellular calcium dynamics.

    Science.gov (United States)

    Ye, Jingxin; Rozdeba, Paul J; Morone, Uriel I; Daou, Arij; Abarbanel, Henry D I

    2014-06-01

    We investigate the dynamics of a conductance-based neuron model coupled to a model of intracellular calcium uptake and release by the endoplasmic reticulum. The intracellular calcium dynamics occur on a time scale that is orders of magnitude slower than voltage spiking behavior. Coupling these mechanisms sets the stage for the appearance of chaotic dynamics, which we observe within certain ranges of model parameter values. We then explore the question of whether one can, using observed voltage data alone, estimate the states and parameters of the voltage plus calcium (V+Ca) dynamics model. We find the answer is negative. Indeed, we show that voltage plus another observed quantity must be known to allow the estimation to be accurate. We show that observing both the voltage time course V(t) and the intracellular Ca time course will permit accurate estimation, and from the estimated model state, accurate prediction after observations are completed. This sets the stage for how one will be able to use a more detailed model of V+Ca dynamics in neuron activity in the analysis of experimental data on individual neurons as well as functional networks in which the nodes (neurons) have these biophysical properties.

  20. Extracellular and Intracellular Mechanisms Mediating Metastatic Activity of Exogenous Osteopontin

    Science.gov (United States)

    Mandelin, Jami; Lin, Emme C. K.; Hu, Dana D.; Knowles, Susan K.; Do, Kim-Anh; Wang, Xuemei; Sage, E. Helene; Smith, Jeffrey W.; Arap, Wadih; Pasqualini, Renata

    2009-01-01

    BACKGROUND Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. We used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo designed to test extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS In the extracellular environment, we confirm that soluble osteopontin is required for its pro-metastatic effects; this phenomenon is specific, RGD-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induces rapid Tyr-418 dephosphorylation of c-Src, with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation is followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSION Our results reveal a complex molecular interaction as well as a dual role for osteopontin in metastasis that is dependent on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to anti-metastasis strategies. PMID:19224553

  1. Metabolic responses of primary and transformed cells to intracellular Listeria monocytogenes.

    Directory of Open Access Journals (Sweden)

    Nadine Gillmaier

    Full Text Available The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using (13C-labelled glucose or glutamine as carbon tracers. The (13C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.

  2. The β-hemolysin and intracellular survival of Streptococcus agalactiae in human macrophages.

    Science.gov (United States)

    Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara

    2013-01-01

    S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.

  3. The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

    Science.gov (United States)

    Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara

    2013-01-01

    S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches. PMID:23593170

  4. The β-hemolysin and intracellular survival of Streptococcus agalactiae in human macrophages.

    Directory of Open Access Journals (Sweden)

    Anubha Sagar

    Full Text Available S. agalactiae (group B streptococci, GBS is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.

  5. INTRACELLULAR COPPER ACCUMULATION ENHANCES THE GROWTH OF KINEOCOCCUS RADIOTOLERANS DURING CHRONIC IRRADIATION

    International Nuclear Information System (INIS)

    Bagwell, C; Charles Milliken, C

    2007-01-01

    The actinobacteria Kineococcus radiotolerans is highly resistant to ionizing radiation, desiccation, and oxidative stress; though the underlying biochemical mechanisms are unknown. The purpose of this study was to explore a possible linkage between the uptake of transition metals and extreme resistance to ionizing radiation and oxidative stress. The effects of 6 different divalent cationic metals on growth were examined in the absence of ionizing radiation. None of the metals tested were stimulatory, though cobalt was inhibitory to growth. In contrast, copper supplementation dramatically increased cell growth during chronic irradiation. K. radiotolerans exhibited specific uptake and intracellular accumulation of copper compared to only a weak response to both iron and manganese supplementation. Copper accumulation sensitized cells to hydrogen peroxide. Acute irradiation induced DNA damage was similar between the copper-loaded culture as the age-synchronized no copper control culture, though low molecular weight DNA was more persistent during post-irradiation recovery in the Cu-loaded culture. Still, the estimated times for genome restoration differed by only 1 hr between treatments. While we cannot discount the possibility that copper fulfills an unexpectedly important biochemical role in a radioactive environment; K. radiotolerans has a high capacity for intracellular copper sequestration, and presumably efficiently coordinated oxidative stress defenses and detoxification systems, which confers cross-protection from the damaging affects ionizing radiation

  6. Intracellular zinc activates KCNQ channels by reducing their dependence on phosphatidylinositol 4,5-bisphosphate.

    Science.gov (United States)

    Gao, Haixia; Boillat, Aurélien; Huang, Dongyang; Liang, Ce; Peers, Chris; Gamper, Nikita

    2017-08-01

    M-type (Kv7, KCNQ) potassium channels are proteins that control the excitability of neurons and muscle cells. Many physiological and pathological mechanisms of excitation operate via the suppression of M channel activity or expression. Conversely, pharmacological augmentation of M channel activity is a recognized strategy for the treatment of hyperexcitability disorders such as pain and epilepsy. However, physiological mechanisms resulting in M channel potentiation are rare. Here we report that intracellular free zinc directly and reversibly augments the activity of recombinant and native M channels. This effect is mechanistically distinct from the known redox-dependent KCNQ channel potentiation. Interestingly, the effect of zinc cannot be attributed to a single histidine- or cysteine-containing zinc-binding site within KCNQ channels. Instead, zinc dramatically reduces KCNQ channel dependence on its obligatory physiological activator, phosphatidylinositol 4,5-bisphosphate (PIP 2 ). We hypothesize that zinc facilitates interactions of the lipid-facing interface of a KCNQ protein with the inner leaflet of the plasma membrane in a way similar to that promoted by PIP 2 Because zinc is increasingly recognized as a ubiquitous intracellular second messenger, this discovery might represent a hitherto unknown native pathway of M channel modulation and provide a fresh strategy for the design of M channel activators for therapeutic purposes.

  7. Enhanced Intracellular Delivery and Tissue Retention of Nanoparticles by Mussel-Inspired Surface Chemistry.

    Science.gov (United States)

    Chen, Kai; Xu, Xiaoqiu; Guo, Jiawei; Zhang, Xuelin; Han, Songling; Wang, Ruibing; Li, Xiaohui; Zhang, Jianxiang

    2015-11-09

    Nanomaterials have been broadly studied for intracellular delivery of diverse compounds for diagnosis or therapy. Currently it remains challenging for discovering new biomolecules that can prominently enhance cellular internalization and tissue retention of nanoparticles (NPs). Herein we report for the first time that a mussel-inspired engineering approach may notably promote cellular uptake and tissue retention of NPs. In this strategy, the catechol moiety is covalently anchored onto biodegradable NPs. Thus, fabricated NPs can be more effectively internalized by sensitive and multidrug resistant tumor cells, as well as some normal cells, resulting in remarkably potentiated in vitro activity when an antitumor drug is packaged. Moreover, the newly engineered NPs afford increased tissue retention post local or oral delivery. This biomimetic approach is promising for creating functional nanomaterials for drug delivery, vaccination, and cell therapy.

  8. Large margin classification with indefinite similarities

    KAUST Repository

    Alabdulmohsin, Ibrahim; Cisse, Moustapha; Gao, Xin; Zhang, Xiangliang

    2016-01-01

    Classification with indefinite similarities has attracted attention in the machine learning community. This is partly due to the fact that many similarity functions that arise in practice are not symmetric positive semidefinite, i.e. the Mercer

  9. Testing Self-Similarity Through Lamperti Transformations

    KAUST Repository

    Lee, Myoungji; Genton, Marc G.; Jun, Mikyoung

    2016-01-01

    extensively, while statistical tests for self-similarity are scarce and limited to processes indexed in one dimension. This paper proposes a statistical hypothesis test procedure for self-similarity of a stochastic process indexed in one dimension and multi

  10. Personality similarity and life satisfaction in couples

    OpenAIRE

    Furler Katrin; Gomez Veronica; Grob Alexander

    2013-01-01

    The present study examined the association between personality similarity and life satisfaction in a large nationally representative sample of 1608 romantic couples. Similarity effects were computed for the Big Five personality traits as well as for personality profiles with global and differentiated indices of similarity. Results showed substantial actor and partner effects indicating that both partners' personality traits were related to both partners' life satisfaction. Personality similar...

  11. Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris.

    Science.gov (United States)

    Meza, Beatriz; de-Bashan, Luz E; Bashan, Yoav

    2015-01-01

    Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  12. New insights into valve-related intramural and intracellular bacterial diversity in infective endocarditis.

    Science.gov (United States)

    Oberbach, Andreas; Schlichting, Nadine; Feder, Stefan; Lehmann, Stefanie; Kullnick, Yvonne; Buschmann, Tilo; Blumert, Conny; Horn, Friedemann; Neuhaus, Jochen; Neujahr, Ralph; Bagaev, Erik; Hagl, Christian; Pichlmaier, Maximilian; Rodloff, Arne Christian; Gräber, Sandra; Kirsch, Katharina; Sandri, Marcus; Kumbhari, Vivek; Behzadi, Armirhossein; Behzadi, Amirali; Correia, Joao Carlos; Mohr, Friedrich Wilhelm; Friedrich, Maik

    2017-01-01

    In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE. Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM). Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified. The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may

  13. Diffusive spatio-temporal noise in a first-passage time model for intracellular calcium release

    KAUST Repository

    Flegg, Mark B.

    2013-01-01

    The intracellular release of calcium from the endoplasmic reticulum is controlled by ion channels. The resulting calcium signals exhibit a rich spatio-temporal signature, which originates at least partly from microscopic fluctuations. While stochasticity in the gating transition of ion channels has been incorporated into many models, the distribution of calcium is usually described by deterministic reaction-diffusion equations. Here we test the validity of the latter modeling approach by using two different models to calculate the frequency of localized calcium signals (calcium puffs) from clustered IP3 receptor channels. The complexity of the full calcium system is here limited to the basic opening mechanism of the ion channels and, in the mathematical reduction simplifies to the calculation of a first passage time. Two models are then studied: (i) a hybrid model, where channel gating is treated stochastically, while calcium concentration is deterministic and (ii) a fully stochastic model with noisy channel gating and Brownian calcium ion motion. The second model utilises the recently developed two-regime method [M. B. Flegg, S. J. Chapman, and R. Erban, "The two-regime method for optimizing stochastic reaction-diffusion simulations," J. R. Soc., Interface 9, 859-868 (2012)] in order to simulate a large domain with precision required only near the Ca2+ absorbing channels. The expected time for a first channel opening that results in a calcium puff event is calculated. It is found that for a large diffusion constant, predictions of the interpuff time are significantly overestimated using the model (i) with a deterministic non-spatial calcium variable. It is thus demonstrated that the presence of diffusive noise in local concentrations of intracellular Ca2+ ions can substantially influence the occurrence of calcium signals. The presented approach and results may also be relevant for other cell-physiological first-passage time problems with small ligand concentration

  14. DMPD: Intracellular DNA sensors in immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18573338 Intracellular DNA sensors in immunity. Takeshita F, Ishii KJ. Curr Opin Im...munol. 2008 Aug;20(4):383-8. Epub 2008 Jun 23. (.png) (.svg) (.html) (.csml) Show Intracellular DNA sensors ...in immunity. PubmedID 18573338 Title Intracellular DNA sensors in immunity. Authors Takeshita F, Ishii KJ. P

  15. DMPD: NOD-like receptors (NLRs): bona fide intracellular microbial sensors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18585455 NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Shaw...tml) (.csml) Show NOD-like receptors (NLRs): bona fide intracellular microbial sensors. PubmedID 18585455 Ti...tle NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Authors

  16. TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

    Directory of Open Access Journals (Sweden)

    Kwang-Hyung Kim

    2009-11-01

    Full Text Available The regulation of intracellular levels of reactive oxygen species (ROS is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we

  17. Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells.

    Science.gov (United States)

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2016-09-30

    Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis.

  18. BLAST and FASTA similarity searching for multiple sequence alignment.

    Science.gov (United States)

    Pearson, William R

    2014-01-01

    BLAST, FASTA, and other similarity searching programs seek to identify homologous proteins and DNA sequences based on excess sequence similarity. If two sequences share much more similarity than expected by chance, the simplest explanation for the excess similarity is common ancestry-homology. The most effective similarity searches compare protein sequences, rather than DNA sequences, for sequences that encode proteins, and use expectation values, rather than percent identity, to infer homology. The BLAST and FASTA packages of sequence comparison programs provide programs for comparing protein and DNA sequences to protein databases (the most sensitive searches). Protein and translated-DNA comparisons to protein databases routinely allow evolutionary look back times from 1 to 2 billion years; DNA:DNA searches are 5-10-fold less sensitive. BLAST and FASTA can be run on popular web sites, but can also be downloaded and installed on local computers. With local installation, target databases can be customized for the sequence data being characterized. With today's very large protein databases, search sensitivity can also be improved by searching smaller comprehensive databases, for example, a complete protein set from an evolutionarily neighboring model organism. By default, BLAST and FASTA use scoring strategies target for distant evolutionary relationships; for comparisons involving short domains or queries, or searches that seek relatively close homologs (e.g. mouse-human), shallower scoring matrices will be more effective. Both BLAST and FASTA provide very accurate statistical estimates, which can be used to reliably identify protein sequences that diverged more than 2 billion years ago.

  19. Quantification of the Intracellular Life Time of Water Molecules to Measure Transport Rates of Human Aquaglyceroporins.

    Science.gov (United States)

    Palmgren, Madelene; Hernebring, Malin; Eriksson, Stefanie; Elbing, Karin; Geijer, Cecilia; Lasič, Samo; Dahl, Peter; Hansen, Jesper S; Topgaard, Daniel; Lindkvist-Petersson, Karin

    2017-12-01

    Orthodox aquaporins are transmembrane channel proteins that facilitate rapid diffusion of water, while aquaglyceroporins facilitate the diffusion of small uncharged molecules such as glycerol and arsenic trioxide. Aquaglyceroporins play important roles in human physiology, in particular for glycerol metabolism and arsenic detoxification. We have developed a unique system applying the strain of the yeast Pichia pastoris, where the endogenous aquaporins/aquaglyceroporins have been removed and human aquaglyceroporins AQP3, AQP7, and AQP9 are recombinantly expressed enabling comparative permeability measurements between the expressed proteins. Using a newly established Nuclear Magnetic Resonance approach based on measurement of the intracellular life time of water, we propose that human aquaglyceroporins are poor facilitators of water and that the water transport efficiency is similar to that of passive diffusion across native cell membranes. This is distinctly different from glycerol and arsenic trioxide, where high glycerol transport efficiency was recorded.

  20. Testing Self-Similarity Through Lamperti Transformations

    KAUST Repository

    Lee, Myoungji

    2016-07-14

    Self-similar processes have been widely used in modeling real-world phenomena occurring in environmetrics, network traffic, image processing, and stock pricing, to name but a few. The estimation of the degree of self-similarity has been studied extensively, while statistical tests for self-similarity are scarce and limited to processes indexed in one dimension. This paper proposes a statistical hypothesis test procedure for self-similarity of a stochastic process indexed in one dimension and multi-self-similarity for a random field indexed in higher dimensions. If self-similarity is not rejected, our test provides a set of estimated self-similarity indexes. The key is to test stationarity of the inverse Lamperti transformations of the process. The inverse Lamperti transformation of a self-similar process is a strongly stationary process, revealing a theoretical connection between the two processes. To demonstrate the capability of our test, we test self-similarity of fractional Brownian motions and sheets, their time deformations and mixtures with Gaussian white noise, and the generalized Cauchy family. We also apply the self-similarity test to real data: annual minimum water levels of the Nile River, network traffic records, and surface heights of food wrappings. © 2016, International Biometric Society.

  1. Similarity increases altruistic punishment in humans.

    Science.gov (United States)

    Mussweiler, Thomas; Ockenfels, Axel

    2013-11-26

    Humans are attracted to similar others. As a consequence, social networks are homogeneous in sociodemographic, intrapersonal, and other characteristics--a principle called homophily. Despite abundant evidence showing the importance of interpersonal similarity and homophily for human relationships, their behavioral correlates and cognitive foundations are poorly understood. Here, we show that perceived similarity substantially increases altruistic punishment, a key mechanism underlying human cooperation. We induced (dis)similarity perception by manipulating basic cognitive mechanisms in an economic cooperation game that included a punishment phase. We found that similarity-focused participants were more willing to punish others' uncooperative behavior. This influence of similarity is not explained by group identity, which has the opposite effect on altruistic punishment. Our findings demonstrate that pure similarity promotes reciprocity in ways known to encourage cooperation. At the same time, the increased willingness to punish norm violations among similarity-focused participants provides a rationale for why similar people are more likely to build stable social relationships. Finally, our findings show that altruistic punishment is differentially involved in encouraging cooperation under pure similarity vs. in-group conditions.

  2. Women's Handwork: Stories of Similarity and Diversity.

    Science.gov (United States)

    Kellman, Julia

    1996-01-01

    Characterizes women's handwork (needlepoint, beadwork, embroidery, crocheting) as a valid means of artistic expression and an integral part of women's culture. Discusses the varieties of handwork and their relationships within local cultures. Recommends using these objects as focal points for studying art, local history, or social studies. (MJP)

  3. Transient fluctuations of intracellular zinc ions in cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuan [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Maret, Wolfgang, E-mail: womaret@utmb.edu [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2009-08-15

    Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn{sup 2+}). By employing a fluorescent Zn{sup 2+} probe, FluoZin-3 acetoxymethyl ester, intracellular Zn{sup 2+} concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn{sup 2+} concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn{sup 2+} concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn{sup 2+} concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn{sup 2+} concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn{sup 2+} concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn{sup 2+} concentrations, suggest a role for Zn{sup 2+} in the control of the cell cycle. Interventions targeted at these picomolar Zn{sup 2+} fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.

  4. The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling.

    Science.gov (United States)

    Lou, Hsin-Ya; Zhao, Wenting; Zeng, Yongpeng; Cui, Bianxiao

    2018-05-15

    Over the past decade, there has been growing interest in developing biosensors and devices with nanoscale and vertical topography. Vertical nanostructures induce spontaneous cell engulfment, which enhances the cell-probe coupling efficiency and the sensitivity of biosensors. Although local membranes in contact with the nanostructures are found to be fully fluidic for lipid and membrane protein diffusions, cells appear to actively sense and respond to the surface topography presented by vertical nanostructures. For future development of biodevices, it is important to understand how cells interact with these nanostructures and how their presence modulates cellular function and activities. How cells recognize nanoscale surface topography has been an area of active research for two decades before the recent biosensor works. Extensive studies show that surface topographies in the range of tens to hundreds of nanometers can significantly affect cell functions, behaviors, and ultimately the cell fate. For example, titanium implants having rough surfaces are better for osteoblast attachment and host-implant integration than those with smooth surfaces. At the cellular level, nanoscale surface topography has been shown by a large number of studies to modulate cell attachment, activity, and differentiation. However, a mechanistic understanding of how cells interact and respond to nanoscale topographic features is still lacking. In this Account, we focus on some recent studies that support a new mechanism that local membrane curvature induced by nanoscale topography directly acts as a biochemical signal to induce intracellular signaling, which we refer to as the curvature hypothesis. The curvature hypothesis proposes that some intracellular proteins can recognize membrane curvatures of a certain range at the cell-to-material interface. These proteins then recruit and activate downstream components to modulate cell signaling and behavior. We discuss current technologies

  5. Polymeric gel nanoparticle pH sensors for intracellular measurements

    OpenAIRE

    Almdal, Kristoffer; Andresen, Thomas Lars; Benjaminsen, Rikke Vicki; Christensen, Nynne Meyn; Henriksen, Jonas Rosager; Sun, Honghao

    2011-01-01

    Precise measurements of pH in cells and intracellular compartments are of importance to both the fundamental understanding of metabolism and to the development of drugs that are released from the endosomes-lysome pathway. We have developed polymer gel nanoparticles as carriers of covalently bound fluorophores for ratiometric measurements of pH. One pH insensitive fluorophore serves as a reference while one or more pH sensitive fluorophores serve to give the desired pH dependence of the output...

  6. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    Science.gov (United States)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

  7. Dependence of cerebral arterial contractions on intracellularly stored Ca++.

    Science.gov (United States)

    Sasaki, T; Kassell, N F; Zuccarello, M

    1986-01-01

    The purpose of the present study was to evaluate the dependence of the arterial contractions induced by different vasoactive agents upon intracellularly stored calcium in canine versus monkey cerebral arteries. The potency for inducing contractions in Ca++-free media was in the order of 9,11-epithio-11,12-metano-thromboxane A2 (STXA2) greater than prostaglandin F2 alpha (PGF2 alpha) much greater than serotonin greater than K+ in canine basilar arteries, and STXA2 greater than PGF2 alpha much greater than serotonin = K+ in monkey basilar arteries.

  8. Oxygen effect and intracellular oxygen content (adaptation hypothesis)

    Energy Technology Data Exchange (ETDEWEB)

    Yarmonenko, S P; Ehpshtejn, I M [Akademiya Meditsinskikh Nauk SSSR, Moscow. Onkologicheskij Tsentr

    1977-01-01

    Experimental data indicating that a radiomodifying action of hypoxia is dependent on the ''prehistory'' of the irradiated object are considered. This dependence manifests itself in a decreased protective action of acute hypoxia on the hypoxia-adapted objects. To explain this a hypothesis is proposed connecting a degree of cell radiosensitivity modification, determined by the oxygen effect, with the intracellular oxygen content. The latter, in accord with current ideas, is regulated by variations in the diffusion resistance to oxygen shown by the cytoplasmic membranes depending on the energy level of the cell and the degree of its oxygenation.

  9. Oxygen effect and intracellular oxygen content (adaptation hypothesis)

    International Nuclear Information System (INIS)

    Yarmonenko, S.P.; Ehpshtejn, I.M.

    1977-01-01

    Experimental data indicating that a radiomodifying action of hypoxia is dependent on the ''prehistory'' of the irradiated object are considered. This dependence manifests itself in a decreased protective action of acute hypoxia on the hypoxia-adapted objects. To explain this a hypothesis is proposed connecting a degree of cell radiosensitivity modification, determined by the oxygen effect, with the intracellular oxygen content. The latter, in accord with current ideas, is regulated by variations in the diffusion resistance to oxygen shown by the cytoplasmic membranes depending on the energy level of the cell and the degree of its oxygenation

  10. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    Wagner, G.J.

    1979-01-01

    The mechanisms underlying heavy metal accumulation, toxicity, and tolerance in higher plants are poorly understood. Since subcellular processes are undoubtedly involved in all these phenomena, it is of interest to study the extent, subcellular site and nature of intracellularly accumulated cadmium in higher plants. Whole plants supplied 109 CdCl 2 or 112 CdSO 4 accumulated Cd into roots and aerial tissues. Preparation of protoplasts from aerial tissues followed by subcellular fractionation of the protoplasts to obtain intact vacuoles, chloroplasts and cytosol revealed the presence of Cd in the cytosol but not in vacuoles or chloroplasts. No evidence was obtained for the production of volatile Cd complexes in tobacco

  11. Long-term inhibition of cyclophilin D results in intracellular translocation of calcein AM from mitochondria to lysosomes.

    Science.gov (United States)

    Shinohe, Daisuke; Kobayashi, Asuka; Gotoh, Marina; Tanaka, Kotaro; Ohta, Yoshihiro

    2017-01-01

    Cyclophilin D is a peptidyl-prolyl cis-trans isomerase localized in the mitochondrial matrix. Although its effects on mitochondrial characteristics have been well studied, its relation to the uptake of molecules by mitochondria remains unknown. Here, we demonstrated the effects of cyclophilin D on the intracellular translocation of calcein AM. Following addition of calcein AM to control cells or cells overexpressing wild-type cyclophilin D, calcein fluorescence was observed in mitochondria. However, long-term inhibition of cyclophilin D in these cells altered the localization of calcein fluorescence from mitochondria to lysosomes without changing mitochondrial esterase activity. In addition, depletion of glucose from the medium recovered calcein localization from lysosomes to mitochondria. This is the first demonstration of the effects of cyclophilin D on the intracellular translocation of molecules other than proteins and suggests that cyclophilin D may modify mitochondrial features by inducing the translocation of molecules to the mitochondria through the mechanism associated with cellular energy metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Notions of similarity for systems biology models.

    Science.gov (United States)

    Henkel, Ron; Hoehndorf, Robert; Kacprowski, Tim; Knüpfer, Christian; Liebermeister, Wolfram; Waltemath, Dagmar

    2018-01-01

    Systems biology models are rapidly increasing in complexity, size and numbers. When building large models, researchers rely on software tools for the retrieval, comparison, combination and merging of models, as well as for version control. These tools need to be able to quantify the differences and similarities between computational models. However, depending on the specific application, the notion of 'similarity' may greatly vary. A general notion of model similarity, applicable to various types of models, is still missing. Here we survey existing methods for the comparison of models, introduce quantitative measures for model similarity, and discuss potential applications of combined similarity measures. To frame model comparison as a general problem, we describe a theoretical approach to defining and computing similarities based on a combination of different model aspects. The six aspects that we define as potentially relevant for similarity are underlying encoding, references to biological entities, quantitative behaviour, qualitative behaviour, mathematical equations and parameters and network structure. We argue that future similarity measures will benefit from combining these model aspects in flexible, problem-specific ways to mimic users' intuition about model similarity, and to support complex model searches in databases. © The Author 2016. Published by Oxford University Press.

  13. Similar speaker recognition using nonlinear analysis

    International Nuclear Information System (INIS)

    Seo, J.P.; Kim, M.S.; Baek, I.C.; Kwon, Y.H.; Lee, K.S.; Chang, S.W.; Yang, S.I.

    2004-01-01

    Speech features of the conventional speaker identification system, are usually obtained by linear methods in spectral space. However, these methods have the drawback that speakers with similar voices cannot be distinguished, because the characteristics of their voices are also similar in spectral space. To overcome the difficulty in linear methods, we propose to use the correlation exponent in the nonlinear space as a new feature vector for speaker identification among persons with similar voices. We show that our proposed method surprisingly reduces the error rate of speaker identification system to speakers with similar voices

  14. Localized superconductors

    International Nuclear Information System (INIS)

    Ma, M.; Lee, P.A.

    1985-01-01

    We study the effects of Anderson localization on superconductivity by using a Bardeen-Cooper-Schrieffer (BCS)-type trial wave function which pairs electrons in exact time-reversed eigenstates of the single-particle Hamiltonian. Within this approximation, and neglecting localization effects on the effective Coulomb repulsion and the electron-phonon coupling, we find that superconductivity persists below the mobility edge. In fact, Anderson's theorem is valid in the localized phase as long as rhoΔ 0 L/sup d/ > 1 (rho is the density of states averaged over +- Δ 0 of the Fermi energy, Δ 0 the BCS gap parameter, and L the localization length). Hence the gap order parameter Δ(r) remains uniform in space at the BCS value Δ 0 . The superfluid density and response to electromagnetic perturbations, however, show marked differences from the ''dirty superconductor'' regime. For rhoΔ 0 L/sup d/ < 1, Δ(r) fluctuates spatially and eventually drops to zero. In the limit when states are site localized, the system crosses over into the ''Anderson negative-U glass.'' Considerations beyond the trial wave-function approximation will speed up the destruction of superconductivity. The superconductor formed from localized states has the property that its quasiparticle excitations are also localized. Such excitations can be probed by observing the normal current in a tunneling junction

  15. The alteration of intracellular enzymes. III. The effect of temperature on the kinetics of altered and unaltered yeast catalase.

    Science.gov (United States)

    FRASER, M J; KAPLAN, J G

    1955-03-20

    1. The very large increase in catalase activity (Euler effect) which follows treatment of yeast cells with CHCl(3), UV and n-propanol is accompanied by highly significant changes in kinetic properties. With respect to the enzymatic decomposition of H(2)O(2), the thermodynamic constants of the activation process micro, DeltaHdouble dagger, DeltaSdouble dagger, DeltaFdouble dagger, decrease, following treatment of the intracellular enzyme, by 4.5 kcal., 4.5 kcal., 10.1 e.u. and 1.7 kcal., respectively, all these differences being significant at the 1 per cent level. 2. Similar differences exist between the untreated, intracellular enzyme on the one hand, and the extracted yeast and crystalline beef liver catalases on the other. Significant differences in these thermodynamic constants do not exist among the treated intracellular, extracted yeast, and crystalline liver catalases. 3. These data provide unequivocal confirmation of the phenomenon of enzyme alteration reported previously, and confirm previous evidence that the extracted and crystalline enzymes have also undergone enzyme alteration and have properties which are identical with, or very similar to, those of the catalase altered in situ. 4. With respect to the process of heat destruction of catalase, the greatly diminished stability to heat of the altered enzymes, previously reported, has been confirmed. The thermodynamic constants of activation of this process have likewise changed following alteration, in the case of micro, DeltaHdouble dagger, and DeltaSdouble dagger an increase of 20.6 kcal., 20.6 kcal., and 70 e.u., respectively, and of DeltaFdouble dagger a decrease of 2.8 kcal. 5. All these data have been shown to be consistent with, and in some cases predictable from, the interfacial hypothesis, which states that the unaltered catalase exists within the cell adsorbed to some interface, in a partially, but reversibly, unfolded configuration of relatively low specificity; enzyme alteration consists, in

  16. On self-similar Tolman models

    International Nuclear Information System (INIS)

    Maharaj, S.D.

    1988-01-01

    The self-similar spherically symmetric solutions of the Einstein field equation for the case of dust are identified. These form a subclass of the Tolman models. These self-similar models contain the solution recently presented by Chi [J. Math. Phys. 28, 1539 (1987)], thereby refuting the claim of having found a new solution to the Einstein field equations

  17. Mining Diagnostic Assessment Data for Concept Similarity

    Science.gov (United States)

    Madhyastha, Tara; Hunt, Earl

    2009-01-01

    This paper introduces a method for mining multiple-choice assessment data for similarity of the concepts represented by the multiple choice responses. The resulting similarity matrix can be used to visualize the distance between concepts in a lower-dimensional space. This gives an instructor a visualization of the relative difficulty of concepts…

  18. Similarity indices I: what do they measure

    International Nuclear Information System (INIS)

    Johnston, J.W.

    1976-11-01

    A method for estimating the effects of environmental effusions on ecosystems is described. The characteristics of 25 similarity indices used in studies of ecological communities were investigated. The type of data structure, to which these indices are frequently applied, was described as consisting of vectors of measurements on attributes (species) observed in a set of samples. A general similarity index was characterized as the result of a two-step process defined on a pair of vectors. In the first step an attribute similarity score is obtained for each attribute by comparing the attribute values observed in the pair of vectors. The result is a vector of attribute similarity scores. These are combined in the second step to arrive at the similarity index. The operation in the first step was characterized as a function, g, defined on pairs of attribute values. The second operation was characterized as a function, F, defined on the vector of attribute similarity scores from the first step. Usually, F was a simple sum or weighted sum of the attribute similarity scores. It is concluded that similarity indices should not be used as the test statistic to discriminate between two ecological communities

  19. On distributional assumptions and whitened cosine similarities

    DEFF Research Database (Denmark)

    Loog, Marco

    2008-01-01

    Recently, an interpretation of the whitened cosine similarity measure as a Bayes decision rule was proposed (C. Liu, "The Bayes Decision Rule Induced Similarity Measures,'' IEEE Trans. Pattern Analysis and Machine Intelligence, vol. 29, no. 6, pp. 1086-1090, June 2007. This communication makes th...

  20. Self-Similar Traffic In Wireless Networks

    OpenAIRE

    Jerjomins, R.; Petersons, E.

    2005-01-01

    Many studies have shown that traffic in Ethernet and other wired networks is self-similar. This paper reveals that wireless network traffic is also self-similar and long-range dependant by analyzing big amount of data captured from the wireless router.

  1. Similarity Structure of Wave-Collapse

    DEFF Research Database (Denmark)

    Rypdal, Kristoffer; Juul Rasmussen, Jens; Thomsen, Kenneth

    1985-01-01

    Similarity transformations of the cubic Schrödinger equation (CSE) are investigated. The transformations are used to remove the explicit time variation in the CSE and reduce it to differential equations in the spatial variables only. Two different methods for similarity reduction are employed and...

  2. Similarity indices I: what do they measure.

    Energy Technology Data Exchange (ETDEWEB)

    Johnston, J.W.

    1976-11-01

    A method for estimating the effects of environmental effusions on ecosystems is described. The characteristics of 25 similarity indices used in studies of ecological communities were investigated. The type of data structure, to which these indices are frequently applied, was described as consisting of vectors of measurements on attributes (species) observed in a set of samples. A general similarity index was characterized as the result of a two-step process defined on a pair of vectors. In the first step an attribute similarity score is obtained for each attribute by comparing the attribute values observed in the pair of vectors. The result is a vector of attribute similarity scores. These are combined in the second step to arrive at the similarity index. The operation in the first step was characterized as a function, g, defined on pairs of attribute values. The second operation was characterized as a function, F, defined on the vector of attribute similarity scores from the first step. Usually, F was a simple sum or weighted sum of the attribute similarity scores. It is concluded that similarity indices should not be used as the test statistic to discriminate between two ecological communities.

  3. Crystallographic study of FABP5 as an intracellular endocannabinoid transporter

    International Nuclear Information System (INIS)

    Sanson, Benoît; Wang, Tao; Sun, Jing; Wang, Liqun; Kaczocha, Martin; Ojima, Iwao; Deutsch, Dale; Li, Huilin

    2014-01-01

    FABP5 was recently found to intracellularly transport endocannabinoid signaling lipids. The structures of FABP5 complexed with two endocannabinoids and an inhibitor were solved. Human FABP5 was found to dimerize via a domain-swapping mechanism. This work will help in the development of inhibitors to raise endocannabinoid levels. In addition to binding intracellular fatty acids, fatty-acid-binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2-AG and the inhibitor BMS-309403 were determined. These ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5–endocannabinoid interactions and may be useful for future efforts in the development of small-molecule inhibitors to raise endocannabinoid levels

  4. Imaging the intracellular degradation of biodegradable polymer nanoparticles

    Directory of Open Access Journals (Sweden)

    Anne-Kathrin Barthel

    2014-10-01

    Full Text Available In recent years, the development of smart drug delivery systems based on biodegradable polymeric nanoparticles has become of great interest. Drug-loaded nanoparticles can be introduced into the cell interior via endocytotic processes followed by the slow release of the drug due to degradation of the nanoparticle. In this work, poly(L-lactic acid (PLLA was chosen as the biodegradable polymer. Although common degradation of PLLA has been studied in various biological environments, intracellular degradation processes have been examined only to a very limited extent. PLLA nanoparticles with an average diameter of approximately 120 nm were decorated with magnetite nanocrystals and introduced into mesenchymal stem cells (MSCs. The release of the magnetite particles from the surface of the PLLA nanoparticles during the intracellular residence was monitored by transmission electron microscopy (TEM over a period of 14 days. It was demonstrated by the release of the magnetite nanocrystals from the PLLA surface that the PLLA nanoparticles do in fact undergo degradation within the cell. Furthermore, even after 14 days of residence, the PLLA nanoparticles were found in the MSCs. Additionally, the ultrastructural TEM examinations yield insight into the long term intercellular fate of these nanoparticles. From the statistical analysis of ultrastructural details (e.g., number of detached magnetite crystals, and the number of nanoparticles in one endosome, we demonstrate the importance of TEM studies for such applications in addition to fluorescence studies (flow cytometry and confocal laser scanning microscopy.

  5. Fluorescent nanosensors for intracellular measurements: synthesis, characterisation, calibration and measurement

    Directory of Open Access Journals (Sweden)

    Arpan Shailesh Desai

    2014-01-01

    Full Text Available Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer and a pH-insensitive reference fluorophore (internal standard immobilised in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesised using standard laboratory equipment and are detectable by non-invasive widely accessibly imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: 1 synthesis and characterisation of polyacrylamide and silica based nanosensors 2 nanosensor calibration and 3 performing measurements using fluorescence microscopy.

  6. Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

    Energy Technology Data Exchange (ETDEWEB)

    Dobay, M. P. D., E-mail: maria.pamela.david@physik.uni-muenchen.de; Alberola, A. Piera; Mendoza, E. R.; Raedler, J. O., E-mail: joachim.raedler@physik.uni-muenchen.de [Ludwig-Maximilians University, Faculty of Physics, Center for NanoScience (Germany)

    2012-03-15

    Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

  7. Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

    International Nuclear Information System (INIS)

    Dobay, M. P. D.; Alberola, A. Piera; Mendoza, E. R.; Rädler, J. O.

    2012-01-01

    Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

  8. Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

    Science.gov (United States)

    Dobay, M. P. D.; Alberola, A. Piera; Mendoza, E. R.; Rädler, J. O.

    2012-03-01

    Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

  9. Molecular characterization of a novel intracellular ADP-ribosyl cyclase.

    Directory of Open Access Journals (Sweden)

    Dev Churamani

    2007-08-01

    Full Text Available ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates.Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1 is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained.Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.

  10. Light irradiation helps magnetotactic bacteria eliminate intracellular reactive oxygen species.

    Science.gov (United States)

    Li, Kefeng; Wang, Pingping; Chen, Chuanfang; Chen, Changyou; Li, Lulu; Song, Tao

    2017-09-01

    Magnetotactic bacteria (MTB) demonstrate photoresponse. However, little is known about the biological significance of this behaviour. Magnetosomes exhibit peroxidase-like activity and can scavenge reactive oxygen species (ROS). Magnetosomes extracted from the Magnetospirillum magneticum strain AMB-1 show enhanced peroxidase-like activity under illumination. The present study investigated the effects of light irradiation on nonmagnetic (without magnetosomes) and magnetic (with magnetosomes) AMB-1 cells. Results showed that light irradiation did not affect the growth of nonmagnetic and magnetic cells but significantly increased magnetosome synthesis and reduced intracellular ROS level in magnetic cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyse the expression level of magnetosome formation-associated genes (mamA, mms6, mms13 and mmsF) and stress-related genes (recA, oxyR, SOD, amb0664 and amb2684). Results showed that light irradiation upregulated the expression of mms6, mms13 and mmsF. Furthermore, light irradiation upregulated the expression of stress-related genes in nonmagnetic cells but downregulated them in magnetic cells. Additionally, magnetic cells exhibited stronger phototactic behaviour than nonmagnetic ones. These results suggested that light irradiation could heighten the ability of MTB to eliminate intracellular ROS and help them adapt to lighted environments. This phenomenon may be related to the enhanced peroxidase-like activity of magnetosomes under light irradiation. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Cell fate reprogramming by control of intracellular network dynamics

    Science.gov (United States)

    Zanudo, Jorge G. T.; Albert, Reka

    Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell's fate, such as disease therapeutics and stem cell reprogramming. Although the topic of controlling the dynamics of a system has a long history in control theory, most of this work is not directly applicable to intracellular networks. Here we present a network control method that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our control method takes advantage of certain function-dependent network components and their relation to steady states in order to identify control targets, which are guaranteed to drive any initial state to the target state with 100% effectiveness and need to be applied only transiently for the system to reach and stay in the desired state. We illustrate our method's potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. This work was supported by NSF Grant PHY 1205840.

  12. Transient light-induced intracellular oxidation revealed by redox biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Kolossov, Vladimir L., E-mail: viadimer@illinois.edu [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Beaudoin, Jessica N. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Hanafin, William P. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); DiLiberto, Stephen J. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Kenis, Paul J.A. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801 (United States); Rex Gaskins, H. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Avenue, Urbana, IL 61801 (United States); Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States)

    2013-10-04

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

  13. Downregulation of transferrin receptor surface expression by intracellular antibody

    International Nuclear Information System (INIS)

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

    2007-01-01

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

  14. Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels.

    Science.gov (United States)

    Muro-Pastor, M I; Reyes, J C; Florencio, F J

    2001-10-12

    The regulatory circuits that control nitrogen metabolism are relatively well known in several bacterial model groups. However, much less is understood about how the nitrogen status of the cell is perceived in vivo. In cyanobacteria, the transcription factor NtcA is required for regulation (activation or repression) of an extensive number of genes involved in nitrogen metabolism. In contrast, how NtcA activity is regulated is largely unknown. Assimilation of ammonium by most microorganisms occurs through the sequential action of two enzymes: glutamine synthetase (GS) and glutamate synthase. Interestingly, regulation of the expression of NtcA-dependent genes in the cyanobacterium Synechocystis sp. PCC 6803 is altered in mutants with modified levels of GS activity. Two types of mutants were analyzed: glnA null mutants that lack GS type I and gif mutants unable to inactivate GS in the presence of ammonium. Changes in the intracellular pools of 19 different amino acids and the keto acid 2-oxoglutarate were recorded in wild-type and mutant strains under different nitrogen conditions. Our data strongly indicate that the nitrogen status in cyanobacteria is perceived as changes in the intracellular 2-oxoglutarate pool.

  15. Optochemokine Tandem for Light-Control of Intracellular Ca2.

    Directory of Open Access Journals (Sweden)

    Katrin Feldbauer

    Full Text Available An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C, CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.

  16. Horizontal Transmission of Intracellular Insect Symbionts via Plants

    Directory of Open Access Journals (Sweden)

    Ewa Chrostek

    2017-11-01

    Full Text Available Experimental evidence is accumulating that endosymbionts of phytophagous insects may transmit horizontally via plants. Intracellular symbionts known for manipulating insect reproduction and altering fitness (Rickettsia, Cardinium, Wolbachia, and bacterial parasite of the leafhopper Euscelidius variegatus have been found to travel from infected insects into plants. Other insects, either of the same or different species can acquire the symbiont from the plant through feeding, and in some cases transfer it to their progeny. These reports prompt many questions regarding how intracellular insect symbionts are delivered to plants and how they affect them. Are symbionts passively transported along the insect-plant-insect path, or do they actively participate in the process? How widespread are these interactions? How does symbiont presence influence the plant? And what conditions are required for the new infection to establish in an insect? From an ecological, evolutionary, and applied perspective, this mode of horizontal transmission could have profound implications if occurring frequently enough or if new stable symbiont infections are established. Transmission of symbionts through plants likely represents an underappreciated means of infection, both in terms of symbiont epidemiology and the movement of symbionts to new host species.

  17. Transient light-induced intracellular oxidation revealed by redox biosensor

    International Nuclear Information System (INIS)

    Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J.A.; Rex Gaskins, H.

    2013-01-01

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition

  18. Crystallographic study of FABP5 as an intracellular endocannabinoid transporter

    Energy Technology Data Exchange (ETDEWEB)

    Sanson, Benoît; Wang, Tao [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Sun, Jing; Wang, Liqun; Kaczocha, Martin [Stony Brook University, Stony Brook, NY 11794-5213 (United States); Ojima, Iwao [Stony Brook University, Stony Brook, NY 1794-3400 (United States); Stony Brook University, Stony Brook, NY 11794-3400 (United States); Deutsch, Dale, E-mail: dale.deutsch@stonybrook.edu [Stony Brook University, Stony Brook, NY 11794-5213 (United States); Stony Brook University, Stony Brook, NY 11794-3400 (United States); Li, Huilin, E-mail: dale.deutsch@stonybrook.edu [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Stony Brook University, Stony Brook, NY 11794-5213 (United States); Stony Brook University, Stony Brook, NY 11794-3400 (United States)

    2014-02-01

    FABP5 was recently found to intracellularly transport endocannabinoid signaling lipids. The structures of FABP5 complexed with two endocannabinoids and an inhibitor were solved. Human FABP5 was found to dimerize via a domain-swapping mechanism. This work will help in the development of inhibitors to raise endocannabinoid levels. In addition to binding intracellular fatty acids, fatty-acid-binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2-AG and the inhibitor BMS-309403 were determined. These ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5–endocannabinoid interactions and may be useful for future efforts in the development of small-molecule inhibitors to raise endocannabinoid levels.

  19. CIRRHOSIS INDUCES APOPTOSIS IN RENAL TISSUE THROUGH INTRACELLULAR OXIDATIVE STRESS

    Directory of Open Access Journals (Sweden)

    Keli Cristina Simões da SILVEIRA

    2015-03-01

    Full Text Available Background Renal failure is a frequent and serious complication in patients with decompensated cirrhosis. Objectives We aimed to evaluate the renal oxidative stress, cell damage and impaired cell function in animal model of cirrhosis. Methods Secondary biliary cirrhosis was induced in rats by ligation of the common bile duct. We measured TBARS, ROS and mitochondrial membrane potential in kidney as markers of oxidative stress, and activities of the antioxidant enzymes. Relative cell viability was determined by trypan blue dye-exclusion assay. Annexin V-PE was used with a vital dye, 7-AAD, to distinguish apoptotic from necrotic cells and comet assay was used for determined DNA integrity in single cells. Results In bile duct ligation animals there was significant increase in the kidney lipoperoxidation and an increase of the level of intracellular ROS. There was too an increase in the activity of all antioxidant enzymes evaluated in the kidney. The percentage viability was above 90% in the control group and in bile duct ligation was 64.66% and the dominant cell death type was apoptosis. DNA damage was observed in the bile duct ligation. There was a decreased in the mitochondrial membrane potential from 71.40% ± 6.35% to 34.48% ± 11.40% in bile duct ligation. Conclusions These results indicate that intracellular increase of ROS cause damage in the DNA and apoptosis getting worse the renal function in cirrhosis.

  20. Information filtering based on transferring similarity.

    Science.gov (United States)

    Sun, Duo; Zhou, Tao; Liu, Jian-Guo; Liu, Run-Ran; Jia, Chun-Xiao; Wang, Bing-Hong

    2009-07-01

    In this Brief Report, we propose an index of user similarity, namely, the transferring similarity, which involves all high-order similarities between users. Accordingly, we design a modified collaborative filtering algorithm, which provides remarkably higher accurate predictions than the standard collaborative filtering. More interestingly, we find that the algorithmic performance will approach its optimal value when the parameter, contained in the definition of transferring similarity, gets close to its critical value, before which the series expansion of transferring similarity is convergent and after which it is divergent. Our study is complementary to the one reported in [E. A. Leicht, P. Holme, and M. E. J. Newman, Phys. Rev. E 73, 026120 (2006)], and is relevant to the missing link prediction problem.

  1. Self-similar continued root approximants

    International Nuclear Information System (INIS)

    Gluzman, S.; Yukalov, V.I.

    2012-01-01

    A novel method of summing asymptotic series is advanced. Such series repeatedly arise when employing perturbation theory in powers of a small parameter for complicated problems of condensed matter physics, statistical physics, and various applied problems. The method is based on the self-similar approximation theory involving self-similar root approximants. The constructed self-similar continued roots extrapolate asymptotic series to finite values of the expansion parameter. The self-similar continued roots contain, as a particular case, continued fractions and Padé approximants. A theorem on the convergence of the self-similar continued roots is proved. The method is illustrated by several examples from condensed-matter physics.

  2. Correlation between social proximity and mobility similarity.

    Science.gov (United States)

    Fan, Chao; Liu, Yiding; Huang, Junming; Rong, Zhihai; Zhou, Tao

    2017-09-20

    Human behaviors exhibit ubiquitous correlations in many aspects, such as individual and collective levels, temporal and spatial dimensions, content, social and geographical layers. With rich Internet data of online behaviors becoming available, it attracts academic interests to explore human mobility similarity from the perspective of social network proximity. Existent analysis shows a strong correlation between online social proximity and offline mobility similarity, namely, mobile records between friends are significantly more similar than between strangers, and those between friends with common neighbors are even more similar. We argue the importance of the number and diversity of common friends, with a counter intuitive finding that the number of common friends has no positive impact on mobility similarity while the diversity plays a key role, disagreeing with previous studies. Our analysis provides a novel view for better understanding the coupling between human online and offline behaviors, and will help model and predict human behaviors based on social proximity.

  3. Scalar Similarity for Relaxed Eddy Accumulation Methods

    Science.gov (United States)

    Ruppert, Johannes; Thomas, Christoph; Foken, Thomas

    2006-07-01

    The relaxed eddy accumulation (REA) method allows the measurement of trace gas fluxes when no fast sensors are available for eddy covariance measurements. The flux parameterisation used in REA is based on the assumption of scalar similarity, i.e., similarity of the turbulent exchange of two scalar quantities. In this study changes in scalar similarity between carbon dioxide, sonic temperature and water vapour were assessed using scalar correlation coefficients and spectral analysis. The influence on REA measurements was assessed by simulation. The evaluation is based on observations over grassland, irrigated cotton plantation and spruce forest. Scalar similarity between carbon dioxide, sonic temperature and water vapour showed a distinct diurnal pattern and change within the day. Poor scalar similarity was found to be linked to dissimilarities in the energy contained in the low frequency part of the turbulent spectra ( definition.

  4. Surf similarity and solitary wave runup

    DEFF Research Database (Denmark)

    Fuhrman, David R.; Madsen, Per A.

    2008-01-01

    The notion of surf similarity in the runup of solitary waves is revisited. We show that the surf similarity parameter for solitary waves may be effectively reduced to the beach slope divided by the offshore wave height to depth ratio. This clarifies its physical interpretation relative to a previ...... functional dependence on their respective surf similarity parameters. Important equivalencies in the runup of sinusoidal and solitary waves are thus revealed.......The notion of surf similarity in the runup of solitary waves is revisited. We show that the surf similarity parameter for solitary waves may be effectively reduced to the beach slope divided by the offshore wave height to depth ratio. This clarifies its physical interpretation relative...... to a previous parameterization, which was not given in an explicit form. Good coherency with experimental (breaking) runup data is preserved with this simpler parameter. A recasting of analytical (nonbreaking) runup expressions for sinusoidal and solitary waves additionally shows that they contain identical...

  5. Similarity in Bilateral Isolated Internal Orbital Fractures.

    Science.gov (United States)

    Chen, Hung-Chang; Cox, Jacob T; Sanyal, Abanti; Mahoney, Nicholas R

    2018-04-13

    In evaluating patients sustaining bilateral isolated internal orbital fractures, the authors have observed both similar fracture locations and also similar expansion of orbital volumes. In this study, we aim to investigate if there is a propensity for the 2 orbits to fracture in symmetrically similar patterns when sustaining similar trauma. A retrospective chart review was performed studying all cases at our institution of bilateral isolated internal orbital fractures involving the medial wall and/or the floor at the time of presentation. The similarity of the bilateral fracture locations was evaluated using the Fisher's exact test. The bilateral expanded orbital volumes were analyzed using the Wilcoxon signed-rank test to assess for orbital volume similarity. Twenty-four patients with bilateral internal orbital fractures were analyzed for fracture location similarity. Seventeen patients (70.8%) had 100% concordance in the orbital subregion fractured, and the association between the right and the left orbital fracture subregion locations was statistically significant (P < 0.0001). Fifteen patients were analyzed for orbital volume similarity. The average orbital cavity volume was 31.2 ± 3.8 cm on the right and 32.0 ± 3.7 cm on the left. There was a statistically significant difference between right and left orbital cavity volumes (P = 0.0026). The data from this study suggest that an individual who suffers isolated bilateral internal orbital fractures has a statistically significant similarity in the location of their orbital fractures. However, there does not appear to be statistically significant similarity in the expansion of the orbital volumes in these patients.

  6. Similar temperature dependencies of glycolytic enzymes: an evolutionary adaptation to temperature dynamics?

    Directory of Open Access Journals (Sweden)

    Cruz Luisa Ana B

    2012-12-01

    Full Text Available Abstract Background Temperature strongly affects microbial growth, and many microorganisms have to deal with temperature fluctuations in their natural environment. To understand regulation strategies that underlie microbial temperature responses and adaptation, we studied glycolytic pathway kinetics in Saccharomyces cerevisiae during temperature changes. Results Saccharomyces cerevisiae was grown under different temperature regimes and glucose availability conditions. These included glucose-excess batch cultures at different temperatures and glucose-limited chemostat cultures, subjected to fast linear temperature shifts and circadian sinoidal temperature cycles. An observed temperature-independent relation between intracellular levels of glycolytic metabolites and residual glucose concentration for all experimental conditions revealed that it is the substrate availability rather than temperature that determines intracellular metabolite profiles. This observation corresponded with predictions generated in silico with a kinetic model of yeast glycolysis, when the catalytic capacities of all glycolytic enzymes were set to share the same normalized temperature dependency. Conclusions From an evolutionary perspective, such similar temperature dependencies allow cells to adapt more rapidly to temperature changes, because they result in minimal perturbations of intracellular metabolite levels, thus circumventing the need for extensive modification of enzyme levels.

  7. Melanin targeting for intracellular drug delivery: Quantification of bound and free drug in retinal pigment epithelial cells.

    Science.gov (United States)

    Rimpelä, Anna-Kaisa; Hagström, Marja; Kidron, Heidi; Urtti, Arto

    2018-05-31

    Melanin binding affects drug distribution and retention in pigmented ocular tissues, thereby affecting drug response, duration of activity and toxicity. Therefore, it is a promising possibility for drug targeting and controlled release in the pigmented cells and tissues. Intracellular unbound drug concentrations determine pharmacological and toxicological actions, but analyses of unbound vs. total drug concentrations in pigmented cells are lacking. We studied intracellular binding and cellular drug uptake in pigmented retinal pigment epithelial cells and in non-pigmented ARPE-19 cells with five model drugs (chloroquine, propranolol, timolol, diclofenac, methotrexate). The unbound drug fractions in pigmented cells were 0.00016-0.73 and in non-pigmented cells 0.017-1.0. Cellular uptake (i.e. distribution ratio Kp), ranged from 1.3 to 6300 in pigmented cells and from 1.0 to 25 in non-pigmented cells. Values for intracellular bioavailability, F ic , were similar in both cells types (although larger variation in pigmented cells). In vitro melanin binding parameters were used to predict intracellular unbound drug fraction and cell uptake. Comparison of predictions with experimental data indicates that other factors (e.g. ion-trapping, lipophilicity-related binding to other cell components) also play a role. Melanin binding is a major factor that leads to cellular uptake and unbound drug fractions of a range of 3-4 orders of magnitude indicating that large reservoirs of melanin bound drug can be generated in the cells. Understanding melanin binding has important implications on retinal drug targeting, efficacy and toxicity. Copyright © 2017. Published by Elsevier B.V.

  8. Localized Scleroderma

    Science.gov (United States)

    ... How Is Localized Scleroderma Diagnosed? Doctors who are familiar with scleroderma, or who are experts at examining ... systemic treatment with a medication or other treatment interventions (for example, ultraviolet light), are reserved for more ...

  9. Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

    Science.gov (United States)

    Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

    2018-01-01

    Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Directory of Open Access Journals (Sweden)

    Unzueta U

    2012-08-01

    Full Text Available Ugutz Unzueta,1–3 María Virtudes Céspedes,3,4 Neus Ferrer-Miralles,1–3 Isolda Casanova,3,4 Juan Cedano,5 José Luis Corchero,1–3 Joan Domingo-Espín,1–3 Antonio Villaverde,1–3 Ramón Mangues,3,4 Esther Vázquez1–31Institut de Biotecnologia i de Biomedicina, 2Departamento de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 3CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, Barcelona, 4Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Laboratory of Immunology, Regional Norte, Universidad de la Republica, Salto, UruguayBackground: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4 is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing.Results: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer.Conclusion: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles

  11. Measure of Node Similarity in Multilayer Networks.

    Directory of Open Access Journals (Sweden)

    Anders Mollgaard

    Full Text Available The weight of links in a network is often related to the similarity of the nodes. Here, we introduce a simple tunable measure for analysing the similarity of nodes across different link weights. In particular, we use the measure to analyze homophily in a group of 659 freshman students at a large university. Our analysis is based on data obtained using smartphones equipped with custom data collection software, complemented by questionnaire-based data. The network of social contacts is represented as a weighted multilayer network constructed from different channels of telecommunication as well as data on face-to-face contacts. We find that even strongly connected individuals are not more similar with respect to basic personality traits than randomly chosen pairs of individuals. In contrast, several socio-demographics variables have a significant degree of similarity. We further observe that similarity might be present in one layer of the multilayer network and simultaneously be absent in the other layers. For a variable such as gender, our measure reveals a transition from similarity between nodes connected with links of relatively low weight to dis-similarity for the nodes connected by the strongest links. We finally analyze the overlap between layers in the network for different levels of acquaintanceships.

  12. Notions of similarity for computational biology models

    KAUST Repository

    Waltemath, Dagmar

    2016-03-21

    Computational models used in biology are rapidly increasing in complexity, size, and numbers. To build such large models, researchers need to rely on software tools for model retrieval, model combination, and version control. These tools need to be able to quantify the differences and similarities between computational models. However, depending on the specific application, the notion of similarity may greatly vary. A general notion of model similarity, applicable to various types of models, is still missing. Here, we introduce a general notion of quantitative model similarities, survey the use of existing model comparison methods in model building and management, and discuss potential applications of model comparison. To frame model comparison as a general problem, we describe a theoretical approach to defining and computing similarities based on different model aspects. Potentially relevant aspects of a model comprise its references to biological entities, network structure, mathematical equations and parameters, and dynamic behaviour. Future similarity measures could combine these model aspects in flexible, problem-specific ways in order to mimic users\\' intuition about model similarity, and to support complex model searches in databases.

  13. Trajectory similarity join in spatial networks

    KAUST Repository

    Shang, Shuo

    2017-09-07

    The matching of similar pairs of objects, called similarity join, is fundamental functionality in data management. We consider the case of trajectory similarity join (TS-Join), where the objects are trajectories of vehicles moving in road networks. Thus, given two sets of trajectories and a threshold θ, the TS-Join returns all pairs of trajectories from the two sets with similarity above θ. This join targets applications such as trajectory near-duplicate detection, data cleaning, ridesharing recommendation, and traffic congestion prediction. With these applications in mind, we provide a purposeful definition of similarity. To enable efficient TS-Join processing on large sets of trajectories, we develop search space pruning techniques and take into account the parallel processing capabilities of modern processors. Specifically, we present a two-phase divide-and-conquer algorithm. For each trajectory, the algorithm first finds similar trajectories. Then it merges the results to achieve a final result. The algorithm exploits an upper bound on the spatiotemporal similarity and a heuristic scheduling strategy for search space pruning. The algorithm\\'s per-trajectory searches are independent of each other and can be performed in parallel, and the merging has constant cost. An empirical study with real data offers insight in the performance of the algorithm and demonstrates that is capable of outperforming a well-designed baseline algorithm by an order of magnitude.

  14. The baryonic self similarity of dark matter

    International Nuclear Information System (INIS)

    Alard, C.

    2014-01-01

    The cosmological simulations indicates that dark matter halos have specific self-similar properties. However, the halo similarity is affected by the baryonic feedback. By using momentum-driven winds as a model to represent the baryon feedback, an equilibrium condition is derived which directly implies the emergence of a new type of similarity. The new self-similar solution has constant acceleration at a reference radius for both dark matter and baryons. This model receives strong support from the observations of galaxies. The new self-similar properties imply that the total acceleration at larger distances is scale-free, the transition between the dark matter and baryons dominated regime occurs at a constant acceleration, and the maximum amplitude of the velocity curve at larger distances is proportional to M 1/4 . These results demonstrate that this self-similar model is consistent with the basics of modified Newtonian dynamics (MOND) phenomenology. In agreement with the observations, the coincidence between the self-similar model and MOND breaks at the scale of clusters of galaxies. Some numerical experiments show that the behavior of the density near the origin is closely approximated by a Einasto profile.

  15. Notions of similarity for computational biology models

    KAUST Repository

    Waltemath, Dagmar; Henkel, Ron; Hoehndorf, Robert; Kacprowski, Tim; Knuepfer, Christian; Liebermeister, Wolfram

    2016-01-01

    Computational models used in biology are rapidly increasing in complexity, size, and numbers. To build such large models, researchers need to rely on software tools for model retrieval, model combination, and version control. These tools need to be able to quantify the differences and similarities between computational models. However, depending on the specific application, the notion of similarity may greatly vary. A general notion of model similarity, applicable to various types of models, is still missing. Here, we introduce a general notion of quantitative model similarities, survey the use of existing model comparison methods in model building and management, and discuss potential applications of model comparison. To frame model comparison as a general problem, we describe a theoretical approach to defining and computing similarities based on different model aspects. Potentially relevant aspects of a model comprise its references to biological entities, network structure, mathematical equations and parameters, and dynamic behaviour. Future similarity measures could combine these model aspects in flexible, problem-specific ways in order to mimic users' intuition about model similarity, and to support complex model searches in databases.

  16. Rapid tissue regeneration induced by intracellular ATP delivery-A preliminary mechanistic study.

    Directory of Open Access Journals (Sweden)

    Harshini Sarojini

    Full Text Available We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery-extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold

  17. Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Mori Isamu

    2006-12-01

    Full Text Available Abstract Background Recently it has been reported that, toll-like receptors (TLRs are expressed on a series of tumor cells, such as colon cancer, breast cancer, prostate cancer, melanoma and lung cancer. Although some cancer cells like melanoma cells are known to respond to lipopolysaccharide (LPS via TLR4, not all cancer cells are positive for TLR4. There is little information on the expression and function of TLR4 in neuroblastoma cells. In this study, we investigated the expression of TLR4 in human neuroblastoma NB-1 cell line. Methods Expression and localization of TLR4 were detected by reverse transcription-polymerase chain reaction (RT-PCR and flow cytometric analysis, respectively. Activation of nuclear factor (NF-κB by LPS was detected by degradation of IκB-α and NF-κB luciferase assay. Activation and expression of mitogen-activated protein (MAP kinase and interferon regulatory factor (IRF-3 was detected by immunoblot analysis. Results Human NB-1 neuroblastoma cells expressed intracellular form of TLR4, but not the cell surface form. Further, NB-1 cells express CD14, MD2 and MyD88, which are required for LPS response. However, LPS did not significantly induce NF-κB activation in NB-1 cells although it slightly degraded IκB-α. NB-1 cells expressed no IRF-3, which plays a pivotal role on the MyD88-independent pathway of LPS signaling. Collectively, NB-1 cells are capable to avoid their response to LPS. Conclusion Although human NB-1 neuroblastoma cells possessed all the molecules required for LPS response, they did not respond to LPS. It might be responsible for intracellular expression of TLR4 or lack of IRF-3.

  18. Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide

    International Nuclear Information System (INIS)

    Hassan, Ferdaus; Islam, Shamima; Tumurkhuu, Gantsetseg; Naiki, Yoshikazu; Koide, Naoki; Mori, Isamu; Yoshida, Tomoaki; Yokochi, Takashi

    2006-01-01

    Recently it has been reported that, toll-like receptors (TLRs) are expressed on a series of tumor cells, such as colon cancer, breast cancer, prostate cancer, melanoma and lung cancer. Although some cancer cells like melanoma cells are known to respond to lipopolysaccharide (LPS) via TLR4, not all cancer cells are positive for TLR4. There is little information on the expression and function of TLR4 in neuroblastoma cells. In this study, we investigated the expression of TLR4 in human neuroblastoma NB-1 cell line. Expression and localization of TLR4 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis, respectively. Activation of nuclear factor (NF)-κB by LPS was detected by degradation of IκB-α and NF-κB luciferase assay. Activation and expression of mitogen-activated protein (MAP) kinase and interferon regulatory factor (IRF)-3 was detected by immunoblot analysis. Human NB-1 neuroblastoma cells expressed intracellular form of TLR4, but not the cell surface form. Further, NB-1 cells express CD14, MD2 and MyD88, which are required for LPS response. However, LPS did not significantly induce NF-κB activation in NB-1 cells although it slightly degraded IκB-α. NB-1 cells expressed no IRF-3, which plays a pivotal role on the MyD88-independent pathway of LPS signaling. Collectively, NB-1 cells are capable to avoid their response to LPS. Although human NB-1 neuroblastoma cells possessed all the molecules required for LPS response, they did not respond to LPS. It might be responsible for intracellular expression of TLR4 or lack of IRF-3

  19. Engineering the Intracellular Micro- and Nano-environment via Magnetic Nanoparticles

    Science.gov (United States)

    Tseng, Peter

    Single cells, despite being the base unit of living organisms, possess a high degree of hierarchical structure and functional compartmentalization. This complexity exists for good reason: cells must respond efficiently and effectively to its surrounding environment by differentiating, moving, interacting, and more in order to survive or inhabit its role in the larger biological system. At the core of these responses is cellular decision-making. Cells process cues internally and externally from the environment and effect intracellular asymmetry in biochemistry and structure in order to carry out the proper biological responses. Functionalized magnetic particles have shown to be a powerful tool in interacting with biological matter, through either cell or biomolecule sorting, and the activation of biological processes. This dissertation reports on techniques utilizing manipulated magnetic nanoparticles (internalized by cells) to spatially and temporally localize intracellular cues, and examines the resulting asymmetry in biological processes generated by our methods. We first examine patterned micromagnetic elements as a simple strategy of rapidly manipulating magnetic nanoparticles throughout the intracellular space. Silicon or silicon dioxide substrates form the base for electroplated NiFe rods, which are repeated at varying size and pitch. A planarizing resin, initially SU-8, is used as the substrate layer for cellular adhesion. We demonstrate that through the manipulations of a simple external magnet, these micro-fabricated substrates can mediate rapid (under 2 s) and precise (submicron), reversible translation of magnetic nanoparticles through cellular space. Seeding cells on substrates composed of these elements allows simultaneous control of ensembles of nanoparticles over thousands of cells at a time. We believe such substrates could form the basis of magnetically based tools for the activation of biological matter. We further utilize these strategies to

  20. Similarity-based pattern analysis and recognition

    CERN Document Server

    Pelillo, Marcello

    2013-01-01

    This accessible text/reference presents a coherent overview of the emerging field of non-Euclidean similarity learning. The book presents a broad range of perspectives on similarity-based pattern analysis and recognition methods, from purely theoretical challenges to practical, real-world applications. The coverage includes both supervised and unsupervised learning paradigms, as well as generative and discriminative models. Topics and features: explores the origination and causes of non-Euclidean (dis)similarity measures, and how they influence the performance of traditional classification alg

  1. Intracellular Trafficking Modulation by Ginsenoside Rg3 Inhibits Brucella abortus Uptake and Intracellular Survival within RAW 264.7 Cells.

    Science.gov (United States)

    Huy, Tran Xuan Ngoc; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2017-03-28

    Ginsenoside Rg3, a saponin extracted from ginseng, has various pharmacological and biological activities; however, its effects against Brucella infection are still unclear. Herein, the inhibitory effects of ginsenoside Rg3 against intracellular parasitic Brucella infection were evaluated through bacterial infection, adherence assays, and LAMP-1 colocalization, as well as immunoblotting and FACS for detecting MAPK signaling proteins and F-actin polymerization, respectively. The internalization, intracellular growth, and adherence of Brucella abortus in Rg3-treated RAW 264.7 cells were significantly decreased compared with the Rg3-untreated control. Furthermore, an apparent reduction of F-actin content and intensity of F-actin fluorescence in Rg3-treated cells was observed compared with B. abortus -infected cells without treatment by flow cytometry analysis and confocal microscopy, respectively. In addition, treating cells with Rg3 decreased the phosphorylation of MAPK signaling proteins such as ERK 1/2 and p38 compared with untreated cells. Moreover, the colocalization of B. abortus -containing phagosomes with LAMP-1 was markedly increased in Rg3-treated cells. These findings suggest that ginsenoside Rg3 inhibits B. abortus infection in mammalian cells and can be used as an alternative approach in the treatment of brucellosis.

  2. Three types of ependymal cells with intracellular calcium oscillation are characterized by distinct cilia beating properties.

    Science.gov (United States)

    Liu, Tongyu; Jin, Xingjian; Prasad, Rahul M; Sari, Youssef; Nauli, Surya M

    2014-09-01

    Ependymal cells are multiciliated epithelial cells that line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia has been associated with various neurological deficits. For the first time, we report three distinct ependymal cell types, I, II, and III, based on their unique ciliary beating frequency and beating angle. These ependymal cells have specific localizations within the third ventricle of the mouse brain. Furthermore, neither ependymal cell types nor their localizations are altered by aging. Our high-speed fluorescence imaging analysis reveals that these ependymal cells have an intracellular pacing calcium oscillation property. Our study further shows that alcohol can significantly repress the amplitude of calcium oscillation and the frequency of ciliary beating, resulting in an overall decrease in volume replacement by the cilia. Furthermore, the pharmacological agent cilostazol could differentially increase cilia beating frequency in type II, but not in type I or type III, ependymal cells. In summary, we provide the first evidence of three distinct types of ependymal cells with calcium oscillation properties. © 2014 Wiley Periodicals, Inc.

  3. BSA-coated nanoparticles for improved SERS-based intracellular pH sensing.

    Science.gov (United States)

    Zheng, Xiao-Shan; Hu, Pei; Cui, Yan; Zong, Cheng; Feng, Jia-Min; Wang, Xin; Ren, Bin

    2014-12-16

    Local microenvironment pH sensing is one of the key parameters for the understanding of many biological processes. As a noninvasive and high sensitive technique, surface-enhanced Raman spectroscopy (SERS) has attracted considerable interest in the detection of the local pH of live cells. We herein develop a facile way to prepare Au-(4-MPy)-BSA (AMB) pH nanosensor. The 4-MPy (4-mercaptopyridine) was used as the pH sensing molecule. The modification of the nanoparticles with BSA not only provides a high sensitive response to pH changes ranging from pH 4.0 to 9.0 but also exhibits a high sensitivity and good biocompatibility, stability, and reliability in various solutions (including the solutions of high ionic strength or with complex composition such as the cell culture medium), both in the aggregation state or after long-term storage. The AMB pH nanosensor shows great advantages for reliable intracellular pH analysis and has been successfully used to monitor the pH distribution of live cells and can address the grand challenges in SERS-based pH sensing for practical biological applications.

  4. Intracellular Immunohistochemical Detection of Tetrodotoxin in Pleurobranchaea maculata (Gastropoda and Stylochoplana sp. (Turbellaria

    Directory of Open Access Journals (Sweden)

    Lauren R. Salvitti

    2015-01-01

    Full Text Available Tetrodotoxin (TTX, is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring.

  5. Intracellular immunohistochemical detection of tetrodotoxin in Pleurobranchaea maculata (Gastropoda) and Stylochoplana sp. (Turbellaria).

    Science.gov (United States)

    Salvitti, Lauren R; Wood, Susanna A; Winsor, Leigh; Cary, Stephen Craig

    2015-01-28

    Tetrodotoxin (TTX), is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring.

  6. Pathway analysis for intracellular Porphyromonas gingivalis using a strain ATCC 33277 specific database

    Directory of Open Access Journals (Sweden)

    Wang Tiansong

    2009-09-01

    provides a more energy rich environment compared to the extracellular milieu. Shifts in the production of cytotoxic fatty acids by intracellular P. gingivalis may play a role in virulence. Moreover, despite extensive genomic re-arrangements between strains W83 and 33277, there is sufficient sequence similarity at the peptide level for proteomic abundance trends to be largely accurate when using the heterologous strain annotated genome as the reference for database searching.

  7. HYPOTHESIS TESTING WITH THE SIMILARITY INDEX

    Science.gov (United States)

    Mulltilocus DNA fingerprinting methods have been used extensively to address genetic issues in wildlife populations. Hypotheses concerning population subdivision and differing levels of diversity can be addressed through the use of the similarity index (S), a band-sharing coeffic...

  8. On self-similarity of crack layer

    Science.gov (United States)

    Botsis, J.; Kunin, B.

    1987-01-01

    The crack layer (CL) theory of Chudnovsky (1986), based on principles of thermodynamics of irreversible processes, employs a crucial hypothesis of self-similarity. The self-similarity hypothesis states that the value of the damage density at a point x of the active zone at a time t coincides with that at the corresponding point in the initial (t = 0) configuration of the active zone, the correspondence being given by a time-dependent affine transformation of the space variables. In this paper, the implications of the self-similarity hypothesis for qusi-static CL propagation is investigated using polystyrene as a model material and examining the evolution of damage distribution along the trailing edge which is approximated by a straight segment perpendicular to the crack path. The results support the self-similarity hypothesis adopted by the CL theory.

  9. Bilateral Trade Flows and Income Distribution Similarity

    Science.gov (United States)

    2016-01-01

    Current models of bilateral trade neglect the effects of income distribution. This paper addresses the issue by accounting for non-homothetic consumer preferences and hence investigating the role of income distribution in the context of the gravity model of trade. A theoretically justified gravity model is estimated for disaggregated trade data (Dollar volume is used as dependent variable) using a sample of 104 exporters and 108 importers for 1980–2003 to achieve two main goals. We define and calculate new measures of income distribution similarity and empirically confirm that greater similarity of income distribution between countries implies more trade. Using distribution-based measures as a proxy for demand similarities in gravity models, we find consistent and robust support for the hypothesis that countries with more similar income-distributions trade more with each other. The hypothesis is also confirmed at disaggregated level for differentiated product categories. PMID:27137462

  10. Discovering Music Structure via Similarity Fusion

    DEFF Research Database (Denmark)

    for representing music structure is studied in a simplified scenario consisting of 4412 songs and two similarity measures among them. The results suggest that the PLSA model is a useful framework to combine different sources of information, and provides a reasonable space for song representation.......Automatic methods for music navigation and music recommendation exploit the structure in the music to carry out a meaningful exploration of the “song space”. To get a satisfactory performance from such systems, one should incorporate as much information about songs similarity as possible; however...... semantics”, in such a way that all observed similarities can be satisfactorily explained using the latent semantics. Therefore, one can think of these semantics as the real structure in music, in the sense that they can explain the observed similarities among songs. The suitability of the PLSA model...

  11. Abundance estimation of spectrally similar minerals

    CSIR Research Space (South Africa)

    Debba, Pravesh

    2009-07-01

    Full Text Available This paper evaluates a spectral unmixing method for estimating the partial abundance of spectrally similar minerals in complex mixtures. The method requires formulation of a linear function of individual spectra of individual minerals. The first...

  12. Lagrangian-similarity diffusion-deposition model

    International Nuclear Information System (INIS)

    Horst, T.W.

    1979-01-01

    A Lagrangian-similarity diffusion model has been incorporated into the surface-depletion deposition model. This model predicts vertical concentration profiles far downwind of the source that agree with those of a one-dimensional gradient-transfer model

  13. Discovering Music Structure via Similarity Fusion

    DEFF Research Database (Denmark)

    Arenas-García, Jerónimo; Parrado-Hernandez, Emilio; Meng, Anders

    Automatic methods for music navigation and music recommendation exploit the structure in the music to carry out a meaningful exploration of the “song space”. To get a satisfactory performance from such systems, one should incorporate as much information about songs similarity as possible; however...... semantics”, in such a way that all observed similarities can be satisfactorily explained using the latent semantics. Therefore, one can think of these semantics as the real structure in music, in the sense that they can explain the observed similarities among songs. The suitability of the PLSA model...... for representing music structure is studied in a simplified scenario consisting of 4412 songs and two similarity measures among them. The results suggest that the PLSA model is a useful framework to combine different sources of information, and provides a reasonable space for song representation....

  14. Outsourced similarity search on metric data assets

    KAUST Repository

    Yiu, Man Lung

    2012-02-01

    This paper considers a cloud computing setting in which similarity querying of metric data is outsourced to a service provider. The data is to be revealed only to trusted users, not to the service provider or anyone else. Users query the server for the most similar data objects to a query example. Outsourcing offers the data owner scalability and a low-initial investment. The need for privacy may be due to the data being sensitive (e.g., in medicine), valuable (e.g., in astronomy), or otherwise confidential. Given this setting, the paper presents techniques that transform the data prior to supplying it to the service provider for similarity queries on the transformed data. Our techniques provide interesting trade-offs between query cost and accuracy. They are then further extended to offer an intuitive privacy guarantee. Empirical studies with real data demonstrate that the techniques are capable of offering privacy while enabling efficient and accurate processing of similarity queries.

  15. Intracellular Crosslinking of Filoviral Nucleoproteins with Xintrabodies Restricts Viral Packaging

    Directory of Open Access Journals (Sweden)

    Tamarand Lee Darling

    2017-09-01

    Full Text Available Viruses assemble large macromolecular repeat structures that become part of the infectious particles or virions. Ribonucleocapsids (RNCs of negative strand RNA viruses are a prime example where repetition of nucleoprotein (NP along the genome creates a core polymeric helical scaffold that accommodates other nucleocapsid proteins including viral polymerase. The RNCs are transported through the cytosol for packaging into virions through association with viral matrix proteins at cell membranes. We hypothesized that RNC would be ideal targets for crosslinkers engineered to promote aberrant protein–protein interactions, thereby blocking their orderly transport and packaging. Previously, we had generated single-domain antibodies (sdAbs against Filoviruses that have all targeted highly conserved C-terminal regions of NP known to be repetitively exposed along the length of the RNCs of Marburgvirus (MARV and Ebolavirus (EBOV. Our crosslinker design consisted of dimeric sdAb expressed intracellularly, which we call Xintrabodies (X- for crosslinking. Electron microscopy of purified NP polymers incubated with purified sdAb constructs showed NP aggregation occurred in a genus-specific manner with dimeric and not monomeric sdAb. A virus-like particle (VLP assay was used for initial evaluation where we found that dimeric sdAb inhibited NP incorporation into VP40-based VLPs whereas monomeric sdAb did not. Inhibition of NP packaging was genus specific. Confocal microscopy revealed dimeric sdAb was diffuse when expressed alone but focused on pools of NP when the two were coexpressed, while monomeric sdAb showed ambivalent partition. Infection of stable Vero cell lines expressing dimeric sdAb specific for either MARV or EBOV NP resulted in smaller plaques and reduced progeny of cognate virus relative to wild-type Vero cells. Though the impact was marginal at later time-points, the collective data suggest that viral replication can be reduced by crosslinking

  16. LDL Receptors as Gateways for Intracellular Porphyrin Uptake

    International Nuclear Information System (INIS)

    Novick, S.; Laster, B.; Quastel, M.

    2004-01-01

    Boronated compounds are currently being studied for possible use in Boron Neutron Capture Therapy (BNCT). We found that one of these agents, BOPP (tetrakis-carborane-carboxylate, esters of 2,4-bis (a,b- dihydroxyethyl) deuteroporphyrin IX), could also be labeled with indium (In-BOPP) and, therefore, could also be used potentially to transport high Z atoms into tumor cell DNA for AET (Auger Electron Therapy). In order to assess the uptake of these agents into cells, the role of the LDL receptor in the intracellular accumulation of BOPP and In-BOPP was investigated. Pre-incubation of V-79 Chinese hamster cells in medium containing delipidized fetal bovine serum (FBS) markedly increased the subsequent uptake of intracellular boron transported by both BOPP and In-BOPP when compared with cells that had been pre-incubated with medium containing 10% normal FBS (lipidized). The increased uptake was characterized by elevated levels of receptor, and greater affinity was shown for both BOPP and In-BOPP, although less marked with the latter. Positive cooperativity was demonstrated by sigmoid saturation curves, Scatchard analysis and Hill plots. Increasing the amount of LDL in the incubation medium had a relatively small effect on the total accumulation of either indium or boron atoms inside the cell. Furthermore, chemical acetylation of LDL did not decrease the intracellular uptake of either boron or indium transported by BOPP or In-BOPP. It is thus concluded that BOPP and In-BOPP preferentially enter the cells directly by way of the LDL receptor and that only a small fraction of these molecules are transported into the cells indirectly using serum LDLs as their carriers. These data suggest a novel way of bringing greater amounts of boron and indium (and perhaps other agents) into tissues. Porphyrins can be used to transport different agents into tumor cells because they are tumor affinic molecules. Tumors express a higher number of LDL receptors than do most normal tissues

  17. Protein structural similarity search by Ramachandran codes

    Directory of Open Access Journals (Sweden)

    Chang Chih-Hung

    2007-08-01

    Full Text Available Abstract Background Protein structural data has increased exponentially, such that fast and accurate tools are necessary to access structure similarity search. To improve the search speed, several methods have been designed to reduce three-dimensional protein structures to one-dimensional text strings that are then analyzed by traditional sequence alignment methods; however, the accuracy is usually sacrificed and the speed is still unable to match sequence similarity search tools. Here, we aimed to improve the linear encoding methodology and develop efficient search tools that can rapidly retrieve structural homologs from large protein databases. Results We propose a new linear encoding method, SARST (Structural similarity search Aided by Ramachandran Sequential Transformation. SARST transforms protein structures into text strings through a Ramachandran map organized by nearest-neighbor clustering and uses a regenerative approach to produce substitution matrices. Then, classical sequence similarity search methods can be applied to the structural similarity search. Its accuracy is similar to Combinatorial Extension (CE and works over 243,000 times faster, searching 34,000 proteins in 0.34 sec with a 3.2-GHz CPU. SARST provides statistically meaningful expectation values to assess the retrieved information. It has been implemented into a web service and a stand-alone Java program that is able to run on many different platforms. Conclusion As a database search method, SARST can rapidly distinguish high from low similarities and efficiently retrieve homologous structures. It demonstrates that the easily accessible linear encoding methodology has the potential to serve as a foundation for efficient protein structural similarity search tools. These search tools are supposed applicable to automated and high-throughput functional annotations or predictions for the ever increasing number of published protein structures in this post-genomic era.

  18. Similarity search processing. Paralelization and indexing technologies.

    Directory of Open Access Journals (Sweden)

    Eder Dos Santos

    2015-08-01

    The next Scientific-Technical Report addresses the similarity search and the implementation of metric structures on parallel environments. It also presents the state of the art related to similarity search on metric structures and parallelism technologies. Comparative analysis are also proposed, seeking to identify the behavior of a set of metric spaces and metric structures over processing platforms multicore-based and GPU-based.

  19. Parallel trajectory similarity joins in spatial networks

    KAUST Repository

    Shang, Shuo

    2018-04-04

    The matching of similar pairs of objects, called similarity join, is fundamental functionality in data management. We consider two cases of trajectory similarity joins (TS-Joins), including a threshold-based join (Tb-TS-Join) and a top-k TS-Join (k-TS-Join), where the objects are trajectories of vehicles moving in road networks. Given two sets of trajectories and a threshold θ, the Tb-TS-Join returns all pairs of trajectories from the two sets with similarity above θ. In contrast, the k-TS-Join does not take a threshold as a parameter, and it returns the top-k most similar trajectory pairs from the two sets. The TS-Joins target diverse applications such as trajectory near-duplicate detection, data cleaning, ridesharing recommendation, and traffic congestion prediction. With these applications in mind, we provide purposeful definitions of similarity. To enable efficient processing of the TS-Joins on large sets of trajectories, we develop search space pruning techniques and enable use of the parallel processing capabilities of modern processors. Specifically, we present a two-phase divide-and-conquer search framework that lays the foundation for the algorithms for the Tb-TS-Join and the k-TS-Join that rely on different pruning techniques to achieve efficiency. For each trajectory, the algorithms first find similar trajectories. Then they merge the results to obtain the final result. The algorithms for the two joins exploit different upper and lower bounds on the spatiotemporal trajectory similarity and different heuristic scheduling strategies for search space pruning. Their per-trajectory searches are independent of each other and can be performed in parallel, and the mergings have constant cost. An empirical study with real data offers insight in the performance of the algorithms and demonstrates that they are capable of outperforming well-designed baseline algorithms by an order of magnitude.

  20. Parallel trajectory similarity joins in spatial networks

    KAUST Repository

    Shang, Shuo; Chen, Lisi; Wei, Zhewei; Jensen, Christian S.; Zheng, Kai; Kalnis, Panos

    2018-01-01

    The matching of similar pairs of objects, called similarity join, is fundamental functionality in data management. We consider two cases of trajectory similarity joins (TS-Joins), including a threshold-based join (Tb-TS-Join) and a top-k TS-Join (k-TS-Join), where the objects are trajectories of vehicles moving in road networks. Given two sets of trajectories and a threshold θ, the Tb-TS-Join returns all pairs of trajectories from the two sets with similarity above θ. In contrast, the k-TS-Join does not take a threshold as a parameter, and it returns the top-k most similar trajectory pairs from the two sets. The TS-Joins target diverse applications such as trajectory near-duplicate detection, data cleaning, ridesharing recommendation, and traffic congestion prediction. With these applications in mind, we provide purposeful definitions of similarity. To enable efficient processing of the TS-Joins on large sets of trajectories, we develop search space pruning techniques and enable use of the parallel processing capabilities of modern processors. Specifically, we present a two-phase divide-and-conquer search framework that lays the foundation for the algorithms for the Tb-TS-Join and the k-TS-Join that rely on different pruning techniques to achieve efficiency. For each trajectory, the algorithms first find similar trajectories. Then they merge the results to obtain the final result. The algorithms for the two joins exploit different upper and lower bounds on the spatiotemporal trajectory similarity and different heuristic scheduling strategies for search space pruning. Their per-trajectory searches are independent of each other and can be performed in parallel, and the mergings have constant cost. An empirical study with real data offers insight in the performance of the algorithms and demonstrates that they are capable of outperforming well-designed baseline algorithms by an order of magnitude.

  1. Are calanco landforms similar to river basins?

    Science.gov (United States)

    Caraballo-Arias, N A; Ferro, V

    2017-12-15

    In the past badlands have been often considered as ideal field laboratories for studying landscape evolution because of their geometrical similarity to larger fluvial systems. For a given hydrological process, no scientific proof exists that badlands can be considered a model of river basin prototypes. In this paper the measurements carried out on 45 Sicilian calanchi, a type of badlands that appears as a small-scale hydrographic unit, are used to establish their morphological similarity with river systems whose data are available in the literature. At first the geomorphological similarity is studied by identifying the dimensionless groups, which can assume the same value or a scaled one in a fixed ratio, representing drainage basin shape, stream network and relief properties. Then, for each property, the dimensionless groups are calculated for the investigated calanchi and the river basins and their corresponding scale ratio is evaluated. The applicability of Hack's, Horton's and Melton's laws for establishing similarity criteria is also tested. The developed analysis allows to conclude that a quantitative morphological similarity between calanco landforms and river basins can be established using commonly applied dimensionless groups. In particular, the analysis showed that i) calanchi and river basins have a geometrically similar shape respect to the parameters Rf and Re with a scale factor close to 1, ii) calanchi and river basins are similar respect to the bifurcation and length ratios (λ=1), iii) for the investigated calanchi the Melton number assumes values less than that (0.694) corresponding to the river case and a scale ratio ranging from 0.52 and 0.78 can be used, iv) calanchi and river basins have similar mean relief ratio values (λ=1.13) and v) calanchi present active geomorphic processes and therefore fall in a more juvenile stage with respect to river basins. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Dynamic Regulation of Ero1α and Peroxiredoxin 4 Localization in the Secretory Pathway*

    Science.gov (United States)

    Kakihana, Taichi; Araki, Kazutaka; Vavassori, Stefano; Iemura, Shun-ichiro; Cortini, Margherita; Fagioli, Claudio; Natsume, Tohru; Sitia, Roberto; Nagata, Kazuhiro

    2013-01-01

    In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis. PMID:23979138

  3. Development of a custom biological scaffold for investigating ultrasound-mediated intracellular delivery

    Energy Technology Data Exchange (ETDEWEB)

    Bui, Loan [Department of Bioengineering, University of Texas at Arlington, Arlington, TX 76010 (United States); Aleid, Adham [Department of Biomedical Technology, King Saud University, Riyadh 12372 (Saudi Arabia); Alassaf, Ahmad [Department of Biomedical Engineering, University of Miami, Coral Gables, FL 33146 (United States); Department of Medical Equipment Technology, Majmaah University, Majmaah City 11952 (Saudi Arabia); Wilson, Otto C.; Raub, Christopher B. [Department of Biomedical Engineering, Catholic University of America, Washington, DC 20064 (United States); Frenkel, Victor, E-mail: vfrenkel@som.umaryland.edu [Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201 (United States)

    2017-01-01

    In vitro investigations of ultrasound mediated, intracellular drug and gene delivery (i.e. sonoporation) are typically carried out in cells cultured in standard plastic well plates. This creates conditions that poorly resemble in vivo conditions, as well as generating unwanted ultrasound phenomena that may confound the interpretation of results. Here, we present our results in the development of a biological scaffold for sonoporation studies. The scaffolds were comprised of cellulose fibers coated with chitosan and gelatin. Scaffold formulation was optimized for adherence and proliferation of mouse fibroblasts in terms of the ratio and relative concentration of the two constituents. The scaffolds were also shown to significantly reduce ultrasound reflections compared to the plastic well plates. A custom treatment chamber was designed and built, and the occurrence of acoustic cavitation in the chamber during the ultrasound treatments was detected; a requirement for the process of sonoporation. Finally, experiments were carried out to optimize the ultrasound exposures to minimize cellular damage. Ultrasound exposure was then shown to enable the uptake of 100 nm fluorescently labeled polystyrene nanoparticles in suspension into the cells seeded on scaffolds, compared to incubation of cell-seeded scaffolds with nanoparticles alone. These preliminary results set the basis for further development of this platform. They also provide motivation for the development of similar platforms for the controlled investigation of other ultrasound mediated cell and tissue therapies. - Highlights: • A custom, biological scaffold was developed, comprised of chitosan and gelatin. • The scaffold formulation was optimized for adhesion and proliferation of fibroblasts. • Investigations showed the scaffolds to be less reflective to ultrasound than plastic well plates. • The scaffolds were found to be suitable for investigations of ultrasound mediated intracellular nanoparticle

  4. Poking cells for efficient vector-free intracellular delivery

    Science.gov (United States)

    Wang, Ying; Yang, Yang; Yan, Li; Kwok, So Ying; Li, Wei; Wang, Zhigang; Zhu, Xiaoyue; Zhu, Guangyu; Zhang, Wenjun; Chen, Xianfeng; Shi, Peng

    2014-07-01

    Techniques for introducing foreign molecules and materials into living cells are of great value in cell biology research. A major barrier for intracellular delivery is to cross the cell membrane. Here we demonstrate a novel platform utilizing diamond nanoneedle arrays to facilitate efficient vector-free cytosolic delivery. Using our technique, cellular membrane is deformed by an array of nanoneedles with a force on the order of a few nanonewtons. We show that this technique is applicable to deliver a broad range of molecules and materials into different types of cells, including primary neurons in adherent culture. Especially, for delivering plasmid DNAs into neurons, our technique produces at least eightfold improvement (~45% versus ~1-5%) in transfection efficiency with a dramatically shorter experimental protocol, when compared with the commonly used lipofection approach. It is anticipated that our technique will greatly benefit basic research in cell biology and also a wide variety of clinical applications.

  5. The intracellular cholesterol landscape: dynamic integrator of the immune response

    Science.gov (United States)

    Fessler, Michael B.

    2016-01-01

    Cholesterol has typically been considered an exogenous, disease-related factor in immunity; however, recent literature suggests that a paradigm shift is in order. Sterols are now recognized to ligate several immune receptors. Altered flux through the mevalonic acid synthesis pathway also appears to be a required event in the antiviral interferon response of macrophages and in the activation, proliferation, and differentiation of T cells. In this review, evidence is discussed that suggests an intrinsic, ‘professional’ role for sterols and oxysterols in macrophage and T cell immunity. Host defense may have been the original selection pressure behind the development of mechanisms for intracellular cholesterol homeostasis. Functional coupling between sterol metabolism and immunity has fundamental implications for health and disease. PMID:27692616

  6. Extraction of intracellular protein from Glaciozyma antarctica for proteomics analysis

    Science.gov (United States)

    Faizura, S. Nor; Farahayu, K.; Faizal, A. B. Mohd; Asmahani, A. A. S.; Amir, R.; Nazalan, N.; Diba, A. B. Farah; Muhammad, M. Nor; Munir, A. M. Abdul

    2013-11-01

    Two preparation methods of crude extracts of psychrophilic yeast Glaciozyma antarctica were compared in order to obtain a good recovery of intracellular proteins. Extraction with mechanical procedures using sonication was found to be more effective for obtaining good yield compare to alkaline treatment method. The procedure is simple, rapid, and produce better yield. A total of 52 proteins were identified by combining both extraction methods. Most of the proteins identified in this study involves in the metabolic process including glycolysis pathway, pentose phosphate pathway, pyruyate decarboxylation and also urea cyle. Several chaperons were identified including probable cpr1-cyclophilin (peptidylprolyl isomerase), macrolide-binding protein fkbp12 and heat shock proteins which were postulate to accelerate proper protein folding. Characteristic of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

  7. Intracellular pH in rat pancreatic ducts

    DEFF Research Database (Denmark)

    Novak, I; Hug, M; Greger, R

    1997-01-01

    In order to study the mechanism of H+ and HCO3- transport in a HCO3- secreting epithelium, pancreatic ducts, we have measured the intracellular pH (pHi) in this tissue using the pH sensitive probe BCECF. We found that exposures of ducts to solutions containing acetate/acetic acid or NH4+/NH3...... buffers (20 mmol/l) led to pHi changes in accordance with entry of lipid-soluble forms of the buffers, followed by back-regulation of pHi by duct cells. In another type of experiment, changes in extracellular pH of solutions containing HEPES or HCO3-/CO2 buffers led to significant changes in pHi that did....... Under some conditions, these exchangers can be invoked to regulate cell pH....

  8. Intracellular Signaling Mediators in the Circulatory and Ventilatory Systems

    CERN Document Server

    Thiriet, Marc

    2013-01-01

    The volumes in this authoritative series present a multidisciplinary approach to modeling and simulation of flows in the cardiovascular and ventilatory systems, especially multiscale modeling and coupled simulations. The cardiovascular and respiratory systems are tightly coupled, as their primary function is to supply oxygen to and remove carbon dioxide from the body's cells. Because physiological conduits have deformable and reactive walls, macroscopic flow behavior and prediction must be coupled to phenomenological models of nano- and microscopic events in a corrector scheme of regulated mechanisms when the vessel lumen caliber varies markedly. Therefore, investigation of flows of blood and air in physiological conduits requires an understanding of the biology, chemistry, and physics of these systems together with the mathematical tools to describe their functioning. Volume 4 is devoted to major sets of intracellular mediators that transmit signals upon stimulation of cell-surface receptors.  Activation of...

  9. Intracellular mediators of potassium-induced aldosterone secretion

    International Nuclear Information System (INIS)

    Ganguly, A.; Chiou, S.; Davis, J.S.

    1990-01-01

    We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in 3 H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium

  10. ATPase and GTPase Tangos Drive Intracellular Protein Transport.

    Science.gov (United States)

    Shan, Shu-Ou

    2016-12-01

    The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

    DEFF Research Database (Denmark)

    Benjaminsen, Rikke Vicki; Sun, Honghao; Henriksen, Jonas Rosager

    2011-01-01

    Particle-based nanosensors have over the last decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors is challenging...... and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle...... quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2-7.0, which was synthesized by adding two pH-sensitive fluorophores with different pKa to each sensor, seem to be a solution...

  12. Intracellular Events and Cell Fate in Filovirus Infection

    Directory of Open Access Journals (Sweden)

    Elena Ryabchikova

    2011-08-01

    Full Text Available Marburg and Ebola viruses cause a severe hemorrhagic disease in humans with high fatality rates. Early target cells of filoviruses are monocytes, macrophages, and dendritic cells. The infection spreads to the liver, spleen and later other organs by blood and lymph flow. A hallmark of filovirus infection is the depletion of non-infected lymphocytes; however, the molecular mechanisms leading to the observed bystander lymphocyte apoptosis are poorly understood. Also, there is limited knowledge about the fate of infected cells in filovirus disease. In this review we will explore what is known about the intracellular events leading to virus amplification and cell damage in filovirus infection. Furthermore, we will discuss how cellular dysfunction and cell death may correlate with disease pathogenesis.

  13. Intracellular Transport and Kinesin Superfamily Proteins: Structure, Function and Dynamics

    Science.gov (United States)

    Hirokawa, N.; Takemura, R.

    Using various molecular cell biological and molecular genetic approaches, we identified kinesin superfamily proteins (KIFs) and characterized their significant functions in intracellular transport, which is fundamental for cellular morphogenesis, functioning, and survival. We showed that KIFs not only transport various membranous organelles, proteins complexes and mRNAs fundamental for cellular functions but also play significant roles in higher brain functions such as memory and learning, determination of important developmental processes such as left-right asymmetry formation and brain wiring. We also elucidated that KIFs recognize and bind to their specific cargoes using scaffolding or adaptor protein complexes. Concerning the mechanism of motility, we discovered the simplest unique monomeric motor KIF1A and determined by molecular biophysics, cryoelectron microscopy and X-ray crystallography that KIF1A can move on a microtubule processively as a monomer by biased Brownian motion and by hydolyzing ATP.

  14. Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus.

    OpenAIRE

    Mliki, A; Zimmermann, W

    1992-01-01

    An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a Km of 17.8 microM and a pH optimum of 5.0. It also showed catalase activity with a Km of 2.07 mM H2O2 and a pH optimum of 8.0. The purified enzyme did not catalyze C alpha-C beta bond cleavage of 1,3-dihydr...

  15. Measuring intracellular redox conditions using GFP-based sensors

    DEFF Research Database (Denmark)

    Björnberg, Olof; Ostergaard, Henrik; Winther, Jakob R

    2006-01-01

    Recent years have seen the development of methods for analyzing the redox conditions in specific compartments in living cells. These methods are based on genetically encoded sensors comprising variants of Green Fluorescent Protein in which vicinal cysteine residues have been introduced at solvent......-exposed positions. Several mutant forms have been identified in which formation of a disulfide bond between these cysteine residues results in changes of their fluorescence properties. The redox sensors have been characterized biochemically and found to behave differently, both spectroscopically and in terms...... of redox properties. As genetically encoded sensors they can be expressed in living cells and used for analysis of intracellular redox conditions; however, which parameters are measured depends on how the sensors interact with various cellular redox components. Results of both biochemical and cell...

  16. 2011 Rita Schaffer lecture: nanoparticles for intracellular nucleic acid delivery.

    Science.gov (United States)

    Green, Jordan J

    2012-07-01

    Nanoparticles are a promising technology for delivery of new types of therapeutics. A polymer library approach has allowed engineering of polymeric particles that are particularly effective for the delivery of DNA and siRNA to human cells. Certain chemical structural motifs, degradable linkages, hydrophobicity, and biophysical properties are key for successful intracellular delivery. Small differences to biomaterial structure, and especially the type of degradable linkage in the polymers, can be critical for successful delivery of siRNA vs. DNA. Furthermore, subtle changes to biomaterial structure can facilitate cell-type gene delivery specificity between human brain cancer cells and healthy cells as well as between human retinal endothelial cells and epithelial cells. These polymeric nanoparticles are effective for nucleic acid delivery in a broad range of human cell types and have applications to regenerative medicine, ophthalmology, and cancer among many other biomedical research areas.

  17. Control of intracellular heme levels: Heme transporters and Heme oxygenases

    Science.gov (United States)

    Khan, Anwar A.; Quigley, John G.

    2011-01-01

    Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number of transporters of heme and heme synthesis intermediates have been described. Here we review aspects of heme metabolism and discuss our current understanding of heme transporters, with emphasis on the function of the cell-surface heme exporter, FLVCR. Knockdown of Flvcr in mice leads to both defective erythropoiesis and disturbed systemic iron homeostasis, underscoring the critical role of heme transporters in mammalian physiology. PMID:21238504

  18. Variety in intracellular diffusion during the cell cycle

    DEFF Research Database (Denmark)

    Selhuber-Unkel, C.; Yde, P.; Berg-Sørensen, Kirstine

    2009-01-01

    During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast...... Schizosaccharomyces Pombe using optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominant