Construction of a series of congenic mice with recombinant chromosome 1 regions surrounding the genetic loci for resistance to intracellular parasites (Ity, Lsh, and Bcg), DNA repair responses (Rep-1), and the cytoskeletal protein villin (Vil).

Science.gov (United States)

Mock, B A; Holiday, D L; Cerretti, D P; Darnell, S C; O'Brien, A D; Potter, M

1994-01-01

The interval of mouse chromosome 1 extending from Idh-1 to Pep-3 harbors the natural resistance gene Ity/Lsh/Bcg; it controls the outcome of infection with Salmonella typhimurium, Leishmania donovani, and several Mycobacterium species. This region also contains a DNA repair gene, Rep-1, which determines the rapidity with which double-strand breaks in chromatin are repaired. BALB/cAnPt and DBA/2N mice differ in their phenotypic expression of these genes. To generate appropriate strains of mice for the study of these genes, a series of 10 C.D2 congenic strains recombinant across a 28-centimorgan interval of mouse chromosome 1 extending from Idh-1 to Pep-3 were derived from crosses of the C.D2-Idh-1 Pep-3 congenic strain back to BALB/cAn. Analyses of these recombinant strains will allow the correlation of biological-immunological phenotypes with defined genetic regions.

Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).

Science.gov (United States)

Meinert, Christian; Gembardt, Florian; Böhme, Ilka; Tetzner, Anja; Wieland, Thomas; Greenberg, Barry; Walther, Thomas

2016-01-01

The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1 h, 3 h, 6 h, and 9 h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1 h, 119 after 3 h, 31 after 6 h, and 86 after 9 h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1 h and 3 h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7) treatment. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.

Lipofection of purified adeno-associated virus Rep68 protein: toward a chromosome-targeting nonviral particle.

Science.gov (United States)

Lamartina, S; Roscilli, G; Rinaudo, D; Delmastro, P; Toniatti, C

1998-09-01

Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.

Variation in extragenic repetitive DNA sequences in Pseudomonas syringae and potential use of modified REP primers in the identification of closely related isolates

Directory of Open Access Journals (Sweden)

Elif Çepni

2012-01-01

Full Text Available In this study, Pseudomonas syringe pathovars isolated from olive, tomato and bean were identified by species-specific PCR and their genetic diversity was assessed by repetitive extragenic palindromic (REP-PCR. Reverse universal primers for REP-PCR were designed by using the bases of A, T, G or C at the positions of 1, 4 and 11 to identify additional polymorphism in the banding patterns. Binding of the primers to different annealing sites in the genome revealed additional fingerprint patterns in eight isolates of P. savastanoi pv. savastanoi and two isolates of P. syringae pv. tomato. The use of four different bases in the primer sequences did not affect the PCR reproducibility and was very efficient in revealing intra-pathovar diversity, particularly in P. savastanoi pv. savastanoi. At the pathovar level, the primer BOX1AR yielded shared fragments, in addition to five bands that discriminated among the pathovars P. syringae pv. phaseolicola, P. savastanoi pv. savastanoi and P. syringae pv. tomato. REP-PCR with a modified primer containing C produced identical bands among the isolates in a pathovar but separated three pathovars more distinctly than four other primers. Although REP-and BOX-PCRs have been successfully used in the molecular identification of Pseudomonas isolates from Turkish flora, a PCR based on inter-enterobacterial repetitive intergenic concensus (ERIC sequences failed to produce clear banding patterns in this study.

Safety and efficacy of REP 2139 and pegylated interferon alfa-2a for treatment-naive patients with chronic hepatitis B virus and hepatitis D virus co-infection (REP 301 and REP 301-LTF): a non-randomised, open-label, phase 2 trial.

Science.gov (United States)

Bazinet, Michel; Pântea, Victor; Cebotarescu, Valentin; Cojuhari, Lilia; Jimbei, Pavlina; Albrecht, Jeffrey; Schmid, Peter; Le Gal, Frédéric; Gordien, Emmanuel; Krawczyk, Adalbert; Mijočević, Hrvoje; Karimzadeh, Hadi; Roggendorf, Michael; Vaillant, Andrew

2017-12-01

REP 2139 clears circulating hepatitis B virus (HBV) surface antigen (HBsAg), enhancing the restoration of functional control of HBV infection by immunotherapy. We assessed the safety and efficacy of REP 2139 and pegylated interferon alfa-2a in patients with chronic HBV and hepatitis D virus (HDV) co-infection. In this open-label, non-randomised, phase 2 trial, patients aged 18-55 years, who were treatment naive, hepatitis B e antigen [HBeAg] negative, anti-hepatitis D antigen [HDAg] positive, and HDV RNA positive, with serum HBsAg concentrations of more than 1000 IU/mL, and a history of HDV infection for 6 months or more before treatment, were recruited at Toma Ciorbă Hospital of Infectious Diseases in Chișinău, Moldova. Patients were excluded if they had HDV superinfection, liver infections other than HBV and HDV, or liver cirrhosis. Patients received 500 mg intravenous REP 2139 once per week for 15 weeks, followed by combined therapy with 250 mg intravenous REP 2139 and 180 μg subcutaneous pegylated interferon alfa-2a once per week for 15 weeks, then monotherapy with 180 μg pegylated interferon alfa-2a once per week for 33 weeks. The primary endpoints assessed at the end of treatment were the safety and tolerability of the treatment regimen, analysed in the intention-to-treat population. Secondary outcomes included the proportion of patients with serum HBsAg less than 50 IU/mL, the proportion of patients with suppressed HBV DNA, and the proportion of patients who maintained these responses through follow-up. The REP 301 trial is registered with ClinicalTrials.gov, number NCT02233075. We also did an additional follow-up at 1 year after the end of treatment, as an interim analysis of the REP 301-LTF trial (planned duration 3 years), registered with ClinicalTrials.gov, number NCT02876419, which is ongoing but not recruiting patients. Between Sept 8, 2014, and Jan 27, 2015, we enrolled 12 patients into the REP 301 study. All 12 patients experienced at least one

Utshitsja drug u druga / Mailis Reps

Index Scriptorium Estoniae

Reps, Mailis, 1975-

2005-01-01

Haridus- ja teadusminister Mailis Reps uuendustest koolide õppekavades, õpetajate täienduskoolituse tähtsusest, kutsehariduse arengust, tasuta toitlustamisest põhi- ja kutsekoolides, õpetajate palkadest

Characterization of the adenoassociated virus Rep protein complex formed on the viral origin of DNA replication

International Nuclear Information System (INIS)

Li Zengi; Brister, J. Rodney; Im, Dong-Soo; Muzyczka, Nicholas

2003-01-01

Interaction between the adenoassociated virus (AAV) replication proteins, Rep68 and 78, and the viral terminal repeats (TRs) is mediated by a DNA sequence termed the Rep-binding element (RBE). This element is necessary for Rep-mediated unwinding of duplex DNA substrates, directs Rep catalyzed cleavage of the AAV origin of DNA replication, and is required for viral transcription and proviral integration. Six discrete Rep complexes with the AAV TR substrates have been observed in vitro, and cross-linking studies suggest these complexes contain one to six molecules of Rep. However, the functional relationship between Rep oligomerization and biochemical activity is unclear. Here we have characterized Rep complexes that form on the AAV TR. Both Rep68 and Rep78 appear to form the same six complexes with the AAV TR, and ATP seems to stimulate formation of specific, higher order complexes. When the sizes of these Rep complexes were estimated on native polyacrylamide gels, the four slower migrating complexes were larger than predicted by an amount equivalent to one or two TRs. To resolve this discrepancy, the molar ratio of protein and DNA was calculated for the three largest complexes. Data from these experiments indicated that the larger complexes included multiple TRs in addition to multiple Rep molecules and that the Rep-to-TR ratio was approximately 2. The two largest complexes were also associated with increased Rep-mediated, origin cleavage activity. Finally, we characterized a second, Rep-mediated cleavage event that occurs adjacent to the normal nicking site, but on the opposite strand. This second site nicking event effectively results in double-stranded DNA cleavage at the normal nicking site

Identification and Characterization of the Novel p97 co-factors, Rep8 and ASPL

DEFF Research Database (Denmark)

Klausen, Louise Kjær

to the ER membrane with the UBX domain situated in the cytosol. Mouse Rep8 is highly tissue-specific and abundant in gonads. In tests, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Additional precipitation experiments revealed that Rep8......The highly conserved and ubiquitin-specific AAA ATPase p97 acts on ubiquitylated substrates in diverse cellular mechanisms such as chromatin-associated degradation, fusion of homotypic membranes and ER-associated degradation. Different p97 cofactors associate with the ATPase, thereby constituting...... that ASPL localizes to the ER membrane and in vitro ASPL leads to disassembly of the p97 hexameric ATPase. Rep8 was found to interact with p97 both in vitro and in vivo, and the binding was mediated through the N-domain of p97 and the UBX domain of Rep8. Localization studies showed that Rep8 localizes...

3D printing with RepRap cookbook

CERN Document Server

Salinas, Richard

2014-01-01

A systematic guide consisting of over 100 recipes which focus on helping you understand the process of 3D printing using RepRap machines. The book aims at providing professionals with a series of working recipes to help make their fuzzy notions into real, saleable projects/objects using 3D printing technology. This book is for novice designers and artists who own a RepRap-based 3D printer, have fundamental knowledge of its working, and who desire to gain better mastery of the printing process. For the more experienced user, it will provide a handy visual resource, with side-by-side comparisons

Regulation and drive system for high rep-rate magnetic-pulse compressors

International Nuclear Information System (INIS)

Birx, D.L.; Cook, E.G.; Hawkins, S.; Meyers, A.; Reginato, L.L.; Schmidt, J.A.; Smith, M.W.

1982-01-01

The essentially unlimited rep-rate capability of non-linear magnetic systems has imposed strict requirements on the drive system which initiates the pulse compression. An order of magnitude increase in the rep-rates achieved by the Advanced Test Accelerator (ATA) gas blown system is not difficult to achieve in the magnetic compressor. The added requirement of having a high degree of regulation at the higher rep-rates places strict requirements on the triggerable switch for charging and de-Queing. A novel feedback technique which applies the proper bias to a magnetic core by comparing a reference voltage to the charging voltage eases considerably the regulation required to achieve low jitter in magnetic compression. The performance of the high rep-rate charging and regulation systems will be described in the following pages

RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

Science.gov (United States)

Zhu, Hai-Ying; Jin, Long; Guo, Qing; Luo, Zhao-Bo; Li, Xiao-Chen; Zhang, Yu-Chen; Xing, Xiao-Xu; Xuan, Mei-Fu; Zhang, Guang-Lei; Luo, Qi-Rong; Wang, Jun-Xia; Cui, Cheng-Du; Li, Wen-Xue; Cui, Zheng-Yun; Yin, Xi-Jun; Kang, Jin-Dan

2017-05-01

To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p  0.05), this was not significant. RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

Deformable M-Reps for 3D Medical Image Segmentation

Science.gov (United States)

Pizer, Stephen M.; Fletcher, P. Thomas; Joshi, Sarang; Thall, Andrew; Chen, James Z.; Fridman, Yonatan; Fritsch, Daniel S.; Gash, Graham; Glotzer, John M.; Jiroutek, Michael R.; Lu, Conglin; Muller, Keith E.; Tracton, Gregg; Yushkevich, Paul; Chaney, Edward L.

2013-01-01

M-reps (formerly called DSLs) are a multiscale medial means for modeling and rendering 3D solid geometry. They are particularly well suited to model anatomic objects and in particular to capture prior geometric information effectively in deformable models segmentation approaches. The representation is based on figural models, which define objects at coarse scale by a hierarchy of figures – each figure generally a slab representing a solid region and its boundary simultaneously. This paper focuses on the use of single figure models to segment objects of relatively simple structure. A single figure is a sheet of medial atoms, which is interpolated from the model formed by a net, i.e., a mesh or chain, of medial atoms (hence the name m-reps), each atom modeling a solid region via not only a position and a width but also a local figural frame giving figural directions and an object angle between opposing, corresponding positions on the boundary implied by the m-rep. The special capability of an m-rep is to provide spatial and orientational correspondence between an object in two different states of deformation. This ability is central to effective measurement of both geometric typicality and geometry to image match, the two terms of the objective function optimized in segmentation by deformable models. The other ability of m-reps central to effective segmentation is their ability to support segmentation at multiple levels of scale, with successively finer precision. Objects modeled by single figures are segmented first by a similarity transform augmented by object elongation, then by adjustment of each medial atom, and finally by displacing a dense sampling of the m-rep implied boundary. While these models and approaches also exist in 2D, we focus on 3D objects. The segmentation of the kidney from CT and the hippocampus from MRI serve as the major examples in this paper. The accuracy of segmentation as compared to manual, slice-by-slice segmentation is reported. PMID

Molecular characterization of Salmonella isolates by REP-PCR and RAPD analysis.

Science.gov (United States)

Albufera, U; Bhugaloo-Vial, P; Issack, M I; Jaufeerally-Fakim, Y

2009-05-01

Eighteen Salmonella isolates from both human and food (non-human) sources (fish, meat, and poultry) were characterized using conventional culture methods, biochemical, serological, and molecular analyses. REP-PCR and RAPD produced DNA profiles for differentiation purposes. Enterobacterial repetitive intergenic consensus (ERIC), repetitive extragenic palindronic (REP) and BOXAIR primers were selected for REP-PCR and two arbitrary primers, namely OPP-16 and OPS-11 were used for RAPD to generate DNA fingerprints from the Salmonella isolates. REP-PCR method showed greater discriminatory power in differentiating closely related strains of the related strains of Salmonella and produced more complex banding patterns as compared with RAPD. A dendogram was constructed with both sets of profiles using SPSS Version 13.0 computer software and showed that most human isolates were separately clustered from the non-human isolates. Two of the human isolates were closely related to some of the non-human isolates. A good correlation was also observed between the serogrouping of the O antigen and the molecular profiles obtained from REP-PCR and RAPD data of the Salmonella isolates. The results of a principal coordinate analysis (PCA) corresponded to the clustering in the dendrogram.

Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

Directory of Open Access Journals (Sweden)

Marizela Delic

2014-10-01

Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

O ethos do repórter de TV da Rede Globo

Directory of Open Access Journals (Sweden)

Marcia Benetti

2017-05-01

Full Text Available O artigo analisa os elementos instituintes das imagens de si de repórteres de televisão da Rede Globo, maior emissora do Brasil. A partir do conceito de ethos discursivo, discutimos como o discurso autorreferente afirma a identidade do jornalista e certos efeitos de verdade a respeito da reportagem. Por meio da análise do discurso de cinco entrevistas de repórteres da Rede Globo concedidas à também jornalista Fátima Bernardes, propomos que o ethos do repórter constrói-se em três dimensões: os atributos do “bom jornalista”, as características do “bom repórter” e as particularidades de pertencimento ao campo jornalístico. Como articuladores do ethos, os valores morais e os aspectos relacionados às rotinas profissionais convocam a audiência a se conectar com certas definições sobre o jornalismo.

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.

Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system.

Science.gov (United States)

Yamamoto, Shinji; Sakai, Ayako; Agustina, Vita; Moriguchi, Kazuki; Suzuki, Katsunori

2018-02-01

Ti and Ri plasmids of pathogenic Agrobacterium strains are stably maintained by the function of a repABC operon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of the Ti2 small RNA and luxAB from Vibrio harveyi. This system distinguished a Ti/Ri plasmid-free cell colony among plasmid-harboring cell colonies by causing the latter colonies to emit light in response to AS. We then constructed new "Ti/Ri eviction plasmids," each of which carries a repABC from one of four Ti/Ri plasmids that belonged to incRh1, incRh2, incRh3, and incRh4 groups in the suicidal plasmid pK18mobsacB and in a broad-host-range pBBR1 vector. Introduction of the new eviction plasmids into Agrobacterium cells harboring the corresponding Ti/Ri plasmids led to Ti/Ri plasmid-free cells in every incRh group. The Ti/Ri eviction was more effective by plasmids with the pBBR1 backbone than by those with the pK18mobsacB backbone. Furthermore, the highly stable cryptic plasmid pAtC58 in A. tumefaciens C58 was effectively evicted by the introduction of a pBBR1 vector containing the repABC of pAtC58. These results indicate that the set of pBBR1-repABC plasmids is a powerful tool for the removal of stable rhizobial plasmids.

Images en mouvement stockage, repérage, indexation

CERN Document Server

Turner, James

1998-01-01

L'avènement puis la fusion des nouveaux modes de communication que sont l'informatique, les télécommunications et l'audiovisuel ont mis à la portée de tous une grande quantité d'images fixes et en mouvement dont la conservation et le repérage risquent de prendre des proportions démesurées. Le présent ouvrage veut offrir aux responsables de collection des repères pour aborder la problématique de l'indexation des images et faciliter l'accès des usagers à ces images.

Genetic Diversity and Evidence for Transmission of Streptococcus mutans by DiversiLab rep-PCR.

Science.gov (United States)

Momeni, Stephanie S; Whiddon, Jennifer; Cheon, Kyounga; Ghazal, Tariq; Moser, Stephen A; Childers, Noel K

2016-09-01

This two-part study investigated the genetic diversity and transmission of Streptococcus mutans using the DiversiLab repetitive extragenic palindromic PCR (rep-PCR) approach. For children with S. mutans and participating household members, analysis for evidence of unrelated child-to-child as well as intra-familial transmission was evaluated based on commonality of genotypes. A total of 169 index children and 425 household family members from Uniontown, Alabama were evaluated for genetic diversity using rep-PCR. Thirty-four unique rep-PCR genotypes were observed for 13,906 S. mutans isolates. For transmission, 117 child and household isolates were evaluated for shared genotype (by child and by genotype cases, multiple matches possible for each child). Overall, children had 1-9 genotypes and those with multiple genotypes were 2.3 times more likely to have caries experience (decayed, missing and filled teeth/surfaces>0). Only 28% of children shared all genotypes within the household, while 72% had at least 1 genotype not shared with anyone in the household. Children had genotype(s) not shared with any household members in 157 cases. In 158 cases children and household members shared a genotype in which 55% (87/158 cases) were shared with more than one family member. Children most frequently shared genotypes with their mothers (54%; 85/158), siblings (46%; 72/158) and cousins (23%; 37/158). A reference library for S. mutans for epidemiological surveillance using the DiversiLab rep-PCR approach is detailed. The genetic diversity of S. mutans in this population demonstrated frequent commonality of genotypes. Evidence for both child-to-child and intra-familial transmission of S. mutans was observed by rep-PCR. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange.

Science.gov (United States)

Montrose, M H; Murer, H

1986-01-01

Suspensions of LLC-PK1 cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na+ and K+ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05 +/- 0.01, n = 5) of cells which have recovered in (pH 7.4) Na+-containing medium is not affected over several minutes by addition of 100 microM amiloride or removal of extracellular Na+ (Na+o less than 1 mM). In contrast, when the cells recover from an acid load (caused by NH4 preincubation and removal), the recovery is largely Na+ dependent and is sensitive to 100 microM amiloride. These results suggest that with resting pH near neutrality, both Na+o/H+i and Na+i/H+o exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na+o/H+i exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H+ efflux or net Na+ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a "set point" of Na+/H+ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.

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.

Mailis Reips : Prezhde vsego neobhodimo natshat / Mailis Reps

Index Scriptorium Estoniae

Reps, Mailis, 1975-

2006-01-01

Haridusminister Mailis Reps kakskeelse hariduse vajalikkusest vene õppekeelega koolides, noorte keeleoskusest, eesti keele õpetamise kvaliteedist, õpetajate eesti keele oskusest, õppetöö kvaliteedist

Development of low-cost open source 3D gel printer "RepRap SWIM-ER"

Science.gov (United States)

Sato, Kei; Basher, Samiul; Ota, Takafumi; Tase, Taishi; Takamatsu, Kyuichiro; Saito, Azusa; Khosla, Ajit; Kawakami, Masaru; Furuawa, Hidemitsu

2017-04-01

Gels are soft and wet materials having low friction, good biocompatibility, and material permeability. It is expected that gel materials will be used as new kinds of industrial materials in the engineering and medical applications. But it cannot build a complicated shape. Soft & Wet Matter Engineering Laboratory developed a 3D gel Printer "SWIM-ER", has enabled modeling of complex shapes of the gel. However, this is expensive. Therefore not all of the gel researchers and the companies have such a device. To solve this problem, we manufacture a low-cost open-source 3D gel printer "RepRap SWIM-ER" from the RepRap. We made the components required to manufacture the "RepRap SWIM-ER" from the 3D printer and chose a light source. In addition, we produced the P-DN gel for RepRap SWIM-ER and conducted the molding test to confirm whether RepRap SWIM-ER can used it.

High-rep-rate Thomson scattering for LHD

Science.gov (United States)

den Hartog, D. J.; Borchardt, M. T.; Holly, D. J.; Schmitz, O.; Yasuhara, R.; Yamada, I.; Funaba, H.; Osakabe, M.; Morisaki, T.

2017-10-01

A high-rep-rate pulse-burst laser system is being built for the LHD Thomson scattering (TS) diagnostic. This laser will have two operating scenarios, a fast-burst sequence of 15 kHz rep rate for at least 15 ms, and a slow-burst sequence of 1 kHz for at least 50 ms. There will be substantial flexibility in burst sequences for tailoring to experimental requirements. This new laser system will operate alongside the existing lasers in the LHD TS diagnostic, and will use the same beamline. This increase in temporal resolution capability complements the high spatial resolution (144 points) of the LHD TS diagnostic, providing unique measurement capability unmatched on any other fusion experiment. The new pulse-burst laser is a straightforward application of technology developed at UW-Madison, consisting of a Nd:YAG laser head with modular flashlamp drive units and a customized control system. Variable pulse-width drive of the flashlamps is accomplished by IGBT (insulated gate bipolar transistor) switching of electrolytic capacitor banks. Direct control of the laser Pockels cell drive enables optimal pulse energy extraction, producing >1.5 J q-switched pulses with 20 ns FWHM. Burst operation of this laser system will be used to capture fast time evolution of the electron temperature and density profiles during events such as ELMs, RMP perturbations, and various MHD modes. This work is supported by the U. S. Department of Energy and the National Institute for Fusion Science (Japan).

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

Expression of Bax in yeast affects not only the mitochondria but also vacuolar integrity and intracellular protein traffic

DEFF Research Database (Denmark)

Dimitrova, Irina; Toby, Garabet G; Tili, Esmerina

2004-01-01

-transferase (BI-GST) leads to aggregation, but not fusion of the mitochondria. In addition, Bax affects the integrity of yeast vacuoles, resulting in the disintegration and eventual loss of the organelles, and the disruption of intracellular protein traffic. While Bcl-2 coexpression only partially corrects...

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.

Discriminação de sorovares de Salmonella spp. isolados de carcaças de frango por REP e ERIC-PCR e fagotipagem do sorovar Enteriditis Discrimination of Salmonella serovars isolated from chicken meat by REP and ERIC-PCR and phagotyping of Enteriditis sorovar

Directory of Open Access Journals (Sweden)

Iliana Alcocer

2006-06-01

Full Text Available Salmonelose é a infecção bacteriana de origem alimentar mais freqüente no Paraná, Brasil, e os surtos estão associados, principalmente, ao consumo de ovos, carne de aves e derivados. Os objetivos deste trabalho foram identificar os sorovares de Salmonella isolados de carcaças de frango e caracterizá-los molecularmente por REP e ERIC-PCR, assim como identificar os fagotipos de Salmonella Enteriditis. Dos 25 isolados de Salmonella spp. analisados, 18 foram identificados como Enteriditis, 4 como Braenderup, 2 como Worthington e 1 como infantis. Dos 18 isolados de Enteriditis, 14 foram PT4, 2 PT4a, 1 PT7 e 1 RDNC, por se tratar de colônia rugosa. REP-PCR forneceu padrão eletroforético distinto de 10 a 13 bandas distribuídas entre 120 e 2072 pb para cada sorovar diferente testado. A ERIC-PCR mostrou um padrão de 4 a 5 bandas entre 180 e 1000 pb e foi menos discriminativa quando comparada à REP-PCR. Os resultados encontrados confirmaram que a fagotipagem é uma ferramenta útil e discriminativa para o sorovar Enteriditis. Apesar do pequeno número de sorovares testados, os resultados sugerem que a REP-PCR parece ser um método atrativo a ser utilizado no futuro para a discriminação preliminar de sorovares de Salmonella.Salmonellosis is the most prevalent bacterial food-borne disease in the State of Paraná, Brazil, and the outbreaks are often associated with consumption of poultry products. The aim of this study was to serotype Salmonella strains isolated from chicken carcasses and characterize them molecularly using REP and ERIC-PCR. The phage types of Salmonella Enteriditis were also identified. Of the 25 Salmonella strains analysed, 18 were identified as Enteriditis, 4 as Braenderup, 2 as Worthington and 1 as infantis. Of the 18 Enteriditis isolates, 14 were PT4, 2 PT4a, 1 PT7 and 1 "reacted, but did not conform" - RDNC. Distinct REP-PCR profiles with 10 to 13 fragments distributed between 120 and 2072 pb were easily obtained for

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

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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

CARACTERIZACIÓN MOLECULAR MEDIANTE rep-PCR DE AISLADOS NATIVOS DE Bacillus thuringiensis, OBTENIDOS DE MUESTRAS DE SUELO

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Fabián Galvis

2014-01-01

Full Text Available Bacillus thuringiensis es una bacteria Gram-positiva formadora de esporas, que produ - ce cristales parasporales de naturaleza proteica, tóxicos contra diferentes órdenes de insectos y biodegradables e inocuos para otras especies. Esta investigación empleó el modelo experimen - tal, que mediante técnicas de observación permi - tió, la identificación microbiológica y bioquímica de B. thuringiensis a partir de muestras de suelo de los municipios de Cúcuta, El Zulia, Los Patios, San Cayetano y Villa del Rosario, Norte de Santander, Colombia, y su posterior caracteri - zación con los marcadores moleculares Bc-Rep y MB1. Se identificaron microbiológica y bioquí - micamente 10 aislados como B. thuringiensis ; los resultados del análisis filogenético mostraron diferencias significativas en los agrupamientos obtenidos con los marcadores Bc-Rep y MB1. Con Bc-Rep se registró un índice de similaridad bajo (18%, mientras que con el marcador MB1 se obtuvo un índice mayor de similitud, 58%. En este trabajo se evidenció una gran variabilidad genética entre los aislados, que mostraron a los marcadores Bc-Rep y MB1 como altamente efectivos para diferenciar cepas estrechamente relacionadas, convirtiéndose en una herramienta genética de gran valor para estudios de identifi-cación y diversidad en B. thuringiensis.

Democracia y República

OpenAIRE

Toledo, Víctor Fabio

2013-01-01

p. 33-34 En la actualidad se ha reavivado el debate en torno a los conceptos de Democracia y República. Desde el triunfo sobre el nazismo a mediados del siglo XX la democracia se ha constituido en un imperativo occidental y, a partir del derrumbe soviético, en uno cuasi universal. Sin embargo, en algunos casos —y no pocos por cierto— en el reclamo por la democratización se encontraron razones ideales para consagrar la concentración de los poderes del Estado, por paradójico que parezca. Es ...

Yeast identification by sequencing, biochemical kits, MALDI-TOF MS and rep-PCR DNA fingerprinting.

Science.gov (United States)

Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Chan, Jasper F W; Lau, Susanna K P; Kong, Fanrong; Xu, Yingchun; Woo, Patrick C Y

2017-12-08

No study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI-TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, using 71 clinically important yeast isolates (excluding Candida albicans) collected from a single centre, we determined the concordance of 28S nrDNA and ITS sequencing and evaluated the performance of two commercial test kits, two MALDI-TOF MS platforms, and rep-PCR DNA fingerprinting. 28S nrDNA and ITS sequencing showed complete agreement on the identities of the 71 isolates. Using sequencing results as the standard, 78.9% and 71.8% isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively; and 90.1% and 80.3% isolates were correctly identified using the Bruker and Vitek MALDI-TOF MS platforms, respectively. Of the 18 strains belonging to the Candida parapsilosis species complex tested by DiversiLab automated rep-PCR DNA fingerprinting, all were identified only as Candida parapsilosis with similarities ≥93.2%, indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, hierarchical cluster analysis of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters, indicating that this technology can potentially differentiate the three species. To achieve higher accuracies of identification, the databases of commercial biochemical test kits, MALDI-TOF MS platforms, and DiversiLab automated rep-PCR DNA fingerprinting needs further enrichment, particularly for uncommonly encountered yeast species. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

Evolution of REP diversity: a comparative study

Czech Academy of Sciences Publication Activity Database

Nunvář, Jaroslav; Lichá, I.; Schneider, Bohdan

2013-01-01

Roč. 14, č. 385 (2013) ISSN 1471-2164 R&D Projects: GA ČR GAP305/12/1801 Institutional research plan: CEZ:AV0Z50520701 Keywords : REP elements * Stenotrophomonas maltophilia * Pseudomonas fluorescens Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.041, year: 2013

ReRep: Computational detection of repetitive sequences in genome survey sequences (GSS

Directory of Open Access Journals (Sweden)

Alves-Ferreira Marcelo

2008-09-01

Full Text Available Abstract Background Genome survey sequences (GSS offer a preliminary global view of a genome since, unlike ESTs, they cover coding as well as non-coding DNA and include repetitive regions of the genome. A more precise estimation of the nature, quantity and variability of repetitive sequences very early in a genome sequencing project is of considerable importance, as such data strongly influence the estimation of genome coverage, library quality and progress in scaffold construction. Also, the elimination of repetitive sequences from the initial assembly process is important to avoid errors and unnecessary complexity. Repetitive sequences are also of interest in a variety of other studies, for instance as molecular markers. Results We designed and implemented a straightforward pipeline called ReRep, which combines bioinformatics tools for identifying repetitive structures in a GSS dataset. In a case study, we first applied the pipeline to a set of 970 GSSs, sequenced in our laboratory from the human pathogen Leishmania braziliensis, the causative agent of leishmaniosis, an important public health problem in Brazil. We also verified the applicability of ReRep to new sequencing technologies using a set of 454-reads of an Escheria coli. The behaviour of several parameters in the algorithm is evaluated and suggestions are made for tuning of the analysis. Conclusion The ReRep approach for identification of repetitive elements in GSS datasets proved to be straightforward and efficient. Several potential repetitive sequences were found in a L. braziliensis GSS dataset generated in our laboratory, and further validated by the analysis of a more complete genomic dataset from the EMBL and Sanger Centre databases. ReRep also identified most of the E. coli K12 repeats prior to assembly in an example dataset obtained by automated sequencing using 454 technology. The parameters controlling the algorithm behaved consistently and may be tuned to the properties

The tissue-specific Rep8/UBXD6 tethers p97 to the endoplasmic reticulum membrane for degradation of misfolded proteins

DEFF Research Database (Denmark)

Madsen, Louise; Kriegenburg, Franziska; Lages Lino Vala, Andrea

2011-01-01

is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p......The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded...... protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8...

El concepto de República en José Martí

OpenAIRE

Dr. Ibrahim Hidalgo

2015-01-01

El acercamiento a una visión martiana sobre el concepto de la república es la esencia del artículo, donde se parte del análisis de sus textos, situando el concepto de república en el centro de su pensamiento y su actuar políticos e ideológicos, que han devenido objeto de análisis y valoraciones desde diversos ángulos, perspectivas y proyecciones por autores que a lo largo del siglo XX, y en la actualidad. El elemento esencial de la concepción es el ser individualmente considerado, cuya unión ...

Obliquamente, os mitos. (República, II

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Loraine Oliveira

2016-12-01

Full Text Available A República é um texto repleto de mitos, que ora são condenados, ora integrados à educação na cidade. No livro II a discussão mostra quais são condenáveis e quais são úteis. E o motivo dessa posição ambivalente de Platão a respeito do mito é um só: os mitos se imprimem na alma. Sendo assim, eles são formadores do caráter. O problema é que há mitos pseûdos, ou seja, falsos, mentirosos, fictícios. E há mitos que possuem um elemento de verdade e um elemento pseûdos. Mas eles jamais são completamente verdadeiros. Ora, qual a função do pseûdos, considerando que os mitos formam o caráter? Um discurso, seja ficcional, ou mítico, pode ser filosófico? Se isso for possível, o texto da República não é ele mesmo uma ficção? Um mito? Por breves apontamentos, este estudo segue essas questões.

Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat (MIRU-VNTR) Genotyping of Mycobacterium intracellulare for Strain Comparison with Establishment of a PCR-Based Database

Science.gov (United States)

Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A.; Falkinham, Joseph O.; Williams, Myra D.; Vasireddy, Ravikiran; Wilson, Rebecca W.; Turenne, Christine

2013-01-01

Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the “gold standard” of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible. PMID:23175249

Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping of mycobacterium intracellulare for strain comparison with establishment of a PCR-based database.

Science.gov (United States)

Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A; Falkinham, Joseph O; Williams, Myra D; Vasireddy, Ravikiran; Wilson, Rebecca W; Turenne, Christine; Wallace, Richard J

2013-02-01

Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the "gold standard" of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible.

Modulation of Acid-sensing Ion Channel 1a by Intracellular pH and Its Role in Ischemic Stroke.

Science.gov (United States)

Li, Ming-Hua; Leng, Tian-Dong; Feng, Xue-Chao; Yang, Tao; Simon, Roger P; Xiong, Zhi-Gang

2016-08-26

An important contributor to brain ischemia is known to be extracellular acidosis, which activates acid-sensing ion channels (ASICs), a family of proton-gated sodium channels. Lines of evidence suggest that targeting ASICs may lead to novel therapeutic strategies for stroke. Investigations of the role of ASICs in ischemic brain injury have naturally focused on the role of extracellular pH in ASIC activation. By contrast, intracellular pH (pHi) has received little attention. This is a significant gap in our understanding because the ASIC response to extracellular pH is modulated by pHi, and activation of ASICs by extracellular protons is paradoxically enhanced by intracellular alkalosis. Our previous studies show that acidosis-induced cell injury in in vitro models is attenuated by intracellular acidification. However, whether pHi affects ischemic brain injury in vivo is completely unknown. Furthermore, whereas ASICs in native neurons are composed of different subunits characterized by distinct electrophysiological/pharmacological properties, the subunit-dependent modulation of ASIC activity by pHi has not been investigated. Using a combination of in vitro and in vivo ischemic brain injury models, electrophysiological, biochemical, and molecular biological approaches, we show that the intracellular alkalizing agent quinine potentiates, whereas the intracellular acidifying agent propionate inhibits, oxygen-glucose deprivation-induced cell injury in vitro and brain ischemia-induced infarct volume in vivo Moreover, we find that the potentiation of ASICs by quinine depends on the presence of the ASIC1a, ASIC2a subunits, but not ASIC1b, ASIC3 subunits. Furthermore, we have determined the amino acids in ASIC1a that are involved in the modulation of ASICs by pHi. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of Brucella melitensis

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Aseem Pandey

2018-04-01

Full Text Available Brucella spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR sensor IRE1α (inositol-requiring enzyme 1 and ER-associated autophagy confer susceptibility to Brucella melitensis and Brucella abortus intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from Ern1 (encoding IRE1 conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to Brucella intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of Brucella intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of Brucella.

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

Hariduspoliitikas ilmnevad üksmeele märgid / Mailis Reps

Index Scriptorium Estoniae

Reps, Mailis, 1975-

2006-01-01

Ilmunud ka: Severnoje Poberezhje 7. juuni lk. 2, Nädaline 10. juuni lk. 2. Haridusminister Mailis Reps Res Publica ja Isamaaliidu ühisest haridusprogrammist ning juba teostumas hariduspoliitilistest eesmärkidest

Difference between Extra- and Intracellular T1 Values of Carboxylic Acids Affects the Quantitative Analysis of Cellular Kinetics by Hyperpolarized NMR

DEFF Research Database (Denmark)

Karlsson, Magnus; Jensen, Pernille Rose; Ardenkjær-Larsen, Jan Henrik

2016-01-01

on the quantification of intracellular metabolicactivity. It is expected that the significantly shorter T1valueof the carboxylic moieties inside cells is a result of macro-molecular crowding. An artificial cytosol has been preparedand applied to predict the T1of other carboxylic acids. Wedemonstrate the value......Incomplete knowledge of the longitudinal relaxationtime constant (T1) leads to incorrect assumptions in quantita-tive kinetic models of cellular systems, studied by hyper-polarized real-time NMR. Using an assay that measures theintracellular signal of small carboxylic acids in living cells...

Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival.

Science.gov (United States)

Massimi, Mara; Cardarelli, Silvia; Galli, Francesca; Giardi, Maria Federica; Ragusa, Federica; Panera, Nadia; Cinque, Benedetta; Cifone, Maria Grazia; Biagioni, Stefano; Giorgi, Mauro

2017-06-01

Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 μM) and DC-TA-46 (0.5 μM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. J. Cell. Biochem. 118: 1401-1411, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Therapeutic Antiviral Effect of the Nucleic Acid Polymer REP 2055 against Persistent Duck Hepatitis B Virus Infection

Science.gov (United States)

Noordeen, Faseeha; Scougall, Catherine A.; Grosse, Arend; Qiao, Qiao; Ajilian, Behzad B.; Reaiche-Miller, Georget; Finnie, John; Werner, Melanie; Broering, Ruth; Schlaak, Joerg F.; Vaillant, Andrew; Jilbert, Allison R.

2015-01-01

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055. Ducks with persistent DHBV infection were treated with NAP 2055 to determine if the post-entry inhibitory activity exhibited by NAPs could provide a therapeutic effect against established DHBV infection in vivo. In all REP 2055-treated ducks, 28 days of treatment lead to initial rapid reductions in serum DHBsAg and DHBV DNA and increases in anti-DHBs antibodies. After treatment, 6/11 ducks experienced a sustained virologic response: DHBsAg and DHBV DNA remained at low or undetectable levels in the serum and no DHBsAg or DHBV core antigen positive hepatocytes and only trace amounts of DHBV total and covalently closed circular DNA (cccDNA) were detected in the liver at 9 or 16 weeks of follow-up. In the remaining 5/11 REP 2055-treated ducks, all markers of DHBV infection rapidly rebounded after treatment withdrawal: At 9 and 16 weeks of follow-up, levels of DHBsAg and DHBcAg and DHBV total and cccDNA in the liver had rebounded and matched levels observed in the control ducks treated with normal saline which remained persistently infected with DHBV. These data demonstrate that treatment with the NAP REP 2055 can lead to sustained control of persistent DHBV infection. These effects may be related to the unique ability of REP 2055 to block release of DHBsAg from infected hepatocytes. PMID:26560490

Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors.

Science.gov (United States)

Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

2014-07-17

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. © 2014 The Authors.

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

Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1.

Science.gov (United States)

Marr, A K; Jenssen, H; Moniri, M Roshan; Hancock, R E W; Panté, N

2009-01-01

Although both lactoferrin (Lf), a component of the innate immune system of living organisms, and its N-terminal pepsin cleavage product lactoferricin (Lfcin) have anti-herpes activity, the precise mechanisms by which Lf and Lfcin bring about inhibition of herpes infections are not fully understood. In the present study, experiments were carried out to characterize the activity of bovine Lf and Lfcin (BLf and BLfcin) against the Herpes simplex virus-1 (HSV-1). HSV-1 cellular uptake and intracellular trafficking were studied by immunofluorescence microscopy. In comparison to the untreated infected control cells, both the BLf- and BLfcin-treated cells showed a significant reduction in HSV-1 cellular uptake. The few virus particles that were internalized appeared to have a delayed intracellular trafficking. Thus, in addition to their interference with the uptake of the virus into host cells, Lf and Lfcin also exert their antiviral effect intracellularly.

The tissue-specific Rep8/UBXD6 tethers p97 to the endoplasmic reticulum membrane for degradation of misfolded proteins.

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Louise Madsen

Full Text Available The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded secretory proteins via the ER-associated degradation pathway. This functional diversity of p97 depends on its association with various cofactors, and to further our understanding of p97 function it is important that these cofactors are identified and analyzed. Here, we isolate and characterize the human protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8 is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p97 to the ER membrane for efficient ER-associated degradation.

Interlaboratory reproducibility of DiversiLab rep-PCR typing and clustering of Acinetobacter baumannii isolates.

Science.gov (United States)

Higgins, Paul G; Hujer, Andrea M; Hujer, Kristine M; Bonomo, Robert A; Seifert, Harald

2012-01-01

We have investigated the reproducibility of DiversiLab rep-PCR fingerprints between two laboratories with the aim of determining if the fingerprints and clustering are laboratory-specific or portable. One-hundred non-duplicate Acinetobacter baumannii isolates were used in this study. DNA isolation and rep-PCR were each performed separately in two laboratories and rep-PCR patterns generated in laboratory A were compared with those from laboratory B. Twelve A. baumannii isolates processed in laboratory A showed ≥98 % pattern similarity with the corresponding 12 isolates tested in laboratory B and were considered identical. Sixty-four isolates showed 95-97.9 % similarity with their corresponding isolates. Twenty-three isolates showed 90-94 % similarity with the corresponding isolates, while one isolate showed only 87.4 % similarity. However, intra-laboratory clustering was conserved: isolates that clustered in laboratory A also clustered in laboratory B. While clustering was conserved and reproducible at two different laboratories, demonstrating the robustness of rep-PCR, interlaboratory comparison of individual isolate fingerprints showed more variability. This comparison allows conclusions regarding clonality to be reached independent of the laboratory where the analysis is performed.

El concepto de República en José Martí

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Dr. Ibrahim Hidalgo

2015-10-01

Full Text Available El acercamiento a una visión martiana sobre el concepto de la república es la esencia del artículo, donde se parte del análisis de sus textos, situando el concepto de república en el centro de su pensamiento y su actuar políticos e ideológicos, que han devenido objeto de análisis y valoraciones desde diversos ángulos, perspectivas y proyecciones por autores que a lo largo del siglo XX, y en la actualidad. El elemento esencial de la concepción es el ser individualmente considerado, cuya unión constituye el pueblo, que deviene así no un ente abstracto y amorfo, sino un conglomerado de personas

Intracellular calcium chelation and pharmacological SERCA inhibition of Ca2+ pump in the insular cortex differentially affect taste aversive memory formation and retrieval.

Science.gov (United States)

Miranda, María Isabel; González-Cedillo, Francisco J; Díaz-Muñoz, Mauricio

2011-09-01

Variation in intracellular calcium concentration regulates the induction of long-term synaptic plasticity and is associated with a variety of memory/retrieval and learning paradigms. Accordingly, impaired calcium mobilization from internal deposits affects synaptic plasticity and cognition in the aged brain. During taste memory formation several proteins are modulated directly or indirectly by calcium, and recent evidence suggests the importance of calcium buffering and the role of intracellular calcium deposits during cognitive processes. Thus, the main goal of this research was to study the consequence of hampering changes in cytoplasmic calcium and inhibiting SERCA activity by BAPTA-AM and thapsigargin treatments, respectively, in the insular cortex during different stages of taste memory formation. Using conditioned taste aversion (CTA), we found differential effects of BAPTA-AM and thapsigargin infusions before and after gustatory stimulation, as well as during taste aversive memory consolidation; BAPTA-AM, but not thapsigargin, attenuates acquisition and/or consolidation of CTA, but neither compound affects taste aversive memory retrieval. These results point to the importance of intracellular calcium dynamics in the insular cortex during different stages of taste aversive memory formation. Copyright © 2011 Elsevier Inc. All rights reserved.

Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution.

Science.gov (United States)

Ishii, Satoshi; Sadowsky, Michael J

2009-04-01

A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or 'fingerprints' of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.

Investigation of Genetic Diversity of Wilsonomyces carpophilus in Khorasan Razavi Using rep-PCR Marker

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R. Barazandeh Aq Kariz

2017-08-01

Full Text Available Introduction: Shot hole disease of stone fruit trees resulted from Wilsonomyces carpophilus can weaken the trees and reduce the quantity and quality of the crops worldwide particularly in semi-arid regions. Coryneum blight or shot hole disease infects all the stone fruit trees including peach, nectarine, apricot, sour cherry, plum, cherry, and almond. One of the most important strategies to manage any plant disease is to use resistant cultivars. In this way, it is very important to have knowledge about the status of genetic diversity and to determine the relationship between isolates of the causal agent fungus. The main objective of the present research was to study the genetic diversity of W. carpophilus in Khorasan Razavi province using the rep-PCR molecular fingerprinting method. Materials and Methods: Sampling was performed from peach, nectarine, plum, apricot and cherry orchards of Quchan, Torqabeh, Shandiz, Chenaran, Neishabur, Kalat, Torbat Heidarieh and Mashhad during spring and summer of 2012 and 2013. Mono-conidial isolates were recovered from infected leaves, fruits, and twigs of different parts of orchards. Infected collected leaves, twigs, and fruits were transferred to the laboratory. By using techniques of Klimesova and Prasil (1989 and Mehta (1998 from the cut parts between infected and healthy tissues of each isolate, cuts of 2-3 mm from leaf, fruit and twig were prepared by the scalpel. These pieces were surface sterilized with 1% sodium hypochlorite liquid about 1 to 3 minutes based on the thickness of tissue. Then, the samples were cultured on PDA, MEA, and WA media and incubated at 18, 20, and 25 °C. The isolated fungi were purified and identified. The research was performed on 20 fungal isolates collected from different stone fruit trees. Genomic DNA was amplified using BOX A1R, ERIC2, ERIC1R, REP2-I, and REP1R-I primers. Thirty-eight of 39 fragments amplified were polymorphic for 100 to 5000 base pairs. Similarity matrix

A Comparison of Molecular Typing Methods Applied to Enterobacter cloacae complex: hsp60 Sequencing, Rep-PCR, and MLST

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Roberto Viau

2017-02-01

Full Text Available Molecular typing using repetitive sequenced-based PCR (rep-PCR and hsp60 sequencing were applied to a collection of diverse Enterobacter cloacae complex isolates. To determine the most practical method for reference laboratories, we analyzed 71 E. cloacae complex isolates from sporadic and outbreak occurrences originating from 4 geographic areas. While rep-PCR was more discriminating, hsp60 sequencing provided a broader and a more objective geographical tracking method similar to multilocus sequence typing (MLST. In addition, we suggest that MLST may have higher discriminative power compared to hsp60 sequencing, although rep-PCR remains the most discriminative method for local outbreak investigations. In addition, rep-PCR can be an effective and inexpensive method for local outbreak investigation.

Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

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Marion Le Coadic

Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

Identification of Tilletia species using rep-PCR fingerprinting technique

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Župunski Vesna

2011-01-01

Full Text Available Analyzing 167 non-processed seed samples of wheat, it was found that 145 samples (86.8 % were contaminated with Tilletia species, while 22 (13.2 % samples were not contaminated. By using rep-PCR fingerprinting technique, it was found that DNA isolates of T. tritici originated from Serbian wheat samples had 80 % similarity with positive control for T. tritici. One isolate shared similarity of 60% with T. tritici, T. controversa and T. laevis. It was supposed that this isolate belongs to T. bromi. Isolate of T. laevis shared a similarity of 70 % with isolates of T. tritici and T. controversa, while T. walkeri was more than 10 % similar with T. tritici, T. controversa and T. laevis. Although T. controversa and T. tritici had high percent of genetic similarity, they were clustered separately. Our results suggest that rep-PCR fingerprinting could be a useful tool for monitoring presence of morphologically similar Tilletia species in wheat production areas.

Open-Source Wax RepRap 3-D Printer for Rapid Prototyping Paper-Based Microfluidics.

Science.gov (United States)

Pearce, J M; Anzalone, N C; Heldt, C L

2016-08-01

The open-source release of self-replicating rapid prototypers (RepRaps) has created a rich opportunity for low-cost distributed digital fabrication of complex 3-D objects such as scientific equipment. For example, 3-D printable reactionware devices offer the opportunity to combine open hardware microfluidic handling with lab-on-a-chip reactionware to radically reduce costs and increase the number and complexity of microfluidic applications. To further drive down the cost while improving the performance of lab-on-a-chip paper-based microfluidic prototyping, this study reports on the development of a RepRap upgrade capable of converting a Prusa Mendel RepRap into a wax 3-D printer for paper-based microfluidic applications. An open-source hardware approach is used to demonstrate a 3-D printable upgrade for the 3-D printer, which combines a heated syringe pump with the RepRap/Arduino 3-D control. The bill of materials, designs, basic assembly, and use instructions are provided, along with a completely free and open-source software tool chain. The open-source hardware device described here accelerates the potential of the nascent field of electrochemical detection combined with paper-based microfluidics by dropping the marginal cost of prototyping to nearly zero while accelerating the turnover between paper-based microfluidic designs. © 2016 Society for Laboratory Automation and Screening.

The effect of high pressure on the intracellular trehalose synthase activity of Thermus aquaticus.

Science.gov (United States)

Dong, Yongsheng; Ma, Lei; Duan, Yuanliang

2016-01-01

To understand the effect of high pressure on the intracellular trehalose synthase activity, Thermus aquaticus (T. aquaticus) in the logarithmic growth phase was treated with high-pressure air, and its intracellular trehalose synthase (TSase) activity was determined. Our results indicated that pressure is a factor strongly affecting the cell growth. High pressure significantly attenuated the growth rate of T. aquaticus and shortened the duration of stationary phase. However, after 2 h of culture under 1.0 MPa pressure, the activity of intracellular TSase in T. aquaticus reached its maximum value, indicating that pressure can significantly increase the activity of intracellular TSase in T. aquaticus. Thus the present study provides an important guide for the enzymatic production of trehalose.

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.

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.

La república conservadora

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Matías Muraca

2011-12-01

Full Text Available Este artigo tem como objetivo revisar e analisar como aparece a discussão sobre República ao longo dos anos de redemocratização na Argentina, articulando com outra categoria da teoria política: o populismo. Os conteúdos e sentidos da demanda republicana que se conjuga com populismo são analisados nas edições de um dos mais importantes jornais argentinos durante o segundo governo de Carlos Saúl Menem (1995-1999 e o governo de Aliança (1999-2001. Especificamente, é revisado nas Colunas de Opinião do Diário “La Nación” a primeira etapa das críticas republicanas centradas em um dos seus componentes principais: a questão da corrupção associada a problemática da moral.

Identification of a Paralog-Specific Notch1 Intracellular Domain Degron

OpenAIRE

Broadus, Matthew R.; Chen, Tony W.; Neitzel, Leif R.; Ng, Victoria H.; Jodoin, Jeanne; Lee, Laura A.; Salic, Adrian; Robbins, David J.; Capobianco, Anthony J.; Patton, James G.; Huppert, Stacey S.; Lee, Ethan

2016-01-01

Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD), which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box). We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box...

A novel intracellular pool of LFA-1 is critical for asymmetric CD8+ T cell activation and differentiation.

Science.gov (United States)

Capece, Tara; Walling, Brandon L; Lim, Kihong; Kim, Kyun-Do; Bae, Seyeon; Chung, Hung-Li; Topham, David J; Kim, Minsoo

2017-11-06

The integrin lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is a key T cell adhesion receptor that mediates stable interactions with antigen-presenting cell (APC), as well as chemokine-mediated migration. Using our newly generated CD11a-mYFP knock-in mice, we discovered that naive CD8 + T cells reserve a significant intracellular pool of LFA-1 in the uropod during migration. Intracellular LFA-1 quickly translocated to the cell surface with antigenic stimulus. Importantly, the redistribution of intracellular LFA-1 at the contact with APC was maintained during cell division and led to an unequal inheritance of LFA-1 in divided T cells. The daughter CD8 + T cells with disparate LFA-1 expression showed different patterns of migration on ICAM-1, APC interactions, and tissue retention, as well as altered effector functions. In addition, we identified Rab27 as an important regulator of the intracellular LFA-1 translocation. Collectively, our data demonstrate that an intracellular pool of LFA-1 in naive CD8 + T cells plays a key role in T cell activation and differentiation. © 2017 Capece et al.

A república e o sonho The republic and its dream

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Maria Tereza Chaves de Mello

2011-06-01

Full Text Available O desencanto com a República brasileira tem sido tema de reflexões desde os seus momentos iniciais. Buscando os conteúdos concretos dos sonhos republicanos na década de 1880 e na Primeira República vamos achar uma importante defasagem entre eles não exatamente em função do marco cronológico, mas do grupo social que, respectivamente, os acalentou.The disillusion with the Brazilian Republic since its first days has been a topic of academician reflexions. Examinating the concrete contents of the 1880`s and the First Republic republican dreams we will find a substantive difference between them. To be exact, this difference must not be related to the chronological demarcation but to the social group that embraced each of them.

Production of recombinant AAV vectors encoding insulin-like growth factor I is enhanced by interaction among AAV rep regulatory sequences

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Dilley Robert

2009-01-01

Full Text Available Abstract Background Adeno-associated virus (AAV vectors are promising tools for gene therapy. Currently, their potential is limited by difficulties in producing high vector yields with which to generate transgene protein product. AAV vector production depends in part upon the replication (Rep proteins required for viral replication. We tested the hypothesis that mutations in the start codon and upstream regulatory elements of Rep78/68 in AAV helper plasmids can regulate recombinant AAV (rAAV vector production. We further tested whether the resulting rAAV vector preparation augments the production of the potentially therapeutic transgene, insulin-like growth factor I (IGF-I. Results We constructed a series of AAV helper plasmids containing different Rep78/68 start codon in combination with different gene regulatory sequences. rAAV vectors carrying the human IGF-I gene were prepared with these vectors and the vector preparations used to transduce HT1080 target cells. We found that the substitution of ATG by ACG in the Rep78/68 start codon in an AAV helper plasmid (pAAV-RC eliminated Rep78/68 translation, rAAV and IGF-I production. Replacement of the heterologous sequence upstream of Rep78/68 in pAAV-RC with the AAV2 endogenous p5 promoter restored translational activity to the ACG mutant, and restored rAAV and IGF-I production. Insertion of the AAV2 p19 promoter sequence into pAAV-RC in front of the heterologous sequence also enabled ACG to function as a start codon for Rep78/68 translation. The data further indicate that the function of the AAV helper construct (pAAV-RC, that is in current widespread use for rAAV production, may be improved by replacement of its AAV2 unrelated heterologous sequence with the native AAV2 p5 promoter. Conclusion Taken together, the data demonstrate an interplay between the start codon and upstream regulatory sequences in the regulation of Rep78/68 and indicate that selective mutations in Rep78/68 regulatory elements

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

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.

Deletion of GPR40 Impairs Glucose-Induced Insulin Secretion In Vivo in Mice Without Affecting Intracellular Fuel Metabolism in Islets

Energy Technology Data Exchange (ETDEWEB)

Alquier, Thierry; Peyot, Marie-Line; Latour, M. G.; Kebede, Melkam; Sorensen, Christina M.; Gesta, Stephane; Kahn, C. R.; Smith, Richard D.; Jetton, Thomas L.; Metz, Thomas O.; Prentki, Marc; Poitout, Vincent J.

2009-11-01

The G protein-coupled receptor GPR40 mediates fatty-acid potentiation of glucose-stimulated insulin secretion, but its contribution to insulin secretion in vivo and mechanisms of action remain uncertain. This study was aimed to ascertain whether GPR40 controls insulin secretion in vivo and modulates intracellular fuel metabolism in islets. We observed that glucose- and arginine-stimulated insulin secretion, assessed by hyperglycemic clamps, was decreased by approximately 60% in GPR40 knock-out (KO) fasted and fed mice, without changes in insulin sensitivity assessed by hyperinsulinemic-euglycemic clamps. Glucose and palmitate metabolism were not affected by GPR40 deletion. Lipid profiling revealed a similar increase in triglyceride and decrease in lysophosphatidylethanolamine species in WT and KO islets in response to palmitate. These results demonstrate that GPR40 regulates insulin secretion in vivo not only in response to fatty acids but also to glucose and arginine, without altering intracellular fuel metabolism.

Direct Application of Rep-PCR on Type I Sourdough Matrix to Monitor the Dominance and Persistence of a Lactobacillus plantarum Starter Throughout Back-Slopping.

Science.gov (United States)

Dolci, Paola; Cocolin, Luca

2017-08-01

This study describes the optimization and application of repetitive element-PCR (rep-PCR) technique directly on microbial DNA extracted from type I sourdoughs for fast monitoring of a Lb. plantarum starter strain (P1FMC) throughout daily back-slopping. The challenge was to follow and study the performance of a starter culture directly in sourdoughs without cultivation on selective media. The extraction of good quality microbial DNA suitable for amplification from a complex matrix such as dough was the first target. In addition, the objective to obtain a clear rep-PCR profile referable to a specific starter strain among a microbial community was pursued. Co-inoculum trials, in flour matrix, with Lb. plantarum P1FMC and L. lactis LC71 strains and, subsequently, type I sourdough back-slopping trials were performed. The rep-PCR amplification profiles obtained were clearly referable to that of Lb. plantarum P1FMC starter in both co-inoculum trials (also when it was present with one order of magnitude less with respect to L. lactis LC71) and back-slopping trials where it dominated the fermentation process with loads of 10 8 cfu g -1 and prevailed on the autochthonous microbiota. Thus, the approach proposed in this paper could be considered a methodological advancement, based on a culture-independent one-step rep-PCR, suitable for fast monitoring of starter performance. © 2017 Institute of Food Technologists®.

Key mediators of intracellular amino acids signaling to mTORC1 activation.

Science.gov (United States)

Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

2015-05-01

Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

Sodium transport and intracellular sodium activity in cultured human nasal epithelium

DEFF Research Database (Denmark)

Willumsen, Niels J.; Boucher, Richard C.

1991-01-01

 human nasal epithelium (HNE). Under control conditions, intracellular Na+ activity (acNa) was 23 +/- 1 mM (n = 44 preparations, 393 impalements).Amiloride (10(-4) M) hyperpolarized the apical membrane and increased the fractional apical membrane resistance but did not affect acNa. Exposure to...

El Ecoturismo en los Humedales: Análisis de las Potencialidades de República Dominicana

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Francisco Orgaz Agüera

2014-04-01

Full Text Available El turismo se configura como uno de los principales motores económicos de República Dominicana. En este sentido, el turismo de sol y playa es la principal tipología turística en el país. Aunque, República Dominicana cuenta con numerosas potencialidades turísticas para poner en marchas nuevas formas de turismo: Turismo gastronómico, turismo ornitológico, turismo cultural, ecoturismo, etc. El principal objetivo de esta investigación es analizar las potencialidades existentes en República Dominicana para desarrollar el ecoturismo en las zonas de humedales. La metodología ha consistido en un trabajo de campo, a través de las entrevistas, los grupos de discusión y la observación participante. Los resultados muestran las grandes potencialidades que tiene el país para desarrollar el ecoturismo en los humedales, y contribuir así, al desarrollo socioeconómico de las comunidades locales. 

Identification of new isolates of Bacillus thuringiensis using rep-PCR products and delta-endotoxin electron microscopy

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A.S.G. Lima

2002-01-01

Full Text Available PCR has been used to analyze the distribution of REP (Repetitive Extragenic Palindromic and ERIC (Enterobacterial Repetitive Intergenic Consensus sequences (rep-PCR found within the genome of the bacterium Bacillus thuringiensis, with the purpose to analyze the genetic similarities among 56 subspecies samples and 95 field isolates. The PCR products were analyzed by EB-AGE (ethidium bromide-agarose electrophoresis and then submitted to banding comparisons, based on the Phyllip software algorithm. When the banding similarities were considered for comparison purposes among all the strains, the phylogenic tree patterns varied according to the rep-PCR primers considered, but, from a broader point of view, the ERIC sequences produced better results, which, together with electron microscopy analysis of the released parasporal bodies and colony morphology characteristics, allowed to detect two possible new subspecies of B. thuringiensis.

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

Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

Science.gov (United States)

Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

2015-09-01

Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Repérage d’Images Ordinaires : Analyse des Requêtes des Chercheurs d’Images

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Elaine Ménard

2009-06-01

Full Text Available Abstract For many years, the Web became an important media for the diffusion of multilingual resources. However, linguistic differences still form a major obstacle to scientific, cultural, and educational exchange. Besides this linguistic diversity, a multitude of databases and collections now contain documents in various formats, which may also adversely affect the retrieval process. For example, image collections have many areas of application. The need to retrieve images from a particular collection is shared by several user communities. Until recently, image collections were difficult to access due to limitations in dissemination and duplication procedures. The advent of the Web highlighted the pressing need to develop suitable tools for the description of digital images, since these can be found in the majority of available resources. This research compares image retrieval within two contexts: a monolingual context, i.e., where the language of the query (French is the same as the indexing language (French; and a multilingual context, i.e., where the language of the query (French is different from the indexing language (English.This article presents the results of the analysis of the queries used to retrieve a set of ordinary images representing everyday-life objects in a multilingual retrieval context. The analysis of the terms contained in the queries of the image searchers illustrates the terminological, perceptual, and structural trends. In general, participants employ simple queries containing only one word. Moreover, the queries are often generic in nature. The analysis also underlines that participants frequently use functional attributes to retrieve ordinary images. On the structural level, generic relations dominate other types of relations. The contributions of this research project are manifold. At the theoretical level, this research identifies the characteristics essential to the indexing process of digital images. This study

REP Executive Education focuses on Midwest plant off-site emergency preparedness

OpenAIRE

Center for Homeland Defense and Security

2015-01-01

Center for Homeland Defense and Security News and Stories, PRESS RELEASES The Center for Homeland Defense and Security burgeoning Radiological Emergency Preparedness (REP) Executive Education Program convened June 8-12 with a special focus on emergency preparedness policy and strategic issues for...

Memantine, an antagonist of the NMDA glutamate receptor, affects cell proliferation, differentiation and the intracellular cycle and induces apoptosis in Trypanosoma cruzi.

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Flávia Silva Damasceno

2014-02-01

Full Text Available Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and affects approximately 10 million people in endemic areas of Mexico and Central and South America. Currently available chemotherapies are limited to two compounds: Nifurtimox and Benznidazole. Both drugs reduce the symptoms of the disease and mortality among infected individuals when used during the acute phase, but their efficacy during the chronic phase (during which the majority of cases are diagnosed remains controversial. Moreover, these drugs have several side effects. The aim of this study was to evaluate the effect of Memantine, an antagonist of the glutamate receptor in the CNS of mammals, on the life cycle of T. cruzi. Memantine exhibited a trypanocidal effect, inhibiting the proliferation of epimastigotes (IC50 172.6 µM. Furthermore, this compound interfered with metacyclogenesis (approximately 30% reduction and affected the energy metabolism of the parasite. In addition, Memantine triggered mechanisms that led to the apoptosis-like cell death of epimastigotes, with extracellular exposure of phosphatidylserine, increased production of reactive oxygen species, decreased ATP levels, increased intracellular Ca(2+ and morphological changes. Moreover, Memantine interfered with the intracellular cycle of the parasite, specifically the amastigote stage (IC50 31 µM. Interestingly, the stages of the parasite life cycle that require more energy (epimastigote and amastigote were more affected as were the processes of differentiation and cell invasion.

RiskREP: Risk-Based Security Requirements Elicitation and Prioritization (extended version)

NARCIS (Netherlands)

Herrmann, Andrea; Morali, A.

2010-01-01

Today, companies are required to be in control of the security of their IT assets. This is especially challenging in the presence of limited budgets and conflicting requirements. Here, we present Risk-Based Requirements Elicitation and Prioritization (RiskREP), a method for managing IT security

Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

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Norkin Leonard C

2004-07-01

Full Text Available Abstract Background Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function. Methods We utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions. Results We show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp, C. caviae (GPIC, and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi. Conclusion Caveolin-2 associates with the chlamydial

Averróis e a República de Platão

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Pereira, Rosalie Helena de Souza

2007-01-01

Full Text Available Sobre República é o único comentário de uma obra de Platão deixado por Averróis, o comentador por antonomásia, diga-se de Aristóteles. Embora se tenha perdido o original árabe, a teles. Embora se tenha perdido o original árabe, a posteridade recebeu uma versão hebraíca e duas latinas. O comentário (ou paráfrase a República de Platão pode ser considerado uma obra original - divida em três livros - já que apenas um terço dela corresponde ao texto platônico. Além de apresentar conceitos aristotélicos retirados da Ética Nicomaquéia e de obras políticas de Al-Farabi, Averróis apresenta reflexões próprias e faz críticas à sociedade em que vive

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.

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

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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.

El montaje de la transición argentina. Un análisis de los films La República perdida, La República perdida II y Evita, quien quiera oír que oiga

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Paola Judith Margulis

2012-01-01

Full Text Available El presente artículo se propone reconstruir una zona de la historia del documental argentino a partir del abordaje de tres films que tuvieron un alto impacto en Argentina hacia la transición democrática: La República perdida (Miguel Pérez, 1983, su secuela La República perdida II (Miguel Pérez, 1986 y Evita, quien quiera oír que oiga (Eduardo Mignogna, 1984. Dada la importancia de estos films -tanto en términos políticos como de impacto en el público-, intentaremos situarlos en contexto y analizar algunos aspectos inherentes a su lógica fílmica.

CoolRep H22: Synthesis report on R and D results on geological disposal up to 2009

International Nuclear Information System (INIS)

Umeki, Hiroyuki; Hioki, Kazumasa; Osawa, Hideaki; Fujita, Tomoo; Shibata, Masahiro; Makino, Hitoshi; Takeuchi, Shinji; Ishimaru, Tsuneari

2011-02-01

The Japan Atomic Energy Agency (JAEA) has been performing research and development on geological disposal technology of high level radioactive waste. At the end of fiscal year 2009, the Geological Isolation Research and Development Directorate of JAEA made publicly available the 'CoolRep H22', which is a web-based report that summarizes the R and D results, on its website. This document reports the contents of CoolRep H22. (author)

An N-terminal region of a Myb-like protein is involved in its intracellular localization and activation of a gibberellin-inducible proteinase gene in germinated rice seeds.

Science.gov (United States)

Sutoh, Keita; Washio, Kenji; Imai, Ryozo; Wada, Masamitsu; Nakai, Tomonori; Yamauchi, Daisuke

2015-01-01

The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213 N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1.

Iberia : capital federal de la IIª República Española (Un proyecto de Rubio i Tuduri

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Carlos Sambricio

1996-01-01

Full Text Available A los pocos meses de proclamarse, en 1931, la II República Española, Nicolás Rubio i Tuduri presentaba —en el marco de la Exposición organizada por la Asociación de Arquitectos de Cataluña, celebrada en la barcelonesa Galería Maragall— una propuesta un tanto sorprendente: el proyecto de una nueva Capital Federal de la República Española que él denominaba Iberia.

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.

Intracellular β-Glucosidases CEL1a and CEL1b Are Essential for Cellulase Induction on Lactose in Trichoderma reesei

Science.gov (United States)

Xu, Jintao; Zhao, Guolei; Kou, Yanbo; Zhang, Weixin; Zhou, Qingxin; Chen, Guanjun

2014-01-01

Lactose (1,4-O-β-d-galacto-pyranosyl-d-glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular β-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei. We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular β-glucosidases abolished cbh1 gene expression on lactose. d-Galactose restored the efficient cellulase gene induction in the Δcel1a strain independently of its reductive metabolism, but not in the Δcel1a Δcel1b strain. A further comparison of the transcriptional responses of the Δcel1a Δcel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the Δcel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis β-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular β-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes. PMID:24879125

Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

Science.gov (United States)

Tong, Jie; Sun, Lijun; Zhu, Bin; Fan, Yun; Ma, Xingfeng; Yu, Liyin; Zhang, Jianbao

2017-10-01

Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

Energy Technology Data Exchange (ETDEWEB)

Hipp, Katharina; Rau, Peter; Schäfer, Benjamin [Institut für Biomaterialien und biomolekulare Systeme, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart (Germany); Gronenborn, Bruno [Institut des Sciences du Végétal, CNRS, 91198 Gif-sur-Yvette (France); Jeske, Holger, E-mail: holger.jeske@bio.uni-stuttgart.de [Institut für Biomaterialien und biomolekulare Systeme, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart (Germany)

2014-08-15

Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis.

The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

International Nuclear Information System (INIS)

Hipp, Katharina; Rau, Peter; Schäfer, Benjamin; Gronenborn, Bruno; Jeske, Holger

2014-01-01

Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis

RiskREP: Risk-Based Security Requirements Elicitation and Prioritization

OpenAIRE

Herrmann, Andrea; Morali, A.; Etalle, Sandro; Wieringa, Roelf J.; Niedrite, Laila; Strazdina, Renate; Wangler, Benkt

2011-01-01

Companies are under pressure to be in control of their assets but at the same time they must operate as efficiently as possible. This means that they aim to implement “good-enough security‿ but need to be able to justify their security investment plans. In this paper, we present a Risk-Based Requirements Prioritization method (RiskREP) that extends misuse case-based methods with IT architecture based risk assessment and countermeasure definition and prioritization. Countermeasure prioritizati...

Neurodegeneration in Alzheimer Disease: Role of Amyloid Precursor Protein and Presenilin 1 Intracellular Signaling

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Mario Nizzari

2012-01-01

Full Text Available Alzheimer disease (AD is a heterogeneous neurodegenerative disorder characterized by (1 progressive loss of synapses and neurons, (2 intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3 amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP and presenilin 1 and 2 (PS1 and PS2. The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration.

Mechanism of the negative force-frequency relationship in physiologically intact rat ventricular myocardium. Studies by intracellular Ca2+ monitor with iodo-1 and by 31P-nuclear magnetic resonance spectroscopy

International Nuclear Information System (INIS)

Morii, Isao; Kihara, Yasuki; Sasayama, Shigetake; Konishi, Takashi; Inubushi, Toshiro.

1996-01-01

We studied the subcellular mechanisms of the negative force-frequency relationship in rat myocardium by measuring intracellular Ca 2+ transients by indo-1 fluorometry and intracellular pH (pH i ) and phosphate compounds with 31 P-nuclear magnetic resonance (NMR). The data were compared with those from guinea pig hearts, which show a positive force-frequency relationship. By increasing the pacing rate from 3 Hz to 5 Hz, the peak positive first derivative of left ventricular pressure (LVdP/dt) in rat heart decreased by 10±1% (n=6). In contrast to this negative inotropic response, simultaneously measured peak Ca 2+ transients increased by 6±1%. Guinea pig heart (n=6) showed an increase in peak positive LVdP/dt (33±1%) which was associated with an increase in peak Ca 2+ transients (8±1%). Under equivalent experimental conditions in an NMR spectrometer, this increase in the pacing rate did not affect intracellular levels of phosphate compounds in either rat (n=6) or guinea pig heart (n=6). In contrast, pH i showed a decrease of 0.031±0.006 pH units in rat heart, while no changes were observed in guinea pig heart. These results suggest that in physiological rat myocardium, pH i is susceptible to changes in the stimulus frequency and may affect the Ca 2+ -responsiveness of contractile proteins, which results in the negative force-frequency relationship. (author)

Revolución versus reforma educativa en la segunda república española. Elementos de ruptura

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Juan Manuel FERNÁNDEZ SORIA

2010-03-01

Full Text Available RESUMEN: Están fuera de toda duda las conexiones existentes entre la República de 1931-35 y la de 1936-39. En este sentido estamos de acuerdo con quienes afirman que el estallido de la guerra del 36 es el resultado de un proceso que arranca del mismo año en que se proclama la II República, iniciándose ya entonces —como sostiene Andrés M. Kramer— la «mecánica de guerra civil». La educación republicana en tiempo de guerra no puede quedar fuera de esta consideración, porque aquélla sumerge sus cimientos más profundos en la política educativa de 1931-35. Efectivamente, muchas de las acciones educativas y culturales llevadas a cabo durante la guerra no tendrían fácil explicación si no se examina previamente la tarea de la República del 31 en estos mismos aspectos.

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis.

Science.gov (United States)

Wang, Jing-Yu; Wu, Ning; Liu, Wan-Hua; Ren, Juan-Juan; Tang, Pan; Qiu, Yuan-Hao; Wang, Chi-Young; Chang, Ching-Dong; Liu, Hung-Jen

2014-01-01

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the 'Bruce ladder' multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Progressive Conversion from B-rep to BSP for Streaming Geometric Modeling.

Science.gov (United States)

Bajaj, Chandrajit; Paoluzzi, Alberto; Scorzelli, Giorgio

2006-01-01

We introduce a novel progressive approach to generate a Binary Space Partition (BSP) tree and a convex cell decomposition for any input triangles boundary representation (B-rep), by utilizing a fast calculation of the surface inertia. We also generate a solid model at progressive levels of detail. This approach relies on a variation of standard BSP tree generation, allowing for labeling cells as in, out and fuzzy, and which permits a comprehensive representation of a solid as the Hasse diagram of a cell complex. Our new algorithm is embedded in a streaming computational framework, using four types of dataflow processes that continuously produce, transform, combine or consume subsets of cells depending on their number or input/output stream. A varied collection of geometric modeling techniques are integrated in this streaming framework, including polygonal, spline, solid and heterogeneous modeling with boundary and decompositive representations, Boolean set operations, Cartesian products and adaptive refinement. The real-time B-rep to BSP streaming results we report in this paper are a large step forward in the ultimate unification of rapid conceptual and detailed shape design methodologies.

Análisis de la relación comercial entre la República de Colombia y la República de Costa Rica

OpenAIRE

Romero Escobar, Juán Sebastián; Bermudez Monroy, María Gabriela

2012-01-01

Dadas las relaciones de cooperación internacional que presentan las Repúblicas de Colombia y Costa Rica, que se evidencia en los fuertes vínculos comerciales existentes entre las dos naciones, Proexport por medio de su oficina comercial en Costa Rica, ha mantenido al tanto la situación en la que se desenvuelve la alianza comercial entre Colombia y Costa Rica. Él último de estos informes, demuestra la potencialidad de Costa Rica como un mercado para los bienes colombianos y una posibilidad par...

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

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.

Ultra Fast, High Rep Rate, High Voltage Spark Gap Pulser

Science.gov (United States)

1995-07-01

current rise time. The spark gap was designed to have a coaxial geometry reducing its inductance. Provisions were made to pass flowing gas between the...ULTRA FAST, HIGH REP RATE, HIGH VOLTAGE SPARK GAP PULSER Robert A. Pastore Jr., Lawrence E. Kingsley, Kevin Fonda, Erik Lenzing Electrophysics and...Modeling Branch AMSRL-PS-EA Tel.: (908)-532-0271 FAX: (908)-542-3348 U.S. Army Research Laboratory Physical Sciences Directorate Ft. Monmouth

Variation in a surface-exposed region of the Mycoplasma pneumoniae P40 protein as a consequence of homologous DNA recombination between RepMP5 elements.

Science.gov (United States)

Spuesens, Emiel B M; van de Kreeke, Nick; Estevão, Silvia; Hoogenboezem, Theo; Sluijter, Marcel; Hartwig, Nico G; van Rossum, Annemarie M C; Vink, Cornelis

2011-02-01

Mycoplasma pneumoniae is a human pathogen that causes a range of respiratory tract infections. The first step in infection is adherence of the bacteria to the respiratory epithelium. This step is mediated by a specialized organelle, which contains several proteins (cytadhesins) that have an important function in adherence. Two of these cytadhesins, P40 and P90, represent the proteolytic products from a single 130 kDa protein precursor, which is encoded by the MPN142 gene. Interestingly, MPN142 contains a repetitive DNA element, termed RepMP5, of which homologues are found at seven other loci within the M. pneumoniae genome. It has been hypothesized that these RepMP5 elements, which are similar but not identical in sequence, recombine with their counterpart within MPN142 and thereby provide a source of sequence variation for this gene. As this variation may give rise to amino acid changes within P40 and P90, the recombination between RepMP5 elements may constitute the basis of antigenic variation and, possibly, immune evasion by M. pneumoniae. To investigate the sequence variation of MPN142 in relation to inter-RepMP5 recombination, we determined the sequences of all RepMP5 elements in a collection of 25 strains. The results indicate that: (i) inter-RepMP5 recombination events have occurred in seven of the strains, and (ii) putative RepMP5 recombination events involving MPN142 have induced amino acid changes in a surface-exposed part of the P40 protein in two of the strains. We conclude that recombination between RepMP5 elements is a common phenomenon that may lead to sequence variation of MPN142-encoded proteins.

How Often Is p[subscript rep] Close to the True Replication Probability?

Science.gov (United States)

Trafimow, David; MacDonald, Justin A.; Rice, Stephen; Clason, Dennis L.

2010-01-01

Largely due to dissatisfaction with the standard null hypothesis significance testing procedure, researchers have begun to consider alternatives. For example, Killeen (2005a) has argued that researchers should calculate p[subscript rep] that is purported to indicate the probability that, if the experiment in question were replicated, the obtained…

Human Ubc9 is involved in intracellular HIV-1 Env stability after trafficking out of the trans-Golgi network in a Gag dependent manner.

Directory of Open Access Journals (Sweden)

Christopher R Bohl

Full Text Available The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER and its trafficking to the trans-Golgi network (TGN were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.

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

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.

Birmingham Rep, Youth and Community, and the Products and Possibilities of Precarity

Science.gov (United States)

Cochrane, Claire

2017-01-01

Birmingham Rep, a leading producing theatre based in the UK's "second city", has historically had a complex relationship with the cultural priorities of its home city. In recent years, Birmingham City Council has faced multiple challenges represented by debt burden, government-imposed cuts in public funding, scandals linked to failing…

Eukaryotic translation initiation factor 3 subunit e controls intracellular calcium homeostasis by regulation of cav1.2 surface expression.

Directory of Open Access Journals (Sweden)

Pawel Buda

Full Text Available Inappropriate surface expression of voltage-gated Ca(2+channels (CaV in pancreatic ß-cells may contribute to the development of type 2 diabetes. First, failure to increase intracellular Ca(2+ concentrations at the sites of exocytosis impedes insulin release. Furthermore, excessive Ca(2+ influx may trigger cytotoxic effects. The regulation of surface expression of CaV channels in the pancreatic β-cells remains unknown. Here, we used real-time 3D confocal and TIRFM imaging, immunocytochemistry, cellular fractionation, immunoprecipitation and electrophysiology to study trafficking of L-type CaV1.2 channels upon β-cell stimulation. We found decreased surface expression of CaV1.2 and a corresponding reduction in L-type whole-cell Ca(2+ currents in insulin-secreting INS-1 832/13 cells upon protracted (15-30 min stimulation. This internalization occurs by clathrin-dependent endocytosis and could be prevented by microtubule or dynamin inhibitors. eIF3e (Eukaryotic translation initiation factor 3 subunit E is part of the protein translation initiation complex, but its effect on translation are modest and effects in ion channel trafficking have been suggested. The factor interacted with CaV1.2 and regulated CaV1.2 traffic bidirectionally. eIF3e silencing impaired CaV1.2 internalization, which resulted in an increased intracellular Ca(2+ load upon stimulation. These findings provide a mechanism for regulation of L-type CaV channel surface expression with consequences for β-cell calcium homeostasis, which will affect pancreatic β-cell function and insulin production.

Intracellular S1P Generation Is Essential for S1P-Induced Motility of Human Lung Endothelial Cells: Role of Sphingosine Kinase 1 and S1P Lyase

Science.gov (United States)

Berdyshev, Evgeny V.; Gorshkova, Irina; Usatyuk, Peter; Kalari, Satish; Zhao, Yutong; Pyne, Nigel J.; Pyne, Susan; Sabbadini, Roger A.; Garcia, Joe G. N.; Natarajan, Viswanathan

2011-01-01

Background Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P1 receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility. Methodology/Principal Findings Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1Pint, and attenuated S1Pext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1Pint and potentiated motility of HPAECs to S1Pext or serum. S1Pext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1Pext or 4-deoxypyridoxine-dependent endothelial cell motility. Conclusions/Significance These results suggest S1Pext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL. PMID:21304987

Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase.

Directory of Open Access Journals (Sweden)

Evgeny V Berdyshev

Full Text Available BACKGROUND: Earlier we have shown that extracellular sphingosine-1-phosphate (S1P induces migration of human pulmonary artery endothelial cells (HPAECs through the activation of S1P(1 receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs and S1P lyase (S1PL, that regulate intracellular S1P accumulation, in HPAEC motility. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino-4-(p-Chlorophenyl Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1P(int, and attenuated S1P(ext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P(int and potentiated motility of HPAECs to S1P(ext or serum. S1P(ext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting "inside-out" signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs. Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1P(ext or 4-deoxypyridoxine-dependent endothelial cell motility. CONCLUSIONS/SIGNIFICANCE: These results suggest S1P(ext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.

Development of an in vitro photosafety evaluation method utilizing intracellular ROS production in THP-1 cells.

Science.gov (United States)

Toyoda, Akemi; Itagaki, Hiroshi

2018-01-01

Photoreactive compounds that may experience exposure to ultraviolet (UV) radiation can lead to the intracellular production of reactive oxygen species (ROS), which may cause phototoxic and photoallergenic responses. Here, we developed a novel in vitro photosafety assay and investigated whether it could be used to predict phototoxicity and photosensitivity by measuring changes in intracellular ROS production. THP-1 cells that had previously taken up 5-(and-6)-carboxy-2',7'-difluorodihydrofluorescein diacetate (carboxy-H 2 DFFDA), a ROS-sensitive fluorescent reagent, were exposed to photoreactive substances such as phototoxic and photoallergenic materials and then subjected to with UV-A irradiation (5 J/cm 2 ). The fluorescence intensity was subsequently measured using a flow cytometer, and the intracellular ROS production was calculated. A statistically significant increase in ROS following treatment with photoreactive substances was observed in cells irradiated with UV-A. In contrast, no significant increase was observed for non-photoreactive substances in comparison to the control solution. Next, to confirm the impact of intracellular ROS on the photosensitive response, changes in CD86 and CD54 expression were measured following quencher addition during the photo human cell line activation test (photo h-CLAT). The results confirmed the reduction of CD86 and CD54 expression in response to photoallergenic substances following quencher addition. Together, these findings suggest that intracellular ROS production is involved in photosensitizing reactions. Therefore, we suggest that the developed method utilizing intracellular ROS production as an index may be useful as a novel in vitro evaluation tool for photoreactive substances.

Control conception for REP 1300 MW units of the Electricite de France

International Nuclear Information System (INIS)

Blanc, P.; Guesnier, G.

1986-01-01

The paper describes the control equipment for the REP 1300 MW units including the reactor protection system, the control rods drive system and nuclear instrumentation, the control of the auxiliaries in the systems important for the reactor safety and the safety survey computer. Finally, the digital connections between different control systems are presented

Leptospirosis porcina: prevalencia serológica en establecimientos productores de la República Argentina

OpenAIRE

Petrakovsky M, Jessica; Tinao, Julio; Esteves M, Jorge

2013-01-01

RESUMENObjetivo. Detectar la presencia de anticuerpos contra Leptospira sp en cerdos que no presentaban sintomatología clínica, pertenecientes a establecimientos de producción porcina ubicados en todo el territorio de la República Argentina. Materiales y métodos. La asignación de predios y muestras se realizó en forma proporcional a los registrados en cada provincia a lo largo de todo el país. En total se estudiaron 1.524 predios, de los cuales se tomaron 19.872 muestras de suero y en la prim...

SEATURTLE: Sustained Engagement Autonomous Tracking of Underwater RepTiLEs

OpenAIRE

Budd, Johnathan

2015-01-01

While oceans cover the majority of our planet, these vast expanses remain relatively unexplored. Among the most interesting parts of the ocean are the shallow reef systems, which contain a huge amount of the planet’s biodiversity. The Sustained Engagement Autonomous Tracking of Underwater RepTiLEs or SEATURTLE is a low cost Autonomous Underwater Vehicle designed to carry out missions in these shallow environments. Its small displacement and precise movement make it ideal for navigating tight ...

Transepithelial SCFA fluxes link intracellular and extracellular pH regulation of mouse colonocytes.

Science.gov (United States)

Chu, S; Montrose, M H

1997-10-01

We have studied pH regulation in both intracellular and extracellular compartments of mouse colonic crypts, using distal colonic mucosa with intact epithelial architecture. In this work, we question how transepithelial SCFA gradients affect intracellular pH (pHi) and examine interactions between extracellular pH (pHo) and pHi regulation in crypts of distal colonic epithelium from mouse. We studied pH regulation in three adjacent compartments of distal colonic epithelium (crypt lumen, crypt epithelial cell cytosol, and lamina propria) with SNARF-1 (a pH sensitive fluorescent dye), digital imaging microscopy (for pHi), and confocal microscopy (for pHo). Combining results from the three compartments allows us to find how pHi and pHo are regulated and related under the influence of physiological transepithelial SCFA gradients, and develop a better understanding of pH regulation mechanisms in colonic crypts. Results suggest a complex interdependency between SCFA fluxes and pHo values, which can directly affect how strongly SCFAs acidify colonocytes.

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

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.

Structural characterization and antioxidant property of released exopolysaccharides from Lactobacillus delbrueckii ssp. bulgaricus SRFM-1.

Science.gov (United States)

Tang, Weizhi; Dong, Mingsheng; Wang, Weilu; Han, Shuo; Rui, Xin; Chen, Xiaohong; Jiang, Mei; Zhang, Qiuqin; Wu, Junjun; Li, Wei

2017-10-01

Three released exopolysaccharide fractions (r-EPS1, r-EPS2 and r-EPS3) were isolated from the fermented milk of Lactobacillus delbrueckii ssp. bulgaricus SRFM-1 and purified by anion exchange chromatography, and characterizations of the structures were conducted. The r-EPS1 and r-EPS2 were homogenous with the average molecular weights of 3.97×10 5 Da and 3.86×10 5 Da, respectively. Three r-EPS fractions were composed of galactose and glucose with a molar ratio of 1.23: 1.00, 1.33: 1.00 and 1.00: 1.34, respectively. Structural characterization indicated that the r-EPS1 contained a backbone of →6-β-d-Galp-(1→4)-β-d-Glcp-(1→4)-α-d-Galp-(1→4)-β-d-Galp-(1→6)-β-d-Galp-(1→4)-β-d-Glcp-(1→4)-α-d-Galp-(1→4)-β-d-Galp-(1→4)-α-d-Glcp-(1→, and had three branching points which existed in terminal with D-Glcp residues with α/β-d-(1→6) linkages. The r-EPS2 was composed of →6-β-d-Galp-(1→4)-β-d-Glcp-(1→6)-α-d-Galp-(1→ as the backbone chain with a branching point which also existed in terminal D-Glcp residue with β-(1→6) linkage. In addition, three r-EPS fractions exhibited strong scavenging activities on superoxide radical, hydroxyl radical, DPPH radical and chelating activity on ferrous ion, and their antioxidant activities decreased in the order of r-EPS1>r-EPS2>r-EPS3. Copyright © 2017 Elsevier Ltd. All rights reserved.

Overview of RepLab 2014: Author Profiling and Reputation Dimensions for Online Reputation Management

NARCIS (Netherlands)

Amigó, E.; Carrillo-de-Albornoz, J.; Chugur, I.; Corujo, A.; Gonzalo, J.; Meij, E.; de Rijke, M.; Spina, D.; Kanoulas, E.; Lupu, M.; Clough, P.; Sanderson, M.; Hall, M.; Hanbury, A.; Toms, E.

2014-01-01

This paper describes the organisation and results of RepLab 2014, the third competitive evaluation campaign for Online Reputation Management systems. This year the focus lied on two new tasks: reputation dimensions classification and author profiling, which complement the aspects of reputation

Identification of a Paralog-Specific Notch1 Intracellular Domain Degron

Directory of Open Access Journals (Sweden)

Matthew R. Broadus

2016-05-01

Full Text Available Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD, which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box. We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box enhances Notch1 activity in cultured human cells and zebrafish embryos. Human cancer mutations within the N1-Box enhance Notch1 signaling in transgenic zebrafish, highlighting the physiological relevance of this destruction signal. We find that binding of the Notch nuclear factor, CSL, to the N1-Box blocks NICD1 turnover. Our studies reveal a mechanism by which degradation of NICD1 is regulated by the N1-Box to minimize stochastic flux and to establish a threshold for Notch1 pathway activation.

Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

International Nuclear Information System (INIS)

Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

2012-01-01

Highlights: ► Adeno-associated virus (AAV) is capable of targeted integration in human cells. ► Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. ► A targeted integration system of IDRV DNA using the AAV integration mechanism. ► Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

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...

Automated Production of High Rep Rate Foam Targets

Science.gov (United States)

Hall, F.; Spindloe, C.; Haddock, D.; Tolley, M.; Nazarov, W.

2016-04-01

Manufacturing low density targets in the numbers needed for high rep rate experiments is highly challenging. This report summarises advances from manual production to semiautomated and the improvements that follow both in terms of production time and target uniformity. The production process is described and shown to be improved by the integration of an xyz robot with dispensing capabilities. Results are obtained from manual and semiautomated production runs and compared. The variance in the foam thickness is reduced significantly which should decrease experimental variation due to target parameters and could allow for whole batches to be characterised by the measurement of a few samples. The work applies to both foil backed and free standing foam targets.

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-01-15

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

RepExplore: addressing technical replicate variance in proteomics and metabolomics data analysis.

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Glaab, Enrico; Schneider, Reinhard

2015-07-01

High-throughput omics datasets often contain technical replicates included to account for technical sources of noise in the measurement process. Although summarizing these replicate measurements by using robust averages may help to reduce the influence of noise on downstream data analysis, the information on the variance across the replicate measurements is lost in the averaging process and therefore typically disregarded in subsequent statistical analyses.We introduce RepExplore, a web-service dedicated to exploit the information captured in the technical replicate variance to provide more reliable and informative differential expression and abundance statistics for omics datasets. The software builds on previously published statistical methods, which have been applied successfully to biomedical omics data but are difficult to use without prior experience in programming or scripting. RepExplore facilitates the analysis by providing a fully automated data processing and interactive ranking tables, whisker plot, heat map and principal component analysis visualizations to interpret omics data and derived statistics. Freely available at http://www.repexplore.tk enrico.glaab@uni.lu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

Glucocorticoids can affect Pseudomonas aeruginosa (ATCC 27853) internalization and intracellular calcium concentration in cystic fibrosis bronchial epithelial cells.

Science.gov (United States)

Hussain, Rashida; Shahror, Rami; Karpati, Ferenc; Roomans, Godfried M

2015-01-01

Glucocorticoids (GCs) are anti-inflammatory agents, but their use in cystic fibrosis (CF) is controversial. In CF, the early colonization with Pseudomonas aeruginosa is mainly due to nonmucoid strains that can internalize, and induce apoptosis in the epithelial cells. Uptake of P. aeruginosa by the epithelial cells and subsequent apoptosis may prevent colonization of P. aeruginosa in CF airways. In the airway epithelia, several other biological effects, including an anti-secretory role by decreasing intracellular Ca(2+) concentration have been described for this anti-inflammatory drug. However, the effects of GCs on the nonmucoid P. aeruginosa internalization and intracellular Ca(2+) in CF bronchial epithelial cells have not been evaluated. We used cultured human CF bronchial airway epithelial cell (CFBE) monolayers to determine P. aeruginosa internalization, apoptosis, and intracellular Ca(2+)concentration in CF bronchial epithelial cells. Cells were treated with IL-6, IL-8, dexamethasone, betamethasone, or budesonide. GCs in co-treatments with IL-6 reversed the effect of IL-6 by decreasing the internalization of P. aeruginosa in the CFBE cells. GCs decreased the extent of apoptosis in CFBE cells infected with internalized P. aeruginosa, and increased the intracellular Ca(2+) concentration. These findings suggest that if internalization of P. aeruginosa reduces infection, GC therapy would increase the risk of pulmonary infection by decreasing the internalization of P. aeruginosa in CF cells, but GCs may improve airway hydration by increasing the intracellular Ca(2+) concentration. Whether the benefits of GC treatment outweigh the negative effects is questionable, and further clinical studies need to be carried out.

A method and software for segmentation of anatomic object ensembles by deformable m-reps

International Nuclear Information System (INIS)

Pizer, Stephen M.; Fletcher, P. Thomas; Joshi, Sarang; Gash, A. Graham; Stough, Joshua; Thall, Andrew; Tracton, Gregg; Chaney, Edward L.

2005-01-01

Deformable shape models (DSMs) comprise a general approach that shows great promise for automatic image segmentation. Published studies by others and our own research results strongly suggest that segmentation of a normal or near-normal object from 3D medical images will be most successful when the DSM approach uses (1) knowledge of the geometry of not only the target anatomic object but also the ensemble of objects providing context for the target object and (2) knowledge of the image intensities to be expected relative to the geometry of the target and contextual objects. The segmentation will be most efficient when the deformation operates at multiple object-related scales and uses deformations that include not just local translations but the biologically important transformations of bending and twisting, i.e., local rotation, and local magnification. In computer vision an important class of DSM methods uses explicit geometric models in a Bayesian statistical framework to provide a priori information used in posterior optimization to match the DSM against a target image. In this approach a DSM of the object to be segmented is placed in the target image data and undergoes a series of rigid and nonrigid transformations that deform the model to closely match the target object. The deformation process is driven by optimizing an objective function that has terms for the geometric typicality and model-to-image match for each instance of the deformed model. The success of this approach depends strongly on the object representation, i.e., the structural details and parameter set for the DSM, which in turn determines the analytic form of the objective function. This paper describes a form of DSM called m-reps that has or allows these properties, and a method of segmentation consisting of large to small scale posterior optimization of m-reps. Segmentation by deformable m-reps, together with the appropriate data representations, visualizations, and user interface, has been

Engineered bacterial hydrophobic oligopeptide repeats in a synthetic yeast prion, [REP-PSI+

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Fátima eGasset-Rosa

2015-04-01

Full Text Available The yeast translation termination factor Sup35p, by aggregating as the [PSI+] prion, enables ribosomes to read-through stop codons, thus expanding the diversity of the Saccharomyces cerevisiae proteome. Yeast prions are functional amyloids that replicate by templating their conformation on native protein molecules, then assembling as large aggregates and fibers. Prions propagate epigenetically from mother to daughter cells by fragmentation of such assemblies. In the N-terminal prion-forming domain, Sup35p has glutamine/asparagine-rich oligopeptide repeats (OPRs, which enable propagation through chaperone-elicited shearing. We have engineered chimeras by replacing the polar OPRs in Sup35p by up to five repeats of a hydrophobic amyloidogenic sequence from the synthetic bacterial prionoid RepA-WH1. The resulting hybrid, [REP-PSI+], i was functional in a stop codon read-through assay in S. cerevisiae; ii generates weak phenotypic variants upon both its expression or transformation into [psi-] cells; iii these variants correlated with high molecular weight aggregates resistant to SDS during electrophoresis; and iv according to fluorescence microscopy, the fusion of the prion domains from the engineered chimeras to the reporter protein mCherry generated perivacuolar aggregate foci in yeast cells. All these are signatures of bona fide yeast prions. As assessed through biophysical approaches, the chimeras assembled as oligomers rather than as the fibers characteristic of [PSI+]. These results suggest that it is the balance between polar and hydrophobic residues in OPRs what determines prion conformational dynamics. In addition, our findings illustrate the feasibility of enabling new propagation traits in yeast prions by engineering OPRs with heterologous amyloidogenic sequence repeats.

Intracellular Localization of Arabidopsis Sulfurtransferases1

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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

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

Use of a rep-PCR system to predict species in the Aspergillus section Nigri

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The Aspergillus niger aggregate within the A. section Nigri, is a group of black-spored aspergilli which taxonomy has been elusive. REP-PCR has become a rapid and cost-effective method for genotyping fungi and bacteria. In the present study, we evaluated the discriminatory power of a semi-automate...

Non-recessive Bt toxin resistance conferred by an intracellular cadherin mutation in field-selected populations of cotton bollworm.

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Haonan Zhang

Full Text Available Transgenic crops producing Bacillus thuringiensis (Bt toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.

Hydroxyhydroquinone, a by-product of coffee bean roasting, increases intracellular Ca2+ concentration in rat thymic lymphocytes.

Science.gov (United States)

Kamae, Risa; Nojima, Shoko; Akiyoshi, Kenji; Setsu, Shoki; Honda, Sari; Masuda, Toshiya; Oyama, Yasuo

2017-04-01

Hydroxyhydroquinone (HHQ) is generated during coffee bean roasting. A cup of coffee contains 0.1-1.7 mg of HHQ. The actions of HHQ on mammalian DNA were examined because HHQ is a metabolite of benzene, which causes leukemia. Currently, information on the cellular actions of HHQ is limited. We examined the effects of sublethal levels of HHQ on the concentration of intracellular Ca 2+ in rat thymic lymphocytes by using a flow cytometric technique with fluorescent probes. HHQ at 10 μM or more significantly elevated intracellular Ca 2+ levels by increasing the membrane permeability of divalent cations, resulting in hyperpolarization via the activation of Ca 2+ -dependent K + channels. HHQ-induced changes in the intracellular Ca 2+ concentration and membrane potential may affect the cell functions of lymphocytes. HHQ-reduced coffee may be preferable in order to avoid the possible adverse effects of HHQ. Copyright © 2017 Elsevier Ltd. All rights reserved.

Utilisation of Rep-PCR to track microbes in aerosols collected adjacent to their source, a saline lake in Victoria, Australia.

Science.gov (United States)

Munday, Chris I; O'Loingsigh, Tadhg; Tapper, Nigel J; De Deckker, Patrick; Allison, Gwen E

2013-04-15

Dust storms are a major source of aerosolized bacteria, especially in the drought conditions experienced in Australia in the decade to 2009. The major aims of this project were to identify the culturable bacteria in environmental samples and to genetically fingerprint all isolates using repetitive element PCR (Rep-PCR) to investigate the possibility of tracking isolates from their source into the atmosphere. Four field trips were conducted to a dry lake in western Victoria, Australia to sample aerosols and sediments. Aerosols were collected at heights up to 150 m using vacuum pumps with filters attached to a tethered helium balloon, while corresponding sediments were collected in sterile polypropylene tubes. Isolates were cultivated on Tryptic Soy Agar, R2 Agar and Marine Agar, and grown in dark conditions at ambient temperature. By sequencing the 16S rRNA gene of 270 isolates, fifteen different bacterial families were identified, with both the aerosols and sediments dominated by the Bacillaceae family. Four sets of Rep-PCR primers were tested, with the ERIC and (GTG)5 primers proving to be the most suitable for fingerprinting the cultured taxa. Rep-PCR revealed very high strain diversity in the samples collected, however some strains were still able to be tracked from sediments up to 150 m in height. This shows the potential of Rep-PCR, however very large reference databases would be required for the technique to be more useful. Copyright © 2013 Elsevier B.V. All rights reserved.

The effect of intracellular trafficking of CD1d on the formation of TCR repertoire of NKT cells.

Science.gov (United States)

Shin, Jung Hoon; Park, Se-Ho

2014-05-01

CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to αβ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of αβ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant Vα14-Jα18 TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire.

Tris-hydroxymethyl-aminomethane enhances capsaicin-induced intracellular Ca2+ influx through transient receptor potential V1 (TRPV1 channels

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Satoshi Murakami

2016-02-01

Full Text Available Non-selective transient receptor potential vanilloid (TRPV cation channels are activated by various insults, including exposure to heat, acidity, and the compound capsaicin, resulting in sensations of pain in the skin, visceral organs, and oral cavity. Recently, TRPV1 activation was also demonstrated in response to basic pH elicited by ammonia and intracellular alkalization. Tris-hydroxymethyl aminomethane (THAM is widely used as an alkalizing agent; however, the effects of THAM on TRPV1 channels have not been defined. In this study, we characterized the effects of THAM-induced TRPV1 channel activation in baby hamster kidney cells expressing human TRPV1 (hTRPV1 and the Ca2+-sensitive fluorescent sensor GCaMP2 by real-time confocal microscopy. Notably, both capsaicin (1 μM and pH 6.5 buffer elicited steep increases in the intracellular Ca2+ concentration ([Ca2+]i, while treatment with THAM (pH 8.5 alone had no effect. However, treatment with THAM (pH 8.5 following capsaicin application elicited a profound, long-lasting increase in [Ca2+]i that was completely inhibited by the TRPV1 antagonist capsazepine. Taken together, these results suggest that hTRPV1 pre-activation is required to provoke enhanced, THAM-induced [Ca2+]i increases, which could be a mechanism underlying pain induced by basic pH.

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

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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.

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

Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency

International Nuclear Information System (INIS)

Lan, Ke; Murakami, Masanao; Choudhuri, Tathagata; Kuppers, Daniel A.; Robertson, Erle S.

2006-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a predominantly latent infection in the infected host. Importantly, during latency, only a small number of viral encoded genes are expressed. This viral gene expression pattern contributes to the establishment of long-term infection as well as the ability of the virus to evade the immune system. Previous studies have been shown that the replication and transcription activator (RTA) encoded by ORF50 activates it downstream genes and initiates viral lytic reactivation through functional interaction with RBP-Jκ, the major downstream effector of the Notch signaling pathway. This indicates that RTA can usurp the conserved Notch signaling pathway and mimic the activities of intracellular Notch1 to modulate gene expression. In this report, we show that the activated intracellular domain of Notch1 (ICN) is aberrantly accumulated in KSHV latently infected pleural effusion lymphoma (PEL) cells. ICN activated the RTA promoter in a dose-dependent manner, and forced expression of ICN in latently infected KSHV-positive cells initiated full blown lytic replication with the production of infectious viral progeny. However, latency-associated nuclear antigen (LANA) which is predominantly expressed during latency can specifically down-modulate ICN-mediated transactivation of RTA and so control KSHV for lytic reactivation. These results demonstrate that LANA can inhibit viral lytic replication by antagonizing ICN function and suggest that LANA is a critical component of the regulatory control mechanism for switching between viral latent and lytic replication by directly interacting with effectors of the conserved cellular Notch1 pathway

Cold exposure affects carbohydrates and lipid metabolism, and induces Hog1p phosphorylation in Dekkera bruxellensis strain CBS 2499.

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Galafassi, Silvia; Toscano, Marco; Vigentini, Ileana; Zambelli, Paolo; Simonetti, Paolo; Foschino, Roberto; Compagno, Concetta

2015-05-01

Dekkera bruxellensis is a yeast known to affect the quality of wine and beer. This species, due to its high ethanol and acid tolerance, has been reported also to compete with Saccharomyces cerevisiae in distilleries producing fuel ethanol. In order to understand how this species responds when exposed to low temperatures, some mechanisms like synthesis and accumulation of intracellular metabolites, changes in lipid composition and activation of the HOG-MAPK pathway were investigated in the genome sequenced strain CBS 2499. We show that cold stress caused intracellular accumulation of glycogen, but did not induce accumulation of trehalose and glycerol. The cellular fatty acid composition changed after the temperature downshift, and a significant increase of palmitoleic acid was observed. RT-PCR analysis revealed that OLE1 encoding for Δ9-fatty acid desaturase was up-regulated, whereas TPS1 and INO1 didn't show changes in their expression. In D. bruxellensis Hog1p was activated by phosphorylation, as described in S. cerevisiae, highlighting a conserved role of the HOG-MAP kinase signaling pathway in cold stress response.

The Effect of Curcumin on Intracellular pH (pHi), Membrane Hyperpolarization and Sperm Motility.

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Naz, Rajesh K

2014-04-01

Curcumin has shown to affect sperm motility and function in vitro and fertility in vivo. The molecular mechanism(s) by which curcumin affects sperm motility has not been delineated. Since modulation of intracellular pH (pHi) and plasma membrane polarization is involved in sperm motility, the present study was conducted to investigate the effect of curcumin on these sperm (human and murine) parameters. The effect of curcumin on sperm forward motility was examined by counting percentages of forward moving sperm. The effect of curcumin on intracellular pH (pHi) was measured by the fluorescent pH indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein-acetoxymethyl ester (BCECF-AM). The effect of curcumin on plasma membrane polarization was examined using the fluorescence sensitive dye bis (1,3-dibarbituric acid)-trimethine oxanol [DiBAC4(3)]. Curcumin caused a concentration-dependent (ppHi) in both human and mouse sperm. Curcumin induced significant (ppHi and membrane polarization that affect sperm forward motility. These exciting findings will have application in deciphering the signal transduction pathway involved in sperm motility and function and in development of a novel non-steroidal contraceptive for infertility.

Rep-PCR typing of Staphylococcus spp. strains in meat paste production line and identification of their origin

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Ivan Manga

2015-05-01

Full Text Available A meat paste production line and its microbial parameters have been evaluated in single Czech company. The raw meat paste samples before heat treatment were tested positively for the presence of three staphylococci species: Staphylococcus aureus, Staphylococcus haemolyticus and Staphylococcus epidermidis. Subsequent microbial analysis of meat paste components and ingredients (fresh meat, water, spices, equipment identified only the spices used as positive for S. aureus (coriander, cinnamon, badian, mustard – (10 - 40 cfu/g and S. haemolyticus strains (juniper, ginger. The collection of sixteen collected strains (S. aureus (n = 4, S. haemolyticus (n = 4, S. epidermidis (n = 8 has been typed with the rep-PCR method utilising (GTG5 primer. Analysis of the fingerprints using the unweighted pair-group method using arithmetic averages (UPGMA clustering method revealed presence of eleven strain clusters with similarity lower than 90%: two fingerprint clusters of S. aureus, three individual clusters characteristic for S. haemolyticus and six different S. epidermidis specific clusters. The S. aureus strains from different types of spice were identical, resp. very similar. Molecular tracking composed from the rep-PCR analysis of acquired isolates and comparison among all collected fingerprints confirmed the spices to be the source of both S. aureus and S. haemolyticus strains identified in raw meat paste. The additional rep-PCR analysis of the S. epidermidis collection confirmed usability and performance of this method. The antibiotic susceptibility to fourteen individual antibiotics has been examined among the collected staphylococci strains. The predominant erythromycin resistance (68.8% was followed with the resistance to amoxicillin/clavulanic acid (56.2%. Other resistances observed were less frequent (clindamycin – 12.5%, oxacillin – 6.3%, tetracycline – 6.3%, sulphamethoxazole-trimethoprim – 6.3%, chloramphenicol – 6.3%, novobiocin – 6

Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

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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

MetaRep, an extended CMAS 3D program to visualize mafic (CMAS, ACF-S, ACF-N) and pelitic (AFM-K, AFM-S, AKF-S) projections

Science.gov (United States)

France, Lydéric; Nicollet, Christian

2010-06-01

MetaRep is a program based on our earlier program CMAS 3D. It is developed in MATLAB ® script. MetaRep objectives are to visualize and project major element compositions of mafic and pelitic rocks and their minerals in the pseudo-quaternary projections of the ACF-S, ACF-N, CMAS, AFM-K, AFM-S and AKF-S systems. These six systems are commonly used to describe metamorphic mineral assemblages and magmatic evolutions. Each system, made of four apices, can be represented in a tetrahedron that can be visualized in three dimensions with MetaRep; the four tetrahedron apices represent oxides or combination of oxides that define the composition of the projected rock or mineral. The three-dimensional representation allows one to obtain a better understanding of the topology of the relationships between the rocks and minerals and relations. From these systems, MetaRep can also project data in ternary plots (for example, the ACF, AFM and AKF ternary projections can be generated). A functional interface makes it easy to use and does not require any knowledge of MATLAB ® programming. To facilitate the use, MetaRep loads, from the main interface, data compiled in a Microsoft Excel ™ spreadsheet. Although useful for scientific research, the program is also a powerful tool for teaching. We propose an application example that, by using two combined systems (ACF-S and ACF-N), provides strong confirmation in the petrological interpretation.

Calcium influx affects intracellular transport and membrane repair following nanosecond pulsed electric field exposure.

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Thompson, Gary Lee; Roth, Caleb C; Dalzell, Danielle R; Kuipers, Marjorie; Ibey, Bennett L

2014-05-01

The cellular response to subtle membrane damage following exposure to nanosecond pulsed electric fields (nsPEF) is not well understood. Recent work has shown that when cells are exposed to nsPEF, ion permeable nanopores (2  nm) created by longer micro- and millisecond duration pulses. Nanoporation of the plasma membrane by nsPEF has been shown to cause a transient increase in intracellular calcium concentration within milliseconds after exposure. Our research objective is to determine the impact of nsPEF on calcium-dependent structural and repair systems in mammalian cells. Chinese hamster ovary (CHO-K1) cells were exposed in the presence and absence of calcium ions in the outside buffer to either 1 or 20, 600-ns duration electrical pulses at 16.2  kV/cm, and pore size was determined using propidium iodide and calcium green. Membrane organization was observed with morphological changes and increases in FM1-43 fluorescence. Migration of lysosomes, implicated in membrane repair, was followed using confocal microscopy of red fluorescent protein-tagged LAMP1. Microtubule structure was imaged using mEmerald-tubulin. We found that at high 600-ns PEF dosage, calcium-induced membrane restructuring and microtubule depolymerization coincide with interruption of membrane repair via lysosomal exocytosis.

Impacto de la inmigración haitiana sobre el mercado laboral y las finanzas públicas de la República Dominicana

OpenAIRE

Aristy-Escuder, Jaime

2010-01-01

La pobreza del pueblo haitiano influye de forma determinante sobre los flujos migratorios hacia la República Dominicana. Este artículo se propone estimar el impacto de la inmigración haitiana sobre el mercado laboral dominicano, así como sobre sus finanzas públicas. Luego de un exhaustivo análisis teórico y aplicado, el artículo concluye que la mano de obra haitiana es complementaria al capital y a la mano de obra calificada de la República Dominicana. En cambio, es sustituta d...

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.

Intracellular crowding effects on the self-association of the bacterial cell division protein FtsZ.

Science.gov (United States)

Naddaf, Lamis; Sayyed-Ahmad, Abdallah

2014-12-15

The dimerization rate of the bacterial cell division protein FtsZ is strongly affected by the intracellular crowding. Yet the complexity of the intracellular environment makes it difficult to investigate via all-atom molecular dynamics or other detailed theoretical methods. We study the crowding effect on FtsZ dimerization which is the first step of an oligomerization process that results in more elaborate supramolecular structures. In particular, we consider the effect of intracellular crowding on the reaction rates, and their dependence on the different concentrations of crowding agents. We achieved this goal by using Brownian dynamics (BD) simulation techniques and a modified post-processing approach in which we decompose the rate constant in crowded media as a product of the rate constant in the dilute solution times a factor that incorporates the crowding effect. The latter factor accounts for the diffusion reduction and crowder induced energy. In addition we include the crowding effects on water viscosity in the BD simulations of crowded media. We finally show that biomolecular crowding has a considerable effect on the FtsZ dimerization by increasing the dimerization rate constant from 2.6×10(7)M(-1)s(-1) in the absence of crowders to 1.0×10(8)M(-1)s(-1) at crowding level of 0.30. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

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Dawei Yu

Full Text Available Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α (without secretory signal sequence gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT. Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9% became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS, which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

International Nuclear Information System (INIS)

Arai, Roberto J.; Ogata, Fernando T.; Batista, Wagner L.; Masutani, Hiroshi; Yodoi, Junji; Debbas, Victor; Augusto, Ohara; Stern, Arnold; Monteiro, Hugo P.

2008-01-01

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases

Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

Science.gov (United States)

Tu, Yu-Hsuan; Ho, Yu-Hsuan; Chuang, Ying-Chih; Chen, Po-Chung; Chen, Chien-Sheng

2011-01-01

Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

Directory of Open Access Journals (Sweden)

Yu-Hsuan Tu

Full Text Available Lactoferricin B (LfcinB is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP assays. Sixteen proteins were identified, and an E. coli interaction database (EcID analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

La República de los historiadores del Paraguay posbélico

OpenAIRE

Telesca, Ignacio

2015-01-01

En ocasión del bicentenario de la adopción de la República como sistema de gobierno, e independiente tanto de España como de Buenos Aires, este artículo reflexiona sobre como los historiadores paraguayos de fines de siglo XIX reflexionaron sobre el tema, relacionándolo con la adopción de un nuevo sistema de gobierno con la constitución de 1870 (calco de la argentina). Fil: Telesca, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET ...

Internalisation of the protease-activated receptor 1: role of the third intracellular loop and of the cytoplasmic tail.

Science.gov (United States)

Chen, X; Berrou, J; Vigneau, C; Delarue, F; Rondeau, E

2001-06-01

To analyse the mechanisms of PAR-1 internalisation, we constructed several PAR-1 mutants and stably expressed them in CHO cells. Our study shows that the Ser(306)-->Ala mutation (S306A), which eliminates a potential site of phosphorylation by PKC in the third intracellular loop of PAR-1, did not change the rate of phosphorylation but reduced the rate of thrombin-induced internalisation of the PAR-1 mutant (58 versus 78% of membrane PAR-1 in 15 min, pinternalisation upon activation. This deletion also inhibited the PMA-induced and the agonist-independent internalisation of the receptor. The Tyr(371)--> Ala mutation (Y371A), in a NPXXY motif of the seventh transmembrane domain of the receptor had no effect on the receptor behaviour. Our results indicate that both the C-tail and the third intracellular loop are involved in PAR-1 internalisation induced by thrombin while only the C-tail plays a role in the PMA-induced and in the agonist-independent PAR-1 internalisation.

Protección contra la violencia sexual en la República Democrática del Congo

OpenAIRE

Mosely, Sarah; Cetinoglu, Talita; Glad, Marit

2010-01-01

Si bien la prestación de servicios a los supervivientes de la violencia sexual es la principal prioridad en la República Democrática del Congo, la protección para las mujeres y niñas también puede mejorarse.

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

75 FR 78694 - Proposed Residential Exchange Program Settlement Agreement Proceeding (REP-12); Public Hearing...

Science.gov (United States)

2010-12-16

..., 2010, on BPA's Web site at http://www.bpa.gov/corporate/ratecase/2012/rep-12.cfm . Hard copies of these.... Bonneville Power Admin., 928 F.2d 905 (9th Cir. 1991). Due to the burdensome, expensive and contentious... Electric v. Bonneville Power Admin., 501 F.3d 1009 (9th Cir. 2007) (PGE), the Court found that BPA had...

El Museo de Arte del Banco de la República

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Boletín Cultural y Bibliográfico Banco de la República

2003-05-01

Full Text Available El Banco de la República desde sus comienzos, en los años veinte y treinta del siglo pasado, ha apoyado la cultura. De esas épocas datan los inicios de las colecciones de numismática y orfebrería, creadas con la intención de preservar y defender el patrimonio artístico y cultural colombiano. A ese atesoramiento se une el desarrollo de otras colecciones que son referencia obligada para el conocimiento de la historia de Colombia: la colección documental (bibliográfica, hemerográfica, audiovisual, la de arte, a partir de 1957 y la de filatelia.

Liberalismo, iluminismo y romanticismo: el problema de la libertad en dos novelas de la República Restaurada

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Verónica Hernández Landa Valencia

2016-07-01

Full Text Available El artículo propone una reflexión en torno a la forma como influyen tres corrientes de pensamiento, el liberalismo, el iluminismo y el romanticismo, en la representación del problema de la libertad en dos novelas históricas publicadas en la República Restaurada: Monja y casada, virgen y mártir, de Vicente Riva Palacio, y El pecado del siglo, de José Tomás de Cuéllar. Se busca mostrar que dichas corrientes se entrecruzan en un solo texto, que ello contribuye a la conformación de un concepto de libertad restringida y que la forma específica que adquiere ese concepto se explica a partir de la postura ideológica y política de los autores en el contexto de la República Restaurada.

Comparison of semi-automated commercial rep-PCR fingerprinting, spoligotyping, 12-locus MIRU-VNTR typing and single nucleotide polymorphism analysis of the embB gene as molecular typing tools for Mycobacterium bovis.

Science.gov (United States)

Armas, Federica; Camperio, Cristina; Coltella, Luana; Selvaggini, Serena; Boniotti, Maria Beatrice; Pacciarini, Maria Lodovica; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia

2017-08-04

Highly discriminatory genotyping strategies are essential in molecular epidemiological studies of tuberculosis. In this study we evaluated, for the first time, the efficacy of the repetitive sequence-based PCR (rep-PCR) DiversiLab Mycobacterium typing kit over spoligotyping, 12-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and embB single nucleotide polymorphism (SNP) analysis for Mycobacterium bovis typing. A total of 49 M. bovis animal isolates were used. DNA was extracted and genomic DNA was amplified using the DiversiLab Mycobacterium typing kit. The amplified fragments were separated and detected using a microfluidics chip with Agilent 2100. The resulting rep-PCR-based DNA fingerprints were uploaded to and analysed using web-based DiversiLab software through Pearson's correlation coefficient. Rep-PCR DiversiLab grouped M. bovis isolates into ten different clusters. Most isolates sharing identical spoligotype, MIRU-VNTR profile or embB gene polymorphism were grouped into different rep-PCR clusters. Rep-PCR DiversiLab displayed greater discriminatory power than spoligotyping and embB SNP analysis but a lower resolution power than the 12-locus MIRU-VNTR analysis. MIRU-VNTR confirmed that it is superior to the other PCR-based methods tested here. In combination with spoligotyping and 12-locus MIRU-VNTR analysis, rep-PCR improved the discriminatory power for M. bovis typing.

31P MR spectroscopic measurement of intracellular pH in normal human hearts

International Nuclear Information System (INIS)

Kwon, Jae Hyun; Lee, Hui Joong; Jang, Yong Min

2002-01-01

To assess the usefulness of intracellular pH (pHi), calculated by determining the shift of a high-energy metabolite such as inorganic phosphate (Pi) of γ-ATP after performing MRS with ECG-gated two-dimensional 31 P CSI (chemical shift imaging), as a parameter for the overall state of the intracellular milieu. Proto decoupled 31 P CSI was performed on a 1.5-T scanner using a 1 H 31 P dual-tuned surface coil. Cardiac MRS data were obtained from eight normal volunteers aged 24-32 years with no history of heart disease. From the spectra obtained from several regions of the heart, peack position and peak area were estimated. The metabolic ratios of α-, β-, γ-ATP, PCr, Pi, phosphodiester and diphosphoglycerate were calculated, and pHi was estimated from the chemical shift of Pi and γ-ATP resonance. We then compared the data for the anterior myocardium with those previously published. The major phosphorous metabolites identified in these human hearts were as follows: PCr, at -0.1 to +0.1 ppm; three phosphate peaks from ATP, with a chemical shift centered at about -2.7 ppm (γ-ATP), -7.8 ppm (α-ATP), and -16.3 ppm (β-ATP); and phosphodiester (PDE) at 2-3 ppm, inorganic phosphate (Pi) at 4.5-5.4 ppm, and diphosphoglycerate (DPG) at 5.4-6.3 ppm. The PCr/β-ATP ratio was 2.20±0.17 and the PDE/β-ATP ratio, 1.04±0.09 pHi readings were 7.31±0.23 (calculated by the shift of Pi) and 6.81±0.20 (calculated by the shift of γ-ATP). Pi/PCR was 0.539, a ratio higher than that mentioned in previously published reports. The measurement of intracellular metabolism was affected by various kinds of factors. We believe, however, that pHi readings indicate the overall state of the cardiac intracellular milieu. An unexpected pHi readings, seen at MRS, may reflect errors in the MR procedure itself and, or in the analytical method

Topology reconstruction for B-Rep modeling from 3D mesh in reverse engineering applications

Science.gov (United States)

Bénière, Roseline; Subsol, Gérard; Gesquière, Gilles; Le Breton, François; Puech, William

2012-03-01

Nowadays, most of the manufactured objects are designed using CAD (Computer-Aided Design) software. Nevertheless, for visualization, data exchange or manufacturing applications, the geometric model has to be discretized into a 3D mesh composed of a finite number of vertices and edges. But, in some cases, the initial model may be lost or unavailable. In other cases, the 3D discrete representation may be modified, for example after a numerical simulation, and does not correspond anymore to the initial model. A reverse engineering method is then required to reconstruct a 3D continuous representation from the discrete one. In previous work, we have presented a new approach for 3D geometric primitive extraction. In this paper, to complete our automatic and comprehensive reverse engineering process, we propose a method to construct the topology of the retrieved object. To reconstruct a B-Rep model, a new formalism is now introduced to define the adjacency relations. Then a new process is used to construct the boundaries of the object. The whole process is tested on 3D industrial meshes and bring a solution to recover B-Rep models.

Insulin-Like Growth Factor I (IGF-1) Deficiency Ameliorates Sex Difference in Cardiac Contractile Function and Intracellular Ca2+ Homeostasis

Science.gov (United States)

Ceylan-Isik, Asli F.; Li, Qun; Ren, Jun

2011-01-01

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (± dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), fura-fluorescence intensity (FFI) and intracellular Ca2+ clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ± dL/dt, longer TPS, TR90 and intracellular Ca2+ clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na+-Ca2+ exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca2+ regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca2+ regulation. PMID:21763763

Insulin-like growth factor I (IGF-1) deficiency ameliorates sex difference in cardiac contractile function and intracellular Ca(2+) homeostasis.

Science.gov (United States)

Ceylan-Isik, Asli F; Li, Qun; Ren, Jun

2011-10-10

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (±dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), fura-fluorescence intensity (FFI) and intracellular Ca(2+) clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ±dL/dt, longer TPS, TR(90) and intracellular Ca(2+) clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na(+)-Ca(2+) exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca(2+) regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca(2+) regulation. Copyright © 2011 Elsevier Ireland

Detection of ubiquitinated huntingtin species in intracellular aggregates

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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.

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.

Nano hydroxyapatite-blasted titanium surface affects pre-osteoblast morphology by modulating critical intracellular pathways.

Science.gov (United States)

Bezerra, Fábio; Ferreira, Marcel R; Fontes, Giselle N; da Costa Fernandes, Célio Jr; Andia, Denise C; Cruz, Nilson C; da Silva, Rodrigo A; Zambuzzi, Willian F

2017-08-01

Although, intracellular signaling pathways are proposed to predict the quality of cell-surface relationship, this study addressed pre-osteoblast behavior in response to nano hydroxyapatite (HA)-blasted titanium (Ti) surface by exploring critical intracellular pathways and pre-osteoblast morphological change. Physicochemical properties were evaluated by atomic force microscopy (AFM) and wettability considering water contact angle of three differently texturized Ti surfaces: Machined (Mac), Dual acid-etching (DAE), and nano hydroxyapatite-blasted (nHA). The results revealed critical differences in surface topography, impacting the water contact angle and later the osteoblast performance. In order to evaluate the effect of those topographical characteristics on biological responses, we have seeded pre-osteoblast cells on the Ti discs for up to 4 h and subjected the cultures to biological analysis. First, we have observed pre-osteoblasts morphological changes resulting from the interaction with the Ti texturized surfaces whereas the cells cultured on nHA presented a more advanced spreading process when compared with the cells cultured on the other surfaces. These results argued us for analyzing the molecular machinery and thus, we have shown that nHA promoted a lower Bax/Bcl2 ratio, suggesting an interesting anti-apoptotic effect, maybe explained by the fact that HA is a natural element present in bone composition. Thereafter, we investigated the potential effect of those surfaces on promoting pre-osteoblast adhesion and survival signaling by performing crystal violet and immunoblotting approaches, respectively. Our results showed that nHA promoted a higher pre-osteoblast adhesion supported by up-modulating FAK and Src activations, both signaling transducers involved during eukaryotic cell adhesion. Also, we have shown Ras-Erk stimulation by the all evaluated surfaces. Finally, we showed that all Ti-texturing surfaces were able to promote osteoblast differentiation

Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial

NARCIS (Netherlands)

Maclaren, R.E.; Groppe, M.; Barnard, A.R.; Cottriall, C.L.; Tolmachova, T.; Seymour, L.; Clark, K.; During, M.J.; Cremers, F.P.M.; Black, G.C.M.; Lotery, A.J.; Downes, S.M.; Webster, A.R.; Seabra, M.C.

2014-01-01

BACKGROUND: Choroideremia is an X-linked recessive disease that leads to blindness due to mutations in the CHM gene, which encodes the Rab escort protein 1 (REP1). We assessed the effects of retinal gene therapy with an adeno-associated viral (AAV) vector encoding REP1 (AAV.REP1) in patients with

Repérer des sites similaires à une recherche par Google

OpenAIRE

Ginouvès, Véronique

2006-01-01

Repérer des sites similaires à une recherche par Google Afficher graphiquement les liens d’un site avec d’autres sites similaires TouchGraph GoogleBrowser crée des agrégations de sites à partir de l’interrogation de Google. Vous visualisez ainsi des nébuleuses de sites à l’intérieur desquelles vous pouvez naviguer facilement, en sautant d'un groupe à l'autre afin de voir les nuages autour de sujets ou de communautés de sujets. Cet outil a été développé par Alex Shapiro, à partir de la version...

Effect of curing ingredients and vacuum packaging on the physico-chemical and storage quality of ready-to-eat Vawksa rep (smoked pork product) during refrigerated storage.

Science.gov (United States)

Deuri, Deepshikha; Hazarika, Pragati; Singh, Tarun Pal; Chhangte, Lalchamliani; Singh, Parminder; Talukder, Suman

2016-06-01

The present study was conducted for the development of ready-to-eat Vawksa rep (smoked pork product) and to study the synergistic effect of curing ingredients and vacuum packaging on the physico-chemical and storage quality during refrigerated storage at (4°C±1°C) for 15 days. Four different batches of Vawksa rep samples were prepared, i.e., T-1 (uncured, first cooked at 121°C for 15 min, and then smoked at 120°C for 30 min), T-2 (uncured, cooked, and smoked simultaneously at 120°C for 45 min), T-3 (cured, first cooked at 121°C for 15 min, and then smoked at 120°C for 30 min), and T-4 (cured, cooked, and smoked simultaneously at 120°C for 45 min). Cooking yield was significantly higher (pproduct) could be prepared easily with little technology up-gradation and with a negligible escalation of production cost.

Summary Record of the First Meeting of the Radioactive Waste Repository Metadata Management (RepMet) Initiative

International Nuclear Information System (INIS)

2014-01-01

National radioactive waste repository programmes are collecting large amounts of data to support the long-term management of their nations' radioactive wastes. The data and related records increase in number, type and quality as programmes proceed through the successive stages of repository development: pre-siting, siting, characterisation, construction, operation and finally closure. Regulatory and societal approvals are included in this sequence. Some programmes are also documenting past repository projects and facing a challenge in allowing both current and future generations to understand actions carried out in the past. Metadata allows context to be stored with data and information so that it can be located, used, updated and maintained. Metadata helps waste management organisations better utilise their data in carrying out their statutory tasks and can also help verify and demonstrate that their programmes are appropriately driven. The NEA Radioactive Waste Repository Metadata Management (RepMet) initiative aims to bring about a better understanding of the identification and administration of metadata - a key aspect of data management - to support national programmes in managing their radioactive waste repository data, information and records in a way that is both harmonised internationally and suitable for long-term management and use. This is a summary record of the 1. meeting of the RepMet initiative. The actions and decisions from this meeting were sent separately to the group after the meeting, but are also included in this document (Annex A). The list of participants is attached as well (Annex B)

Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

International Nuclear Information System (INIS)

Majumdar, S.; Basu, S.K.

1991-01-01

p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections

Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

DEFF Research Database (Denmark)

Aakerlund, L; Gether, U; Fuhlendorff, J

1990-01-01

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused...

Pacific ciguatoxin-1b effect over Na+ and K+ currents, inositol 1,4,5-triphosphate content and intracellular Ca2+ signals in cultured rat myotubes

Science.gov (United States)

Hidalgo, Jorge; Liberona, José Luis; Molgó, Jordi; Jaimovich, Enrique

2002-01-01

The action of the main ciguatoxin involved in ciguatera fish poisoning in the Pacific region (P-CTX-1b) was studied in myotubes originated from rat skeletal muscle cells kept in primary culture. The effect of P-CTX-1b on sodium currents at short times of exposure (up to 1 min) showed a moderate increase in peak Na+ current. During prolonged exposures, P-CTX-1b decreased the peak Na+ current. This action was always accompanied by an increase of leakage currents, tail currents and outward Na+ currents, resulting in an intracellular Na+ accumulation. This effect is blocked by prior exposure to tetrodotoxin (TTX) and becomes evident only after washout of TTX. Low to moderate concentrations of P-CTX-1b (2–5 nM) partially blocked potassium currents in a manner that was dependent on the membrane potential. P-CTX-1b (2–12 nM) caused a small membrane depolarization (3–5 mV) and an increase in the frequency of spontaneous action potential discharges that reached in general low frequencies (0.1–0.5 Hz). P-CTX-1b (10 nM) caused a transient increase of intracellular inositol 1,4,5-trisphosphate (IP3) mass levels, which was blocked by TTX. In the presence of P-CTX-1b (10 nM) and in the absence of external Ca2+, the intracellular Ca2+ levels show a transient increase in the cytoplasm as well as in the nuclei. The time course of this effect may reflect the action of IP3 over internal stores activated by P-CTX-1b-induced membrane depolarization. PMID:12429578

Repše: välislaenuabi katkedes on aasta lõpuks raha läbi / Mikk Salu

Index Scriptorium Estoniae

Salu, Mikk, 1975-

2009-01-01

Läti rahandusminister Einars Repše hoiatas, et kui Lätil ei õnnestu täita oma kohustusi rahvusvaheliste kreeditoride ees ja vähendada 2010. a. eelarvedefitsiiti poole miljardi lati võrra, võib riigikassa rahast tühjaks joosta juba enne aasta lõppu

{sup 31}P MR spectroscopic measurement of intracellular pH in normal human hearts

Energy Technology Data Exchange (ETDEWEB)

Kwon, Jae Hyun; Lee, Hui Joong; Jang, Yong Min [Kyungpook National Univ., Taegu (Korea, Republic of)] [and others

2002-05-01

To assess the usefulness of intracellular pH (pHi), calculated by determining the shift of a high-energy metabolite such as inorganic phosphate (Pi) of {gamma}-ATP after performing MRS with ECG-gated two-dimensional {sup 31}P CSI (chemical shift imaging), as a parameter for the overall state of the intracellular milieu. Proto decoupled {sup 31}P CSI was performed on a 1.5-T scanner using a {sup 1}H{sup 31}P dual-tuned surface coil. Cardiac MRS data were obtained from eight normal volunteers aged 24-32 years with no history of heart disease. From the spectra obtained from several regions of the heart, peack position and peak area were estimated. The metabolic ratios of {alpha}-, {beta}-, {gamma}-ATP, PCr, Pi, phosphodiester and diphosphoglycerate were calculated, and pHi was estimated from the chemical shift of Pi and {gamma}-ATP resonance. We then compared the data for the anterior myocardium with those previously published. The major phosphorous metabolites identified in these human hearts were as follows: PCr, at -0.1 to +0.1 ppm; three phosphate peaks from ATP, with a chemical shift centered at about -2.7 ppm ({gamma}-ATP), -7.8 ppm ({alpha}-ATP), and -16.3 ppm ({beta}-ATP); and phosphodiester (PDE) at 2-3 ppm, inorganic phosphate (Pi) at 4.5-5.4 ppm, and diphosphoglycerate (DPG) at 5.4-6.3 ppm. The PCr/{beta}-ATP ratio was 2.20{+-}0.17 and the PDE/{beta}-ATP ratio, 1.04{+-}0.09 pHi readings were 7.31{+-}0.23 (calculated by the shift of Pi) and 6.81{+-}0.20 (calculated by the shift of {gamma}-ATP). Pi/PCR was 0.539, a ratio higher than that mentioned in previously published reports. The measurement of intracellular metabolism was affected by various kinds of factors. We believe, however, that pHi readings indicate the overall state of the cardiac intracellular milieu. An unexpected pHi readings, seen at MRS, may reflect errors in the MR procedure itself and, or in the analytical method.

Effect of curing ingredients and vacuum packaging on the physicochemical and storage quality of ready-to-eat Vawksa rep (smoked pork product during refrigerated storage

Directory of Open Access Journals (Sweden)

Deepshikha Deuri

2016-06-01

Full Text Available Aim: The present study was conducted for the development of ready-to-eat Vawksa rep (smoked pork product and to study the synergistic effect of curing ingredients and vacuum packaging on the physico-chemical and storage quality during refrigerated storage at (4°C±1°C for 15 days. Materials and Methods: Four different batches of Vawksa rep samples were prepared, i.e., T-1 (uncured, first cooked at 121°C for 15 min, and then smoked at 120°C for 30 min, T-2 (uncured, cooked, and smoked simultaneously at 120°C for 45 min, T-3 (cured, first cooked at 121°C for 15 min, and then smoked at 120°C for 30 min, and T-4 (cured, cooked, and smoked simultaneously at 120°C for 45 min. Results: Cooking yield was significantly higher (p<0.05 for the T-4. The pH of T-3 and T-4 samples was significantly higher (p<0.05 on day 15. The tyrosine value of all the samples increased significantly (p<0.05 among the different days of analysis. Thiobarbituric acid value was significantly (p<0.05 lower in T-3 sample both at the beginning and at the end of storage period. In microbiological profile, total plate count was lower in T-3 and T-4 than T-1 and T-2. However, Escherichia coli count was negative for T-3 and T-4 samples throughout the storage period. Among sensory attributes, T-3 and T-4 samples registered superior scores for color, flavor, texture, juiciness, and overall acceptability. Conclusion: Furthermore, Vawksa rep (smoked pork product could be prepared easily with little technology up-gradation and with a negligible escalation of production cost.

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+.

Hacia una república conservadora : el programa político del Partido Agrario Español

Directory of Open Access Journals (Sweden)

Luis Teófilo Gil Cuadrado

2005-01-01

Full Text Available El Partido Agrario Español (PAE, fundado en enero de 1934 y presidido por José Martínez de Velasco, representó, dentro de la II República española, una alternativa conservadora y, al tiempo, leal al régimen. El programa político del PAE aceptaba la República —a diferencia de otros partidos derechistas del momento, como la CEDA y los monárquicos— y defendía la unidad nacional, el establecimiento de una segunda Cámara legislativa, la protección de la agricultura y la industria mediante aranceles, el libre comercio dentro de las fronteras nacionales y la revisión de algunas leyes del primer bienio como la Reforma Agraria de 1932. El objetivo era la configuración de una República conservadora distinta, en aspectos fundamentales, a la diseñada por la Constitución de 1931 y la legislación del bienio social-republicano.The Spanish Agrarian Party (Partido Agrario Español, PAE, founded in january of 1934 and presided by José Martínez de Velasco, was, in the political life of the Spanish Second Republic, a conservative and republican option. The agrarian program accepted the republican regime —in opposition to other conservative parties, like the CEDA or the monarchists— and defended the national union, the establishment of a Senate, the protection of the national agriculture and industry trough tariffs, the free trade in the Spanish market and the revision of some laws voted in the first biennium like the Agrarian Reform, promulgated in 1932. The idea was to establish a conservative Republic, different, in some fundamental aspects, of the regime designed by the Constitution of 1931 and the legislation approved during the socialist-republican biennium.

Tight Coupling of Metabolic Oscillations and Intracellular Water Dynamics in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Thoke, Henrik Seir; Tobiesen, Asger; Brewer, Jonathan R.

2015-01-01

We detected very strong coupling between the oscillating concentration of ATP and the dynamics of intracellular water during glycolysis in Saccharomyces cerevisiae. Our results indicate that: i) dipolar relaxation of intracellular water is heterogeneous within the cell and different from dilute...... conditions, ii) water dipolar relaxation oscillates with glycolysis and in phase with ATP concentration, iii) this phenomenon is scale-invariant from the subcellular to the ensemble of synchronized cells and, iv) the periodicity of both glycolytic oscillations and dipolar relaxation are equally affected by D...

¿Por qué no la Segunda República Argentina?

Directory of Open Access Journals (Sweden)

Manuel ALCÁNTARA SÁEZ

2009-11-01

Full Text Available RESUMEN: El autor analiza en este artículo el proceso de Reforma Constitucional que se está desarrollando en Argentina. Teniendo en cuenta la coyuntura actual del régimen político, el desarollo de este proceso de reforma y los cambios previstos que van a resultar de esta «Refundación de la República» el autor apunta las posibilidades y problemas que estos datos permiten vislumbrar.ABSTRACT: In this article, the author analyzes the present process of Constitutional Reform in Argentina. Studying the current moment of argentinian political regime, the development of this reform process and possible changes of this «Republic Refoundation» the author points out the possibilities and problems that this information indicates.

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.

Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

Science.gov (United States)

Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

2012-04-15

Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

La Segunda República, una coyuntura para las mujeres españolas: Cambios y permanencias en las relaciones de género.

OpenAIRE

Merino Hernández, Rosa María

2016-01-01

[ES] En España, no será hasta el período de la Segunda República (1931-1939), cuando las mujeres conseguirán el reconocimiento de ciudadanas de pleno derecho en igualdad con los hombres, respaldadas por la Constitución de 1931. La Segunda República española da voz y derechos a más de la otra mitad de la población del país, por lo que la convierte en una época única, en una etapa nueva donde convivirán el cambio y la permanencia, el avance hacia una nueva sociedad y, dentro de ésta, la emer...

Firmino Costa, un intelectual de la República y sus propósitos educativos (1907-1937

Directory of Open Access Journals (Sweden)

Juliana Cesário Hamdan

2014-01-01

Full Text Available El presente artículo pretende analizar la actu - ación del educador Firmino Costa, nacido en Minas Gerais, al retomar sus antecedentes his - tóricos que se localizan en la base de las formas de su actuación política e intelectual. Para este análisis, recurrimos al trabajo de Alonso (2008, 1 Oliveira (1999 2 y Carvalho (2003, 3 considera - mos las expresivas contribuciones para su com - prensión en relación con la historia e ideas que circularon en este periodo, comprendido entre los años que antecedieron a las primeras déca - das de la Proclamación de la República en Brasil (I889. Las tres obras, pese a que se escribieron a partir de objetos y objetivos distintos, refuta - ron interpretaciones que, por un lado, tratan el escenario intelectual de la República por medio de tipologías de intelectuales, que estarían ab - sorbiendo pasivamente las teorías europeas y norteamericanas. Y, por otro lado, las que son marcadas por estudios que enfatizan las acciones individuales, empeñadas en llevar adelante sus banderas ideológicas. Al contrario, los autores entienden que los sujetos están inmersos en de - terminado entorno cultural, constituido por las redes de sociabilidad y por el repertorio de ideas en circulación y, por eso mismo, tienen formas distintas de actuación política e intelectual. Y ese escenario condiciona, en gran medida, las posi - bilidades de actuación. Así, este artículo busca establecer conexiones entre algunos elementos del escenario cultural, presentes en los años que antecedieron y en los que sucedieron a la República, relacionándolos a las formas de actuación política e intelectual que informan la acción educacional del educador minero. Ese movimiento analítico, por su vez, exigió que recorriésemos a algunas de las formu - laciones teóricas de Antonio Gramsci, en lo que se refiere a los sentidos de actuación de los inte - lectuales, como forma de comprenderlos a partir del contexto y de las

Influence of intracellular acidosis on contractile function in the working rat heart

International Nuclear Information System (INIS)

Jeffrey, F.M.H.; Malloy, C.R.; Radda, G.K.

1987-01-01

The decrease in myocardial contractility during ischemia, hypoxia, and extracellular acidosis has been attributed to intracellular acidosis. Previous studies of the relationship between pH and contractile state have utilized respiratory or metabolic acidosis to alter intracellular pH. The authors developed a model in the working perfused rat heart to study the effects of intracellular acidosis with normal external pH and optimal O 2 delivery. Intracellular pH and high-energy phosphates were monitored by 31 P nuclear magnetic resonance spectroscopy. Hearts were perfused to a steady state with a medium containing 10 mM NH 4 Cl. Acidosis induced a substantial decrease in aortic flow and stroke volume which was associated with little change in peak systolic pressure. It was concluded that (1) for the same intracellular acidosis the influence on tension development was more pronounced with a combined extra- and intracellular acidosis than with an isolated intracellular acidosis, and (2) stroke volume at constant preload was impaired by intracellular acidosis even though changes in developed pressure were minimal. These observations suggest that isolated intracellular acidosis has adverse effects on diastolic compliance and/or relaxation

In vivo assessment of intracellular redox state in rat liver using hyperpolarized [1-13 C]Alanine.

Science.gov (United States)

Park, Jae Mo; Khemtong, Chalermchai; Liu, Shie-Chau; Hurd, Ralph E; Spielman, Daniel M

2017-05-01

The intracellular lactate to pyruvate concentration ratio is a commonly used tissue assay biomarker of redox, being proportional to free cytosolic [NADH]/[NAD + ]. In this study, we assessed the use of hyperpolarized [1- 13 C]alanine and the subsequent detection of the intracellular products of [1- 13 C]pyruvate and [1- 13 C]lactate as a useful substrate for assessing redox levels in the liver in vivo. Animal experiments were conducted to measure in vivo metabolism at baseline and after ethanol infusion. A solution of 80-mM hyperpolarized [1- 13 C]alanine was injected intravenously at baseline (n = 8) and 45 min after ethanol infusion (n = 4), immediately followed by the dynamic acquisition of 13 C MRS spectra. In vivo rat liver spectra showed peaks from [1- 13 C] alanine and the products of [1- 13 C]lactate, [1- 13 C]pyruvate, and 13 C-bicarbonate. A significantly increased 13 C-lactate/ 13 C-pyruvate ratio was observed after ethanol infusion (8.46 ± 0.58 at baseline versus 13.58 ± 0.69 after ethanol infusion; P alanine is presented, with the validity of the proposed 13 C-pyruvate/ 13 C-lactate metric tested using an ethanol challenge to alter liver redox state. Magn Reson Med 77:1741-1748, 2017. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Legislative Districts, Four layers - One State Assembly district, one State Senate district, one US House of Rep district, and one US Senate district showing the locations in the County of Polk, WI., Published in 2007, 1:24000 (1in=2000ft) scale, Polk County Government.

Data.gov (United States)

NSGIC Local Govt | GIS Inventory — Legislative Districts dataset current as of 2007. Four layers - One State Assembly district, one State Senate district, one US House of Rep district, and one US...

Imaging Intracellular pH in Live Cells with a Genetically-Encoded Red Fluorescent Protein Sensor

OpenAIRE

Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

2011-01-01

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically-encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at...

Reactive Energetic Plasticizers Utilizing Cu-Free Azide-Alkyne 1,3-Dipolar Cycloaddition for In-Situ Preparation of Poly(THF-co-GAP-Based Polyurethane Energetic Binders

Directory of Open Access Journals (Sweden)

Mingyang Ma

2018-05-01

Full Text Available Reactive energetic plasticizers (REPs coupled with hydroxy-telechelic poly(glycidyl azide-co-tetrahydrofuran (PGT-based energetic polyurethane (PU binders for use in solid propellants and plastic-bonded explosives (PBXs were investigated. The generation of gem-dinitro REPs along with a terminal alkyne stemmed from a series of finely designed approaches to not only satisfy common demands as conventional energetic plasticizers, but also to prevent the migration of plasticizers. The miscibility and rheological behavior of a binary mixture of PGT/REP with various REP fractions were quantitatively determined by differential scanning calorimetry (DSC and rheometer, respectively, highlighting the promising performance of REPs in the formulation process. The kinetics on the distinct reactivity of propargyl vs. 3-butynyl species of REPs towards the azide group of the PGT prepolymer in terms of Cu-free azide-alkyne 1,3-dipolar cycloaddition (1,3-DPCA was studied by monitoring 1H nuclear magnetic resonance spectroscopy and analyzing the activation energies (Ea obtained using DSC. The thermal stability of the finally cured energetic binders with the incorporation of REPs indicated that the thermal stability of the REP/PGT-based PUs was maintained independently of the REP content. The tensile strength and modulus of the PUs increased with an increase in the REP content. In addition, the energetic performance and sensitivity of REP and REP triazole species was predicted.

Evolución y relevancia del turismo en la República Dominicana

OpenAIRE

Real Aquino, Héctor Julio

2014-01-01

República Dominicana es un país que durante las últimas décadas ha sufrido grandes transformaciones socioeconómicas, anteriormente su economía dependía casi exclusivamente de la elaboración y exportación de productos agrícolas como: la caña de azúcar, cacao, tabaco, y en menor medida, la industria textil. Ese panorama se ha modificado, debido en gran parte al turismo, dado que este subsector de la economía se ha convertido en la principal actividad económica del país. La evolución y relevanci...

Euclides da Cunha e a República

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Roberto Ventura

1996-04-01

Full Text Available A revisão da República é central na obra de Euclides da Cunha, presente em seus artigos de jornal e na maior parte de seus livros. Analisou o regime republicano não só em Os sertões (1902, em que tratou da guerra de Canudos, como em Contrastes e confrontos (1907 e em Á margem da história (1909. Foi testemunha e intérprete dos rumos do novo regime, que ajudara a fundar com artigos de propaganda política no jornal A Província de S. Paulo.The revision of the Republic is a central concern in the works of Euclides da Cunha. It appears in his newspaper articles and in most of his books. He analysed the republican regime not only in Os sertões (Rebellion in the backlands, 1902, in which he deals with the Canudos War, but also in Contrastes e confrontos (1907 and in À margem da história (1909. He both witnessed and interpreted the trends of the new regime which he had helped to set up with his political articles in the newspaper A Província de S. Paulo.

Intracellular long-chain acyl CoAs activate TRPV1 channels.

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Yi Yu

Full Text Available TRPV1 channels are an important class of membrane proteins that play an integral role in the regulation of intracellular cations such as calcium in many different tissue types. The anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 is a known positive modulator of TRPV1 channels and the negatively charged phosphate groups interact with several basic amino acid residues in the proximal C-terminal TRP domain of the TRPV1 channel. We and other groups have shown that physiological sub-micromolar levels of long-chain acyl CoAs (LC-CoAs, another ubiquitous anionic lipid, can also act as positive modulators of ion channels and exchangers. Therefore, we investigated whether TRPV1 channel activity is similarly regulated by LC-CoAs. Our results show that LC-CoAs are potent activators of the TRPV1 channel and interact with the same PIP2-binding residues in TRPV1. In contrast to PIP2, LC-CoA modulation of TRPV1 is independent of Ca2+i, acting in an acyl side-chain saturation and chain-length dependent manner. Elevation of LC-CoAs in intact Jurkat T-cells leads to significant increases in agonist-induced Ca2+i levels. Our novel findings indicate that LC-CoAs represent a new fundamental mechanism for regulation of TRPV1 channel activity that may play a role in diverse cell types under physiological and pathophysiological conditions that alter fatty acid transport and metabolism such as obesity and diabetes.

Realidades de la independencia del Banco de la República Realities of the independence of the Banco de la República

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Kalmanovitz Salomón

1997-12-01

Full Text Available La autonomía del banco central Colombiano es parte del proceso político que buscaba reducir el exagerado centralismo colombiano. Sin embargo el poder central no fue limitado en grado suficiente por la Constitución del 1991, como muestra el hecho de que el Ministro de Hacienda es el Presidente de la Junta Directiva del Banco de la República. Esto no sucede en ningun banco independiente del mundo por que los objetivos del gobierno son distintos a los del Banco Central y el Ministerio de Finanzas podría desviar al Banco de sus objetivos antiinflacionarios fundamentales. En Colombia, el gobierno no sólo participa en la Junta para acomodar sus múltiples objetivos sino también hace opocisión pública a la política monetaria y así la desacredita. El articulo plantea que el principio de no reelección hace que el horizonte de tiempo sea muy corto para que el gobierno coseche los frutos de una política de estabilización. El gobierno tiende a gastar en exceso para mejorar su imagen pública y el desajuste lo tiene que enfrentar el gobierno siguiente. De modo que la coordinación entre el Banco Central y el gobierno se torna muy conflictiva.The autonomy of the Central Bank of Colombia is part of a political process which seeks to reduce Colombia' s exaggerated centralismo However, central power was not limited to a sufficient degree by the Constitution of 1991, as demonstrated by the fact that the Minister of Finance is the President of the Board of Directors of the Banco de la República. This does not happen in any independent bank in the world, because the objectives of the government are different from those of the central bank, and the Minister of Finance could divert the Bank away from its fundamental anti-inflationary objectives. This article proposes that the principle of no reelection makes the time horizon very short for the government to reap the fruits of a stabilization policy. The government tends to spend in excess in order to

Analysis of a new strain of Euphorbia mosaic virus with distinct replication specificity unveils a lineage of begomoviruses with short Rep sequences in the DNA-B intergenic region

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Argüello-Astorga Gerardo R

2010-10-01

Full Text Available Abstract Background Euphorbia mosaic virus (EuMV is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep and virion-strand replication origin. The first entirely characterized EuMV isolate is native from Yucatan Peninsula, Mexico; subsequently, EuMV was detected in weeds and pepper plants from another region of Mexico, and partial DNA-A sequences revealed significant differences in their putative replication specificity determinants with respect to EuMV-YP. This study was aimed to investigate the replication compatibility between two EuMV isolates from the same country. Results A new isolate of EuMV was obtained from pepper plants collected at Jalisco, Mexico. Full-length clones of both genomic components of EuMV-Jal were biolistically inoculated into plants of three different species, which developed symptoms indistinguishable from those induced by EuMV-YP. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that these viruses do not form infectious reassortants in Nicotiana benthamiana, presumably because of Rep-iteron incompatibility. Sequence analysis of the EuMV-Jal DNA-B intergenic region (IR led to the unexpected discovery of a 35-nt-long sequence that is identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences ranging from 35- to 51-nt in length were identified in all EuMV isolates and in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a ~160-nt non-coding domain highly similar to the CP promoter of begomoviruses belonging to the SLCV clade. Conclusions EuMV strains are not compatible in replication, indicating that this begomovirus species probably is not a replicating lineage in nature. The genomic analysis of EuMV-Jal led to the discovery of a subgroup of SLCV clade viruses that contain in

Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells.

Science.gov (United States)

Vereb, G; Szöllösi, J; Mátyus, L; Balázs, M; Hyun, W C; Feuerstein, B G

1996-05-01

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.

T1 and T2 relaxivity of intracellular and extracellular USPIO at 1.5T and 3T clinical MR scanning

International Nuclear Information System (INIS)

Simon, Gerhard H.; Bauer, Jan; Saborovski, Olaf; Fu, Yanjun; Wendland, Michael F.; Daldrup-Link, Heike E.; Corot, Claire

2006-01-01

In this study we evaluated the effects of intracellular compartmentalization of the ultrasmall superparamagnetic iron oxide (USPIO) ferumoxtran-10 on its proton T1 and T2 relaxivities at 1.5 and 3T. Monocytes were labeled with ferumoxtran-10 by simple incubation. Decreasing quantities of ferumoxtran-10-labeled cells (2.5 x 10 7 -0.3 x 10 7 cells/ml) and decreasing concentrations of free ferumoxtran-10 (without cells) in Ficoll solution were evaluated with 1.5 and 3T clinical magnetic resonance (MR) scanners. Pulse sequences comprised axial spin echo (SE) sequences with multiple TRs and fixed TE and SE sequences with fixed TR and increasing TEs. Signal intensity measurements were used to calculate T1 and T2 relaxation times of all samples, assuming a monoexponential signal decay. The iron content in all samples was determined by inductively coupled plasma atomic emission spectrometry and used for calculating relaxivities. Measurements at 1.5T and 3T showed higher T1 and T2 relaxivity values of free extracellular ferumoxtran-10 as opposed to intracellularly compartmentalized ferumoxtran-10, under the evaluated conditions of homogeneously dispersed contrast agents/cells in Ficoll solution and a cell density of up to 2.5 x 10 7 cells/ml. At 3T, differences in T1-relaxivities between intra- and extracellular USPIO were smaller, while differences in USPIO T2-relaxivities were similar compared with 1.5T. In conclusion, cellular compartmentalization of ferumoxtran-10 changes proton relaxivity. (orig.)

Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane.

Science.gov (United States)

Goodchild, Sophia C; Howell, Michael W; Cordina, Nicole M; Littler, Dene R; Breit, Samuel N; Curmi, Paul M G; Brown, Louise Jennifer

2009-12-01

Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.

Characterization of Leptin Intracellular Trafficking

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E Walum

2009-12-01

Full Text Available Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBRl with an intracellular domain of 303 amino acids and a shorter form (OBRs with an intracellular domain of 34 amino acids. Since OBRl is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3- transferrin containing compartments at 37°C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF and the lysosomal marker protein lamp-1. The transport of leptin was also shown

repDNA: a Python package to generate various modes of feature vectors for DNA sequences by incorporating user-defined physicochemical properties and sequence-order effects.

Science.gov (United States)

Liu, Bin; Liu, Fule; Fang, Longyun; Wang, Xiaolong; Chou, Kuo-Chen

2015-04-15

In order to develop powerful computational predictors for identifying the biological features or attributes of DNAs, one of the most challenging problems is to find a suitable approach to effectively represent the DNA sequences. To facilitate the studies of DNAs and nucleotides, we developed a Python package called representations of DNAs (repDNA) for generating the widely used features reflecting the physicochemical properties and sequence-order effects of DNAs and nucleotides. There are three feature groups composed of 15 features. The first group calculates three nucleic acid composition features describing the local sequence information by means of kmers; the second group calculates six autocorrelation features describing the level of correlation between two oligonucleotides along a DNA sequence in terms of their specific physicochemical properties; the third group calculates six pseudo nucleotide composition features, which can be used to represent a DNA sequence with a discrete model or vector yet still keep considerable sequence-order information via the physicochemical properties of its constituent oligonucleotides. In addition, these features can be easily calculated based on both the built-in and user-defined properties via using repDNA. The repDNA Python package is freely accessible to the public at http://bioinformatics.hitsz.edu.cn/repDNA/. bliu@insun.hit.edu.cn or kcchou@gordonlifescience.org Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Mechanisms of cellular invasion by intracellular parasites.

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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.

La función administrativa ejercida por el Congreso de la República

OpenAIRE

Ortiz, Maribel

2017-01-01

La función administrativa en Colombia, no sólo es desarrollada por la Rama Ejecutiva del poder público, sino que también es ejercida por la Rama Legislativa para el cumplimiento de sus fines. Es a partir de la Constitución de 1991, cuando en desarrollo del mandato Constitucional y de las facultades que tiene el Congreso de la República, se expidieron dos normativas: la ley 3ª de 1992 (por la cual se expiden normas sobre las Comisiones del Congreso de Colombia y se dictan otras disposici...

La radio en la segunda república española: sociedad y publicidad

OpenAIRE

Sebastián Morillas, Sebastián

2006-01-01

El artículo versa sobre la evolución del medio radio durante la II República Española a la vez que analiza la publicidad radiofónica de la época y sus efectos en una sociedad a punto de dividirse en dos bandos enfrentados en armas. This article is about the evolution of the radio broadcast during the Second Spanish Republic. It also analyses the radio commercials in that time, and how this commercials had a great influence on Spanish society, which was close to a civil war.

Feature selection and data sampling methods for learning reputation dimensions: The University of Amsterdam at RepLab 2014

NARCIS (Netherlands)

Gârbacea, C.; Tsagkias, M.; de Rijke, M.

2014-01-01

We report on our participation in the reputation dimension task of the CLEF RepLab 2014 evaluation initiative, i.e., to classify social media updates into eight predefined categories. We address the task by using corpus-based methods to extract textual features from the labeled training data to

Complexation of intracellular cyanide by hydroxocobalamin using a human cellular model.

Science.gov (United States)

Astier, A; Baud, F J

1996-01-01

1. The rational for administering hydroxocobalamin (OHCbl) as an antidote to cyanide poisoning is based on the high affinity of CN ion for cobalt compounds. However, only few data are available on the influence of OHCbl on the intracellular cyanide pool. 2. In human fibroblasts incubated for 10 min with 500 microM of [14C] cyanide, the accumulation ratio was 25 at 37 degrees C (10.45 +/- 1.51 mM) and 11.9 at 4 degrees C. 3. Using the monoblastic U-937 cell line, a rapid uptake of radioactive cyanide was observed with a maximum accumulation ratio of 1.97 at 5 min. 4. A linear relationship between cyanide uptake by U-937 cells and cyanide concentration in incubation medium (10-500 microM; 5 min) was found suggesting a first order process (k = 0.25 min-1). 5. After incubation of fibroblasts with 500 microM of OHCbl, a 75% decrease of intracellular cyanide was observed, with concomittant formation of intracellular cyanocobalamin CNCbl (intracellular/extracellular ratio: 158). 6. These findings suggest that OHCbl is able to penetrate into heavily cyanide loaded cells and to complex cyanide to the non-toxic CNCbl form.

Intracellular Bacterial Infections: A Challenge for Developing Cellular Mediated Immunity Vaccines for Farmed Fish

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Hetron Mweemba Munang’andu

2018-04-01

Full Text Available Aquaculture is one of the most rapidly expanding farming systems in the world. Its rapid expansion has brought with it several pathogens infecting different fish species. As a result, there has been a corresponding expansion in vaccine development to cope with the increasing number of infectious diseases in aquaculture. The success of vaccine development for bacterial diseases in aquaculture is largely attributed to empirical vaccine designs based on inactivation of whole cell (WCI bacteria vaccines. However, an upcoming challenge in vaccine design is the increase of intracellular bacterial pathogens that are not responsive to WCI vaccines. Intracellular bacterial vaccines evoke cellular mediated immune (CMI responses that “kill” and eliminate infected cells, unlike WCI vaccines that induce humoral immune responses whose protective mechanism is neutralization of extracellular replicating pathogens by antibodies. In this synopsis, I provide an overview of the intracellular bacterial pathogens infecting different fish species in aquaculture, outlining their mechanisms of invasion, replication, and survival intracellularly based on existing data. I also bring into perspective the current state of CMI understanding in fish together with its potential application in vaccine development. Further, I highlight the immunological pitfalls that have derailed our ability to produce protective vaccines against intracellular pathogens for finfish. Overall, the synopsis put forth herein advocates for a shift in vaccine design to include CMI-based vaccines against intracellular pathogens currently adversely affecting the aquaculture industry.

La República Holandesa y su posición en el contexto colonial americano después de 1713

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Crespo Solana, Ana

2015-06-01

Full Text Available During the War of Spanish Succession, the Dutch Republic fought alongside the Austrians and their allies against the Bourbon party. Still, and in spite of its alliance with England, the treaties of Utrecht did not leave the Dutch Republic in a good position. In this article, these treaties’ consequences for the commercial and colonial Dutch expansion in the Atlantic world are analyzed.Durante la guerra de Sucesión española, la República Holandesa luchó junto al bloque aliado austracista contra la opción borbónica. Sin embargo, y a pesar de su alianza con Inglaterra, la República Holandesa no salió bien restablecida de los tratados de Utrecht. En este artículo se analizarán las consecuencias que estos tratados tuvieron para la expansión comercial y colonial de Holanda en el Atlántico.

Light irradiation helps magnetotactic bacteria eliminate intracellular reactive oxygen species.

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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.

Homonota fasciata Duméril y Bibron, 1839 (Reptilia, Squamata, Phyllodactylidae. Primer registro para la provincia de Corrientes (República Argentina

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Falcione, Camila

2011-05-01

Full Text Available Homonota fasciata Duméril & Bibron, 1839. República Argentina, Provincia de Corrientes, Departamento Bella Vista, Cuarta Sección, Colonia Progreso, Loma Sur, Toropí (28°35'55" S; 59°03'38" W (Figura 1. Colectores: Eduardo Etchepare, María del Rosario Ingaramo, Camila Falcione y Roberto Aguirre. 05 de abril de 2011. Se colectaron 4 ejemplares adultos (2 machos y 2 hembras y 1 juvenil, depositados en la Colección Herpetológica de la Universidad Nacional del Nordeste (UNNEC-11212, UNNEC-11213, UNNEC- 11214, UNNEC-11215 y UNNEC-11216.

Effects of nitro-heterocyclic derivatives against Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes.

Science.gov (United States)

Petri e Silva, Simone Carolina Soares; Palace-Berl, Fanny; Tavares, Leoberto Costa; Soares, Sandra Regina Castro; Lindoso, José Angelo Lauletta

2016-04-01

Leishmaniasis is an overlooked tropical disease affecting approximately 1 million people in several countries. Clinical manifestation depends on the interaction between Leishmania and the host's immune response. Currently available treatment options for leishmaniasis are limited and induce severe side effects. In this research, we tested nitro-heterocyclic compounds (BSF series) as a new alternative against Leishmania. Its activity was measured in Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes using MTT colorimetric assay. Additionally, we assessed the phosphatidylserine exposure by promastigotes, measured by flow cytometry, as well as nitric oxide production, measured by Griess' method. The nitro-heterocyclic compounds (BSF series) showed activity against L. (L.) infantum promastigotes, inducting the phosphatidylserine exposition by promastigotes, decreasing intracellular amastigotes and increasing oxide nitric production. The selectivity index was more prominent to Leishmania than to macrophages. Compared to amphotericin b, our compounds presented higher IC50, however the selectivity index was more specific to parasite than to amphotericin b. In conclusion, these nitro-heterocyclic compounds showed to be promising as an anti-Leishmania drug, in in vitro studies. Copyright © 2016 Elsevier Inc. All rights reserved.

República, nación y democracia ante el desafío de la diversidad Republic, nation and democracy against the challenge of diversity

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Susana Villavicencio

2010-12-01

Full Text Available La República no es sólo el nombre de una nación estado y su régimen. Es también un modelo nacional de integración basado en la concepción universalista de la ciudadanía que afirma la figura abstracta de la persona jurídica, portadora de iguales derechos y obligaciones, y valores de civismo y civilidad. Este trabajo analiza cómo la emergencia de la diversidad cultural en Argentina pone en cuestión los conceptos modernos de república, nación y democracia. Examina asimismo cómo los legados de la república, que supone una visión homogeneizada de la diversidad, implican una ambivalencia entre la adhesión a principios universalistas de progreso de la humanidad y la realidad de una democracia excluyente que valorizaba una cultura por sobre otras. Se estudian las narrativas de la identidad nacional, mostrando cómo la oposición civilización -barbarie afecta la visión de los desafíos del futuro de la democracia en América Latina.Republic is not only the name of a nation and its regime. It is also a national model of integration based on a universalist conception of citizenship that sustains the abstract figure of the legal person, endowed with equal rights and obligations, and values of civism and civility, the foundation stones of a society that arises from a contract. This paper analyses how the emergency of the cultural diversity in Argentina calls into question modern political concepts like republic, nation and democracy. It is also evaluated how the republic's inheritance, which supposes an homogenized sight of diversity, gives rise to an ambivalence among the adherence to universalist principles of the progress of humanity and the reality of an excluding democracy that gave value to one culture above others. The narratives of national identity are studied describing how the opposition between civilization and barbariy affects the way Latin Americans see the great challenges presented by the future of democracy, and by the

The Dual Half-Arc data structure: towards the universal B-rep data structure

DEFF Research Database (Denmark)

Anton, François; Bugoslawski, P.; Mioc, Darka

2014-01-01

, or more generally, the topology of any topological space. For this purpose, we generalize an existing data structure from 2D to 3D and design a new 3D data structure that realizes the synthesis between an existing 3D data structure (the Dual Half-Edge (See Footonote 1) data structure) and the generalized...... 3D Quad-Arc data structure, (See Footonote 2) and at the same time, improves the Dual Half-Edge towards a simpler and more effective representation of cell complexes through B-rep structures. We generalize the idea of the Quad-Arc data structure from 2D to 3D, but instead of transforming a simple...... edge of the Quad-Edge data structure to an arc with multiple points along it, we group together primal edges of the Dual Half-Edge that have the same dual Half-Edge vertex tags (volume tags) into one Dual Half-Arc whose dual is the common Dual Half-Edge and primal faces corresponding to dual...

La europeización de la República de Moldova: un camino sinuoso e incierto hacia la integración europea

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Aurelia Dercaci

2014-01-01

Full Text Available Después de la proclamación de la independencia en 1991, la República de Moldova (RM ha declarado su firme determinación de emprender su camino hacia Europa, a la que se siente identificada culturalmente. Aunque en las declaraciones y documentos adoptados el Estado nunca ha renunciado a este objetivo, ciertas acciones de sus líderes políticos parecen contradecir dicho compromiso. El trabajo tiene como objetivo analizar el proceso de evolución de la República de Moldova a la Unión Europea (UE, los compromisos asumidos por ambos y su cumplimiento. Además, voy a comparar los progresos realizados por Ucrania y RM hacia el acercamiento a la Unión Europea en los últimos años. Hace algunos años, Ucrania fue el país oriental con la perspectiva fundamental de la futura integración en la UE debido a sus cambios democráticos, hoy República de Moldova ha demostrado ser capaz de hacer más progresos en la promoción de la democracia y la aplicación de las reformas de gobernanza que Ucrania. Por otro lado, la relación entre la RM y la UE sigue siendo influenciada por las medidas adoptadas por la Federación de Rusia en la región donde todavía tiene influencia significativa, y tratar de mantener su presencia. Una de las herramientas fuertes de la influencia es la dependencia del suministro de energía y su falta de diversificación. El conflicto sin resolver de Transnistria es otro factor determinante de la falta de certeza en el proceso de europeización de la RM. El trabajo tiene como objetivo analizar la eficiencia de la europeización de la República de Moldova a través de la Política de Vecindad Europea, utilizando un método combinado constructivista y racionalista.

Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells

NARCIS (Netherlands)

Pandey, R.; Vischer, N.O.E.; Smelt, J.P.P.M.; van Beilen, J.W.A.; Ter Beek, A.; De Vos, W.H.; Brul, S.; Manders, E.M.M.

2016-01-01

Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHi homeostasis. Unfortunately, accurate pHi quantification with existing methods is suboptimal, since measurements are averages across populations of cells,

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

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.

INTRACELLULAR ION CONCENTRATIONS IN BRANCHIAL EPITHELIAL CELLS OF BROWN TROUT (SALMO TRUTTA L.) DETERMINED BY X-RAY MICROANALYSIS

Science.gov (United States)

Morgan; Potts; Oates

1994-09-01

The intracellular concentrations of sodium, chloride, phosphorus and potassium under normal conditions in pavement epithelial (PE) cells of brown trout (Salmo trutta) gill were 66, 51, 87 and 88 mmol l-1 respectively. The concentrations of these elements under identical conditions in mitochondria-rich (MR) cells were not significantly different, except for that of chlorine, which was lower in MR cells (40 mmol l-1). The concentration of sodium in the PE cells decreased slightly after exposure of the fish to low external [Na+] (25 µmol l-1) for 7 days but increased greatly within 5 min of subsequent exposure to 1 mmol l-1 external Na+. These changes in external [Na+] had no significant effect on MR cells. Exposure of fish to low [Cl-] (25 µmol l-1) had no effect on PE or MR cells, but on exposure to 1 mmol l-1 Cl- the concentrations of chlorine, phosphorus and potassium in both types of cells increased, whilst the intracellular sodium concentration decreased only in MR cells. The PE cells were little affected by exposure of the fish to the carbonic anhydrase inhibitor acetazolamide. In contrast, 0.5 mmol l-1 external acetazolamide caused a significant decrease in intracellular phosphorus, chlorine and potassium concentrations in MR cells. This suggests that the PE cells are the sites of sodium uptake in the gills of the brown trout and that chloride uptake occurs via the MR cells. These results are discussed with respect to the sites and possible mechanisms of ionic exchange in freshwater vertebrates.

Intracellular insulin processing is altered in monocytes from patients with type II diabetes mellitus

International Nuclear Information System (INIS)

Trischitta, V.; Benzi, L.; Brunetti, A.; Cecchetti, P.; Marchetti, P.; Vigneri, R.; Navalesi, R.

1987-01-01

We studied total cell-associated A14-[ 125 I]insulin radioactivity (including surface-bound and internalized radioactivity), insulin internalization, and its intracellular degradation at 37 C in monocytes from nonobese type II untreated diabetic patients (n = 9) and normal subjects (n = 7). Total cell-associated radioactivity was decreased in diabetic patients [2.65 +/- 1.21% (+/- SD) vs. 4.47 +/- 1.04% of total radioactivity. Insulin internalization was also reduced in diabetic patients (34.0 +/- 6.8% vs. 59.0 +/- 11.3% of cell-associated radioactivity. Using high performance liquid chromatography six intracellular forms of radioactivity derived from A14-[ 125 I] insulin were identified; 10-20% of intracellular radioactivity had approximately 300,000 mol wt and was identified as radioactivity bound to the insulin receptor, and the remaining intracellular radioactivity included intact A14-[ 125 I]insulin, [ 125 I]iodide, or [ 125 I]tyrosine, and three intermediate compounds. A progressive reduction of intact insulin and a corresponding increase in iodine were found when the incubation time was prolonged. Intracellular insulin degradation was reduced in monocytes from diabetic patients; intracellular intact insulin was 65.6 +/- 18.1% vs. 37.4 +/- 18.0% of intracellular radioactivity after 2 min and 23.6 +/- 22.3% vs. 3.9 +/- 2.3% after 60 min in diabetic patients vs. normal subjects, respectively. In conclusion, 1) human monocytes internalize and degrade insulin in the intracellular compartment in a stepwise time-dependent manner; and 2) in monocytes from type II diabetic patients total cell-associated radioactivity, insulin internalization, and insulin degradation are significantly reduced. These defects may be related to the cellular insulin resistance present in these patients

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.

Regulation of the glutamine transporter SN1 by extracellular pH and intracellular sodium ions

International Nuclear Information System (INIS)

Broeer, A.; Broeer, S.; Setiawan, I.; Lang, F.

2001-01-01

Full text: SN1 has recently been identified as one of the major glutamine transporters in hepatocytes and brain astrocytes. It appears to be the molecular correlate of the system N amino acid transporter. Two different transport mechanisms have been proposed for this transporter. Either an electroneutral mechanism, in which glutamine uptake is coupled to an exchange of 1Na + and 1H + , or an electrogenic mechanism coupled to the exchange of 2Na + against 1H + . This study was performed to solve the discrepancies and to investigate the reversibility of the transporter. When expressed in Xenopus laevis oocytes glutamine uptake activity increased strongly with increasing pH. In agreement with the pH-dependence we found that uptake of glutamine was accompanied by an alkalization of the cytosol, indicating that SN1 mediates Glutamine/H + -Antiport. Uptake of glutamine into oocytes was Na + -dependent. Analysis of the Na + -dependence of glutamine transport and Flux studies using 22 Na + indicated that two or more sodium ions were cotransported together with glutamine. However, at the same time intracellular Na + was exchanged against extracellular Na + . Taken together with the results of the pH-dependence it is proposed that SN1 mediates a Na + /Na + -exchange and a Na + /H + -exchange, both being coupled to the transport of glutamine. In agreement with this mechanism we found that acidic pH caused a reversal of the transporter. To investigate the source of the glutamine-induced inward currents, we compared inward currents generated by the 1Na + /glutamine cotransporter ATA1 with those generated by SN1. Currents induced by glutamine uptake in SN1 expressing oocytes were only a fraction of the currents induced by glutamine in ATA1 expressing oocytes, indicating that they were not generated by a stoichiometric uptake of ions. It is concluded that SN1 is tightly regulated by pH and intracellular Na + -ions and is capable of mediating glutamine uptake and release

Jinlida granule inhibits palmitic acid induced-intracellular lipid accumulation and enhances autophagy in NIT-1 pancreatic β cells through AMPK activation.

Science.gov (United States)

Wang, Dingkun; Tian, Min; Qi, Yuan; Chen, Guang; Xu, Lijun; Zou, Xin; Wang, Kaifu; Dong, Hui; Lu, Fuer

2015-02-23

Jinlida granule (JLDG), composed of seventeen Chinese medical herbs, is a widely used Chinese herbal prescription for treating diabetes mellitus. However, the mechanism underlying this effect remains unclear. To determine the main components in JLDG and to explore the effect of JLDG on autophagy and lipid accumulation in NIT-1 pancreatic β cells exposed to politic acid (PA) through AMP activated protein kinase (AMPK) signaling pathway. JLDG was prepared and the main components contained in the granules were identified by ultra performance liquid chromatography (UPLC) fingerprint. Intracellular lipid accumulation in NIT-1 cells was induced by culturing with medium containing PA. Intracellular lipid droplets were observed by Oil Red O staining and triglyceride (TG) content was measured by colorimetric assay. The formation of autophagosomes was observed under transmission electron microscope. The expression of AMPK and phospho-AMPK (pAMPK) proteins as well as its downstream fatty acid metabolism-related proteins (fatty acid synthase, FAS; acetyl-coA carboxylase, ACC; carnitine acyltransferase 1, CPT-1) and autophagy-related genes (mammal target of rapamycin, mTOR; tuberous sclerosis complex 1, TSC1; microtubule-associated protein 1 light chain 3, LC3-II) were determined by Western blot. The expression of sterol regulating element binding protein 1c (SREBP-1c) mRNA was examined by real time PCR (RT-PCR). Our data showed that JLDG could significantly reduce PA-induced intracellular lipid accumulation in NIT-1 pancreatic β cells. This effect was associated with increased protein expression of pAMPK and AMPK in NIT-1 cells. Treatment with JLDG also decreased the expression of AMPK downstream lipogenic genes (SREBP-1c mRNA, FAS and ACC proteins) whereas increased the expression of fatty acid oxidation gene (CPT-1 protein). Additionally, JLDG-treated cells displayed a markedly increase in the number of autophagosomes which was accompanied by the down-regulation of m

Genotipificación de aislamientos de Bartonella bacilliformis por amplificación de elementos repetitivos mediante el uso de REP-PCR y ERIC-PCR

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Carlos Padilla R

2003-07-01

Full Text Available Objetivos: Genotipificar los aislamientos de Bartonella bacilliformis a través de la amplificación de elementos repetitivos mediante el uso de ERIC-PCR y REP-PCR, y determinar si existe variabilidad genética entre aislamientos de varias zonas endémicas. Materiales y Métodos: Se evaluaron mediante el uso del ERIC-PCR y REP-PCR 17 aislamientos de B. bacilliformis de Lima, Cusco y Ancash. Los aislamientos fueron realizados durante los años 1998 y 1999. Para el análisis de los patrones de bandas se usó el software GelCompar 4,0. Resultados: Fueron identificados en los 17 aislamientos 10 genotipos. Los genotipos D, E y H fueron detectados en Cusco; mientras que los genotipos B, C, G, J e I en Lima; y el genotipo F en Ancash. Conclusiones: Nuestros resultados sugieren que REP-PCR y ERIC-PCR son métodos útiles para genotipificar aislamientos de B. bacilliformis. La variabilidad genética debe ser tomada en cuenta en estudios epidemiológicos y clínicos de Bartonelosis; así como el desarrollo de nuevas técnicas diagnósticas y de vacunas.

HFE mRNA expression is responsive to intracellular and extracellular iron loading: short communication.

Science.gov (United States)

Mehta, Kosha J; Farnaud, Sebastien; Patel, Vinood B

2017-10-01

In liver hepatocytes, the HFE gene regulates cellular and systemic iron homeostasis by modulating cellular iron-uptake and producing the iron-hormone hepcidin in response to systemic iron elevation. However, the mechanism of iron-sensing in hepatocytes remain enigmatic. Therefore, to study the effect of iron on HFE and hepcidin (HAMP) expressions under distinct extracellular and intracellular iron-loading, we examined the effect of holotransferrin treatment (1, 2, 5 and 8 g/L for 6 h) on intracellular iron levels, and mRNA expressions of HFE and HAMP in wild-type HepG2 and previously characterized iron-loaded recombinant-TfR1 HepG2 cells. Gene expression was analyzed by real-time PCR and intracellular iron was measured by ferrozine assay. Data showed that in the wild-type cells, where intracellular iron content remained unchanged, HFE expression remained unaltered at low holotransferrin treatments but was upregulated upon 5 g/L (p HFE and HAMP expressions were elevated only at low 1 g/L treatment (p HFE (p HFE mRNA was independently elevated by extracellular and intracellular iron-excess. Thus, it may be involved in sensing both, extracellular and intracellular iron. Repression of HAMP expression under simultaneous intracellular and extracellular iron-loading resembles non-hereditary iron-excess pathologies.

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.

Elecciones 2016 en República Dominicana: Características, desafíos y retos desde la perspectiva electoral

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Roberto Rosario Márquez

2015-01-01

Full Text Available Expone la reforma electoral del año 2010, los aspectos importantes que se presentaron en las elecciones de 2012 y los retos que la Junta Central Electoral de la República Dominicana enfrenta en la organización y administración del proceso electoral del año 2016

Conventional Morphology Versus PCR Sequencing, rep-PCR, and MALDI-TOF-MS for Identification of Clinical Aspergillus Isolates Collected Over a 2-Year Period in a University Hospital at Kayseri, Turkey.

Science.gov (United States)

Atalay, Altay; Koc, Ayse Nedret; Suel, Ahmet; Sav, Hafize; Demir, Gonca; Elmali, Ferhan; Cakir, Nuri; Seyedmousavi, Seyedmojtaba

2016-09-01

Aspergillus species cause a wide range of diseases in humans, including allergies, localized infections, or fatal disseminated diseases. Rapid detection and identification of Aspergillus spp. facilitate effective patient management. In the current study we compared conventional morphological methods with PCR sequencing, rep-PCR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the identification of Aspergillus strains. A total of 24 consecutive clinical isolates of Aspergillus were collected during 2012-2014. Conventional morphology and rep-PCR were performed in our Mycology Laboratory. The identification, evaluation, and reporting of strains using MALDI-TOF-MS were performed by BioMérieux Diagnostic, Inc. in Istanbul. DNA sequence analysis of the clinical isolates was performed by the BMLabosis laboratory in Ankara. Samples consisted of 18 (75%) lower respiratory tract specimens, 3 otomycosis (12.5%) ear tissues, 1 sample from keratitis, and 1 sample from a cutaneous wound. According to DNA sequence analysis, 12 (50%) specimens were identified as A. fumigatus, 8 (33.3%) as A. flavus, 3 (12.5%) as A. niger, and 1 (4.2%) as A. terreus. Statistically, there was good agreement between the conventional morphology and rep-PCR and MALDI-TOF methods; kappa values were κ = 0.869, 0.871, and 0.916, respectively (P < 0.001). The good level of agreement between the methods included in the present study and sequence method could be due to the identification of Aspergillus strains that were commonly encountered. Therefore, it was concluded that studies conducted with a higher number of isolates, which include other Aspergillus strains, are required. © 2016 Wiley Periodicals, Inc.

Modulatory Effect of the Intracellular Content of Lactobacillus casei CRL 431 Against the Aflatoxin B1-Induced Oxidative Stress in Rats.

Science.gov (United States)

Aguilar-Toalá, J E; Astiazarán-García, H; Estrada-Montoya, M C; Garcia, H S; Vallejo-Cordoba, B; González-Córdova, A F; Hernández-Mendoza, A

2018-06-03

It has been recognized that lactic acid bacteria exhibit antioxidant properties, which have been mainly endorsed to the intact viable bacteria. However, recent studies have shown that intracellular content (IC) may also be good sources of antioxidative metabolites, which may potentially contribute to oxidative homeostasis in vivo. Hence, the modulatory effect of the intracellular content of Lactobacillus casei CRL 431 (IC431) on aflatoxin B 1 (AFB 1 )-induced oxidative stress in rats was evaluated on the basis of its influence on hepatic lipid peroxidation (LPO), antioxidant status-antioxidant capacity (TAC), catalase (CAT), and glutathione peroxidase (GPx) activities; and on the oxidative stress index (OSi). Results demonstrated that CAT and GPx activities, and TAC, determined in plasma samples, were significantly (P < 0.05) higher in rats treated with AFB 1 plus IC431 (3.98 μM/min/mg protein, 1.88 μM/min/mg protein, and 238.7 μM Trolox equivalent, respectively) than AFB 1 -treated rats (3.47 μM/min/mg protein, 1.46 μM/min/mg protein, and 179.7 μM Trolox equivalent, respectively). Furthermore, plasma and liver tissue samples from rats treated with AFB 1 plus IC431 showed significantly (P < 0.05) lower LPO values (52 and 51%, respectively) and OSi (59 and 51%, respectively) than AFB 1 -treated rats. Hence, our results proved that the intracellular content of Lact. casei CRL 431 contains metabolites that are capable to modulate the antioxidant defense systems in living organism, which may help to ameliorate the damage associated to AFB 1 -induced oxidative stress.

Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein-Barr Virus Latent Phase Protein LMP1.

Science.gov (United States)

Lomakin, Ya A; Shmidt, A A; Bobik, T V; Chernov, A S; Pyrkov, A Yu; Aleksandrova, N M; Okunola, D O; Vaskina, M I; Ponomarenko, N A; Telegin, G B; Dubina, M V; Belogurov, A A

2017-10-01

Intracellular fragments of latent phase protein LMP1 of Epstein-Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein.

B-Vitamin Competition: Intracellular and Dissolved B-Vitamins Provide Insight into Marine Microbial Community Dynamics

Science.gov (United States)

Suffridge, C.; Gomez-Consarnau, L.; Qu, P.; Tenenbaum, N.; Fu, F.; Hutchins, D. A.; Sanudo-Wilhelmy, S. A.

2016-02-01

The availability of B-vitamins has the ability to directly affect the dynamics of the marine microbial community. Here we show, for the first time, the connection between dissolved and intracellular B-vitamins in a marine environmental community. Two incubation experiments were conducted at a long-term study site (SPOT) in the San Pedro Basin off the coast of Los Angeles, CA. Experiments were conducted in oligotrophic, preupwelling conditions. Due to the 2015 El Niño event, the seasonal upwelling at SPOT did not occur, creating unusually nutrient depleted conditions. Vitamins B1, B7, and B12 were added in addition to macronutrients at concentrations similar to typical SPOT upwelling conditions. Intracellular and dissolved B-vitamin analyses were conducted to determine shifts in cellular B-vitamin requirements as a function of growth rate. We observed a significant bacterioplankton and phytoplankton growth responses with the addition of B-vitamins in a manner that appears to match the enzymatic requirements for these compounds (e.g. B1>B7>B12). Intracellular B-vitamin analysis of T0 samples support this observation, as all four forms of B12 were not detectable within cells, yet multiple forms of B1 and B7 were detected at or near levels previously reported. Treatments with B12 and macronutrients were observed to have the greatest growth rates. This finding, in addition to the apparent lack of intracellular B12 in the initial community, appears to indicate that the initial microbial community was limited by B12. The addition of each vitamin caused a distinct shift in the blooming microbial community. Our results demonstrate that B-vitamins strongly influence not only the growth rate, but also the species composition and species succession of the microbial community as a whole. Large-scale changes to upwelling regimes are predicted in the future ocean; our results indicate that B-vitamins will have a substantial role in controlling microbial community dynamics under

Camphene, a Plant Derived Monoterpene, Exerts Its Hypolipidemic Action by Affecting SREBP-1 and MTP Expression.

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Ioanna Vallianou

Full Text Available The control of hyperlipidemia plays a central role in cardiovascular disease. Previously, we have shown that camphene, a constituent of mastic gum oil, lowers cholesterol and triglycerides (TG in the plasma of hyperlipidemic rats without affecting HMG-CoA reductase activity, suggesting that its hypocholesterolemic and hypotriglyceridemic effects are associated with a mechanism of action different than that of statins. In the present study, we examine the mechanism by which camphene exerts its hypolipidemic action. We evaluated the effect of camphene on the de novo synthesis of cholesterol and TG from [14C]-acetate in HepG2 cells, along with the statin mevinolin. Camphene inhibited the biosynthesis of cholesterol in a concentration-dependent manner, and a maximal inhibition of 39% was observed at 100 μM while mevinolin nearly abolished cholesterol biosynthesis. Moreover, treatment with camphene reduced TG by 34% and increased apolipoprotein AI expression. In contrast, mevinolin increased TG by 26% and had a modest effect on apolipoprotein AI expression. To evaluate the mode of action of camphene, we examined its effects on the expression of SREBP-1, which affects TG biosynthesis and SREBP-2, which mostly affects sterol synthesis. Interestingly, camphene increased the nuclear translocation of the mature form of SREBP-1 while mevinolin was found to increase the amount of the mature form of SREBP-2. The effect of camphene is most likely regulated through SREBP-1 by affecting MTP levels in response to a decrease in the intracellular cholesterol. We propose that camphene upregulates SREBP-1 expression and MTP inhibition is likely to be a probable mechanism whereby camphene exerts its hypolipidemic effect.

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.

RePS: a sequence assembler that masks exact repeats identified from the shotgun data

DEFF Research Database (Denmark)

Wang, Jun; Wong, Gane Ka-Shu; Ni, Peixiang

2002-01-01

We describe a sequence assembler, RePS (repeat-masked Phrap with scaffolding), that explicitly identifies exact 20mer repeats from the shotgun data and removes them prior to the assembly. The established software is used to compute meaningful error probabilities for each base. Clone......-end-pairing information is used to construct scaffolds that order and orient the contigs. We show with real data for human and rice that reasonable assemblies are possible even at coverages of only 4x to 6x, despite having up to 42.2% in exact repeats. Udgivelsesdato: 2002-May...

El Gobierno de la República en el exilio : crónica de un imposible retorno

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Xavier Flores

2001-01-01

Full Text Available El triunfo de los aliados en la Segunda Guena Mundial hizo que los republicanos españoles exiliados en 1939 concibiesen esperanzas de un inmediato retomo de la República a su país con el apoyo aliado. Con esta idea se reconstituyeron las instituciones de la República Española en el exilio en México D. R, en agosto de 1945. En este artículo se analiza el proceso de esa reconstitución y los sucesivos gobiernos que se sucedieron hasta la disolución de las instituciones en junio de 1977, después de un imposible retorno.The victory of the salles in the Second World war did that the Spanish republicans exiled in 1939 were conceiving hopes ofan immediate return ofthe Republic to his(its country with the allied support. With this idea there were re-constituted the institutions of the Spanish Republic in the exile in México D.F., in August, 1945. In this article there are analyzed the process of this reconstitution and the successive governments that happened up to the dissolution of the institutions in June, 1977, afíer impossible I come back.

Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

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Hurwitz, Stephanie N; Cheerathodi, Mujeeb R; Nkosi, Dingani; York, Sara B; Meckes, David G

2018-03-01

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major

Horizontal Transmission of Intracellular Insect Symbionts via Plants

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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.

P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites.

OpenAIRE

Pal, S K; Chattoraj, D K

1988-01-01

The unit-copy plasmid replicon mini-P1 consists of an origin, a gene for an initiator protein, RepA, and a control locus, incA. Both the origin and the incA locus contain repeat sequences that bind RepA. It has been proposed that the incA repeats control replication by sequestering the rate-limiting RepA initiator protein. Here we show that when the concentration of RepA was increased about fourfold beyond its normal physiological level from an inducible source in trans, the copy number of a ...

Estudo de práticas midiáticas a partir do produto: o jogo de espelhos de Profissão Repórter

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Mirian Meliani

2013-06-01

Full Text Available Analisar todas as nuances do discurso e das práticas adotadas pelo programa jornalístico Profissão Repórter, produzido pela Rede Globo, foi a tarefa realizada por jornalistas, pro- fessores e pós-graduandos ligados ao grupo de pesquisa MidiAto – Grupo de Estudos de Linguagem: Práticas Midiáticas, da Escola de Comunicações e Artes (ECA/USP. O resultado é uma exposição, bastante ampla, dos limites, das contradições e do papel desempenhado pelo programa na grade da emissora líder de audiência no Brasil, transcrita no livro Profissão Repórter em diálogo por meio de dezessete artigos.

Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening.

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Esther Bettiol

Full Text Available The development of new drugs against Chagas disease is a priority since the currently available medicines have toxic effects, partial efficacy and are targeted against the acute phase of disease. At present, there is no drug to treat the chronic stage. In this study, we have optimized a whole cell-based assay for high throughput screening of compounds that inhibit infection of mammalian cells by Trypanosoma cruzi trypomastigotes. A 2000-compound chemical library was screened using a recombinant T. cruzi (Tulahuen strain expressing beta-galactosidase. Three hits were selected for their high activity against T. cruzi and low toxicity to host cells in vitro: PCH1, NT1 and CX1 (IC(50: 54, 190 and 23 nM, respectively. Each of these three compounds presents a different mechanism of action on intracellular proliferation of T. cruzi amastigotes. CX1 shows strong trypanocidal activity, an essential characteristic for the development of drugs against the chronic stage of Chagas disease where parasites are found intracellular in a quiescent stage. NT1 has a trypanostatic effect, while PCH1 affects parasite division. The three compounds also show high activity against intracellular T. cruzi from the Y strain and against the related kinetoplastid species Leishmania major and L. amazonensis. Characterization of the anti-T. cruzi activity of molecules chemically related to the three library hits allowed the selection of two compounds with IC(50 values of 2 nM (PCH6 and CX2. These values are approximately 100 times lower than those of the medicines used in patients against T. cruzi. These results provide new candidate molecules for the development of treatments against Chagas disease and leishmaniasis.

Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening.

Science.gov (United States)

Bettiol, Esther; Samanovic, Marie; Murkin, Andrew S; Raper, Jayne; Buckner, Frederick; Rodriguez, Ana

2009-01-01

The development of new drugs against Chagas disease is a priority since the currently available medicines have toxic effects, partial efficacy and are targeted against the acute phase of disease. At present, there is no drug to treat the chronic stage. In this study, we have optimized a whole cell-based assay for high throughput screening of compounds that inhibit infection of mammalian cells by Trypanosoma cruzi trypomastigotes. A 2000-compound chemical library was screened using a recombinant T. cruzi (Tulahuen strain) expressing beta-galactosidase. Three hits were selected for their high activity against T. cruzi and low toxicity to host cells in vitro: PCH1, NT1 and CX1 (IC(50): 54, 190 and 23 nM, respectively). Each of these three compounds presents a different mechanism of action on intracellular proliferation of T. cruzi amastigotes. CX1 shows strong trypanocidal activity, an essential characteristic for the development of drugs against the chronic stage of Chagas disease where parasites are found intracellular in a quiescent stage. NT1 has a trypanostatic effect, while PCH1 affects parasite division. The three compounds also show high activity against intracellular T. cruzi from the Y strain and against the related kinetoplastid species Leishmania major and L. amazonensis. Characterization of the anti-T. cruzi activity of molecules chemically related to the three library hits allowed the selection of two compounds with IC(50) values of 2 nM (PCH6 and CX2). These values are approximately 100 times lower than those of the medicines used in patients against T. cruzi. These results provide new candidate molecules for the development of treatments against Chagas disease and leishmaniasis.

Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication.

Science.gov (United States)

Hirata, Hisae; Yamaji, Yasuyuki; Komatsu, Ken; Kagiwada, Satoshi; Oshima, Kenro; Okano, Yukari; Takahashi, Shuichiro; Ugaki, Masashi; Namba, Shigetou

2010-09-01

The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal -1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed. Copyright (c) 2010. Published by Elsevier B.V.

A Primeira República Portuguesa (1910-1926: partidos e sistema político

Directory of Open Access Journals (Sweden)

Baiôa, Manuel

2014-04-01

Full Text Available For historical, organisational, clientelist, violent and constitutional reasons the Portuguese Republican Party became the dominant party in the political system of the Portuguese First Republic (1910-1926. The opposition felt alternation via elections would be difficult to achieve so resorted to violent and unconstitutional practices to reach government. The Portuguese political parties modernised themselves in this period, but failed to turn into mass parties. In major cities they acted as a cadre party and in the rural area as a party of notables. The First Republic failed to take solid steps to democratise the political system were And Portugal remained tied to the liberal and elitist tradition of the nineteenth century, although in a republican version.El Partido Republicano Portugués se convirtió en el partido dominante del sistema político de la Primera República Portuguesa (1910-1926 debido a causas históricas, organizacionales, clientelistas, violentas y constitucionales. La oposición sentía que difícilmente conseguiría la alternancia política a través de elecciones, y por lo tanto, recurre a prácticas violentas e inconstitucionales para llegar al gobierno. Los partidos políticos portugueses se modernizaron en este período, pero no se convirtieron en partidos de masas. En las principales ciudades actuaban como partidos de cuadros y en las zonas rurales como partidos de notables. Durante la Primera República no se dieron pasos consistentes para democratizar el sistema político. Portugal se mantuvo ancorado a la tradición liberal y elitista del siglo XIX, aunque en una versión republicana.

O leitor e as ‘fronteiras’ do universo fantástico na obra A República 3000, de Menotti del Picchia = The reader and the ‘borders’ of the fantastic universe in the book A República 3000, from Menotti del Picchia

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Cristiano Mello de Oliveira

2012-01-01

Full Text Available No presente artigo pretendemos descortinar as relações entre os elementos fantásticos na obra A República 3000, do escritor Menotti del Picchia. Primeiramente iremos realizar uma breve recapitulação de alguns aspectos da literatura fantástica no Brasil. Posteriormente faremos recortes textuais, objetivando assim discutir aqueles que aproximem a linguagem com o universo fantástico. Tomaremos como referencial teórico os estudos de autores como Todorov e Carvalho. Também teceremos considerações sobre o comportamento do leitor diante dessa obra ímpar.This article aims to scrutinize the relationship of the fantastic elements in Menotti del Picchia’s work A República 3000. Firstly, we will briefly look at some aspects inherent to the Fantastic Literature in Brazil. Then we will select some excerpts whose language are closely related to the fantastic world. The theoretical framework draws on Todorov and Carvalho. We will also consider the possible reader’s degree of behavior in relation to the aforementioned masterpiece.

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...

Disruption of Intracellular ATP Generation and Tight Junction Protein Expression during the Course of Brain Edema Induced by Subacute Poisoning of 1,2-Dichloroethane

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Gaoyang Wang

2018-01-01

Full Text Available The aim of this study was to explore changes in intracellular ATP generation and tight junction protein expression during the course of brain edema induced by subacute poisoning of 1,2-dichloroethane (1,2-DCE. Mice were exposed to 1.2 g/m3 1,2-DCE for 3.5 h per day for 1, 2, or 3 days, namely group A, B, and C. Na+-K+-ATPase and Ca2+-ATPase activity, ATP and lactic acid content, intracellular free Ca2+ concentration and ZO-1 and occludin expression in the brain were measured. Results of present study disclosed that Ca2+-ATPase activities in group B and C, and Na+/K+-ATPase activity in group C decreased, whereas intracellular free Ca2+ concentrations in group B and C increased significantly compared with control. Moreover, ATP content decreased, whereas lactic acid content increased significantly in group C compared with control. On the other hand, expressions of ZO-1 and occludin at both the protein and gene levels in group B and C decreased significantly compared with control. In conclusion, findings from this study suggest that calcium overload and depressed expression of tight junction associated proteins, such as ZO-1 and occludin might play an important role in the early phase of brain edema formation induced by subacute poisoning of 1,2-DCE.

Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery.

Science.gov (United States)

Wang, Huabin; Chen, Ligang; Sun, Xianchao; Fu, Ailing

2017-09-01

Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.

[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.

MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

Science.gov (United States)

Wang, Jinli; Yang, Kun; Zhou, Lin; Minhaowu; Wu, Yongjian; Zhu, Min; Lai, Xiaomin; Chen, Tao; Feng, Lianqiang; Li, Meiyu; Huang, Chunyu; Zhong, Qiu; Huang, Xi

2013-01-01

Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

Directory of Open Access Journals (Sweden)

Jinli Wang

Full Text Available Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7 reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb, a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

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

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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.

Presence of pRI1:

DEFF Research Database (Denmark)

Migura, Lourdes Garcia; Hasman, Henrik; Jensen, Lars Bogø

2009-01-01

This study focused on the molecular characterization of a small cryptic, mobilizable plasmid (6038 bp) sequenced from an E. faecium 9631160-1 of poultry origin. Sequence analysis of pRI1 revealed seven open reading frames. pRI1 contained an IS 100% identical to ISEfa4. This insertion element...... almost identical to the repA from the pEFNP1 and pKQ10 plasmids from E. faecium was also identified. Presence of the pRI1 replication initiation gene (rep) was analyzed in a panel of 159 E. faecium isolates of human and animal origin from different European countries, of which 60 tested positive...

Intracellular Targeting of CEA Results in Th1-Type Antibody Responses Following Intradermal Genetic Vaccination by a Needle-Free Jet Injection Device

Directory of Open Access Journals (Sweden)

Susanne Johansson

2007-01-01

Full Text Available The route and method of immunization, as well as the cellular localization of the antigen, can influence the generation of an immune response. In general, intramuscular immunization results in Th1 responses, whereas intradermal delivery of DNA by gene gun immunization often results in more Th2 responses. Here we investigate how altering the cellular localization of the tumor antigen CEA (carcinoembryonic antigen affects the quality and amplitude of DNA vaccine-induced antibody responses in mice following intradermal delivery of DNA by a needle-free jet injection device (Biojector. CEA was expressed either in a membrane-bound form (wild-type CEA or in two truncated forms (CEA6 and CEA66 with cytoplasmic localization, where CEA66 was fused to a promiscuous T-helper epitope from tetanus toxin. Repeated intradermal immunization of BALB/c mice with DNA encoding wild-type CEA produced high antibody titers of a mixed IgG1/IgG2a ratio. In contrast, utilizing the DNA construct that resulted in intracellular targeting of CEA led to a reduced capacity to induce CEA-specific antibodies, but instead induced a Th1-biased immune response.

High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap.

Science.gov (United States)

Conway, J E; Rhys, C M; Zolotukhin, I; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

1999-06-01

Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27. 1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.

Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

International Nuclear Information System (INIS)

Rabenstein, D.L.; Isab, A.A.

1982-01-01

A method is described for determining the intracellular pH of intact erythrocytes by 1 H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bounded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from 1 H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the 1 H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-or-saturation by cross-relaxation method

DMPD: Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 16982211 Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Wullaer...vg) (.html) (.csml) Show Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. PubmedID 1698221...1 Title Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Author

Intracellular trafficking of VP22 in bovine herpesvirus-1 infected cells

International Nuclear Information System (INIS)

Lobanov, Vladislav A.; Babiuk, Lorne A.; Drunen Littel-van den Hurk, Sylvia van

2010-01-01

The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 131 PRPR 134 NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.

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

Science.gov (United States)

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-10-01

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 bacterial colonies in infected host cells (Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy, Ernstsen et al., 2017 [1]). The infected cells were imaged with a 10× objective and number of intracellular bacterial colonies, their size distribution and the number of cell nuclei were automatically quantified using a spot detection-tool. The spot detection-output was exported to Excel, where data analysis was performed. In this article, micrographs and spot detection data are made available to facilitate implementation of the method.

Rediseño y fabricación de una impresora 3D RepRap.

OpenAIRE

Huertas González, Joan

2014-01-01

El objetivo principal de este proyecto es conseguir las habilidades necesarias para fabricar una impresora 3D del tipo Rep Rap, una tipología de impresoras Open source. Una vez conseguido este objetivo se rediseñan piezas de la propia impresora con el objetivo de mejorar el diseño actual. Se plantea el proyecto como iniciación al mundo de la impresión 3D y de los materiales plásticos con los que se puede trabajar, PLA, HDPE, ABS o NYLON. Se instruye al lector para que tambié...

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

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

Science.gov (United States)

Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

2013-01-01

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

Macau no Contexto da Estratégia de Integração da República Popular da China no Mercado Mundial

OpenAIRE

Murteira, Mário

1994-01-01

Série II Especulação sobre o futuro de Macau perante a forte estratégia de intergração da República Popular da China no mercado mundial (com o desempenho de um papel influente por parte de Macau e Taiwan).

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

Science.gov (United States)

Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

2018-06-11

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

Science.gov (United States)

Wavre-Shapton, Silène T; Tolmachova, Tanya; Lopes da Silva, Mafalda; da Silva, Mafalda Lopes; Futter, Clare E; Seabra, Miguel C

2013-01-01

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox), Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox), Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

Directory of Open Access Journals (Sweden)

Silène T Wavre-Shapton

Full Text Available The retinal pigment epithelium (RPE is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1. REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox, Tyr-Cre+. Transmission electron microscopy (TEM was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

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Micheva-Viteva, Sofiya N; Shou, Yulin; Ganguly, Kumkum; Wu, Terry H; Hong-Geller, Elizabeth

2017-01-01

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis , we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host

Ordem e Progresso: Augusto Comte e as influências do Positivismo na educação das mulheres na Primeira República brasileira

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Loraine Lopes de Oliveira

2018-04-01

Full Text Available O presente artigo tem como objetivo apresentar uma reflexão acerca da influência do Positivismo na educação das mulheres no contexto da Primeira República brasileira (1889-1930, também chamada de República Velha. Faz-se uma breve abordagem inicial a respeito de Augusto Comte e o surgimento da filosofia positivista e posteriormente se discorre sobre a influência do Positivismo na educação das mulheres, a qual era vista como um elemento chave para a concretização da nova sociedade que se projetava, para o progresso e a modernização. A pesquisa tem caráter bibliográfico e fundamenta-se nos pressupostos teóricos do materialismo histórico-dialético, que busca compreender os fenômenos sociais e históricos a partir da existência material dos homens. A discussão prioriza a relação da educação com os fatores econômicos, políticos e sociais, já que se compreende que o objeto de pesquisa não pode ser entendido como uma situação isolada de seu contexto histórico. Palavras-chave: Positivismo. Primeira República Brasileira. Educação das mulheres.   Order and Progress: Augusto Comte and the influences of Positivism in the education of women in the First Brazilian Republic ABSTRACT The present article aims to present a reflection on the influence of positivism in the education of women in the context of the First Brazilian Republic (1889-1930, also called the Old Republic. A brief initial approach is given to Augusto Comte and the emergence of the positivist philosophy and later it talks about the influence of positivism in the education of women, a qualitative perspective as a key element for a concretization of the new society which was projected, for progress and modernization. The research has a bibliographic character and is based on the theoretical assumptions of historical-dialectical materialism, which seeks social and historical phenomena from the material reality of men. The discussion prioritizes the relationship

Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines.

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Suijker, Johnny; Oosting, Jan; Koornneef, Annemarie; Struys, Eduard A; Salomons, Gajja S; Schaap, Frank G; Waaijer, Cathelijn J F; Wijers-Koster, Pauline M; Briaire-de Bruijn, Inge H; Haazen, Lizette; Riester, Scott M; Dudakovic, Amel; Danen, Erik; Cleton-Jansen, Anne-Marie; van Wijnen, Andre J; Bovée, Judith V M G

2015-05-20

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.

A method for functional trans-complementation of intracellular Francisella tularensis.

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Shaun Steele

Full Text Available Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional

Melatonin-Mediated Intracellular Insulin during 2-Deoxy-d-glucose Treatment Is Reduced through Autophagy and EDC3 Protein in Insulinoma INS-1E Cells

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Han Sung Kim

2016-01-01

Full Text Available 2-DG triggers glucose deprivation without altering other nutrients or metabolic pathways and then activates autophagy via activation of AMPK and endoplasmic reticulum (ER stress. We investigated whether 2-DG reduced intracellular insulin increased by melatonin via autophagy/EDC3 in insulinoma INS-1E cells. p-AMPK and GRP78/BiP level were significantly increased by 2-DG in the presence/absence of melatonin, but IRE1α level was reduced in 2-DG treatment. Levels of p85α, p110, p-Akt (Ser473, Thr308, and p-mTOR (Ser2481 were also significantly reduced by 2-DG in the presence/absence of melatonin. Mn-SOD increased with 2-DG plus melatonin compared to groups treated with/without melatonin alone. Bcl-2 was decreased and Bax increased with 2-DG plus melatonin. LC3II level increased with 2-DG treatment in the presence/absence of melatonin. Intracellular insulin production increased in melatonin plus 2-DG but reduced in treatment with 2-DG with/without melatonin. EDC3 was increased by 2-DG in the presence/absence of melatonin. Rapamycin, an mTOR inhibitor, increased GRP78/BiP and EDC3 levels in a dose-dependent manner and subsequently resulted in a decrease in intracellular production of insulin. These results suggest that melatonin-mediated insulin synthesis during 2-DG treatment involves autophagy and EDC3 protein in rat insulinoma INS-1E cells and subsequently results in a decrease in intracellular production of insulin.

Resolution of intracellular calcium metabolism in intact segments of rabbit aorta

International Nuclear Information System (INIS)

Phair, R.D.; Hai, C.M.

1986-01-01

A new method, based on computer-assisted kinetic analysis of 45 Ca efflux data, was used to measure calcium contents and fluxes for extracellular and intracellular compartments in intact segments of rabbit aorta. After a 1-hour loading period, efflux data were collected for 8 hours using a flow-through tissue chamber. These long-term effluxes were necessary because information on intracellular calcium metabolism was concentrated in the slow components of the efflux curves while earlier components appeared to be dominated by washout of extracellular calcium. Intracellular compartments were identified as those whose calcium contents were altered by 10 microM phenylephrine. This method complements previous approaches by providing simultaneous estimates of compartmental calcium contents and fluxes without requiring the assumption of isotopic equilibrium and without recourse to standard wash techniques for removal of extracellular calcium. In normal, calcium-containing, bicarbonate-buffered physiological salt solution these compartments contained a total of approximately 300 nmol Ca/g wet aorta. Of this total, 55 nmol/g were associated with the slowest resolvable compartment whose turnover time was 170 minutes and whose exchange flux was 0.32 nmol min-1g-1. Two other intracellular compartments had turnover times of 30 minutes. One of these was phenylephrine releasable and contained 145 nmol/g; it exchanged calcium at 4.9 nmol min-1g-1. In normal physiological salt solution the plasma membrane was, surprisingly, not rate limiting for Ca efflux; and in 10 microM phenylephrine the membrane Ca flux was even greater, increasing 3.5-fold compared to control

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.

Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

Energy Technology Data Exchange (ETDEWEB)

Velho, Albertina M. [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); Jarvis, Simon M., E-mail: S.M.Jarvis@westminster.ac.uk [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); University of Westminster, School of Biosciences, London W1W 6UW (United Kingdom)

2009-08-01

The Na{sup +}-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 ({approx} 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated ({approx} 68-90 kDa) with only 31-65% of WT L-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered ({approx} 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at {approx} 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 {sup o}C or 37 {sup o}C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.

Objectif Express 1 le monde professionnel en français A1/A2

CERN Document Server

Tauzin, Béatrice

2013-01-01

Avec un nouveau look et des contenus actualisés pour prendre en compte les évolutions du monde de l’entreprise française, la nouvelle édition d'Objectif Express 1 propose une parfaite association entre le français de tous les jours et le français de l'entreprise. 10 unités basées sur des thématiques de l'entreprise : Entrez en contact ; Organisez votre journée ; Trouvez un emploi ... Chaque unité propose : - 4 leçons d'apprentissage (1 leçon = 1 double page) basées sur des tâches concrètes et simples, ancrées dans le monde du travail francophone - 1 double page Outils linguistiques - 1 page Entrainez-vous - 1 page Testez-vous - 2 pages Repères dans chaque unité : des repères culturels et des repères professionnels Toutes les 3 unités, un scénario professionnel pour une mise en œuvre des acquis langagiers et une mobilisation des compétences professionnelles de l'apprenant. Une préparation aux diplômes DFP A2, DELF Pro A1 et A2 et au test BULATS En annexes : des tableaux d...

[Valitsusliidus olevad Reformierakond ning Isamaa ja Res Publica Liit on kavandatava koolireformi suhtes eri meelt] / Mailis Reps, Tiiu Kuurme, Karin Kütt

Index Scriptorium Estoniae

Reps, Mailis, 1975-

2010-01-01

Reformierakonna ettepaneku kohta jätta omavalitsustele õiguse otsustada, kas gümnaasium ja põhikool lahutatakse või jätkatakse ühendkooliga avaldavad arvamust riigikogu liige Mailis Reps, Tallinna Ülikooli dotsent Tiiu Kuurme ja Rannu keskkooli direktor Karin Kütt

Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system.

Science.gov (United States)

Popławski, Piotr; Wiśniewski, Jacek R; Rijntjes, Eddy; Richards, Keith; Rybicka, Beata; Köhrle, Josef; Piekiełko-Witkowska, Agnieszka

2017-01-01

Type 1 iodothyronine deiodinase (DIO1) contributes to deiodination of 3,5,3',5'-tetraiodo-L-thyronine (thyroxine, T4) yielding of 3,5,3'-triiodothyronine (T3), a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The 'downregulated' group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2) that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2), enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2), sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10). DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression and changes

Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system.

Directory of Open Access Journals (Sweden)

Piotr Popławski

Full Text Available Type 1 iodothyronine deiodinase (DIO1 contributes to deiodination of 3,5,3',5'-tetraiodo-L-thyronine (thyroxine, T4 yielding of 3,5,3'-triiodothyronine (T3, a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The 'downregulated' group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2 that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2, enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2, sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10. DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression

Drosophila VAMP7 regulates Wingless intracellular trafficking.

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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.

Short-term acute hypercapnia affects cellular responses to trace metals in the hard clams Mercenaria mercenaria.

Science.gov (United States)

Ivanina, Anna V; Beniash, Elia; Etzkorn, Markus; Meyers, Tiffany B; Ringwood, Amy H; Sokolova, Inna M

2013-09-15

Estuarine and coastal habitats experience large fluctuations of environmental factors such as temperature, salinity, partial pressure of CO2 ( [Formula: see text] ) and pH; they also serve as the natural sinks for trace metals. Benthic filter-feeding organisms such as bivalves are exposed to the elevated concentrations of metals in estuarine water and sediments that can strongly affect their physiology. The effects of metals on estuarine organisms may be exacerbated by other environmental factors. Thus, a decrease in pH caused by high [Formula: see text] (hypercapnia) can modulate the effects of trace metals by affecting metal bioavailability, accumulation or binding. To better understand the cellular mechanisms of interactions between [Formula: see text] and trace metals in marine bivalves, we exposed isolated mantle cells of the hard clams (Mercenaria mercenaria) to different levels of [Formula: see text] (0.05, 1.52 and 3.01 kPa) and two major trace metal pollutants - cadmium (Cd) and copper (Cu). Elevated [Formula: see text] resulted in a decrease in intracellular pH (pHi) of the isolated mantle cells from 7.8 to 7.4. Elevated [Formula: see text] significantly but differently affected the trace metal accumulation by the cells. Cd uptake was suppressed at elevated [Formula: see text] levels while Cu accumulation has greatly accelerated under hypercapnic conditions. Interestingly, at higher extracellular Cd levels, labile intracellular Cd(2+) concentration remained the same, while intracellular levels of free Zn(2+) increased suggesting that Cd(2+) substitutes bound Zn(2+) in these cells. In contrast, Cu exposure did not affect intracellular Zn(2+) but led to a profound increase in the intracellular levels of labile Cu(2+) and Fe(2+). An increase in the extracellular concentrations of Cd and Cu led to the elevated production of reactive oxygen species under the normocapnic conditions (0.05 kPa [Formula: see text] ); surprisingly, this effect was mitigated in

78 FR 53774 - Guide for the Evaluation of Alert and Notification Systems for Nuclear Power Plants, FEMA-REP-10...

Science.gov (United States)

2013-08-30

... available when FEMA REP-10 was first developed, such as smartphones and social media. Removes technical... proposed policy does not have the force or effect of law. FEMA seeks comment on the proposed policy, which... the force or effect of law. Authority: 6 U.S.C. 313-314a; Sec. 109, Pub. L. 96-295; E.O. 12148; 44 CFR...

Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa

DEFF Research Database (Denmark)

Blomberg Jensen, Martin; Bjerrum, Poul J; Jessen, Torben E

2011-01-01

BACKGROUND The vitamin D receptor (VDR) is expressed in human spermatozoa, and VDR-knockout mice and vitamin D (VD) deficiency in rodents results in impaired fertility, low sperm counts and a low number of motile spermatozoa. We investigated the role of activated VD (1,25(OH)(2)D(3)) in human...... spermatozoa and whether VD serum levels are associated with semen quality. METHODS Cross-sectional association study of semen quality and VD serum level in 300 men from the general population, and in vitro studies on spermatozoa from 40 men to investigate the effects of VD on intracellular calcium, sperm......M). 1,25(OH)(2)D(3) increased intracellular calcium concentration in human spermatozoa through VDR-mediated calcium release from an intracellular calcium storage, increased sperm motility and induced the acrosome reaction in vitro. CONCLUSIONS 1,25(OH)(2)D(3) increased intracellular calcium...

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.

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.

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

Science.gov (United States)

Wu, Yu; Pons, Valérie; Goudet, Amélie; Panigai, Laetitia; Fischer, Annette; Herweg, Jo-Ana; Kali, Sabrina; Davey, Robert A; Laporte, Jérôme; Bouclier, Céline; Yousfi, Rahima; Aubenque, Céline; Merer, Goulven; Gobbo, Emilie; Lopez, Roman; Gillet, Cynthia; Cojean, Sandrine; Popoff, Michel R; Clayette, Pascal; Le Grand, Roger; Boulogne, Claire; Tordo, Noël; Lemichez, Emmanuel; Loiseau, Philippe M; Rudel, Thomas; Sauvaire, Didier; Cintrat, Jean-Christophe; Gillet, Daniel; Barbier, Julien

2017-11-14

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

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

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.

Involvement of intracellular free Ca2+ in enhanced release of herpes simplex virus by hydrogen peroxide

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Ogawa Yuzo

2006-08-01

Full Text Available Abstract Background It was reported that elevation of the intracellular concentration of free Ca2+ ([Ca2+]i by a calcium ionophore increased the release of herpes simplex virus type 1 (HSV-1. Freely diffusible hydrogen peroxide (H2O2 is implied to alter Ca2+ homeostasis, which further enhances abnormal cellular activity, causing changes in signal transduction, and cellular dysfunction. Whether H2O2 could affect [Ca2+]i in HSV-1-infected cells had not been investigated. Results H2O2 treatment increased the amount of cell-free virus and decreased the proportion of viable cells. After the treatment, an elevation in [Ca2+]i was observed and the increase in [Ca2+]i was suppressed when intracellular and cytosolic Ca2+ were buffered by Ca2+ chelators. In the presence of Ca2+ chelators, H2O2-mediated increases of cell-free virus and cell death were also diminished. Electron microscopic analysis revealed enlarged cell junctions and a focal disintegration of the plasma membrane in H2O2-treated cells. Conclusion These results indicate that H2O2 can elevate [Ca2+]i and induces non-apoptotic cell death with membrane lesions, which is responsible for the increased release of HSV-1 from epithelial cells.

Mycobacterium intracellulare Pleurisy Identified on Liquid Cultures of the Pleural Fluid and Pleural Biopsy.

Science.gov (United States)

Lim, Jong Gu; O, Sei Won; Lee, Ki Dong; Suk, Dong Keun; Jung, Tae Young; Shim, Tae Sun; Chon, Gyu Rak

2013-03-01

Pleural effusion is a rare complication in non-tuberculous mycobacterial infection. We report a case of Mycobacterium intracellulare pleuritis with idiopathic pulmonary fibrosis in a 69-year-old man presenting with dyspnea. Pleural effusion revealed lymphocyte dominant exudate. M. intracellulare was identified using a polymerase chain reaction-restriction fragment length polymorphism method and liquid cultures of pleural effusion and pleural biopsy. After combination therapy for M. intracellulare pulmonary disease, the patient was clinically well at a 1-month follow-up.

The Equine Antimicrobial Peptide eCATH1 Is Effective against the Facultative Intracellular Pathogen Rhodococcus equi in Mice

Science.gov (United States)

Schlusselhuber, Margot; Torelli, Riccardo; Martini, Cecilia; Leippe, Matthias; Cattoir, Vincent; Leclercq, Roland; Laugier, Claire; Grötzinger, Joachim; Sanguinetti, Maurizio

2013-01-01

Rhodococcus equi, the causal agent of rhodococcosis, is a major pathogen of foals and is also responsible for severe infections in immunocompromised humans. Of great concern, strains resistant to currently used antibiotics have emerged. As the number of drugs that are efficient in vivo is limited because of the intracellular localization of the bacterium inside macrophages, new active but cell-permeant drugs will be needed in the near future. In the present study, we evaluated, by in vitro and ex vivo experiments, the ability of the alpha-helical equine antimicrobial peptide eCATH1 to kill intracellular bacterial cells. Moreover, the therapeutic potential of the peptide was assessed in experimental rhodococcosis induced in mice, while the in vivo toxicity was evaluated by behavioral and histopathological analysis. The study revealed that eCATH1 significantly reduced the number of bacteria inside macrophages. Furthermore, the bactericidal potential of the peptide was maintained in vivo at doses that appeared to have no visible deleterious effects for the mice even after 7 days of treatment. Indeed, daily subcutaneous injections of 1 mg/kg body weight of eCATH1 led to a significant reduction of the bacterial load in organs comparable to that obtained after treatment with 10 mg/kg body weight of rifampin. Interestingly, the combination of the peptide with rifampin showed a synergistic interaction in both ex vivo and in vivo experiments. These results emphasize the therapeutic potential that eCATH1 represents in the treatment of rhodococcosis. PMID:23817377

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

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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

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

Sphingosine-1-phosphate receptors: Zooming in on ligand-induced intracellular trafficking and its functional implications

NARCIS (Netherlands)

Verzijl, Dennis; Peters, Stephan L. M.; Alewijnse, Astrid E.

2010-01-01

Regulatory processes including receptor phosphorylation and intracellular trafficking, also referred to as receptor internalization, are important processes to terminate G protein-coupled receptor (GPCR) signaling. Compelling evidence now indicates that internalization of a receptor is not

Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

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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.

La disputa por la república en el siglo XIX y la prensa en México

OpenAIRE

Toussaint, Florence

2006-01-01

A partir de la consumación de la independencia, se inicia la disputa por definir el tipo de gobierno que había de asumir el país. Esta oscila entre la República federal y la centralista durante casi todo el tiempo, aunque hay dos momentos en los cuales la forma monárquica aparece como opción. Se conoce a estos periodos como el Primer Imperio y el Segundo Imperio. No duran muchos años. Sin embargo, dan lugar a enconadas luchas internas, sobre todo durante la presencia de...

Política externa do vizinho distante : estudo de caso da República Cooperativa da Guiana

OpenAIRE

Lima, Erick Cavalcanti Linhares

2012-01-01

Esta tese trata das relações externas da República Cooperativa da Guiana, num recorte temporal de quarenta e quatro anos, que vão desde a independência, em 1966 até 2010, período no qual se desenvolveram importantes transformações mundiais, bem como no relacionamento político-diplomático. Remontam a essa época, a formação do marco institucional de representação dos interesses sociais e produtivos, tanto na economia guianense, quanto no âmbito internacional. O objetivo foi traç...

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor.

Science.gov (United States)

Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

2011-07-06

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.

Intracellular amyloid formation in muscle cells of Aβ-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification

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Bush Ashley I

2009-01-01

Full Text Available Abstract Background The amyloid β-peptide is a ubiquitous peptide, which is prone to aggregate forming soluble toxic oligomers and insoluble less-toxic aggregates. The intrinsic and external/environmental factors that determine Aβ aggregation in vivo are poorly understood, as well as the cellular meaning of this process itself. Genetic data as well as cell biological and biochemical evidence strongly support the hypothesis that Aβ is a major player in the onset and development of Alzheimer's disease. In addition, it is also known that Aβ is involved in Inclusion Body Myositis, a common myopathy of the elderly in which the peptide accumulates intracellularly. Results In the present work, we found that intracellular Aβ aggregation in muscle cells of Caenorhabditis elegans overexpressing Aβ peptide is affected by two single amino acid substitutions, E22G (Arctic and V18A (NIC. Both variations show decrease intracellular amyloidogenesis compared to wild type Aβ. We show that intracellular amyloid aggregation of wild type Aβ is accelerated by Cu2+ and diminished by copper chelators. Moreover, we demonstrate through toxicity and behavioral assays that Aβ-transgenic worms display a higher tolerance to Cu2+ toxic effects and that this resistance may be linked to the formation of amyloid aggregates. Conclusion Our data show that intracellular Aβ amyloid aggregates may trap excess of free Cu2+ buffering its cytotoxic effects and that accelerated intracellular Aβ aggregation may be part of a cell protective mechanism.

Epigallocatechin-3-gallate increases intracellular [Ca2+] in U87 cells mainly by influx of extracellular Ca2+ and partly by release of intracellular stores.

Science.gov (United States)

Kim, Hee Jung; Yum, Keun Sang; Sung, Jong-Ho; Rhie, Duck-Joo; Kim, Myung-Jun; Min, Do Sik; Hahn, Sang June; Kim, Myung-Suk; Jo, Yang-Hyeok; Yoon, Shin Hee

2004-02-01

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [3H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca2+] ([Ca2+]i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca2+]i. The EGCG-induced [Ca2+]i increases were reduced to 20.9% of control by removal of extracellular Ca2+. The increases were also inhibited markedly by treatment with the non-specific Ca2+ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca2+ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca2+-ATPase thapsigargin (1 micro M) also significantly inhibited the EGCG-induced [Ca2+]i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 micro M) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 micro M) had no effect. EGCG increased [3H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 micro M) and flufenamic acid (100 micro M), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca2+]i increase in non-treated and thapsigargin-treated cells but indomethacin (100 micro M) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca2+]i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca2+ and partly by mobilizing intracellular Ca2+ stores by PLC activation. The EGCG-induced [Ca2+]i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

Science.gov (United States)

Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

2017-06-01

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

Intracellular renin disrupts chemical communication between heart cells. Pathophysiological implications

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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.

Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum.

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Jose C Garcia-Garcia

2009-06-01

Full Text Available Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1 expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.

Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing.

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Fujiwara, Toshihiro; Dohi, Teruyuki; Maan, Zeshaan N; Rustad, Kristine C; Kwon, Sun Hyung; Padmanabhan, Jagannath; Whittam, Alexander J; Suga, Hirotaka; Duscher, Dominik; Rodrigues, Melanie; Gurtner, Geoffrey C

2017-07-04

Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-β1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The effect of amyotrophic lateral sclerosis-linked exogenous SOD1-G93A on electrophysiological properties and intracellular calcium in cultured rat astrocytes.

Science.gov (United States)

Milošević, Milena; Bataveljić, Danijela; Nikolić, Ljiljana; Bijelić, Dunja; Andjus, Pavle

2016-01-01

Over 150 mutations in the SOD1 gene that encodes Cu/Zn superoxide dismutase (SOD1) cause 20-25% of familial ALS, albeit without a known gain-of-function mechanism. ALS is also non-cell-autonomous, the interactions between motor neurons and their glial neighbours being implicated in disease progression. The aim here was to investigate the biophysical effects of the exogenous human mutant SOD1-G93A on rat astrocytes in culture. Primary cortical astrocyte cultures were treated with recombinant human apo- mSOD1-G93A vs. wild-type control (wtSOD1) and recorded by patch-clamp and calcium imaging. Results showed that exogenous mSOD1 as well as wtSOD1 induced a decrease of membrane resistance, the effect being persistent (up to 13 min) only for the mutant form. Similarly, whole-cell inward currents in astrocytes were augmented by both wt and mSOD1, but the effect was twice larger and only progressed continuously for the latter. Both forms of SOD1 also induced a rise in intracellular Ca(2+) activity, the effect being dependent on external Ca(2+) and again only persisted with mSOD1, becoming significantly different from wtSOD1 only at longer times (14 min). In conclusion, this study points to membrane permeability and Ca(2+) signalling as processes affected by SOD1-G93A that presents the humoral factor triggering the role of astrocytes in ALS pathophysiology.

Rising Intracellular Zinc by Membrane Depolarization and Glucose in Insulin-Secreting Clonal HIT-T15 Beta Cells

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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.

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,

KLF5 overexpression attenuates cardiomyocyte inflammation induced by oxygen-glucose deprivation/reperfusion through the PPARγ/PGC-1α/TNF-α signaling pathway.

Science.gov (United States)

Li, Yang; Li, Jian; Hou, Zhiwen; Yu, Yang; Yu, Bo

2016-12-01

The primary physiological function of Krüppel-like zinc-finger transcription factor (KLF5) is the regulation of cardiovascular remodeling. Vascular remodeling is closely related to the amelioration of various ischemic diseases. However, the underlying correlation of KLF5 and ischemia is not clear. In this study, we aim to investigate the role of KLF5 in myocardial ischemia reperfusion (IR) injury and the potential mechanisms involved. Cultured H9C2 cells were subjected to oxygen-glucose deprivation/reperfusion (OGD/Rep) to mimic myocardial IR injury in vivo. Expressions of KLF5 and PPARγ were distinctly inhibited, and PGC-1α expression was activated at 24h after myocardial OGD/Rep injury. After myocardial OGD/Rep injury, we found that KLF5 overexpression down-regulated levels of TNF-α, IL-1β, IL-6 and IL-8. Through the analysis of lactate dehydrogenase (LDH) release, we demonstrate that KLF5 overexpression reduced the release of OGD/Rep-induced LDH. KLF5 overexpression significantly enhanced cell activity and decreased cell apoptosis during OGD/Rep injury. Compared with the OGD/Rep group, cells overexpressing KLF5 showed anti-apoptotic effects, such as decreased expression of Bax and cleaved caspase-3 as well as increased Bcl-2 expression. KLF5 overexpression activated PPARγ, a protein involved in OGD/Rep injury, and increased levels of PGC-1α, while TNF-α expression was remarkably inhibited. In addition, GW9662, a PPARγ receptor antagonist, reversed the expression of PPARγ/PGC-1α/TNF-α and cell activity induced by KLF5 overexpression. The effects of KLF5 overexpression on PPARγ/PGC-1α/TNF-α and cell activity were abolished by co-treatment with GW9662. Taken together, these results suggest that KLF5 can efficiently alleviate OGD/Rep-induced myocardial injury, perhaps through regulation of the PPARγ/PGC-1α/TNF-α pathway. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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.

Intracellular thiol levels and radioresistance: Studies with glutathione and glutathione mono ethyl ester

International Nuclear Information System (INIS)

Astor, M.B.; Meister, A.; Anderson, M.E.

1987-01-01

Intracellular thiols such as glutathione (GSH) protect cells against free radicals formed during oxidative metabolism or from exposure to drugs or ionizing radiation. The role of intracellular GSH in the repair of radiation induced free radical damage was studied using GSH or its analog glutathione mono ethyl ester (GEE), which readily penetrates into the cell. Chinese hamster V79 cells with normal GSH levels were afforded equal protection under aerated and hypoxic conditions (DMF = 1.2 OER = 3.7) by both 10 mM GSH and GEE although GEE had raised interacellular GSH levels three-fold. Growth of V79 cells in cysteine free media resulted in undetectable levels of GSH and OER of 2.2 with no change in aerated survival. Restoration of intracellular GSH by 10 mM GEE resulted in an increase of the OER from 2.2. to 3.8 (DMF = 1.7). Only 14% of the intracellular GSH needs to be repleted to give an OER of 3.0. These experiments provide evidence that thiols do play a role in the oxygen effect and are present at levels in excess of what is necessary for maximal radioprotection

Maquiavel Versus Rousseau: as Divisões Sociais e Seu Papel em uma República Bem-Ordenada

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Renato Moscateli

2015-01-01

Full Text Available RESUMO:As relações de conflito entre os grupos sociais constituem um tópico relevante para a filosofia política, e as maneiras distintas como elas são interpretadas dependem de uma visão mais ampla sobre as condições apropriadas a um Estado bem-ordenado. Maquiavel, por exemplo, ao refletir sobre o caso da Roma Antiga, procurou refutar aqueles que condenavam os tumultos entre os nobres e a plebe da cidade, como se eles tivessem provocado apenas males à república. Para o autor, tais tumultos estavam entre as principais causas da liberdade romana, visto que a diferença nos “humores” dos grandes e do povo resultou em embates que deram origem às leis favoráveis à liberdade. Rousseau, por sua vez, descreveu a existência de “associações particulares” dentro da sociedade civil como algo potencialmente nocivo à harmonia da república, pois cada uma delas contém um interesse particular passível de se sobrepor ao bem comum, nas deliberações públicas, prejudicando o prevalecimento da vontade geral. Ainda para o genebrino, a ocorrência de longos debates e de tumultos nas assembleias populares poderia ser um sinal de divisões internas capazes de ocasionar a ruína do Estado. Frente a essas duas maneiras de conceber o papel político dos conflitos sociais, o objetivo deste trabalho é realizar uma análise comparativa das ideias de Maquiavel e de Rousseau.

Las enseñanzas de sordomudos durante la II República Española. Una perspectiva histórica

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Alfredo ALCINA MADUEÑO

2011-07-01

Full Text Available Las enseñanzas de sordomudos en el periodo de la II República española es un tema poco estudiado e inédito en determinados aspectos. Se puede decir que participa de las características de la educación general de dicha etapa: esfuerzo económico y presupuestario de los gobiernos, renovación metodológica, modernización del sistema, ampliación de la red escolar, formación del magisterio, etc., si bien presenta una idiosincrasia muy marcada e incluso contradictoria, no solo por la conocida contraposición de los gobiernos de un bienio y otro (31/33 y 34/35, sino también por las decisiones de política educativa que se toman por los mismos gobiernos de la coalición republicano-socialista. El legado de la aportación republicana es más documental que de realizaciones concretas, y tendrá mayor aplicación en el régimen político que aniquila y sucede a la República que en ella misma. La consideración de fuentes primarias (legales y documentales fundamentan principalmente las conclusiones de esta exposición.

Gene conversion homogenizes the CMT1A paralogous repeats

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Hurles Matthew E

2001-12-01

Full Text Available Abstract Background Non-allelic homologous recombination between paralogous repeats is increasingly being recognized as a major mechanism causing both pathogenic microdeletions and duplications, and structural polymorphism in the human genome. It has recently been shown empirically that gene conversion can homogenize such repeats, resulting in longer stretches of absolute identity that may increase the rate of non-allelic homologous recombination. Results Here, a statistical test to detect gene conversion between pairs of non-coding sequences is presented. It is shown that the 24 kb Charcot-Marie-Tooth type 1A paralogous repeats (CMT1A-REPs exhibit the imprint of gene conversion processes whilst control orthologous sequences do not. In addition, Monte Carlo simulations of the evolutionary divergence of the CMT1A-REPs, incorporating two alternative models for gene conversion, generate repeats that are statistically indistinguishable from the observed repeats. Bounds are placed on the rate of these conversion processes, with central values of 1.3 × 10-4 and 5.1 × 10-5 per generation for the alternative models. Conclusions This evidence presented here suggests that gene conversion may have played an important role in the evolution of the CMT1A-REP paralogous repeats. The rates of these processes are such that it is probable that homogenized CMT1A-REPs are polymorphic within modern populations. Gene conversion processes are similarly likely to play an important role in the evolution of other segmental duplications and may influence the rate of non-allelic homologous recombination between them.

El poder local en la provincia de Albacete durante la II República y el intervencionismo del gobierno, 1931-1936

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Manuel REQUENA

2016-05-01

Full Text Available Este artículo estudia los diversos procesos electorales de carácter municipal durante la II República en la provincia de Albacete, las constantes interferencias del Gobierno de turno sustituyendo los consistorios democráticamente elegidos por gubernamentales, la lucha por el control del poder local y las medidas adoptadas para resolver los problemas locales y la conflictividad generada.

Carbon black nanoparticles promote endothelial activation and lipid accumulation in macrophages independently of intracellular ROS production

DEFF Research Database (Denmark)

Cao, Yi; Roursgaard, Martin; Danielsen, Pernille Høgh

2014-01-01

, the concentrations of CB to induce lipid accumulation were lower than the concentrations to promote intracellular ROS production in THP-1a cells. In conclusion, exposure to nano-sized CB induced endothelial dysfunction and foam cell formation, which was not dependent on intracellular ROS production....... and WST-1 assays, especially in THP-1 and THP-1a cells. The CB exposure decreased the glutathione (GSH) content in THP-1 and THP-1a cells, whereas GSH was increased in HUVECs. The reactive oxygen species (ROS) production was increased in all cell types after CB exposure. A reduction of the intracellular...... GSH concentration by buthionine sulfoximine (BSO) pre-treatment further increased the CB-induced ROS production in THP-1 cells and HUVECs. The expression of adhesion molecules ICAM-1 and VCAM-1, but not adhesion of THP-1 to HUVECs or culture dishes, was elevated by CB exposure, whereas these effects...

Acute effects of nitroglycerin depend on both plasma and intracellular sulfhydryl compound levels in vivo. Effect of agents with different sulfhydryl-modulating properties

DEFF Research Database (Denmark)

Boesgaard, S; Poulsen, H E; Aldershvile, J

1993-01-01

in SH group concentrations (cysteine and glutathione [GSH]) affect the responsiveness to NTG in vivo. METHODS AND RESULTS: GSH and cysteine levels in plasma, vena cava, and aorta were measured after administration of N-acetylserine (placebo, n = 6), N-acetylcysteine (NAC, extracellular and intracellular......BACKGROUND: Changes in sulfhydryl (SH) compound availability may alter the hemodynamic effect of nitroglycerin (NTG). Data on the relation between NTG effect and thiol levels are, however, limited to in vitro experiments. The present study investigates how intracellular and extracellular changes...... SH donor, n = 6), oxothiazolidine (OXO, intracellular SH donor, n = 6), buthionine sulfoximine (BSO, intracellular GSH-depleting agent, n = 6), BSO+NAC (n = 6), and BSO+OXO (n = 6) in chronically catheterized conscious rats. In addition, the effect of 2.5 mg NTG/kg i.v. on mean arterial pressure (MAP...

Alejandro Bunge y el problema de la vivienda obrera en la República Argentina (1910-1915

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Jorge A. Núñez

2010-02-01

Full Text Available Este trabajo pretende indagar, a partir del estudio de la figura del economista Alejandro Bunge, en la problemática de la vivienda obrera en la República Argentina en las primeras décadas de la pasada centuria. Aquí se argumentará que los estudios realizados porAlejandro Bunge como director de la División Estadísticas del Departamento Nacional de Trabajo en el período 1910-1915 contribuyeron de manera notable a la elaboración del proyectoy posterior sanción de la Ley de Casas Baratas para Obreros (n°9677.

La República peregrina: hombres de armas y letras en América Andina, 1810-1884

OpenAIRE

Thibaud, Clément

2014-01-01

Del 18 al 20 de mayo de 2005 se realizó en el Instituto de Estudios Peruanos el coloquio internacional «La República peregrina: Hombres de armas y letras en América Andina, 1810-1884». El evento, co-organizado por el Instituto Francés de Estudios Andinos y el IEP, reunió durante tres días a una veintena de historiadores especialistas en el siglo XIX, provenientes de todos los países andinos —de Chile hasta Venezuela, pasando por Argentina—, pero también de Inglaterra, Francia y Estados Unidos...

A Grande Guerra e as relações internacionais de Portugal: Da Monarquia à República

OpenAIRE

Castaño, David

2014-01-01

Neste artigo procuramos recuperar algumas das ideias que ajudam a explicar a participação portuguesa na Grande Guerra e a opção do Partido Democrático pelo envolvimento do País na frente europeia. Um pleno entendimento da participação portuguesa na Grande Guerra exige não apenas um recuo temporal, mas uma visão abrangente entre dinâmicas internas e externas. Nas primeiras, importa ter em consideração o binómio monarquia/república, e também as profundas divergências que rapidamente dividiram o...

Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

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Sandy Ibanes

Full Text Available When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2 vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

Canine Adenovirus Type 2 Vector Generation via I-Sce1-Mediated Intracellular Genome Release

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Ibanes, Sandy; Kremer, Eric J.

2013-01-01

When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation. PMID:23936483

Single-cell intracellular nano-pH probes†

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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

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.

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....

Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation.

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Hayashi, Kentaro; Yamamoto, Takamasa S; Ueno, Naoto

2018-02-05

During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca 2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca 2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca 2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca 2+ signals play an essential role in the active cell migration during gastrulation.

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

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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

Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures.

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Wesley-Smith, James; Berjak, Patricia; Pammenter, N W; Walters, Christina

2014-03-01

Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival. Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro. Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm(2) in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure. The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches.

Intracellular nitrate in sediments of an oxygen-deficient marine basin is linked to pelagic diatoms

DEFF Research Database (Denmark)

Kamp, Anja; Petro, Caitlin; Røy, Hans

2018-01-01

Intracellular nitrate is an important electron acceptor in oxygen-deficient aquatic environments, either for the nitrate-storing microbes themselves, or for ambient microbial communities through nitrate leakage. This study links the spatial distribution of intracellular nitrate with the abundance...... and identity of nitrate-storing microbes in sediments of the Bornholm Basin, an environmental showcase for severe hypoxia. Intracellular nitrate (up to 270 nmol cm−3 sediment) was detected at all 18 stations along a 35-km transect through the basin and typically extended as deep as 1.6 cm into the sediment...

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein.

Science.gov (United States)

Zhi, Pei; Chia, Pei Zhi Cheryl; Chia, Cheryl; Gleeson, Paul A

2011-09-01

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention. Substantive evidence now indicates that the processing of APP by BACE1 is regulated by the intracellular sorting of the enzyme and, moreover, perturbations in these intracellular trafficking pathways have been linked to late-onset AD. In this review, we highlight the recent advances in the understanding of the regulation of the intracellular sorting of BACE1 and APP and illustrate why the trafficking of these cargos represent a key issue for understanding the membrane-mediated events associated with the generation of the neurotoxic Aβ products in AD. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

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Yi-Chieh Lin

Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

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)

Mapa educativo nacional. Una experiencia en la República Argentina

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Horacio Rodolfo Bozzano

2006-01-01

Full Text Available El Proyecto Mapa Educativo Nacional (MEN es un SIG generado a partir del desarrollo e integración de 24 Mapas Educativos Provinciales sobre la base de un modelo conceptual general, con una visión dinámica de los 49.000 establecimientos educativos, así como de su contexto territorial. Los objetivos del trabajo son: a sistematizar los aportes al Proyecto MEN desde la teoría cartográfica, reconociendo instancias de concepción, construcción y comunicación de los mapas que lo integran, así como vinculaciones entre territorio, espacio y espacialidades emergentes de la concreción de la tarea; y b identificar en qué medida la concreción del Proyecto MEN en la República Argentina contribuye a fortalecer un enfoque dialéctico conocimiento-realidad sobre el territorio y la gestión aplicado a un objeto de estudio e intervención concreto de desarrollo institucional, con el propósito de promover un círculo virtuoso de transformación entre sujetos de cambio y agentes de desarrollo.

Cell internalizable and intracellularly degradable cationic polyurethane micelles as a potential platform for efficient imaging and drug delivery.

Science.gov (United States)

Ding, Mingming; Zeng, Xin; He, Xueling; Li, Jiehua; Tan, Hong; Fu, Qiang

2014-08-11

A cell internalizable and intracellularly degradable micellar system, assembled from multiblock polyurethanes bearing cell-penetrating gemini quaternary ammonium pendent groups in the side chain and redox-responsive disulfide linkages throughout the backbone, was developed for potential magnetic resonance imaging (MRI) and drug delivery. The nanocarrier is featured as a typical "cleavable core-internalizable shell-protective corona" architecture, which exhibits small size, positive surface charge, high loading capacity, and reduction-triggered destabilization. Furthermore, it can rapidly enter tumor cells and release its cargo in response to an intracellular level of glutathione, resulting in enhanced drug efficacy in vitro. The magnetic micelles loaded with superparamagnetic iron oxide (SPIO) nanoparticles demonstrate excellent MRI contrast enhancement, with T2 relaxivity found to be affected by the morphology of SPIO-clustering inside the micelle core. The multifunctional carrier with good cytocompatibility and nontoxic degradation products can serve as a promising theranostic candidate for efficient intracellular delivery of anticancer drugs and real-time monitoring of therapeutic effect.

Intracellular Nitrate of Marine Diatoms as a Driver of Anaerobic Nitrogen Cycling in Sinking Aggregates

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Anja Kamp

2016-11-01

Full Text Available Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations > 60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2-3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations.

Hombres de letras en la provincia. Producción y comercio de libros en la República de Colombia, 1821-1874

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Rafael E. Acevedo P.

2016-01-01

Full Text Available Han sido pocos los estudios en la historiografía nacional sobre el proceso de producción y comercialización de libros en la República de Colombia durante el siglo xix. A partir del análisis de las actividades de los impresores, los escritores de textos, los comerciantes y las autoridades políticas en Cartagena, en este artículo se demostrará que la difusión de las letras en la sociedad republicana estuvo sujeta a la dinámica de impresión y comercialización de obras de educación impulsada por los hombres de letras desde las provincias. Esta dinámica se constituyó en la base inicial de la formación de un pequeño mercado editorial en la República y fue posible gracias a los usos de la libertad de imprenta, las demandas de la alfabetización y las negociaciones de los autores con los gobiernos locales y nacionales. Se busca así contribuir al análisis de un problema sobre el que existen escasos trabajos en el contexto regional colombiano.

Intracellular pH and inorganic phosphate content of heart in vivo: A 31P-NMR study

International Nuclear Information System (INIS)

Katz, L.A.; Swain, J.A.; Portman, M.A.; Balaban, R.S.

1988-01-01

Studies were performed to determine the contribution of red blood cells to the 31 P-nuclear magnetic resonance (NMR) spectrum of the canine heart in vivo and the feasibility of measuring myocardial intracellular phosphate and pH. This was accomplished by replacing whole blood with a perfluorochemical perfusion emulsion blood substitute, Oxypherol, and noting the difference in the 31 P-NMR spectrum of the heart. NMR data were collected with a NMR transmitter-receiver coil on the surface of the distal portion of the left ventricle. These studies demonstrated that a small contribution from 2,3-diphosphoglycerate (2,3-DPG) and phosphodiesters in the blood could be detected. The magnitude and shift of these blood-borne signals permitted the relative quantification of intracellular inorganic phosphate (P i ) content as well as intracellular pH. Under resting conditions, the intracellular ATP/P i was 7.0 ± 0.08. This corresponds to a free intracellular P 1 content of ∼ 0.8 μmol./g wet wt. The intracellular pH was 7.10 ± 0.01. Acute respiratory alkalosis and acidosis, with the arterial pH ranging from ∼7.0 to 7.7, resulted in only small changes in the intracellular pH. These latter results demonstrate an effective myocardial intracellular proton-buffering mechanism in vivo

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

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.

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.

Pharmacodynamic evaluation of the activity of antibiotics against hemin- and menadione-dependent small-colony variants of Staphylococcus aureus in models of extracellular (broth) and intracellular (THP-1 monocytes) infections.

Science.gov (United States)

Garcia, L G; Lemaire, S; Kahl, B C; Becker, K; Proctor, R A; Denis, O; Tulkens, P M; Van Bambeke, F

2012-07-01

Staphylococcus aureus small-colony variants (SCVs) persist intracellularly, which may contribute to persistence/recurrence of infections and antibiotic failure. We have studied the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent SCVs, respectively) of the COL methicillin-resistant S. aureus (MRSA) strain and the antibiotic pharmacodynamic profile against extracellular (broth) and intracellular (human THP-1 monocytes) bacteria. Compared to the parental strain, SCVs showed slower extracellular growth (restored upon medium supplementation with menadione or hemin), reduced phagocytosis, and, for the menD SCV, lower intracellular counts at 24 h postinfection. Against extracellular bacteria, daptomycin, gentamicin, rifampin, moxifloxacin, and oritavancin showed similar profiles of activity against all strains, with a static effect obtained at concentrations close to their MICs and complete eradication as maximal effect. In contrast, vancomycin was not bactericidal against SCVs. Against intracellular bacteria, concentration-effect curves fitted sigmoidal regressions for vancomycin, daptomycin, gentamicin, and rifampin (with maximal effects lower than a 2-log decrease in CFU) but biphasic regressions (with a maximal effect greater than a 3-log decrease in CFU) for moxifloxacin and oritavancin, suggesting a dual mode of action against intracellular bacteria. For all antibiotics, these curves were indistinguishable between the strains investigated, except for the menD mutant, which systematically showed a lower amplitude of the concentration-effect response, with markedly reduced minimal efficacy (due to slower growth) but no change in maximal efficacy. The data therefore show that the maximal efficacies of antibiotics are similar against normal-phenotype and menadione- and hemin-dependent strains despite their different intracellular fates, with oritavancin, and to some extent moxifloxacin, being the most effective.

Co-stimulation through 4-1BB/CD137 improves the expansion and function of CD8(+ melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy.

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Jessica Ann Chacon

Full Text Available Adoptive T-cell therapy (ACT using tumor-infiltrating lymphocytes (TIL can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the "rapid expansion protocol" (REP were not designed to enhance the generation of optimal effector-memory CD8(+ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8(+ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8(+ T cells, on the yield, phenotype, and functional activity of expanded CD8(+ T cells during the REP. We found that CD8(+ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG4 (BMS 663513 to the REP significantly enhanced the frequency and total yield of CD8(+ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8(+ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8(+ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.

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.

João do Rio, Repórter da Pobreza na Cidade

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Denise da Costa Oliveira Siqueira

2004-06-01

Full Text Available Tendo como modelo a Belle Époque parisiense, o Rio de Janeiro, na virada do século XIX para o XX, sofria a transição para uma ordem capitalista urbana. Nesse contexto, o escritor e repórter João Paulo Barreto - o João do Rio - vagou pela cidade, observou parcelas da sociedade que raramente figuravam nas páginas de livros e jornais. No livro A Alma Encantadora das Ruas dedicou um capítulo às várias formas de pobreza e exploração presentes na capital federal. A partir dessa temática, o objetivo deste artigo é estudar as representações da pobreza como apresentadas no livro de João do Rio e paralelamente abordar a questão do texto jornalístico como mediador simbólico - reflexo de um tempo e de uma cultura.

Mesurements of intracellular ATP provide new insight into the regulation of glycolysis in the yeast Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Ytting, Cecilie Karkov; Fuglsang, Anja Thoe; Hiltunen, J. Kalervo

2012-01-01

Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F0F1 ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating...... of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal...... activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose...

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

Heat and exercise acclimation increases intracellular levels of Hsp72 and inhibits exercise-induced increase in intracellular and plasma Hsp72 in humans.

Science.gov (United States)

Magalhães, Flávio de Castro; Amorim, Fabiano Trigueiro; Passos, Renata L Freitas; Fonseca, Michele Atalla; Oliveira, Kenya Paula Moreira; Lima, Milene Rodrigues Malheiros; Guimarães, Juliana Bohen; Ferreira-Júnior, João Batista; Martini, Angelo R P; Lima, Nilo R V; Soares, Danusa Dias; Oliveira, Edilamar Menezes; Rodrigues, Luiz Oswaldo Carneiro

2010-11-01

In order to verify the effects of heat and exercise acclimation (HA) on resting and exercise-induced expression of plasma and leukocyte heat shock protein 72 (Hsp72) in humans, nine healthy young male volunteers (25.0 ± 0.7 years; 80.5 ± 2.0 kg; 180 ± 2 cm, mean ± SE) exercised for 60 min in a hot, dry environment (40 ± 0°C and 45 ± 0% relative humidity) for 11 days. The protocol consisted of running on a treadmill using a controlled hyperthermia technique in which the work rate was adjusted to elevate the rectal temperature by 1°C in 30 min and maintain it elevated for another 30 min. Before and after the HA, the volunteers performed a heat stress test (HST) at 50% of their individual maximal power output for 90 min in the same environment. Blood was drawn before (REST), immediately after (POST) and 1 h after (1 h POST) HST, and plasma and leukocytes were separated and stored. Subjects showed expected adaptations to HA: reduced exercise rectal and mean skin temperatures and heart rate, and augmented sweat rate and exercise tolerance. In HST1, plasma Hsp72 increased from REST to POST and then returned to resting values 1 h POST (REST: 1.11 ± 0.07, POST: 1.48 ± 0.10, 1 h POST: 1.22 ± 0.11 ng mL(-1); p  0.05). HA increased resting levels of intracellular Hsp72 (HST1: 1 ± 0.02 and HST2: 4.2 ± 1.2 density units, p  0.05). Regression analysis showed that the lower the pre-exercise expression of intracellular Hsp72, the higher the exercise-induced increase (R = -0.85, p < 0.05). In conclusion, HA increased resting leukocyte Hsp72 levels and inhibited exercise-induced expression. This intracellular adaptation probably induces thermotolerance. In addition, the non-increase in plasma Hsp72 after HA may be related to lower stress at the cellular level in the acclimated individuals.

Lack of association of interferon regulatory factor 1 with severe malaria in affected child-parental trio studies across three African populations.

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Valentina D Mangano

Full Text Available Interferon Regulatory Factor 1 (IRF-1 is a member of the IRF family of transcription factors, which have key and diverse roles in the gene-regulatory networks of the immune system. IRF-1 has been described as a critical mediator of IFN-gamma signalling and as the major player in driving TH1 type responses. It is therefore likely to be crucial in both innate and adaptive responses against intracellular pathogens such as Plasmodium falciparum. Polymorphisms at the human IRF1 locus have been previously found to be associated with the ability to control P. falciparum infection in populations naturally exposed to malaria. In order to test whether genetic variation at the IRF1 locus also affects the risk of developing severe malaria, we performed a family-based test of association for 18 Single Nucleotide Polymorphisms (SNPs across the gene in three African populations, using genotype data from 961 trios consisting of one affected child and his/her two parents (555 from The Gambia, 204 from Kenya and 202 from Malawi. No significant association with severe malaria or severe malaria subphenotypes (cerebral malaria and severe malaria anaemia was observed for any of the SNPs/haplotypes tested in any of the study populations. Our results offer no evidence that the molecular pathways regulated by the transcription factor IRF-1 are involved in the immune-based pathogenesis of severe malaria.

Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

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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.

A Thapsigargin-Resistant Intracellular Calcium Sequestering Compartment in Rat Brain

Science.gov (United States)

2000-03-31

have a major impact on neuronal intracellular signaling. Most of the ER in neurons and glia appears to accumulate calcium by energy driven ion pumps...secretion of exocrine, endocrine, and neurocrine products, regulation of glycogenolysis and gluconeogenesis , intracellular transport, secretion of fluids...the RyRs [140]. Furthermore, the intracellular expression of these receptor-channels in neuronal ER is also reciprocal with RyRs located primarily in

Potestades Administrativas para Denegar Solicitudes de Derechos de Aguas Subterráneas y Retroactividad: Dictamen N° 5.215, de la Contraloría General de la República, de 31 de enero de 2006

OpenAIRE

Vergara Blanco, Alejandro

2007-01-01

Comenta favorablemente el autor un dictamen de la Contraloría General de la República relativo al otorgamiento de derechos de aprovechamiento de aguas por la Dirección General de Aguas, en el cual se precisan las causales de denegación de su concesión y asimismo se aclara la limitada retroactividad de la reciente Ley Nº 20.017, de 2005, modificatoria del Código de Aguas. The autor comments favourably upon a decision from the Contraloría General de la República (the Chilean Authority whose ...

Un ministro para la Defensa de la República desde el exilio : Juan Hernández Saravia

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Manuela Aroca Mohedano

2004-01-01

Full Text Available La nueva situación internacionai surgida tras el final de la II Guerra Mundial propició la recuperación de las instituciones republicanas españolas en el exilio. Juan Hernández Saravia, un militar profesional, azañista y republicano, será el ministro de la Defensa Nacional de la República Española. Entre posibilidades reales y utopías, a caballo entre la defensa a ultranza de la legalidad y la protección de los movimientos clandestinos antifranquistas, en medio de las disensiones de los militares exiliados dispersos en varios continentes, discurrirá la actuación de un Ministerio que tendrá como objetivo principal la recuperación del ejército para la futura restaurada República y que verá truncadas sus expectativas por la aparición en el panorama mundial de los primeros.A Minister in exile for tfie Republic Defence: Juan Hernández Saravia The new International situation arisen after the end of the Second World War led to the recovery of Spanish Republic Institutions in exile. Juan Hernández Saravia, an army officer, republican and adherent to Azaña, became the National Defence Minister of Spanish Republic. Among real possibilities and utopia, halfway between a vigorous legality defence and the protection of clandestino movements against Franco's dictatorship, the main objective of this Ministery was the army recovery for the futuro restoring Republic, but the early Cold War shattered these prospects.

Intensification of Organophosphorus Hydrolase Synthesis by Using Substances with Gas-Transport Function

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Olga Senko

2017-12-01

Full Text Available We have performed studies and comparative analysis of the biosynthesis characteristics of intracellular recombinant enzyme, such as hexahistidine-containing organophosphorus hydrolase (His6-OPH in Escherichia coli SG13009[pREP4] cells when various perfluorocarbon compounds (PFC were introduced into the medium for cell cultivation. The PFC were found to facilitate the biosynthesis of His6-OPH: increased levels of the total OPH-activity (up to 37% were measured upon introduction of 1,1,1,2,2,3,3,4,4,5,5,6,6,6-tetradecafluorohexane (PFH and 4,7,10,13,16,19,22,25,28,31-decaoxaperfluoro-5,8,11,14,17,18,21,24,27,30-decamethyl tetratriacontane (Polyether II into culture medium. We have demonstrated the possibility of effective and multiple (at least five-fold use of PFH for biosynthesis of intracellular recombinant protein His6-OPH, which catalyzes the hydrolysis of organophosphorus pesticides (OP, is widely used in agriculture and can be applied as new antidote for OP-detoxification in vivo. The multiple use of PFH was achieved through recycling of this substance: sediment of Escherichia coli SG13009[pREP4] cell biomass was collected at the end of each culture growing step and disintegrated with ultrasound, and obtained residue containing almost all of the initially introduced PFC was then added to the medium at the start of the following culture growing step.

Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

Science.gov (United States)

Durmaz, Evelyn; Hu, Yan; Aroian, Raffi V.

2015-01-01

The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe. PMID:26682852

Type IV pili in Francisella – A virulence trait in an intracellular pathogen

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Emelie eNäslund Salomonsson

2011-02-01

Full Text Available Francisella tularensis is a highly virulent intracellular human pathogen that is capable of rapid proliferation in the infected host. Mutants affected in intracellular survival and growth are highly attenuated which highlights the importance of the intracellular phase of the infection. Genomic analysis has revealed that Francisella encodes all genes required for expression of functional type IV pili (Tfp, and in this focused review we summarise recent findings regarding this system in the pathogenesis of tularemia. Tfp are dynamic adhesive structures that have been identified as major virulence determinants in several human pathogens, but it is not obvious what role these structures could have in an intracellular pathogen like Francisella. In the human pathogenic strains, genes required for secretion and assembly of Tfp and one pilin, PilA, have shown to be required for full virulence. Importantly, specific genetic differences have been identified between the different Francisella subspecies where in the most pathogenic type A variants all genes are intact while several Tfp genes are pseudogenes in the less pathogenic type B strains. This suggests that there has been a selection for expression of Tfp with different properties in the different subspecies. There is also a possibility that the genetic differences reflect adaption to different environmental niches of the subspecies and plays a role in transmission of tularemia. This is also in line with recent findings where Tfp pilins are found to be glycosylated which could reflect a role for Tfp in the environment to promote survival and transmission. We are still far from understanding the role of Tfp in virulence and transmission of tularemia, but with the genomic information and genetic tools available we are in a good position to address these issues in the future.

Nuclear magnetic resonance studies of intracellular ions in perfused from heart

International Nuclear Information System (INIS)

Burnstein, D.; Fossel, E.T.

1987-01-01

Intracellular sodium, potassium, and lithium were observed in a perfused frog heart by nuclear magnetic resonance (NMR) spectroscopy. A perfusate buffer containing the shift reagent, dysprosium tripolyphosphate, was used in combination with mathematical filtering or presaturation of the extracellular resonance to separate the intra- and extracellular sodium NMR signals. Addition of 10 μM ouabain to the perfusate, perfusion with a zero potassium, low-calcium buffer, and replacement of 66% of the perfusate sodium with lithium resulted in changes in the intracellular sodium levels. An increase of 45% in the intracellular sodium was observed when changing the pacing rate from 0 to 60 beats/min (with proportional changes for intermediate pacing rates). The ratio of intracellular potassium to sodium concentration was determined to be 2.3 by NMR, indicating that a substantial amount of the intracellular potassium is undetectable with these NMR method. In addition, intracellular lithium was observed during perfusion with a lithium-containing perfusate

Impact of adrenaline and metabolic stress on exercise-induced intracellular signaling and PGC-1α mRNA response in human skeletal muscle

DEFF Research Database (Denmark)

Brandt, Nina; Gunnarsson, Thomas Gunnar Petursson; Hostrup, Morten

2016-01-01

This study tested the hypothesis that elevated plasma adrenaline or metabolic stress enhances exercise-induced PGC-1α mRNA and intracellular signaling in human muscle. Trained (VO2-max: 53.8 ± 1.8 mL min(-1) kg(-1)) male subjects completed four different exercise protocols (work load of the legs...... exercise than at rest in all protocols, and higher (P adrenaline nor muscle metabolic stress determines the magnitude of PGC-1α mRNA response in human muscle. Furthermore, higher exercise-induced changes in AMPK, p38, and CREB...

Intracellular pH homeostasis in Leishmania donovani amastigotes and promastigotes

International Nuclear Information System (INIS)

Glaser, T.A.; Baatz, J.E.; Kreishman, G.P.; Mukkada, A.J.

1988-01-01

Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31 P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

Science.gov (United States)

Ericson, Megan E.; Frank, Matthew W.

2016-01-01

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

Identification of CTX-M15-, SHV-28-producing Klebsiella pneumoniae ST15 as an epidemic clone in the Copenhagen area using a semi-automated Rep-PCR typing assay

DEFF Research Database (Denmark)

Nielsen, J B; Skov, M N; Jørgensen, R L

2011-01-01

pneumoniae (K. pneumoniae) producing extended spectrum β-lactamases (ESBLs) and their relation to recognized Danish outbreak strains. PFGE and Rep-PCR produced similar clustering among isolates. Individual isolates from each cluster were further characterized by PCR amplification and sequencing of bla (TEM......), bla (SHV), and bla (CTX-M), and multilocus sequence typing (MLST). Thirty-five out of 52 ESBL-producing K. pneumoniae isolates were ST15 and bla (CTX-M15), bla (SHV-28), and bla (TEM-1) positive by PCR. Ten out of 52 were ST16 and tested positive for bla (CTX-M15), bla (SHV-1), and bla (TEM-1...

U.S. Rep. Dave Weldon outside the U.S. Lab Destiny in the SSPF.

Science.gov (United States)

1999-01-01

In the Space Station Processing Facility, U.S. Rep Dave Weldon (at left) looks at the U.S. Lab, called Destiny. With him are Thomas R. 'Randy' Galloway, with the Space Station Hardware Integration Office, Dana Gartzke, the congressman's chief of staffm and Boeing workers. Weldon is on the House Science Committee and vice chairman of the Space and Aeronautics Subcommittee. Destiny is scheduled to be launched on Space Shuttle Endeavour in early 2000. It will become the centerpiece of scientific research on the ISS, with five equipment racks aboard to provide essential functions for station systems, including high data-rate communications, and to maintain the station's orientation using control gyroscopes launched earlier. Additional equipment and research racks will be installed in the laboratory on subsequent Shuttle flights.

Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

Science.gov (United States)

Bouwer, H. G. Archie; Alberti-Segui, Christine; Montfort, Megan J.; Berkowitz, Nathan D.; Higgins, Darren E.

2006-03-01

We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8+ effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8+ effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens. CD8+ T cell | replication-deficient | Listeria monocytogenes

Controlled intracellular self-assembly of gadolinium nanoparticles as smart molecular MR contrast agents.

Science.gov (United States)

Cao, Chun-Yan; Shen, Ying-Ying; Wang, Jian-Dong; Li, Li; Liang, Gao-Lin

2013-01-01

Herein we developed a new "smart" Gd-based MR contrast agent (i.e., 1) which is susceptive to furin, a protease overexpressed in tumor. Under the action of furin, 1 condenses to form dimers (1-Ds) and the latter self-assemble into gadolinium nanparticles (Gd-NPs). Relaxivity of 1-D is more than 2 folds of those of 1 and magnevist at 1.5 T, and 1.4 folds of that of 1 at 3 T. Intracellular condensation of 1 in furin-overexpressed MDA-MB-468 cells was proven with direct two-photon laser microscopy (TPLM) fluorescence imaging of the cells incubated with the europium analog of 1 (i.e., 2). Intracellular Gd-NPs of 1 were uncovered and characterized for the first time. MRI of MDA-MB-468 tumors showed that 1 has enhanced MR contrast within the tumors than that of its scrambled control 1-Scr.

Conditions With High Intracellular Glucose Inhibit Sensing Through Glucose Sensor Snf3 in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Karhumaa, Kaisa; Wu, B.Q.; Kielland-Brandt, Morten

2010-01-01

as for amino acids. An alternating-access model of the function of transporter-like sensors has been previously suggested based on amino acid sensing, where intracellular ligand inhibits binding of extracellular ligand. Here we studied the effect of intracellular glucose on sensing of extracellular glucose...... through the transporter-like sensor Snf3 in yeast. Sensing through Snf3 was determined by measuring degradation of Mth1 protein. High intracellular glucose concentrations were achieved by using yeast strains lacking monohexose transporters which were grown on maltose. The apparent affinity...... of extracellular glucose to Snf3 was measured for cells grown in non-fermentative medium or on maltose. The apparent affinity for glucose was lowest when the intracellular glucose concentration was high. The results conform to an alternating-access model for transporter-like sensors. J. Cell. Biochem. 110: 920...

Differential intracellular calcium influx, nitric oxide production, ICAM-1 and IL8 expression in primary bovine endothelial cells exposed to nonesterified fatty acids.

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Loaiza, Anitsi; Carretta, María D; Taubert, Anja; Hermosilla, Carlos; Hidalgo, María A; Burgos, Rafael A

2016-02-25

Nonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells. However, the mechanisms underlying these effects are still unknown. The aim of this study was to assess the effects of NEFAs on the intracellular calcium (Ca(2+) i) influx, nitric oxide production, and ICAM-1 and IL-8 expression in primary bovine umbilical vein endothelial cells (BUVECs). Myristic (MA), palmitic (PA), stearic (SA), oleic (OA) and linoleic acid (LA) rapidly increased Ca(2+) i. The calcium response to all tested NEFAs showed an extracellular calcium dependence and only the LA response was significantly inhibited until the intracellular calcium was chelated. The EC50 values for MA and LA were 125 μM and 37 μM, respectively, and the MA and LA effects were dependent on calcium release from the endoplasmic reticulum stores and on the L-type calcium channels. Only the calcium response to MA was significantly reduced by GW1100, a selective G-protein-coupled free fatty acid receptor (GPR40) antagonist. We also detected a functional FFAR1/GPR40 protein in BUVECs by using western blotting and the FFAR1/GPR40 agonist TAK-875. Only LA increased the cellular nitric oxide levels in a calcium-dependent manner. LA stimulation but not MA stimulation increased ICAM-1 and IL-8-expression in BUVECs. This effect was inhibited by GW1100, an antagonist of FFAR1/GPR40, but not by U-73122, a phospholipase C inhibitor. These findings strongly suggest that each individual NEFA stimulates endothelial cells in a different way, with clearly different effects on intracellular calcium mobilization, NO production, and IL-8 and ICAM-1 expression in primary BUVECs. These findings not only extend our understanding of NEFA-mediated diseases in ruminants, but also provide new insight into the different molecular mechanisms involved during endothelial cell activation by NEFAs.

Intracellular Enzymes Contribution to the Biocatalytic Removal of Pharmaceuticals by Trametes hirsuta.

Science.gov (United States)

Haroune, Lounès; Saibi, Sabrina; Cabana, Hubert; Bellenger, Jean-Philippe

2017-01-17

The use of white rot fungi (WRF) for bioremediation of recalcitrant trace organic contaminants (TrOCs) is becoming greatly popular. Biosorption and lignin modifying enzymes (LMEs) are the most often reported mechanisms of action. Intracellular enzymes, such as cytochrome P450 (CYP450), have also been suggested to contribute. However, direct evidence of TrOCs uptake and intracellular transformation is lacking. The aim of this study was to evaluate the relative contribution of biosorption, extracellular LMEs activity, TrOCs uptake, and intracellular CYP450 on the removal of six nonsteroidal anti-inflammatories (NSAIs) by Trametes hirsuta. Results show that for most tested NSAIs, LMEs activity and biosorption failed to explain the observed removal. Most tested TrOCs are quickly taken up and intracellularly transformed. Fine characterization of intracellular transformation using ketoprofen showed that CYP450 is not the sole intracellular enzyme responsible for intracellular transformation. The contribution of CYP450 in further transformation of ketoprofen byproducts is also reported. These results illustrate that TrOCs transformation by WRF is a more complex process than previously reported. Rapid uptake of TrOCs and intracellular transformation through diverse enzymatic systems appears to be important components of WRF efficiency toward TrOCs.

Alterations of the intracellular peptidome in response to the proteasome inhibitor bortezomib.

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Julia S Gelman

Full Text Available Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours, and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50-500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug.

Influencia del campo magnético sobre el crecimiento de microorganismos patógenos ambientales aislados en el Archivo Nacional de la República de Cuba

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Matilde Anaya

2015-09-01

Conclusión. Se concluyó que el campo magnético aplicado tuvo un efecto estimulante sobre los microorganismos estudiados, lo cual potencia el riesgo para la salud del personal y los visitantes del Archivo Nacional de la República de Cuba.

Heme oxygenase-1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells.

Science.gov (United States)

Stepniewski, Jacek; Pacholczak, Tomasz; Skrzypczyk, Aniela; Ciesla, Maciej; Szade, Agata; Szade, Krzysztof; Bidanel, Romain; Langrzyk, Agnieszka; Grochowski, Radoslaw; Vandermeeren, Felix; Kachamakova-Trojanowska, Neli; Jez, Mateusz; Drabik, Grazyna; Nakanishi, Mahito; Jozkowicz, Alicja; Dulak, Jozef

2018-02-01

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1 -/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1 +/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1 +/+ and Hmox1 -/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2 +/+ and Nfe2l2 -/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

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.

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

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

Short-term acute hypercapnia affects cellular responses to trace metals in the hard clams Mercenaria mercenaria

International Nuclear Information System (INIS)

Ivanina, Anna V.; Beniash, Elia; Etzkorn, Markus; Meyers, Tiffany B.; Ringwood, Amy H.; Sokolova, Inna M.

2013-01-01

Highlights: •P CO 2 alters accumulation of Cd and Cu in clam cells. •Accumulation of Cd induces release of free Zn 2+ . •Accumulation of Cu induces an increase in free Cu 2+ and Fe 2+ . •Metal-induced oxidative stress is alleviated at high P CO 2 . •Toxicity of Cu in likely enhanced while that of Cd alleviated by high P CO 2 . -- Abstract: Estuarine and coastal habitats experience large fluctuations of environmental factors such as temperature, salinity, partial pressure of CO 2 (P CO 2 ) and pH; they also serve as the natural sinks for trace metals. Benthic filter-feeding organisms such as bivalves are exposed to the elevated concentrations of metals in estuarine water and sediments that can strongly affect their physiology. The effects of metals on estuarine organisms may be exacerbated by other environmental factors. Thus, a decrease in pH caused by high P CO 2 (hypercapnia) can modulate the effects of trace metals by affecting metal bioavailability, accumulation or binding. To better understand the cellular mechanisms of interactions between P CO 2 and trace metals in marine bivalves, we exposed isolated mantle cells of the hard clams (Mercenaria mercenaria) to different levels of P CO 2 (0.05, 1.52 and 3.01 kPa) and two major trace metal pollutants – cadmium (Cd) and copper (Cu). Elevated P CO 2 resulted in a decrease in intracellular pH (pH i ) of the isolated mantle cells from 7.8 to 7.4. Elevated P CO 2 significantly but differently affected the trace metal accumulation by the cells. Cd uptake was suppressed at elevated P CO 2 levels while Cu accumulation has greatly accelerated under hypercapnic conditions. Interestingly, at higher extracellular Cd levels, labile intracellular Cd 2+ concentration remained the same, while intracellular levels of free Zn 2+ increased suggesting that Cd 2+ substitutes bound Zn 2+ in these cells. In contrast, Cu exposure did not affect intracellular Zn 2+ but led to a profound increase in the intracellular levels

Work-related pain in extrinsic finger extensor musculature of instrumentalists is associated with intracellular pH compartmentation during exercise.

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Angel Moreno-Torres

Full Text Available BACKGROUND: Although non-specific pain in the upper limb muscles of workers engaged in mild repetitive tasks is a common occupational health problem, much is unknown about the associated structural and biochemical changes. In this study, we compared the muscle energy metabolism of the extrinsic finger extensor musculature in instrumentalists suffering from work-related pain with that of healthy control instrumentalists using non-invasive phosphorus magnetic resonance spectroscopy ((31P-MRS. We hypothesize that the affected muscles will show alterations related with an impaired energy metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We studied 19 volunteer instrumentalists (11 subjects with work-related pain affecting the extrinsic finger extensor musculature and 8 healthy controls. We used (31P-MRS to find deviations from the expected metabolic response to exercise in phosphocreatine (PCr, inorganic phosphate (Pi, Pi/PCr ratio and intracellular pH kinetics. We observed a reduced finger extensor exercise tolerance in instrumentalists with myalgia, an intracellular pH compartmentation in the form of neutral and acid compartments, as detected by Pi peak splitting in (31P-MRS spectra, predominantly in myalgic muscles, and a strong association of this pattern with the condition. CONCLUSIONS/SIGNIFICANCE: Work-related pain in the finger extrinsic extensor muscles is associated with intracellular pH compartmentation during exercise, non-invasively detectable by (31P-MRS and consistent with the simultaneous energy production by oxidative metabolism and glycolysis. We speculate that a deficit in energy production by oxidative pathways may exist in the affected muscles. Two possible explanations for this would be the partial and/or local reduction of blood supply and the reduction of the muscle oxidative capacity itself.

Work-Related Pain in Extrinsic Finger Extensor Musculature of Instrumentalists Is Associated with Intracellular pH Compartmentation during Exercise

Science.gov (United States)

Moreno-Torres, Angel; Rosset-Llobet, Jaume; Pujol, Jesus; Fàbregas, Sílvia; Gonzalez-de-Suso, Jose-Manuel

2010-01-01

Background Although non-specific pain in the upper limb muscles of workers engaged in mild repetitive tasks is a common occupational health problem, much is unknown about the associated structural and biochemical changes. In this study, we compared the muscle energy metabolism of the extrinsic finger extensor musculature in instrumentalists suffering from work-related pain with that of healthy control instrumentalists using non-invasive phosphorus magnetic resonance spectroscopy (31P-MRS). We hypothesize that the affected muscles will show alterations related with an impaired energy metabolism. Methodology/Principal Findings We studied 19 volunteer instrumentalists (11 subjects with work-related pain affecting the extrinsic finger extensor musculature and 8 healthy controls). We used 31P-MRS to find deviations from the expected metabolic response to exercise in phosphocreatine (PCr), inorganic phosphate (Pi), Pi/PCr ratio and intracellular pH kinetics. We observed a reduced finger extensor exercise tolerance in instrumentalists with myalgia, an intracellular pH compartmentation in the form of neutral and acid compartments, as detected by Pi peak splitting in 31P-MRS spectra, predominantly in myalgic muscles, and a strong association of this pattern with the condition. Conclusions/Significance Work-related pain in the finger extrinsic extensor muscles is associated with intracellular pH compartmentation during exercise, non-invasively detectable by 31P-MRS and consistent with the simultaneous energy production by oxidative metabolism and glycolysis. We speculate that a deficit in energy production by oxidative pathways may exist in the affected muscles. Two possible explanations for this would be the partial and/or local reduction of blood supply and the reduction of the muscle oxidative capacity itself. PMID:20161738

Intracellular Complement Activation Sustains T Cell Homeostasis and Mediates Effector Differentiation

Science.gov (United States)

Liszewski, M. Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G.; Fara, Antonella F.; Subias, Marta; Pickering, Matthew C.; Drouet, Christian; Meri, Seppo; Arstila, T. Petteri; Pekkarinen, Pirkka T.; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P.; Kemper, Claudia

2013-01-01

Summary Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While “tonic” intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance. PMID:24315997

Expression of the potential therapeutic target CXXC5 in primary acute myeloid leukemia cells - high expression is associated with adverse prognosis as well as altered intracellular signaling and transcriptional regulation.

Science.gov (United States)

Bruserud, Øystein; Reikvam, Håkon; Fredly, Hanne; Skavland, Jørn; Hagen, Karen-Marie; van Hoang, Tuyen Thy; Brenner, Annette K; Kadi, Amir; Astori, Audrey; Gjertsen, Bjørn Tore; Pendino, Frederic

2015-02-20

The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication.

Aliados, tránsfugas y barrilitos: las elecciones legislativas de 2010 en República Dominicana

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Ana Belén BENITO SÁNCHEZ

2011-03-01

Full Text Available Este artículo analiza las claves explicativas de los resultados de las elecciones legislativas y municipales de 2010 en República Dominicana así como de la reciente transformación que ha experimentado el sistema de partidos dominicano, del tripartidismo al bipartidismo plural. El liderazgo del presidente Leonel Fernández, su imagen, popularidad y pragmatismo son la clave del éxito electoral del PLD, favorecido también por el faccionalismo endémico del PRD y la descomposición progresiva del PRSC. En esta contienda, el PLD consolida su dominio electoral gracias al apoyo de los partidos minoritarios y se asegura el control del Legislativo durante los próximos seis años.

El debate entre liberales y conservadores en Centroamérica : distintos medios para un objetivo connún, la construcción de una república de ciudadanos (1821-1900

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Sonia Alda Mejías

2000-01-01

Full Text Available La aspiración común de los liberales y los conservadores en Centroamérica en el siglo XIX fue construir una república representativa de ciudadanos instruidos y formados. La diferencia radico en cómo realizar este proceso de transformación. Este debate determinó en buena parte la evolución de la ciudadanía y los diferentes modelos de república implantados a lo largo del siglo XIX en la región. Mediante el estudio comparado de la legislación electoral y los textos constitucionales se comprueba la existencia de tres modelos de república diferentes en coherencia con la forma en que ambos grupos concibieron la forma, el ritmo y los medios para lograr un objetivo compartido por ambos.The common goal of liberals and conservatives In Centroamérica during the nineteeth century was the construction of a representative republic constituted by formed and instructed citizens. Tfie difference among them was how to perform this transformation process. The debate determined basically the evolution of citizenship and the distinctive modela of republican order implanted during the century. Through the comparative analysis of electoral legislatlon and ttie Cartas Magnas can be preved tfie existence of three different republican models according withi tlie common goal of the two already mentioned groups.

26 CFR 1.806-2 - Taxable years affected.

Science.gov (United States)

2010-04-01

... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Taxable years affected. 1.806-2 Section 1.806-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Investment Income § 1.806-2 Taxable years affected. Section 1.806-1 is applicable only to...

Quantitative measurement of intracellular transport of nanocarriers by spatio-temporal image correlation spectroscopy

Science.gov (United States)

Coppola, S.; Pozzi, D.; Candeloro De Sanctis, S.; Digman, M. A.; Gratton, E.; Caracciolo, G.

2013-03-01

Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method for characterizing the mode of motion of nanocarriers and for quantifying their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP-DOPC/DNA (DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane; DOPC: dioleoylphosphocholine) and DC-Chol-DOPE/DNA (DC-Chol: 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol; DOPE: dioleoylphosphatidylethanolamine) lipoplexes in CHO-K1 (CHO: Chinese hamster ovary) live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient: D ≈ 0.003 μm2 s-1). In the cytosol, the lipoplexes’ motion was characterized by active transport with average velocity v ≈ 0.03 μm2 s-1 and random motion. The method permitted us to generate an intracellular transport map showing several regions of concerted motion of lipoplexes.

Quantitative measurement of intracellular transport of nanocarriers by spatio-temporal image correlation spectroscopy

International Nuclear Information System (INIS)

Coppola, S; Pozzi, D; De Sanctis, S Candeloro; Caracciolo, G; Digman, M A; Gratton, E

2013-01-01

Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method for characterizing the mode of motion of nanocarriers and for quantifying their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP–DOPC/DNA (DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane; DOPC: dioleoylphosphocholine) and DC-Chol–DOPE/DNA (DC-Chol: 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol; DOPE: dioleoylphosphatidylethanolamine) lipoplexes in CHO-K1 (CHO: Chinese hamster ovary) live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient: D ≈ 0.003 μm 2 s −1 ). In the cytosol, the lipoplexes’ motion was characterized by active transport with average velocity v ≈ 0.03 μm 2 s −1 and random motion. The method permitted us to generate an intracellular transport map showing several regions of concerted motion of lipoplexes. (paper)

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

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

International Nuclear Information System (INIS)

Matsumae, Yoshiharu; Takahashi, Yasufumi; Ino, Kosuke; Shiku, Hitoshi; Matsue, Tomokazu

2014-01-01

Graphical abstract: NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells were evaluated by using the menadione–ferrocyanide double mediator system combined with scanning electrochemical microscopy (SECM). - Highlights: • NAD(P)H:quinone oxidoreductase activity of single cells were evaluated with SECM. • Fe(CN) 6 3− /menadione concentrations were optimized for long-term SECM monitoring. • Menadione affect the intracellular levels of reactive oxygen species and GSH. • At 100 μM menadione, the Fe(CN) 6 3− generation rate decreased rapidly within 30 min. - Abstract: We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

Energy Technology Data Exchange (ETDEWEB)

Matsumae, Yoshiharu [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Takahashi, Yasufumi [Advanced Institute for Materials Research, Tohoku University, Katahira 2-1-1, Aoba, Sendai 980-8577 (Japan); Ino, Kosuke [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Advanced Institute for Materials Research, Tohoku University, Katahira 2-1-1, Aoba, Sendai 980-8577 (Japan)

2014-09-09

Graphical abstract: NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells were evaluated by using the menadione–ferrocyanide double mediator system combined with scanning electrochemical microscopy (SECM). - Highlights: • NAD(P)H:quinone oxidoreductase activity of single cells were evaluated with SECM. • Fe(CN){sub 6}{sup 3−}/menadione concentrations were optimized for long-term SECM monitoring. • Menadione affect the intracellular levels of reactive oxygen species and GSH. • At 100 μM menadione, the Fe(CN){sub 6}{sup 3−} generation rate decreased rapidly within 30 min. - Abstract: We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system.

Contribuciones de la Inspección de primera enseñanza a la estructura pedagógico-educativa de la Segunda República española

OpenAIRE

Jiménez Eguizábal, J. Alfredo

2017-01-01

1. Introducción La Segunda República, a juzgar por la corriente de estudios de que es objeto en la actualidad, se revela como un ciclo sumamente fecundo para la sociedad española y, particularmente, para el corpus pedagógico en general por el relieve que la educación adquiere dentro del sistema social, político e, incluso, económico. La comunicación que presentamos aborda un capítulo, a nuestro modo de ver, importante y significativo, al que hasta ahora no se le había prestado la debida atenc...

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...

Study on the inactivation of intracellular enzyme molecules by X-ray irradiation

International Nuclear Information System (INIS)

Lee, S.B.

1977-01-01

Inactivation of the glutamic acid dehydrogenase and glucose-6-phosphate dehydrogenase enzyme molecules in the Ehrlich ascites tumor cells of the mouse were studied. The above mentioned intracellular enzyme molecules were irradiated by the X-ray radiation under the condition of 65 kV, 1 Amp under the atmosphere of nitrogen gases and by 4 0 C. Thereby, irradiation doses were 580 KR/min(error: +-3%). After irradiation, the cell homogentes were prepared through liquid air techniques. There after, the activities of the enzymes were measured with photometric method given by O. Warburg and W. Christian. The dose effect curves of the activities of the two enzymes by the X-ray irradiation showed both exponential and the inactivation doses were 6.5x10 6 and 5.0x10 6 R respectively. These results showed one side that the inactivation process of the intracellular enzyme molecules was one hit reaction after target theory, and the other side that this inactivation process could not be the primary causes of the death through X-ray irradiation of the vertebrate animals, because of the high resistance of the intracellular protein molecules against X-ray irradiation. The one hit reaction by the inactivation process of the irradiated intracellular enzyme molecules was discussed. (author)

Conjugated Bilirubin Differentially Regulates CD4+ T Effector Cells and T Regulatory Cell Function through Outside-In and Inside-Out Mechanisms: The Effects of HAV Cell Surface Receptor and Intracellular Signaling

Science.gov (United States)

Corral-Jara, Karla F.; Gómez-Leyva, Juan F.; Rosenstein, Yvonne; Jose-Abrego, Alexis; Roman, Sonia

2016-01-01

We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1. PMID:27578921

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.

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

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.

Science.gov (United States)

Kang, Yoon-Suk; Kirby, James E

2017-05-01

We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus , B. melitensis , and B. suis , B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila , we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection. Copyright © 2017 American Society for Microbiology.

Leucocins 4010 from Leuconostoc carnosum cause a matrix related decrease in intracellular pH of Listeria monocytogenes

DEFF Research Database (Denmark)

Fang, Weihuan; Budde, Birgitte Bjørn; Siegumfeldt, Henrik

2006-01-01

A mixed culture of single cells of Listeria monocytogenes and the bacteriocin producing Leuconostoc carnosum 4010 showed growth inhibition of L. monocytogenes, although the intracellular pH (pHi) of L. monocytogenes followed by fluorescence ratio imaging microscopy was not affected. Furthermore, L...

Relative effect potency estimates of dioxin-like activity for dioxins, furans, and dioxin-like PCBs in adults based on cytochrome P450 1A1 and 1B1 gene expression in blood

NARCIS (Netherlands)

Wimmerová, Soňa; van den Berg, Martin; Chovancová, Jana; Patayová, Henrieta; Jusko, Todd A; van Duursen, Majorie B M; Palkovičová Murínová, Ľubica; Canton, Rocio F; van Ede, Karin I; Trnovec, Tomáš

2016-01-01

BACKGROUND: In the risk assessment of PCDDs, PCDFs, and dioxin-like (DL) PCBs, regulatory authorities support the use of the toxic equivalency factor (TEF)-scheme derived from a heterogeneous data set of the relative effect potency (REPs) estimates. OBJECTIVES: We sought to determine REPs for

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.

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.

Leptospirosis porcina: prevalencia serológica en establecimientos productores de la República Argentina

Directory of Open Access Journals (Sweden)

Jessica Petrakovsky M.

2013-03-01

Full Text Available Objetivo. Detectar la presencia de anticuerpos contra Leptospira sp en cerdos que no presentaban sintomatología clínica, pertenecientes a establecimientos de producción porcina ubicados en todo el territorio de la República Argentina. Materiales y métodos. La asignación de predios y muestras se realizó en forma proporcional a los registrados en cada provincia a lo largo de todo el país. En total se estudiaron 1.524 predios, de los cuales se tomaron 19.872 muestras de suero y en la primera etapa se procesaron 3.631 de los mismos. Este estudio toma en consideración zonas identificadas por provincia, municipio y predios. Los sueros fueron analizados por la técnica de MAT (prueba de microaglutinación. Resultados. El 30% de las muestras resultaron positivas a la prueba tamiz empleada (primera etapa del MAT. La mayoría presentó coaglutininas a dos o a tres serovariedades. Al realizar la titulación de los sueros (segunda etapa del MAT, el porcentaje de positividad se mantuvo, siendo los serovares de mayor prevalencia Castellonis e Icterohaemorrhagiae. El 90% de las muestras presentó un título final de 1:200. Conclusiones. La existencia de sueros positivos en todas las provincias indica el contacto de los animales con la leptospira a lo largo de todo el país.

Patrimonio arqueológico e identidades en la República Dominicana

Directory of Open Access Journals (Sweden)

Jorge Ulloa Hung

2009-03-01

Full Text Available Se abordan algunos de los principales problemas que presenta el estudio y conservación del patrimonio arqueológico de la República Dominicana a la luz de los nuevos retos y problemas que ha generado la globalización, así como de otras deformaciones impuestas por determinados procesos históricos y políticos dentro de la historia del país. El análisis también intenta hacer un llamado a la conservación y estudio del patrimonio arqueológico dominicano a la luz de nuevos conceptos y categorías usadas para el estudio del tema en el campo de la arqueología y la sociología, en especial, el punto de vista que prioriza la actividad de los agentes sociales. Por último, se intentan puntualizar aspectos que se consideran esenciales para generar una visión distinta en el abordaje del patrimonio arqueológico y que, a larga, podrían redundar en el desarrollo de una percepción distinta sobre la disciplina arqueol��gica en el país.

Rare genomic variants link bipolar disorder to CREB regulated intracellular signaling pathways

Directory of Open Access Journals (Sweden)

Berit eKerner

2013-11-01

Full Text Available Bipolar disorder is a common, complex, and severe psychiatric disorder with cyclical disturbances of mood and a high suicide rate. Here, we describe a family with four siblings, three affected females and one unaffected male. The disease course was characterized by early-onset bipolar disorder and co-morbid anxiety spectrum disorders that followed the onset of bipolar disorder. Genetic risk factors were suggested by the early onset of the disease, the severe disease course, including multiple suicide attempts, and lack of adverse prenatal or early life events. In particular, drug and alcohol abuse did not contribute to the disease onset. Exome sequencing identified very rare, heterozygous, and likely protein-damaging variants in eight brain-expressed genes: IQUB, JMJD1C, GADD45A, GOLGB1, PLSCR5, VRK2, MESDC2, and FGGY. The variants were shared among all three affected family members but absent in the unaffected sibling and in more than 200 controls. The genes encode proteins with significant regulatory roles in the ERK/MAPK and CREB-regulated intracellular signaling pathways. These pathways are central to neuronal and synaptic plasticity, cognition, affect regulation and response to chronic stress. In addition, proteins in these pathways are the target of commonly used mood stabilizing drugs, such as tricyclic antidepressants, lithium and valproic acid. The combination of multiple rare, damaging mutations in these central pathways could lead to reduced resilience and increased vulnerability to stressful life events. Our results support a new model for psychiatric disorders, in which multiple rare, damaging mutations in genes functionally related to a common signaling pathway contribute to the manifestation of bipolar disorder.

Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNA

OpenAIRE

Friew, Yeshitila N; Boyko, Vitaly; Hu, Wei-Shau; Pathak, Vinay K

2009-01-01

Abstract Background Host restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC) to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Ga...

Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

Science.gov (United States)

El-Awady, Ahmed R; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B; Palani, Chithra D; Arce, Roger M; Waller, Jennifer L; Genco, Caroline A; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V; Cutler, Christopher W

2015-02-01

Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

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...

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.

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.

Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions.

Science.gov (United States)

Rimkute, Lina; Kraujalis, Tadas; Snipas, Mindaugas; Palacios-Prado, Nicolas; Jotautis, Vaidas; Skeberdis, Vytenis A; Bukauskas, Feliksas F

2018-01-01

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pH i ) and cytosolic magnesium ion concentration ([Mg 2+ ] i ), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pH i and [Mg 2+ ] i affect junctional conductance (g j ) in an interdependent manner; in other words, intracellular acidification cause increase or decay in g j depending on whether [Mg 2+ ] i is high or low, respectively, and intracellular alkalization cause reduction in g j independently of [Mg 2+ ] i . Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pH i and [Mg 2+ ] i . Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pH i and [Mg 2+ ] i . Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg 2+ ] i , while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36 * E8Q lost the initial increase of g j at low [Mg 2+ ] i and double mutation lost the sensitivity to high [Mg 2+ ] i . These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg 2+ and H + ions.

APP substitutions V715F and L720P alter PS1 conformation and differentially affect Abeta and AICD generation.

Science.gov (United States)

Tesco, Giuseppina; Ginestroni, Andrea; Hiltunen, Mikko; Kim, Minji; Dolios, Georgia; Hyman, Bradley T; Wang, Rong; Berezovska, Oksana; Tanzi, Rudolph E

2005-10-01

The 37-43 amino acid Abeta peptide is the principal component of beta-amyloid deposits in Alzheimer's disease (AD) brain, and is derived by serial proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase. gamma-Secretase also cleaves APP at Val50 in the Abeta numbering (epsilon cleavage), resulting in the release of a fragment called APP intracellular domain (AICD). The aim of this study was to determine whether amino acid substitutions in the APP transmembrane domain differentially affect Abeta and AICD generation. We found that the APPV715F substitution, which has been previously shown to dramatically decrease Abeta40 and Abeta42 while increasing Abeta38 levels, does not affect in vitro generation of AICD. Furthermore, we found that the APPL720P substitution, which has been previously shown to prevent in vitro generation of AICD, completely prevents Abeta generation. Using a fluorescence resonance energy transfer (FRET) method, we next found that both the APPV715F and APPL720P substitutions significantly increase the distance between the N- and C-terminus of presenilin 1 (PS1), which has been proposed to contain the catalytic site of gamma-secretase. In conclusion, both APPV715F and APPL720P change PS1 conformation with differential effects on Abeta and AICD production.