Expression of natural human b1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans
Hesselink, T. ; Rouwendal, G.J.A. ; Henquet, M.G.L. ; Florack, D.E.A. ; Helsper, J.P.F.G. ; Bosch, H.J. - \ 2014
Transgenic Research 23 (2014)5. - ISSN 0962-8819 - p. 717 - 728.
golgi-apparatus - murine beta-1,4-galactosyltransferase - beta 1,4-galactosyltransferase - transgenic plants - gene - cells - localization - antibodies - oligosaccharides - glycoproteins
b1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human b1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of biantennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat a2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.
Time course and role of luteinizing hormone and follicle-stimulating hormone in the expansion of the Leydig cell population at the time of puberty in the rhesus monkey (Macaca mulatta)
Verhagen, I. ; Ramaswamy, S. ; Teerds, K.J. ; Keijer, J. ; Plant, T.M. - \ 2014
Andrology 2 (2014)6. - ISSN 2047-2927 - p. 924 - 930.
gonadotropin-secretion - postnatal-development - human testis - proliferation - testosterone - fsh - differentiation - fascicularis - spermatogonia - localization
In higher primates, development of the adult population of Leydig cells has received little attention. Here, the emergence of 3beta-hydroxysteroid dehydrogenase (HSD3B) positive cells in the testis of the rhesus monkey was examined during spontaneous puberty, and correlated with S-phase labeling in the interstitium at this critical stage of development. In addition, the relative role of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in initiating the pubertal expansion of Leydig cells was studied by precociously stimulating the juvenile testis in vivo with pulsatile 11-day infusions of recombinant LH and FSH, either alone or in combination. At the time of castration, testes were immersion fixed in Bouin's, embedded in paraffin, and sectioned at 5mum. Leydig cells/testis were enumerated using HSD3B as a Leydig cell marker. Leydig cell number per testis increased progressively during puberty to reach values in the adult approximately 10 fold greater than in early-pubertal animals. The rise in cell number was associated with an increase in nuclear diameter. That the pubertal expansion of Leydig cell number was driven primarily by the increase in LH secretion at this stage of development was suggested by the finding that precocious stimulation of mid-juvenile monkeys with LH, either alone or in combination with that of FSH, resulted in a 20-30 fold increase in the number of HSD3B-positive cells. Interestingly, precocious FSH stimulation, alone, also resulted in appearance of Leydig cells as indicated by the occasional HSD3B-positive cell in the interstitium. The nuclear diameter of these Leydig cells, however, was less than that of those generated in response to LH.
Proper Application of Antibodies for Immunohistochemical Detection: Antibody Crimes and How to Prevent Them
Ivell, R. ; Teerds, K.J. ; Hoffman, G.E. - \ 2014
Endocrinology 155 (2014)3. - ISSN 0013-7227 - p. 676 - 687.
hormone neurons - expression - protein - rat - immunocytochemistry - localization - testis - mice
For several decades antibodies raised against specific proteins, peptides, or peptide epitopes have proven to be versatile and very powerful tools to demonstrate molecular identity in cells and tissues. New techniques of immunohistochemistry and immunofluorescence have improved both the optical resolution of such protein identification as well as its sensitivity, particularly through the use of amplification methodology. However, this improved sensitivity has also increased the risks of false-positive and false-negative staining and thereby raised the necessity for proper and adequate controls. In this review, the authors draw on many years of experience to illuminate many of the more common errors and problematic issues in immunohistochemistry, and how these may be avoided. A key factor in all of this is that techniques need to be properly documented and especially antibodies and procedures must be adequately described. Antibodies are a valuable and shared resource within the scientific community; it is essential therefore that mistakes involving antibodies and their controls are not perpetuated through inadequate reporting in the literature.
Image-based particle filtering for navigation in a semi-structured agricultural environment.
Hiremath, S. ; Evert, F.K. van; Braak, C.J.F. ter; Stein, A. ; Heijden, G.W.A.M. van der - \ 2014
Biosystems Engineering 121 (2014). - ISSN 1537-5110 - p. 85 - 95.
automatic guidance - weed-control - robot - vision - segmentation - localization - vehicles - system - detect
Autonomous navigation of field robots in an agricultural environment is a difficult task due to the inherent uncertainty in the environment. The drawback of existing systems is the lack of robustness to these uncertainties. In this study we propose a vision-based navigation method to address these problems. The focus is on navigation through a maize field in an outdoor environment where the robot has to navigate through a corridor formed by two plant rows, detect the end of the rows, navigate the headland and turn into another corridor under natural conditions. The method is based on a Particle Filter (PF) using a novel measurement model, where we construct a model image from the particle and compare it directly with the measurement image after elementary processing, such as down-sampling, excessive-green filtering and thresholding. The new measurement model does not extract features from the image and thus does not suffer from errors associated with the feature extraction process. We show how PF can be used for robust navigation of a robot in a semi-structured agricultural environment such as maize fields with inherent uncertainty. We demonstrate the robustness of the algorithm through experiments in several maize fields with different row patterns, varying plant sizes and diverse lighting conditions. To date we have logged over 5 km of successful test runs in which the robot navigates through the corridor without touching the plant stems, accurately detects the end of the rows and traverses the headland. (C) 2014 IAgrE. Published by Elsevier Ltd. All rights reserved.
Consuming nostalgia? The appreciation of authenticity in local food production
Autio, M. ; Collins, R. ; Wahlen, S. ; Anttila, M. - \ 2013
International Journal of Consumer Studies 37 (2013)5. - ISSN 1470-6423 - p. 564 - 568.
localization - consumption
Many consumers consider local food a more sustainable choice than conventional food because of the shorter transport distances involved as well as the support provided to local economies. In addition, consumers value the perceived safety benefits, ethical associations and improved taste of local food. In this study, we focus on the cultural meanings of locally produced food among Finnish consumers. Based on interviews with 22 consumers, our analysis suggests that, besides consumers valuing sustainable, healthy and tasty locally produced food, they perceived self-produced, self-processed items, including those they have gathered, hunted and fished themselves, as the most authentic local food. Furthermore, local food is associated with craftsmanship and artisan production. We also found that interviewees tended to historicize their relationship to food through local production. Thus, consumers seem to be in search of ‘real’ or ‘true’ food that is embedded in their personal and shared social histories.
Essential C-Terminal region of the baculovirus minor capsid protein VP80 binds DNA
Marek, M. ; Merten, O.W. ; Francis-Devaraj, F. ; Oers, M.M. van - \ 2012
Journal of Virology 86 (2012). - ISSN 0022-538X - p. 1728 - 1738.
nuclear polyhedrosis-virus - f-actin - bombyx-mori - nucleopolyhedrovirus - cytoskeleton - generation - localization - replication - genomics - sequence
The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and in their egress from the virogenic stroma toward the nuclear periphery by a mechanism, which also includes F-actin filaments. We performed functional mapping of VP80 demonstrating that its highly conserved C-terminal region plays a crucial role in virion morphogenesis. Protein database mining identified a putative basic helix-loop-helix (bHLH) domain, a DNA-binding module typical for eukaryotic transcription factors, in the essential C-terminal region of VP80. Using a molecular modeling approach, we predicted the three-dimensional structure of this domain, revealing some unique properties. Biochemical assays proved that VP80 can form homodimers, a critical prerequisite of DNA-binding bHLH proteins. The ability of VP80 to bind DNA was subsequently confirmed by an electrophoretic mobility shift assay. We further show that AcMNPV DNA replication occurs in the absence of VP80. Immunolabeling of VP80 in baculovirus-infected cells rather points toward its involvement in nucleocapsid maturation. The competence of VP80 to interact with both F-actin and DNA provides novel insight into baculovirus morphogenesis.
Distribution, Elimination and Toxicity of Silver Nanoparticles and Silver Ions in Rats after 28-day Oral Exposure
Zande, M. van der; Vandebriel, R.J. ; Doren, E. ; Kramer, E.H.M. ; Herrera-Rivera, Z. ; serrano-Rojero, C.S. ; Gremmer, E.R. ; Mast, J. ; Peters, R.J.B. ; Hollman, P.C.H. ; Hendriksen, P.J.M. ; Marvin, H.J.P. ; Peijnenburg, A.A.C.M. ; Bouwmeester, H. - \ 2012
ACS Nano 6 (2012)8. - ISSN 1936-0851 - p. 7427 - 7442.
sprague-dawley rats - brain-barrier permeability - engineered nanoparticles - inhalation toxicity - tissue distribution - release - translocation - localization - deposition - kinetics
We report the results of a 28-day oral exposure study in rats, exposed to
Genetic characterization of a reciprocal translocation present in a widely grown barley variety
Farré, A. ; Cuadrado, A. ; Lacasa-Benito, I. ; Cistué, L. ; Schubert, I. ; Comadran, J. ; Jansen, J. ; Romagosa, I. - \ 2012
Molecular Breeding 30 (2012)2. - ISSN 1380-3743 - p. 1109 - 1119.
in-situ hybridization - nonhomologous chromosomes - genome duplication - hordeum-vulgare - breakpoints - map - localization - interchange - reduction - markers
Artificially induced translocation stocks have been used to physically map the barley genome; however, natural translocations are extremely uncommon in cultivated genotypes. Albacete is a barley variety widely grown in recent decades in Spain and carrying a reciprocal translocation which obviously does not affect its agronomical fitness. This translocation has been characterized by a combination of cytological and molecular genetic approaches. Firstly, recombination frequencies between markers on chromosomes 1H and 3H were estimated to determine the boundaries of the reciprocal interchange. Secondly, 1H-3H wheat barley telosome addition lines were used to assign selected markers to chromosome arms. Thirdly, fluorescence in situ hybridization (FISH) with rDNA probes (5S and 18S-5.8S-26S) and microsatellite probes [(ACT)5, (AAG)5 and (CAG)5] was used to determine the locations of the translocation breakpoints more precisely. Fourthly, fine-mapping of the regions around the translocation breakpoints was used to increase the marker density for comparative genomics. The results obtained in this study indicate that the translocation is quite large with breakpoints located on the long arms of chromosomes 1H and 3H, between the pericentromeric (AAG)5 bands and above the (ACT)5 interstitial distal bands, resulting in the reciprocal translocation 1HS.1HL-3HL and 3HS.3HL-1HL. The gene content around the translocation breakpoints could be inferred from syntenic relationships observed among different species from the grass family Poaceae (rice, Sorghum and Brachypodium) and was estimated at approximately 1,100 and 710 gene models for 1H and 3H, respectively. Duplicated segments between chromosomes Os01 and Os05 in rice derived from ancestral duplications within the grass family overlap with the translocation breakpoints on chromosomes 1H and 3H in the barley variety Albacete.
Fluorescence and Atomic Force Microscopy Imaging of Wall Teichoic Acids in Lactobacillus plantarum
Andre, G. ; Deghorain, M. ; Bron, P.A. ; Swam, I.I. van; Kleerebezem, M. ; Hols, P. ; Dufrene, Y.F. - \ 2011
Acs Chemical Biology 6 (2011)4. - ISSN 1554-8929 - p. 366 - 376.
gram-positive bacteria - staphylococcus-aureus - cell-wall - lipoteichoic acid - bacillus-subtilis - growth - localization - peptidoglycan - biosynthesis - spectroscopy
Although teichoic acids are major constituents of bacterial cell walls, little is known about the relationships between their spatial localization and their functional roles. Here, we used single-molecule atomic force microscopy (AFM) combined with fluorescence microscopy to image the distribution of wall teichoic acids (WTAs) in. Lactobacillus plantarum, in relation with their physiological roles. Phenotype analysis of the wild-type strain and of mutant strains deficient for the synthesis of WTAs (Delta tagO) or cell wall polysaccharides (Delta cps1-4) revealed that WTAs are required for proper cell elongation and cell division. Nanoscale imaging by AFM showed that strains expressing WTAs have a highly polarized surface morphology, the poles being much smoother than the side walls. AFM and fluorescence imaging with specific lectin probes demonstrated that the polarized surface structure correlates with a heterogeneous distribution of WTAs, the latter being absent from the surface of the poles. These observations indicate that the polarized distribution of WTAs in L. plantarum plays a key role in controlling cell morphogenesis (surface roughness, cell shape, elongation, and division).
Genome-wide BAC-end sequencing of Musa acuminata DH Pahang reveals further insights into the genome organization of banana
Arnago, R.E. ; Togawa, R.C. ; Carpentier, S.C. ; Lintel Hekkert, B. te; Kema, G.H.J. ; Souza, M.T. - \ 2011
Tree Genetics and Genomes 7 (2011)5. - ISSN 1614-2942 - p. 933 - 940.
rice genome - arabidopsis-thaliana - draft sequence - nuclear genome - dna - database - model - identification - localization - proteome
Banana and plantain (Musa spp.) are grown in more than 120 countries in tropical and subtropical regions and constitute an important staple food for millions of people. A Musa acuminata ssp. malaccencis DH Pahang bacterial artificial chromosome (BAC) library (MAMB) was submitted for BAC-end sequencing. MAMB consists of 23,040 clones, with a 140-kbp average insert size, accounting for a five times coverage of the banana genome. A total of 46,080 reads were generated, and 42,750 (92.8%) high-quality sequences were obtained after trimming for vector and quality. Analysis of these data shows a GC content of 41.39%, whereas interspersed repeats comprise 32.3%. The most common repeated sequences found show homology to ribosomal RNA genes, particularly 18S rRNA, while the Ty3/gypsy type monkey retrotransposon is the most common retro element. The sequence data were used to generate a banana-specific repeat library containing 54 new repetitive elements which accounted for 11.86% of the total nucleotides. Simple sequence repeats represent 0.7% of the sequence data and allowed the identification of 2,455 potentially useful marker sites. Functional annotation identified 2,705 sequences that could code for proteins of known function. Microsynteny analysis shows a higher number of co-linear matches to Oryza sativa, in contrast to Arabidopsis thaliana. This database of BAC-end sequences is useful for the assembly of the complete banana genome sequence and is important for identification in functional genomics experiments
Genome-wide identification of Phytophthora sojae SNARE genes and functional characterization of the conserved SNARE PsYKT6
Zhao, W. ; Dong, S. ; Ye, W. ; Hua, C. ; Meijer, H.J.G. ; Dou, X. ; Govers, F. ; Wang, Y. - \ 2011
Fungal Genetics and Biology 48 (2011)3. - ISSN 1087-1845 - p. 241 - 251.
pathogen phytophthora - systematic analysis - root-rot - fusion - proteins - infestans - complex - localization - eukaryotes - mechanisms
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.
Nor-ursodeoxycholic acid reverses hepatocyte-specific nemo-dependent steatohepatitis
Beraza, N. ; Ofner-Ziegenfuss, L. ; Ehedego, H. ; Boekschoten, M.V. ; Bischoff, S.C. ; Müller, M.R. ; Trauner, M. ; Trautwein, C. - \ 2011
Gut 60 (2011). - ISSN 0017-5749 - p. 387 - 396.
trail-mediated cytotoxicity - bile-acids - molecular characterization - liver-regeneration - knockout mice - expression - cholestasis - deletion - localization - mechanisms
Background: Hepatocyte-specific NEMO/NF-¿B deleted mice (NEMO¿hepa) develop spontaneous non-alcoholic steatohepatitis (NASH). Free fatty acids and bile acids promote DR5 expression. TRAIL/NK cell-mediated activation of TRAIL-R2/DR5 plays an important role during acute injury in NEMO¿hepa mice. Aim To inhibit the progression of NASH in the absence of hepatocyte-NEMO/NF-kB signaling. Methods - NEMOf/f and NEMO¿hepa mice were fed with a low-fat diet, and with two anticholestatic diets; UDCA and NorUDCA. The impact of these treatments on the progression of NASH was evaluated. Results - We show that high expression of DR5 in livers from NEMO¿hepa mice is accompanied by an abundant presence of bile acids (BAs), misregulation of BA transporters and significant alteration of lipid metabolism-related genes. Additionally, mice lacking NEMO in hepatocytes spontaneously showed ductular response at young age. Unexpectedly, feeding of NEMO¿hepa mice with low-fat diet failed to improve chronic liver injury. Conversely, anti-cholestatic treatment with nor-ursodeoxycholic acid (NorUDCA), but not with ursodeoxycholic acid (UDCA), led to a significant attenuation of liver damage in NEMO¿hepa mice. The strong therapeutic effect of NorUDCA relied on a significant downregulation of LXR-dependent lipogenesis and the normalisation of BA metabolism through mechanisms involving cross-talk between Cyp7a1 and SHP. This was associated with the significant improvement of liver histology, NEMO¿hepa/NorUDCA-treated mice showed lower apoptosis and reduced CyclinD1 expression, indicating attenuation of the compensatory proliferative response to hepatocellular damage. Finally, fibrosis and ductular reaction markers were significantly reduced in NorUDCA-treated NEMO¿hepa mice. Conclusions - Overall, our work demonstrates the contribution of bile acids metabolism to the progression of NASH in the absence of hepatocyte-NF-kB through mechanisms involving DR5-apoptosis, inflammation and fibrosis. Our work suggests a potential therapeutic effect of NorUDCA in attenuating the progression of NASH
Functional aspects of baculovirus DNA photolyases
Xu, F. - \ 2010
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Monique van Oers. - [S.l. : S.n. - ISBN 9789085857730 - 112
baculoviridae - organismen ingezet bij biologische bestrijding - insectenplagen - fotolyse - chrysodeixis chalcites - kernpolyedervirussen - lyasen - genen - genexpressie - fylogenetica - lokalisatie - ultraviolette straling - eiwitexpressieanalyse - gevoeligheid - baculoviridae - biological control agents - insect pests - photolysis - chrysodeixis chalcites - nuclear polyhedrosis viruses - lyases - genes - gene expression - phylogenetics - localization - ultraviolet radiation - proteomics - sensitivity
Keywords: baculovirus, ChchNPV, CPD photolyase, phylogeny, UV resistance, DNA binding, localization, proteomics
Baculoviruses are insect viruses that are applied as biological control agents due to adequate virulence, host specificity and safety for the environment. Solar light negatively affects field performance of baculoviruses by reducing their infectivity, most likely as a consequence of the formation of cyclobutane pyrimidine dimers (CPDs) in the viral DNA upon ultraviolet (UV) irradiation. CPDs can be repaired by CPD photolyases when exposed to blue light photons, a process called photoreactivation. From previous work it was known that the Cc-phr2 gene of the baculovirus Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) encodes a biochemically active photolyase. The research in this thesis focuses on (i) the degree of conservation of CPD photolyase (phr) genes in a subgroup of baculoviruses, (ii) the localization of baculovirus photolyase proteins in insect cells and occlusion derived virus (ODV), and (iii) the in vivo effect of phr genes on the UV sensitivity of baculoviruses. Homologues of the Cc-phr genes were found in all studied group II NPVs in the genus Alphabaculovirus that infect insects in the subfamily Plusiinae insects. Phylogenetic analysis suggested that these phr-like genes have a common ancestor. Intracellular localization of the two ChchNPV encoded PHR proteins in insect cells was studied using enhanced GFP fusion. Both PHR1 and PHR2 localized in the nucleus and associated with chromosomes, spindle, aster and midbody structures during host cell mitosis. Moreover, Cc-PHR2 co-localized with virogenic stroma, when PHR2-EGFP-transfected cells were infected with Autographa californica (Ac) MNPV. Neither of the two Cc-PHR proteins was identified by LC/MS-MS in the ODVs of ChchNPV. To evaluate the potential of the Cc-PHR2 protein to reduce the UV sensitivity of a baculovirus, the Cc-phr2 gene was incorporated in the genome of Helicoverpa armigera (Hear) NPV, which does not have a UV damage repair system. This resulted in a decreased sensitivity to UV-light compared to wild type HearNPV. A cell line was established from embryos of the insect C. chalcites. This cell line was shown to be permissive for both ChchNPV and the related Trichoplusia ni NPV (TnSNPV). This novel cell line will be a useful tool for making ChchNPV phr mutant viruses to study the impact of DNA repair mediated by photolyases on baculovirus ecology. The collected data support the hypothesis that the Cc- phr2 gene provides a baculovirus with an ecological benefit by increasing the resistance to UV.
Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta
Ribeiro, D.M.O.G. ; Borst, J.W. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2009
Virology 383 (2009)1. - ISSN 0042-6822 - p. 121 - 130.
endoplasmic-reticulum - homotypic interaction - cells - golgi - localization - microscopy - infection - tobacco
Recently, the Tomato Spotted Wilt Virus (TSWV) Gn and Gc glycoproteins were shown to induce the formation of (pseudo-) circular and pleomorphic membrane structures upon transient expression in plant cells. Furthermore, when singly expressed, Gc retains in the ER, while Gn is able to further migrate to the Golgi. Upon co-expression, Gn rescues Gc and co-migrates to the Golgi complex. Here, we have studied the behavior of the glycoproteins in the presence of the viral nucleocapsid (N) protein and in vivo analyzed the occurrence of protein¿protein interactions by fluorescence life time imaging microscopy (FLIM). The analysis demonstrated that N co-localizes and interacts with both glycoproteins, with a preference for Gn. Additionally, it is shown that N causes a dramatic change in the distribution of Gc within the ER, from reticular to punctate spots. The observations are discussed in the context of the virus particle formation during the infection process.
Hydrodynamic flow in the cytoplasm of plant cells
Esseling-Ozdoba, A. ; Houtman, D. ; Lammeren, A.A.M. van; Eiser, E. ; Emons, A.M.C. - \ 2008
Journal of Microscopy 231 (2008)2. - ISSN 0022-2720 - p. 274 - 283.
tobacco by-2 cells - pollen tubes - root hairs - 2,3-butanedione monoxime - actin cytoskeleton - f-actin - myosin - arabidopsis - localization - microtubules
Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study ( Houtman et al., 2007 ) shows that active transport of organelles gives rise to a drag in the cytosol, setting up a hydrodynamic flow, which contributes to a fast distribution of proteins and nutrients in plant cells. Here, we show experimentally that actively transported organelles produce hydrodynamic flow that significantly contributes to the movement of the molecules in the cytosol. We have used fluorescence recovery after photobleaching and show that in tobacco Bright Yellow 2 (BY-2) suspension cells constitutively expressing cytoplasmic green fluorescent protein (GFP), free GFP molecules move faster in cells with active transport of organelles than in cells where this transport has been inhibited with the general myosin inhibitor BDM (2,3-butanedione monoxime). Furthermore, we show that the direction of the GFP movement in the cells with active transport is the same as that of the organelle movement and that the speed of the GFP in the cytosol is proportional to the speed of the organelle movement. In large BY-2 cells with fast cytoplasmic streaming, a GFP molecule reaches the other side of the cell approximately in the similar time frame (about 16 s) as in small BY-2 cells that have slow cytoplasmic streaming. With this, we suggest that hydrodynamic flow is important for efficient transport of cytosolic molecules in large cells. Hydrodynamic flow might also contribute to the movement of larger structures than molecules in the cytoplasm. We show that synthetic lipid (DOPG) vesicles and `stealth' vesicles with PEG phospholipids moved in the cytoplasm
Influence of cellular ER alpha/ER beta ratio on the ER alpha-agonist induced proliferation of human T47D breast cancer cells
Sotoca Covaleda, A.M. ; Berg, H. van den; Vervoort, J.J.M. ; Saag, P. van der; Strom, A. ; Gustafsson, J.A. ; Rietjens, I.M.C.M. ; Murk, A.J. - \ 2008
Toxicological sciences 105 (2008)2. - ISSN 1096-6080 - p. 303 - 311.
estrogen-receptor-beta - gene-expression - in-vitro - osteosarcoma cells - nuclear - phytoestrogens - tamoxifen - localization - heterodimers - selectivity
Breast cancer cells show overexpression of estrogen receptor (ER) relative to ERß compared to normal breast tissues. This observation has lead to the hypothesis that ERß may modulate the proliferative effect of ER. This study investigated how variable cellular expression ratios of the ER and ERß modulate the effects on cell proliferation induced by ER or ERß agonists, respectively. Using human osteosarcoma (U2OS) ER or ERß reporter cells, propyl-pyrazole-triol (PPT) was shown to be a selective ER and diarylpropionitrile (DPN) a preferential ERß modulator. The effects of these selective estrogen receptor modulators (SERMs) and of the model compound E2 on the proliferation of T47D human breast cancer cells with tetracycline-dependent expression of ERß (T47D-ERß) were characterized. E2-induced cell proliferation of cells in which ERß expression was inhibited was similar to that of the T47D wild-type cells, whereas this E2-induced cell proliferation was no longer observed when ERß expression in the T47D-ERß cells was increased. In the T47D-ERß cell line, DPN also appeared to be able to suppress cell proliferation when levels of ERß expression were high. In the T47D-ERß cell line, PPT was unable to suppress cell proliferation at all ratios of ER/ERß expression, reflecting its ability to activate only ER and not ERß. It is concluded that effects of estrogen-like compounds on cell proliferation are dependent on the actual ER/ERß expression levels in these cells or tissues and the potential of the estrogen agonists to activate ER and/or ERß.
NADPH oxidases are involved in differentiation and pathogenicity in Botrytis cinerea
Segmüller, N. ; Kokkelink, L. ; Giesbert, S. ; Odinius, D. ; Kan, J. van; Tudzynski, P. - \ 2008
Molecular Plant-Microbe Interactions 21 (2008)6. - ISSN 0894-0282 - p. 808 - 819.
respiratory burst oxidase - reactive oxygen - functional-analysis - oxidative burst - active oxygen - cell-growth - disease - localization - infection - virulence
Nicotinamide adenine dinucleotide (NADPH) oxidases have been shown to be involved in various differentiation processes in fungi. We investigated the role of two NADPH oxidases in the necrotrophic phytopathogenic fungus, Botrytis cinerea. The genes bcnoxA and bcnoxB were cloned and characterized; their deduced amino acid sequences show high homology to fungal NADPH oxidases. Analyses of single and double knock-out mutants of both NADPH oxidase genes showed that both bcnoxA and bcnoxB are involved in formation of sclerotia. Both genes have a great impact on pathogenicity: whereas bcnoxB mutants showed a retarded formation of primary lesions, probably due to an impaired formation of penetration structures, bcnoxA mutants were able to penetrate host tissue in the same way as the wild type but were much slower in colonizing the host tissue. Double mutants showed an additive effect: they were aberrant in penetration and colonization of plant tissue and, therefore, almost nonpathogenic. To study the structure of the fungal Nox complex in more detail, bcnoxR (encoding a homolog of the mammalian p67phox, a regulatory subunit of the Nox complex) was functionally characterized. The phenotype of ¿bcnoxR mutants is identical to that of ¿bcnoxAB double mutants, providing evidence that BcnoxR is involved in activation of both Bcnox enzymes.
Tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum- and Golgi-derived pleomorphic membrane structures in plant cells
Ribeiro, D.M.O.G. ; Foresti, O. ; Denecke, J. ; Wellink, J. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2008
Journal of General Virology 89 (2008)8. - ISSN 0022-1317 - p. 1811 - 1818.
uukuniemi virus - protein - morphogenesis - bunyaviridae - maturation - localization - complex - tobacco - g1 - proliferation
Tomato spotted wilt virus (TSWV) particles are spherical and enveloped, an uncommon feature among plant infecting viruses. Previous studies have shown that virus particle formation involves the enwrapment of ribonucleoproteins with viral glycoprotein containing Golgi stacks. In this study, the localization and behaviour of the viral glycoproteins Gn and Gc were analysed, upon transient expression in plant protoplasts. When separately expressed, Gc was solely observed in the endoplasmic reticulum (ER), whereas Gn was found both within the ER and Golgi membranes. Upon co-expression, both glycoproteins were found at ER-export sites and ultimately at the Golgi complex, confirming the ability of Gn to rescue Gc from the ER, possibly due to heterodimerization. Interestingly, both Gc and Gn were shown to induce the deformation of ER and Golgi membranes, respectively, also observed upon co-expression of the two glycoproteins. The behaviour of both glycoproteins within the plant cell and the phenomenon of membrane deformation are discussed in light of the natural process of viral infection
Cyclic GMP in the pig vitreous and retina after experimental retinal detachment
Diederen, R. ; Heij, E.C. La; Lemmens, M.A.M. ; Kijlstra, A. ; Vente, J. De; Hendrikse, F. - \ 2008
Molecular Vision 14 (2008). - ISSN 1090-0535 - p. 255 - 261.
soluble guanylate-cyclase - pigment epithelial-cells - oxygen supplementation - subretinal fluid - cgmp - localization - particulate - surgery - pathways - neurons
Purpose: Earlier studies have revealed a decreased level of cGMP in vitreous fluid obtained from patients with a retinal detachment. To further investigate this phenomenon, we developed an experimental retinal detachment model in pigs. Methods: Experimental unilateral retinal detachments were induced in pig eyes by subretinal injection of 0.25% sodium hyaluronate. Fourteen days later the vitreous and retinas were analyzed for cGMP expression. Following enucleation, the retinas were incubated in the presence of a nonselective phosphodiesterase inhibitor (IBMX), and the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). cGMP was visualized in retinal wholemounts by immunochemistry combined with a computer based stereology system. cGMP levels in vitreous were determined by ELISA. Results: The mean vitreous cGMP level in pig eyes with a retinal detachment (1.45 pmol/ml) was significantly lower compared to the mean level of cGMP in healthy pig eyes (4.61 pmol/ml; p= 0.028 was considered significant). In the inner retina, ANP as well as SNP induced cGMP immunoreactivity in both detached and healthy retinas. After incubation with ANP, cGMP could also be detected in the outer nuclear layer of the detached retina, whereas this was not the case in the normal retina. Conclusions: Experimental retinal detachment in the pig eye leads to a decrease of cGMP levels in vitreous similar to that observed in clinical studies. This model may be helpful to analyze the mechanisms involved in cGMP dynamics following retinal detachment.
Biphasic survival analysis of trypanotolerance QTL in mice
Koudante, O.D. ; Thomson, P.C. ; Bovenhuis, H. ; Iraqi, F. ; Gibson, J.P. ; Arendonk, J.A.M. van - \ 2008
Heredity 100 (2008). - ISSN 0018-067X - p. 407 - 414.
trypanosoma-congolense - inbred strains - surface coat - resistance - susceptibility - localization - infection - antigens - antibody - brucei
A marker-assisted introgression (MAI) experiment was conducted to transfer trypanotolerance quantitative trait loci (QTL) from a donor mouse strain, C57BL/6, into a recipient mouse strain, A/J. The objective was to assess the effect of three previously identified chromosomal regions on mouse chromosomes 1 (MMU1), 5 (MMU5) and 17 (MMU17) in different genetic backgrounds on the survival pattern following infection with Trypanosoma congolense. An exploratory data analysis revealed a biphasic pattern of time to death, with highly distinct early and late mortality phases. In this paper, we present survival analysis methods that account for the biphasic mortality pattern and results of reanalyzing the data from the MAI experiment. The analysis with a Weibull mixture model confirmed the biphasic pattern of time to death. Mortality phase, an unobserved variable, appears to be an important factor influencing survival time and is modeled as a binary outcome variable using logistic regression analysis. Accounting for this biphasic pattern in the analysis reveals that a previously observed sex effect on average survival is rather an effect on proportion of mice in the two mortality phases. The C57BL/6 (donor) QTL alleles on MMU1 and MMU17 act dominantly in the late mortality phase while the A/J (recipient) QTL allele on MMU17 acts dominantly in the early mortality phase. From this study, we found clear evidence for a biphasic survival pattern and provided models for its analysis. These models can also be used when studying defense mechanisms against other pathogens. Finally, these approaches provide further information on the nature of gene actions