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Sample records for mylitta cytoplasmic polyhedrosis

  1. Genome segment 5 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes a bona fide guanylyltransferase

    PubMed Central

    2014-01-01

    Background Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects non mulberry Indian silk worm, Antheraea mylitta, and contains eleven segmented double stranded RNA in its genome (S1-S11). Some of its genome segments (S1-S3, and S6-S11) have been previously characterized but genome segment encoding the viral guanylyltransferase which helps in RNA capping has not been characterized. Results In this study genome segment 5 (S5) of AmCPV was converted to cDNA, cloned and sequenced. S5 consisted of 2180 nucleotides, with one long ORF of 1818 nucleotides and could encode a protein of 606 amino acids with molecular mass of ~65 kDa (p65). Bioinformatics analysis showed presence of KLRS and HxnH motifs as observed in some other reoviral guanylyltransferase and suggests that S5 may encodes viral guanylyltransferase. The ORF of S5 was expressed in E. coli as 65 kDa his tagged fusion protein, purified through Ni-NTA chromatography and polyclonal antibody was raised. Immunoblot analysis of virion particles with the purified antibody showed specific immunoreactive band and suggests p65 as a viral structural protein. Functional analysis showed that recombinant p65 possesses guanylyltransferase activity, and transfers GMP moiety to the 5' diphosphate (A/G) ended viral RNA after the formation of p65-GMP complex for capping. Kinetic analysis showed Km of this enzyme for GTP and RNA was 34.24 uM and 98.35 nM, respectively. Site directed mutagenesis at K21A in KLRS motif, and H93A or H105A in HxnH motif completely abolished the autoguanylylation activity and indicates importance of these residues at these sites. Thermodynamic analysis showed p65-GTP interaction was primarily driven by enthalpy (ΔH = -399.1 ± 4.1 kJ/mol) whereas the p65-RNA interaction by favorable entropy (0.043 ± 0.0049 kJ/ mol). Conclusion Viral capping enzymes play a critical role in the post transcriptional or post replication modification in case of

  2. Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases.

    PubMed

    Biswas, Poulomi; Kundu, Anirban; Ghosh, Ananta Kumar

    2015-01-01

    Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment S4 showed that it consists of 3410 nt with a single ORF of 1110 aa which could encode a protein of ~127 kDa (p127). Bioinformatics analysis showed the presence of a 5' RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-l-methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2'-O-methyltransferase (MTase), which suggested that S4 may encode RTPase and MTase. The ORF of S4 was expressed in Escherichia coli as a His-tagged fusion protein and purified by nickel-nitrilotriacetic acid affinity chromatography. Biochemical analysis of recombinant p127 showed its RTPase as well as SAM-dependent guanine N(7)-and ribose 2'-O-MTase activities. A MTase assay using in vitro transcribed AmCPV S2 RNA having a 5' G*pppG end showed that guanine N(7) methylation occurred prior to the ribose 2'-O methylation to yield a m(7)GpppG/m(7)GpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished N(7)- and 2'-O-MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the Km values of N(7)-MTase for SAM and RNA were calculated as 4.41 and 0.39 µM, respectively. These results suggested that AmCPV S4-encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5' end of viral RNA. PMID:25228490

  3. Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

    SciTech Connect

    Ghorai, Suvankar; Chakrabarti, Mrinmay; Roy, Sobhan; Chavali, Venkata Ramana Murthy; Bagchi, Abhisek; Ghosh, Ananta Kumar

    2010-08-15

    Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123 kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123 kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37 deg. C at pH 6.0 in the presence of 3 mM MgCl{sub 2.} Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.

  4. Transcriptome analysis of interactions between silkworm and cytoplasmic polyhedrosis virus

    PubMed Central

    Jiang, Liang; Peng, Zhengwen; Guo, Youbing; Cheng, Tingcai; Guo, Huizhen; Sun, Qiang; Huang, Chunlin; Zhao, Ping; Xia, Qingyou

    2016-01-01

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects silkworm midgut (MG) and multiplication occurs mainly in posterior midgut (PM). In this study, MG and fat body (FB) were extracted at 0, 3, 24, and 72 h after BmCPV infection. The total sequence reads of each sample were more than 1510000, and the mapping ratio exceeded 95.3%. Upregulated transcripts increased in MG during the infection process. Gene ontology (GO) categories showed that antioxidants were all upregulated in FB but not in MG. BGI001299, BGI014434, BGI012068, and BGI009201 were MG-specific genes with transmembrane transport function, the expression of which were induced by BmCPV. BGI001299, BGI014434, and BGI012068 expressed in entire MG and may be involved in BmCPV invasion. BGI009201 expressed only in PM and may be necessary for BmCPV proliferation. BmPGRP-S2 and BGI012452 (a putative serine protease) were induced by BmCPV and may be involved in immune defense against BmCPV. The expression level of BmCPV S1, S2, S3, S6, and S7 was high and there was no expression of S9 in MG 72 h, implying that the expression time of structural protein coding genes is earlier. These results provide insights into the mechanism of BmCPV infection and host defense. PMID:27118345

  5. Cytoplasmic polyhedrosis virus classification by electropherotype; validation by serological analyses and agarose gel electrophoresis.

    PubMed

    Mertens, P P; Crook, N E; Rubinstein, R; Pedley, S; Payne, C C

    1989-01-01

    Serological analyses of several different cytoplasmic polyhedrosis viruses (CPVs), including two type 1 CPVs from Bombyx mori, type 1 CPV from Dendrolimus spectabilis, type 12 CPV from Autographa gamma, type 2 CPV from Inachis io, type 5 CPV from Orgyia pseudotsugata and type 5 CPV from Heliothis armigera, demonstrated a close correlation between the antigenic properties of the polyhedrin or virus particle structural proteins and the genomic dsRNA electropherotypes. The dsRNAs of these viruses were analysed by electrophoresis in 3% and 10% polyacrylamide gels with a discontinuous Tris-HCl/Tris-glycine buffer system or by 1% agarose gel electrophoresis using a continuous Tris-acetate-EDTA buffer system. Electrophoretic analysis in agarose gels was found to be the most suitable for the classification of CPV isolates into electropherotypes, and the results obtained showed a close correlation with the observed antigenic relationships between different virus isolates. However, electrophoretic analysis in 10% polyacrylamide gels was most sensitive for the detection of intra-type variation and the presence of mixed virus isolates. PMID:2499658

  6. “Pretranscriptional capping” in the biosynthesis of cytoplasmic polyhedrosis virus mRNA

    PubMed Central

    Furuichi, Yasuhiro

    1978-01-01

    The in vitro synthesis of cytoplasmic polyhedrosis virus (CPV) mRNA was previously shown to be dependent upon the presence of the methyl donor S-adenosylmethionine (AdoMet). We now find that the competitive inhibitor of methylation, S-adenosylhomocysteine (AdoHcy), also stimulates CPV mRNA synthesis efficiently, resulting in the synthesis of viral mRNAs containing 5′-terminal GpppA and ppA, rather than m7GpppAm as observed with Adomet. In addition to AdoHcy, other AdoMet analogues, including S-adenosylethionine and adenosine, also stimulate CPV mRNA synthesis but to a smaller extent than does AdoHcy or AdoMet. In order to study the relationship between cap formation and mRNA synthesis, nucleoside triphosphates were replaced in the RNA-synthesizing reaction mixture (containing AdoMet) by the corresponding β,γ-imido analogues, which are resistant to nucleotide phosphohydrolase, an enzyme involved in cap formation. Although mRNA synthesis occurred in the presence of UMP-pNHp or GMP-pNHp, none was observed when AMP-pNHp was substituted for ATP. Because the ATP molecule that becomes the 5′-terminal nucleotide of CPV mRNA must be cleaved at the β-γ position during cap formation, the results suggest that, in this viral transcription system, cap formation is prerequisite to mRNA synthesis—i.e., a “pretranscriptional” event. PMID:349555

  7. Transcriptome Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

    PubMed Central

    Kolliopoulou, Anna; Van Nieuwerburgh, Filip; Stravopodis, Dimitrios J.; Deforce, Dieter; Swevers, Luc; Smagghe, Guy

    2015-01-01

    Many insects can be persistently infected with viruses but do not show any obvious adverse effects with respect to physiology, development or reproduction. Here, Bombyx mori strain Daizo, persistently infected with cytoplasmic polyhedrosis virus (BmCPV), was used to study the host’s transcriptional response after pathogenic infection with the same virus in midgut tissue of larvae persistently and pathogenically infected as 2nd and 4th instars. Next generation sequencing revealed that from 13,769 expressed genes, 167 were upregulated and 141 downregulated in both larval instars following pathogenic infection. Several genes that could possibly be involved in B. mori immune response against BmCPV or that may be induced by the virus in order to increase infectivity were identified, whereas classification of differentially expressed transcripts (confirmed by qRT-PCR) resulted in gene categories related to physical barriers, immune responses, proteolytic / metabolic enzymes, heat-shock proteins, hormonal signaling and uncharacterized proteins. Comparison of our data with the available literature (pathogenic infection of persistently vs. non-persistently infected larvae) unveiled various similarities of response in both cases, which suggests that pre-existing persistent infection does not affect in a major way the transcriptome response against pathogenic infection. To investigate the possible host’s RNAi response against BmCPV challenge, the differential expression of RNAi-related genes and the accumulation of viral small RNAs (vsRNAs) were studied. During pathogenic infection, siRNA-like traces like the 2-fold up-regulation of the core RNAi genes Ago-2 and Dcr-2 as well as a peak of 20 nt small RNAs were observed. Interestingly, vsRNAs of the same size were detected at lower rates in persistently infected larvae. Collectively, our data provide an initial assessment of the relative significance of persistent infection of silkworm larvae on the host response following

  8. Genetic analysis of Indian tasar silkmoth (Antheraea mylitta) populations.

    PubMed

    Chakraborty, Saikat; Muthulakshmi, M; Vardhini, Deena; Jayaprakash, P; Nagaraju, J; Arunkumar, K P

    2015-01-01

    Indian tasar silkmoth, Antheraea mylitta is an economically important wild silkmoth species distributed across India. A number of morphologically and ethologically well-defined ecotypes are known for this species that differ in their primary food plant specificity. Most of these ecotypes do not interbreed in nature, but are able to produce offspring under captive conditions. Microsatellite markers were developed for A. mylitta, and out of these, ten well-behaved microsatellite loci were used to analyze the population structure of different ecoraces. A total of 154 individual moths belonging to eight different ecoraces, were screened at each locus. Hierarchical analysis of population structure using Analysis of MOlecular VAriance (AMOVA) revealed significant structuring (FST = 0.154) and considerable inbreeding (FIS = 0.505). A significant isolation by distance was also observed. The number of possible population clusters was investigated using distance method, Bayesian algorithm and self organization maps (SOM). The first two methods revealed two distinct clusters, whereas the SOM showed the different ecoraces not to be clearly differentiated. These results suggest that although there is a large degree of phenotypic variation among the different ecoraces of A. mylitta, genetically they are not very different, and the phenotypic differences may largely be a result of their respective ecology. PMID:26510465

  9. Genetic analysis of Indian tasar silkmoth (Antheraea mylitta) populations

    PubMed Central

    Chakraborty, Saikat; Muthulakshmi, M; Vardhini, Deena; Jayaprakash, P; Nagaraju, J; Arunkumar, K. P.

    2015-01-01

    Indian tasar silkmoth, Antheraea mylitta is an economically important wild silkmoth species distributed across India. A number of morphologically and ethologically well-defined ecotypes are known for this species that differ in their primary food plant specificity. Most of these ecotypes do not interbreed in nature, but are able to produce offspring under captive conditions. Microsatellite markers were developed for A. mylitta, and out of these, ten well-behaved microsatellite loci were used to analyze the population structure of different ecoraces. A total of 154 individual moths belonging to eight different ecoraces, were screened at each locus. Hierarchical analysis of population structure using Analysis of MOlecular VAriance (AMOVA) revealed significant structuring (FST = 0.154) and considerable inbreeding (FIS = 0.505). A significant isolation by distance was also observed. The number of possible population clusters was investigated using distance method, Bayesian algorithm and self organization maps (SOM). The first two methods revealed two distinct clusters, whereas the SOM showed the different ecoraces not to be clearly differentiated. These results suggest that although there is a large degree of phenotypic variation among the different ecoraces of A. mylitta, genetically they are not very different, and the phenotypic differences may largely be a result of their respective ecology. PMID:26510465

  10. Mylitta Fluctus, Venus - Rift-related, centralized volcanism and the emplacement of large-volume flow units

    NASA Technical Reports Server (NTRS)

    Roberts, Kari M.; Guest, John E.; Head, James W.; Lancaster, Michael G.

    1992-01-01

    The flow morphology, stratigraphy, and evoluton of Mylitta Fluctus, a massive lava flow field on Venus, is characterized, and the link between its origin and the local tectonics is examined. The regional setting of the flow field is reviewed. A model for the stratigraphy and emplacement history of Mylitta is developed, flow morphology is discussed, and some preliminary estimates of effusion rates and eruption durations that may have characterized its emplacement are presented. The origin of Mylitta is discussed in relation to local rifting and possible hotspot activity, and the emplacement of Mylitta is compared to the origin of terrestrial flood basalts.

  11. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  12. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  13. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  14. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  15. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  16. Generation of cytotoxic molecules and oxidative stress in haemolymph of pebrinised tasar silkworm Antheraea mylitta Drury.

    PubMed

    Jena, Karmabeer; Pandey, Jay Prakash; Priya, Anshu; Kundu, Papri; Sinha, Ajit Kumar; Yadav, Harendra; Sahay, Alok

    2016-01-01

    The present study was carried out to investigate the effects of microsporidial infection on redox regulation mechanism and oxidative stress in tasar silkworm Antheraea mylitta. High level of superoxide radical (p < 0.05), nitric oxide (p < 0.001) and lipid peroxidation (p < 0.001) was observed in haemolymph of pebrinised larvae, which indicated the resultant generation of cytotoxic molecules and oxidative damage. Increased phenol oxidase (PO) activity in haemolymph of pebrinised larvae indicated the activation of immune defences during pathological conditions. In addition, higher level of glutathione-S-tranferase (GST) activity and reduced glutathione (GSH) level observed in pebrinised larvae indicated adaptive behaviour of tissue against toxic oxyradicals (p < 0.05). Conversely, low level of ascorbic acid (ASA) content suggested that the larvae might have used these compounds to counteract stress in tissues or low uptake under microspridial infection (p < 0.05). Present findings provide new insights into the cellular and biochemical bases of host-pathogen interactions in tasar silkworm A. mylitta. PMID:26930859

  17. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  18. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  19. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  20. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  1. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  2. Crystallization and preliminary X-ray diffraction analysis of a protease inhibitor from the haemolymph of the Indian tasar silkworm Antheraea mylitta

    SciTech Connect

    Roy, Sobhan; Aravind, Penmatsa; Madhurantakam, Chaithanya; Ghosh, Ananta Kumar; Sankaranarayanan, Rajan; Das, Amit Kumar

    2006-07-01

    The crystallization and preliminary X-ray crystallographic analysis of a protease inhibitor from the haemolymph of the Indian tasar silk worm A. mylitta is reported. A protein with inhibitory activity against fungal proteases was purified from the haemolymph of the Indian tasar silkworm Antheraea mylitta and was crystallized using the hanging-drop vapour-diffusion method. Polyethylene glycol 3350 was used as a precipitant. Crystals belonged to space group P6{sub 3}22, with unit-cell parameters a = b = 60.6, c = 85.1 Å. X-ray diffraction data were collected and processed to a maximum resolution of 2.1 Å.

  3. Genome segment 6 of Antheraea mylitta cypovirus encodes a structural protein with ATPase activity

    SciTech Connect

    Chavali, Venkata R.M.; Madhurantakam, Chaithanya; Ghorai, Suvankar; Roy, Sobhan; Das, Amit K.; Ghosh, Ananta K.

    2008-07-20

    The genome segment 6 (S6) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus was converted into cDNA, cloned and sequenced. S6 consisted of 1944 nucleotides with an ORF of 607 amino acids and could encode a protein of 68 kDa, termed P68. Motif scan and molecular docking analysis of P68 showed the presence of two cystathionine beta synthase (CBS) domains and ATP binding sites. The ORF of AmCPV S6 was expressed in E. coli as His-tag fusion protein and polyclonal antibody was raised. Immunoblot analysis of virus infected gut cells and purified polyhedra using raised anti-p68 polyclonal antibody showed that S6 encodes a viral structural protein. Fluorescence and ATPase assay of soluble P68 produced in Sf-9 cells via baculovirus expression system showed its ability to bind and cleave ATP. These results suggest that P68 may bind viral RNA through CBS domains and help in replication and transcription through ATP binding and hydrolysis.

  4. Inactivation of nuclear polyhedrosis virus (Baculovirus subgroup A) by monochromatic UV radiation

    SciTech Connect

    Griego, V.M.; Martignoni, M.E.; Claycomb, A.E.

    1985-03-01

    Monochromatic radiation at wavelengths of 290, 300, 310, and 320 nm inactivated occluded nuclear polyhedrosis virus of the Douglas-fir tussock moth, Orgyia pseudotsugata. Data indicate that all of the wavelengths are capable of causing virus inactivation; much greater fluences are needed for virus inactivation as the wavelength increases.

  5. Generalized Immunoassay for Autographa californica Nuclear Polyhedrosis Virus Infectivity In Vitro

    PubMed Central

    Volkman, Loy E.; Goldsmith, Phyllis A.

    1982-01-01

    A quantitative in vitro immunoassay for the infectivity of Autographa californica nuclear polyhedrosis virus was developed and performed in six different lepidopteran cell lines. The assay was not dependent upon cytopathic effect or polyhedron production, but rather upon viral antigen production and its recognition in a peroxidase-antiperoxidase staining procedure. The importance of using such an assay for accurately assessing infectivity in cell lines which produce polyhedra inefficiently was demonstrated. Differences among the cell lines in sensitivity to viral infection were clearly shown. Differences in the time required to produce infectious progeny were also noted among cells of the same cell line. Images PMID:16346059

  6. [Quantitative determination of the infectivity of nuclear polyhedrosis virus DNA on honeycomb moth (Galleria mellonella) larvae].

    PubMed

    Gorbunova, E E; Makarova, N I; Shchelkunova, G A

    1980-01-01

    The possibility of using honeycomb moth larvae for titration of nuclear polyhedrosis virus (NPV) infectious DNA and determinations of transfection effectiveness was studied. Honeycomb moth larvae were shown to be a sensitive system for NPV DNA titration. DEAE-dextran used as a protector increased NPV DNA infectivity 1000-fold, LD50 in this instance being 2 X 10(8) molecules per larva. The method of NPV DNA infectivity determinations by the number of larvae with polyhedreae in the fatty tissue is more sensitive than infectivity determinations by the number of dead larvae and permits titrations of low DNA concentrations. The curve of DNA titration in the presence of DEAE-dextran by the number of larvae with polyhedrae in the fatty tissue allows to quantitate native DNA within the range of 0.01 to 5 micrograms/ml.

  7. Photoreactivation and ultraviolet-enhanced reactivation of ultraviolet-irradiated nuclear polyhedrosis virus by insect cells.

    PubMed

    Witt, D J

    1984-01-01

    The nuclear polyhedrosis virus (Baculovirus) of Galleria mellonella (Pryalidae: Lepidoptera) was used to investigate the capability of cultured insect cells to repair ultraviolet (UV) induced damage in the viral genome. When assayed by the formation of plaques in the cell line TN-368, the survival of the virus was found to decrease linearly with increased ultraviolet exposure. The infectious capacity of UV-irradiated virions was significantly restored after exposing the TN-368 monolayers to either photoreactivation conditions (white fluorescent and black light) or to UV-enhanced reactivation conditions (far ultraviolet radiation). Using both types of repair sequentially resulted in higher reactivation than when either was used alone. These results indicate that pyrimidine dimers are the major factor responsible for inactivation of this virus by UV radiation but that other photolesions not repairable by photoreactivation partially account for the inactivation of the virus. PMID:6365037

  8. Mechanical properties and structure of silkworm cocoons: a comparative study of Bombyx mori, Antheraea assamensis, Antheraea pernyi and Antheraea mylitta silkworm cocoons.

    PubMed

    Zhang, J; Kaur, J; Rajkhowa, R; Li, J L; Liu, X Y; Wang, X G

    2013-08-01

    As a protective shell against environmental damage and attack by natural predators, the silkworm cocoon has outstanding mechanical properties. In particular, this multilayer non-woven composite structure can be exceptionally tough to enhance the chance of survival for silkworms while supporting their metabolic activity. Peel, out-of-plane compression and nano-indentation tests and micro-structure analysis were performed on four types of silkworm cocoon walls (domesticated Bombyx mori, semi-domesticated Antheraea assamensis and wild Antheraea pernyi and Antheraea mylitta silkworm cocoons) to understand the structure and mechanical property relationships. The wild silkworm cocoons were shown to be uniquely tough composite structures. The maximum work-of-fracture for the wild cocoons (A. pernyi and A. mylitta) was approximately 1000 J/m(2), which was almost 10 times the value for the domesticated cocoon (Bombyx mori) and 3~4 times the value for the semi-domesticated cocoon (A. assamensis). Calcium oxalate crystals were found to deposit on the outer surfaces of the semi-domesticated and wild cocoons. They did not show influence in enhancing the interlaminar adhesion between cocoon layers but exhibited much higher hardness than the cocoon pelades.

  9. Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system.

    PubMed

    Motohashi, Tomoko; Shimojima, Tsukasa; Fukagawa, Tatsuo; Maenaka, Katsumi; Park, Enoch Y

    2005-01-21

    Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. PMID:15596136

  10. Cytoplasmic dynein nomenclature

    PubMed Central

    Pfister, K. Kevin; Fisher, Elizabeth M.C.; Gibbons, Ian R.; Hays, Thomas S.; Holzbaur, Erika L.F.; McIntosh, J. Richard; Porter, Mary E.; Schroer, Trina A.; Vaughan, Kevin T.; Witman, George B.; King, Stephen M.; Vallee, Richard B.

    2005-01-01

    A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms. PMID:16260502

  11. Non-occluded particles of nuclear polyhedrosis virus infecting Galleria mellonella L.: titration in vivo and in vitro. Brief report.

    PubMed

    Komissarenko, S V; Zherebtsova, E N; Sutugina, L P; Primatchenko, L V

    1979-01-01

    Infectivity of non-occluded particles of nuclear polyhedrosis virus infecting Galleria mellonella L. was determined with bioassay technique in vivo and with plaque assay method in vitro using SCLd 135 (Quiot) established cell culture. A plaque-forming virus unit corresponds to about 50 ID50 for Galleria larvae injected into hemocoel for non-cloned and 0.1-1.0 ID50 for a virus isolate cloned from an infective plaque.

  12. Semipermissive replication of a nuclear polyhedrosis virus of Autographa californica in a gypsy moth cell line

    SciTech Connect

    McClintock, J.T.; Dougherty, E.M.; Weiner, R.M.

    1986-01-01

    Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of Autographa californica (AcMNPV). In this report, the authors demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with (/sup 35/S)methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early ..cap alpha.. proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.

  13. Direct effects of recombinant nuclear polyhedrosis viruses on selected nontarget organisms.

    PubMed

    Heinz, K M; McCutchen, B F; Herrmann, R; Parrella, M P; Hammock, B D

    1995-04-01

    A limitation to effective field use of naturally occurring nuclear polyhedrosis viruses (NPVs) is the slow rate at which they kill their host. In making NPVs a more attractive pest management tool, this problem has been addressed by modifying NPVs genetically to express insecticidal proteins resulting in substantial increases in their speed of action. One concern associated with these recombinant NPVs, however, is their effects on nontarget insects associated with pests targeted for control by applications of NPVs. Our studies evaluated the direct effects of wild-type Autographa californica NPV (AcNPV) and a recombinant AcNPV (AcAaIT) on three insects beneficial to production agriculture. The recombinant NPV expresses an insect-selective neurotoxin, AaIT, which was isolated from the scorpion, Androctonus australis Hector. Two generalist predators, Chysoperla carnea Stephens and Orius insidiosus (Say), were not adversely affected by feeding on larvae of Heliothis virescens (F.) infected with AcAaIT. Similarly, no adverse effects were detected in the honey bee, Apis mellifera L., when injected with wild-type or recombinant NPVs. Results from this study may provide a foundation upon which potential risks associated with genetically engineered NPVs may be evaluated on a limited scale in greenhouse or field experiments. PMID:7722081

  14. Multiple early transcripts and splicing of the Autographa californica nuclear polyhedrosis virus IE-1 gene.

    PubMed Central

    Chisholm, G E; Henner, D J

    1988-01-01

    The immediate-early IE-1 gene of Autographa californica nuclear polyhedrosis virus was cloned, and its nucleotide sequence was determined. Sequence analysis indicated that this gene would encode a protein of 582 amino acids with a predicted molecular weight of 66,822. Analysis of IE-1 gene expression during baculovirus infection identified two transcripts. One, 1.9 kilobases (kb), was expressed at constant steady-state levels throughout infection, whereas the other, 2.1 kb, was expressed only early in infection. Analysis of IE-1 cDNA clones demonstrated that the 2.1-kb transcript contained the entire 1.9-kb transcript (exon 1) plus an additional 5' end (exon 0). Genomic Southern analysis placed the exon 0 sequences on the EcoRI B fragment, 4 kilobase pairs upstream of exon 1. Sequencing of the upstream region identified an open reading frame whose 5' end was identical to the exon 0 sequences in the cDNAs. Examination of the genomic DNA sequences around the exon-exon junction revealed sequences similar to published consensus splice acceptor and donor sequences. This is the first example of splicing of any viral transcript during baculovirus infection. Images PMID:3043024

  15. Data for increase of Lymantria dispar male survival after topical application of single-stranded RING domain fragment of IAP-3 gene of its nuclear polyhedrosis virus.

    PubMed

    Oberemok, Volodymyr V; Laikova, Kateryna V; Zaitsev, Aleksei S; Gushchin, Vladimir A; Skorokhod, Oleksii A

    2016-06-01

    This data article is related to the research article entitled "The RING for gypsy moth control: topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide" [1]. This article reports on significantly higher survival of gypsy moth Lymantria dispar male individuals in response to topical application of single-stranded DNA, based on RING (really interesting new gene) domain fragment of LdMNPV (L. dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene and acted as DNA insecticide. PMID:27054151

  16. Data for increase of Lymantria dispar male survival after topical application of single-stranded RING domain fragment of IAP-3 gene of its nuclear polyhedrosis virus

    PubMed Central

    Oberemok, Volodymyr V.; Laikova, Kateryna V.; Zaitsev, Aleksei S.; Gushchin, Vladimir A.; Skorokhod, Oleksii A.

    2016-01-01

    This data article is related to the research article entitled “The RING for gypsy moth control: topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide” [1]. This article reports on significantly higher survival of gypsy moth Lymantria dispar male individuals in response to topical application of single-stranded DNA, based on RING (really interesting new gene) domain fragment of LdMNPV (L. dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene and acted as DNA insecticide. PMID:27054151

  17. DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L. ) larvae

    SciTech Connect

    Keating, S.T.; Burand, J.P.; Elkinton, J.S. )

    1989-11-01

    Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. The hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.

  18. [The effect of fodder on the susceptibility of gypsy moth (Lymantria dispar L.) to nuclear polyhedrosis virus].

    PubMed

    Bakhvalov, S A; Bakhvalova, V N

    2009-01-01

    Gypsy moth (Lymantria dispar L.) growing on different feeding substrates was shown to affect their susceptibility to nuclear polyhedrosis virus (NPV). The insects feeding on birch leaves had the lowest sensitivity to NPV than those on willow leaves, but the insects growing on pine needles showed the highest susceptibility. The sensitivity of the gypsy moths on willow leaves was higher than that of the gypsy moths on birch leaves and lower than that of those on pine needles. At the same time, it did not differ from that of the caterpillars on artificial feeding. The virus polyhedrons formed in the caterpillars on birch or willow leaves were more than those on another fodder.

  19. Cytoplasmic Viral Replication Complexes

    PubMed Central

    den Boon, Johan A.; Diaz, Arturo; Ahlquist, Paul

    2010-01-01

    Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bounded RNA replication complexes and their close coordination with virion assembly and budding. The structure, function and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions, and more general similarities with the replication factories of cytoplasmic DNA viruses. PMID:20638644

  20. A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus

    USGS Publications Warehouse

    Leisy, D.J.; Rasmussen, C.; Owusu, E.O.; Rohrmann, G.F.

    1997-01-01

    The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with Bg/II sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (ple-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1- specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.

  1. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells.

    PubMed

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi

    2006-05-01

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type. PMID:16545777

  2. A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

    PubMed Central

    Ohkawa, T; Majima, K; Maeda, S

    1994-01-01

    Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

  3. Expression of the IE1 transactivator of Autographa californica nuclear polyhedrosis virus during viral infection.

    PubMed

    Choi, J; Guarino, L A

    1995-05-10

    The immediate-early IE1 protein of Autographa californica nuclear polyhedrosis virus (AcMNPV) is an important regulator of viral gene transcription. To provide a tool for further analysis of the expression and function of IE1, a polyclonal antiserum was raised against IE1 expressed in bacteria. Immunoblot analysis of infected cell lysates was used to monitor the accumulation of IE1 throughout the viral life cycle. When extracts were prepared in the presence of phosphatase inhibitors, only one protein band was detected on SDS-polyacrylamide gels. However, in the absence of phosphatase inhibitors, at least four distinct electrophoretic species were detected. Mobility shift assays were conducted using an enhancer DNA probe and whole cell extracts prepared at different times postinfection. Results indicated that the enhancer-binding activity of IE1 increased from 4 to 72 hr postinfection. DNA-protein complexes formed with infected cell extracts migrated more slowly than those formed with transfected cell extracts. This effect was more pronounced with extracts prepared in the presence of phosphatase inhibitors. Supershift experiments with IE1 antiserum confirmed that IE1 was a component of DNA-protein complexes in both transfected and infected cell extracts. A titration experiment was done to determine the minimal amounts of IE1 required for activation of the 39k promoter in the presence and absence of a cis-linked enhancer element. These analyses indicated that the intracellular levels of IE1 are not sufficient for enhancer-independent activation of the 39k promoter during the early phase of viral infection. Quantitative immunoblots revealed that the amount of IE1 in budded virus was less than 0.68 mole per mole of viral DNA, suggesting that IE1 is not a structural protein of AcNPV.

  4. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

    SciTech Connect

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi . E-mail: hiroshi.takaku@it-chiba.ac.jp

    2006-05-05

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

  5. Chloroplast and Cytoplasmic Enzymes

    PubMed Central

    Anderson, Louise E.; Advani, Vimal R.

    1970-01-01

    Three pea (Pisum sativum) leaf chloroplast enzymes—triose phosphate isomerase, glyceric acid 3-phosphate kinase, and fructose 1,6-diphosphate aldolase—have been separated from the corresponding cytoplasmic enzymes by isoelectric focusing. These three enzymes of the reductive pentose phosphate cycle are therefore distinct proteins, not identical with the analogous enzymes of the Embden-Meyerhof-Parnas pathway. PMID:16657347

  6. Cytoplasmic Z-RNA

    SciTech Connect

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-09-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.

  7. Oxidative Damaged Products, Level of Hydrogen Peroxide, and Antioxidant Protection in Diapausing Pupa of Tasar Silk Worm, Antheraea mylitta: A Comparative Study in Two Voltine Groups.

    PubMed

    Sahoo, Alpana; Dandapat, Jagneshwar; Samanta, Luna

    2015-01-01

    The present study demonstrates tissue-specific (hemolymph and fat body) and inter-voltine [bivoltine (BV) and trivoltine (TV)] differences in oxidatively damaged products, H2O2 content, and the relative level of antioxidant protection in the diapausing pupae of Antheraea mylitta. Results suggest that fat body (FB) of both the voltine groups has oxidative predominance, as evident from the high value of lipid peroxidation and H2O2 content, despite better enzymatic defenses in comparison to hemolymph (HL). This may be attributed to the higher metabolic rate of the tissue concerned, concomitant with high lipid content and abundance of polyunsaturated fatty acids (PUFA). Nondetectable catalase activity in the pupal hemolymph of both strains apparently suggests an additional mechanism for H2O2 metabolism in the tissue. Inter-voltine comparison of the oxidative stress indices and antioxidant defense potential revealed that the TV group has a higher oxidative burden, lower activities for the antioxidant enzymes, and compensatory nonenzymatic protection from reduced glutathione and ascorbic acid.

  8. Molecular cloning, sequence analysis and expression of genome segment 7 (S7) of Antheraea mylitta cypovirus (AmCPV) that encodes a viral structural protein.

    PubMed

    Chavali, Venkata Ramana Murthy; Ghosh, Ananta K

    2007-10-01

    The Genome segment 7 (S7) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus (AmCPV) was converted to cDNA, cloned and sequenced. The nucleotide sequence showed that segment 7 consisted of 1789 nucleotides with an ORF of 530 amino acids and could encode a protein of approximately 61 kDa, termed P61. The 5' terminal sequence, AGTAAT and the 3' terminal sequence, AGAGC of the plus strand was found to be the same as genome segment 10 of AmCPV encoding polyhedrin. No sequence similarity was found by searching nucleic acid and protein sequence databases using BLAST. The secondary structure prediction showed the presence of 17 alpha-helices, 18 extended beta-sheets along the entire length of P61. The ORF of segment 7 was expressed in E. coli as His-tagged fusion protein, purified through Ni-NTA chromatography, and polyclonal antibody was raised in rabbit indicating that P61 is immunogenic. Immunoblot analysis using this antibody on viral infected cells as well as purified polyhedra showed that P61 is a viral structural protein. Motif scan search showed some similarity of P61 with Inosine monophosphate dehydrogenase (IMPDH) cystathionine-beta-synthase (CBS) domain at the C-terminus and it was hypothesized that by binding to single stranded viral RNA through its CBS domain P61 may help in virus replication or transcription.

  9. Oxidative Damaged Products, Level of Hydrogen Peroxide, and Antioxidant Protection in Diapausing Pupa of Tasar Silk Worm, Antheraea mylitta: A Comparative Study in Two Voltine Groups.

    PubMed

    Sahoo, Alpana; Dandapat, Jagneshwar; Samanta, Luna

    2015-01-01

    The present study demonstrates tissue-specific (hemolymph and fat body) and inter-voltine [bivoltine (BV) and trivoltine (TV)] differences in oxidatively damaged products, H2O2 content, and the relative level of antioxidant protection in the diapausing pupae of Antheraea mylitta. Results suggest that fat body (FB) of both the voltine groups has oxidative predominance, as evident from the high value of lipid peroxidation and H2O2 content, despite better enzymatic defenses in comparison to hemolymph (HL). This may be attributed to the higher metabolic rate of the tissue concerned, concomitant with high lipid content and abundance of polyunsaturated fatty acids (PUFA). Nondetectable catalase activity in the pupal hemolymph of both strains apparently suggests an additional mechanism for H2O2 metabolism in the tissue. Inter-voltine comparison of the oxidative stress indices and antioxidant defense potential revealed that the TV group has a higher oxidative burden, lower activities for the antioxidant enzymes, and compensatory nonenzymatic protection from reduced glutathione and ascorbic acid. PMID:26816485

  10. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F. William; Rosenberg, Alan H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

  11. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F.W.; Rosenberg, A.H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

  12. Heterogeneity of Maize Cytoplasmic Genomes among Male-Sterile Cytoplasms

    PubMed Central

    Pring, D. R.; Levings, C. S.

    1978-01-01

    Maize mitochondrial and chloroplast DNA's were prepared from normal (fertile) lines or single crosses and from members of the T, C, and S groups of male-sterile cytoplasms. Restriction endonucleases HindIII, BamI, EcoRI, and SalI were used to restrict the DNA, and the resultant fragments were electrophoresed in agarose gels. The results show that the N (fertile), T, C, and S cytoplasms each contained distinct mitochondrial DNA (mtDNA). These distinctive patterns were unaffected by nuclear genotype. No evidence of paternal inheritance of mtDNA was observed. Chloroplast DNA (ctDNA) from the N, C, and T cytoplasms was indistinguishable by HindIII, SalI, or EcoRI endonuclease digestion. The S cytoplasm ctDNA, however, was slightly different from that of other cytoplasms, as indicated by a slight displacement of one band in HindIII digests. The molecular weight of maize ctDNA was estimated to be as high as 88 x 106. Estimates of the minimum molecular weight of maize mtDNA ranged from 116–131 x 106, but the patterns were to complex for an unambiguous determination. Based on HindIII data, a comparison of the molecular weight of mtDNA bands common to the N, T. C, and S cytoplasms suggests that C cytoplasm most closely resembles N cytoplasm. The T and S sources are more divergent from the C and N cytoplasms. These results indicate a possible gradation of relatedness among male-sterile cytoplasms. The marked variation in mtDNA, with apparently less variation in ctDNA, represents circumstantial, but compelling, evidence that mtDNA may be involved in the male sterility and disease susceptibility traits in maize. PMID:17248823

  13. Simulated acid rain reduces the susceptibility of the European pine sawfly (Neodiprion sertifer) to its nuclear polyhedrosis virus.

    PubMed

    Neuvonen, S; Saikkonen, K; Haukioja, E

    1990-06-01

    The study dealt with the effect of simulated acid rain (both H(2)SO(4) and HNO(3); acidities of pH 4 and pH 3) on the susceptibility of the larvae of Neodiprion sertifer to its nuclear polyhedrosis virus. Scots pines growing in a subarctic area with low ambient pollution levels were irrigated with simulated acid rain during two summers. Neodiprion larvae fed with foliage from the experimental trees were infected with a dilute virus suspension. The acid treatment of host trees had a significant effect on the proportion of virus-treated larvae alive 16 days after the virus application: there were almost no differences between the controls and the pH 4 irrigation group, but on the needles of pH 3-treated trees larval survival was twice as high as with other treatments. The direct spraying of acid water on the needles before they were fed to the larvae did not significantly affect the survival of virus infected larvae. Our results suggest that acid rain may reduce the susceptibility of Neodiprion larvae to virus disease via changes in the quality of pine foliage.

  14. Induction of natural killer cell-dependent antitumor immunity by the Autographa californica multiple nuclear polyhedrosis virus.

    PubMed

    Kitajima, Masayuki; Abe, Takayuki; Miyano-Kurosaki, Naoko; Taniguchi, Masaru; Nakayama, Toshinori; Takaku, Hiroshi

    2008-02-01

    Wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a variety of mammalian cell types in vitro, but does not replicate in these cells. We investigated the effects of AcMNPV in the induction of the immune response and tumor metastasis in mice. After intravenous injection, AcMNPV was taken up by the liver and spleen, and preferentially infected dendritic cells (DCs) and B cells in the spleen; costimulatory molecules CD40, CD80, and CD86 were upregulated in the DCs. The hepatic mononuclear cells (MNCs) in these animals were highly cytotoxic to natural killer (NK)-sensitive YAC-1 and B16 melanoma cells, but not to NK-resistant EL4 cells. Intravenous injection of AcMNPV-induced NK cell proliferation in the liver and spleen, and enhanced antitumor immunity in mice with B16 liver metastases. Furthermore, such treatment increased the survival of C57BL/6, J alpha 281 (-/-), and interferon (IFN)-gamma (-/-) mice that were previously injected with B16 tumor cells. AcMNPV injection did not enhance the survival of NK cell-depleted mice. Moreover, one AcMNPV treatment effectively prolonged survival in a B16 liver metastasis model, and was equivalent to five treatments with recombinant interleukin-12 (IL-12) protein. These findings suggest that AcMNPV efficiently stimulates NK cell-mediated antitumor immunity. PMID:18059370

  15. Cellular Subcompartments through Cytoplasmic Streaming.

    PubMed

    Pieuchot, Laurent; Lai, Julian; Loh, Rachel Ann; Leong, Fong Yew; Chiam, Keng-Hwee; Stajich, Jason; Jedd, Gregory

    2015-08-24

    Cytoplasmic streaming occurs in diverse cell types, where it generally serves a transport function. Here, we examine streaming in multicellular fungal hyphae and identify an additional function wherein regimented streaming forms distinct cytoplasmic subcompartments. In the hypha, cytoplasm flows directionally from cell to cell through septal pores. Using live-cell imaging and computer simulations, we identify a flow pattern that produces vortices (eddies) on the upstream side of the septum. Nuclei can be immobilized in these microfluidic eddies, where they form multinucleate aggregates and accumulate foci of the HDA-2 histone deacetylase-associated factor, SPA-19. Pores experiencing flow degenerate in the absence of SPA-19, suggesting that eddy-trapped nuclei function to reinforce the septum. Together, our data show that eddies comprise a subcellular niche favoring nuclear differentiation and that subcompartments can be self-organized as a consequence of regimented cytoplasmic streaming.

  16. Photoreactivation of a Cytoplasmic Virus

    PubMed Central

    Pfefferkorn, E. R.; Boyle, Mary K.

    1972-01-01

    Ultraviolet light-inactivated frog virus 3 is efficiently photoreactivated by chick embryo cells. A cellular enzyme is presumably responsible for this repair of viral deoxyribonucleic acid, for the phenomenon is insensitive to an inhibitor of protein synthesis and is not seen in mammalian cells that are known to lack photoreactivating enzyme. Since frog virus 3 is a cytoplasmic virus, functionally significant amounts of photoreactivating enzyme are probably present in the cytoplasm of chick embryo cells. PMID:5062749

  17. Specificity factors in cytoplasmic polyadenylation.

    PubMed

    Charlesworth, Amanda; Meijer, Hedda A; de Moor, Cornelia H

    2013-01-01

    Poly(A) tail elongation after export of an messenger RNA (mRNA) to the cytoplasm is called cytoplasmic polyadenylation. It was first discovered in oocytes and embryos, where it has roles in meiosis and development. In recent years, however, has been implicated in many other processes, including synaptic plasticity and mitosis. This review aims to introduce cytoplasmic polyadenylation with an emphasis on the factors and elements mediating this process for different mRNAs and in different animal species. We will discuss the RNA sequence elements mediating cytoplasmic polyadenylation in the 3' untranslated regions of mRNAs, including the CPE, MBE, TCS, eCPE, and C-CPE. In addition to describing the role of general polyadenylation factors, we discuss the specific RNA binding protein families associated with cytoplasmic polyadenylation elements, including CPEB (CPEB1, CPEB2, CPEB3, and CPEB4), Pumilio (PUM2), Musashi (MSI1, MSI2), zygote arrest (ZAR2), ELAV like proteins (ELAVL1, HuR), poly(C) binding proteins (PCBP2, αCP2, hnRNP-E2), and Bicaudal C (BICC1). Some emerging themes in cytoplasmic polyadenylation will be highlighted. To facilitate understanding for those working in different organisms and fields, particularly those who are analyzing high throughput data, HUGO gene nomenclature for the human orthologs is used throughout. Where human orthologs have not been clearly identified, reference is made to protein families identified in man.

  18. Probing the structure of cytoplasm

    PubMed Central

    1986-01-01

    We have used size-fractionated, fluorescent dextrans to probe the structure of the cytoplasmic ground substance of living Swiss 3T3 cells by fluorescence recovery after photobleaching and video image processing. The data indicate that the cytoplasm of living cells has a fluid phase viscosity four times greater than water and contains structural barriers that restrict free diffusion of dissolved macromolecules in a size-dependent manner. Assuming these structural barriers comprise a filamentous meshwork, the combined fluorescence recovery after photobleaching and imaging data suggest that the average pore size of the meshwork is in the range of 300 to 400 A, but may be as small as 200 A in some cytoplasmic domains. PMID:2423529

  19. Identification of two independent transcriptional activation domains in the Autographa californica multicapsid nuclear polyhedrosis virus IE1 protein.

    PubMed

    Slack, J M; Blissard, G W

    1997-12-01

    The Autographa californica multicapsid nuclear polyhedrosis virus immediate-early protein, IE1, is a 582-amino-acid phosphoprotein that regulates the transcription of early viral genes. Deletion of N-terminal regions of IE1 in previous studies (G. R. Kovacs, J. Choi, L. A. Guarino, and M. D. Summers, J. Virol. 66:7429-7437, 1992) resulted in the loss of transcriptional activation, suggesting that this region may contain an acidic activation domain. To identify independently functional transcriptional activation domains, we developed a heterologous system in which potential regulatory domains were fused with a modified Escherichia coli Lac repressor protein that contains a nuclear localization signal (NLacR). Transcriptional activation by the resulting NLacR-IE1 chimeras was measured with a basal baculovirus early promoter containing optimized Lac repressor binding sites (lac operators). Chimeras containing IE1 peptides dramatically activated transcription of the basal promoter only when lac operator sequences were present. In addition, transcriptional activation by NLacR-IE1 chimeras was allosterically regulated by the lactose analog, isopropyl-beta-D-thiogalactopyranoside (IPTG). For a more detailed analysis of IE1 regulatory domains, the M1 to T266 N-terminal portion of IE1 was subdivided (on the basis of average amino acid charge) into five smaller regions which were fused in various combinations to NLacR. Regions M1 to N125 and A168 to G222 were identified as independent transcriptional activation domains. Some NLacR-IE1 chimeras exhibited retarded migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. As with wild-type IE1, this aberrant gel mobility was associated with phosphorylation. Mapping studies with the NLacR-IE1 chimeras indicate that the M1 to A168 region of IE1 is necessary for this phosphorylation-associated effect.

  20. [Detection of the nuclear polyhedrosis virus DNA in samples from eggs and caterpillars at different stages of the gypsy moth Lymantria dispar (L.) population dynamics].

    PubMed

    Bakhvalov, S A; Martem'ianov, V V; Bakhvalova, V N; Morozova, O V

    2012-01-01

    The nuclear polyhedrosis virus (NPV) DNA was detected in samples from eggs and caterpillars of the gypsy moth collected in natural populations of the Western Siberia and Ural by means of PCR with primers corresponding to the polyhedrin gene. According to censuring data, the gypsy moth populations of Western Siberia were at the depression stage. The NPV DNA detection frequencies in eggs (8.6 +/- 4.8% - 13.6 +/- 5.2%) and caterpillars (21.0 +/- 6.3% - 22.2 +/- 6.7%) were not significantly differed. In the Urals, collection of the insects was performed in their gradation focus at the phase of maximal abundance. The DNA detection rate in eggs (11.4 +/- 5.0%) was confidently (p < 0.001) lower than in caterpillars (59.8 +/- 5.6%). Consequently, variations of the NPV infection prevalence during ontogenesis of Lymantria dispar (L.) was associated with the gradation cycle of the insect population dynamics.

  1. Single-stranded DNA fragments of insect-specific nuclear polyhedrosis virus act as selective DNA insecticides for gypsy moth control.

    PubMed

    Oberemok, Volodymyr V; Skorokhod, Oleksii A

    2014-07-01

    This paper focuses on the DNA insecticides as a novel preparation against gypsy moth (Lymantria dispar) based on DNA fragments of the anti-apoptotic gene of its nuclear polyhedrosis virus. It was found that the external application of a solution with two single-stranded DNA fragments from BIR and RING domains of LdMNPV (L.dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene induces a significantly higher mortality of gypsy moth caterpillars in comparison with the application of the control solutions. This effect does not depend on the infection of caterpillars with LdMNPV. The results also show that DNA insecticides based on LdMNPV IAP-3 gene fragments can be selective in action, and at least are not harmful to tobacco hornworm (Manduca sexta) and black cutworm (Agrotis ipsilon). Part of the gypsy moth genome cloned with the fragments of BIR and RING domains of LdMNPV IAP-3 gene as primers, has an overlap with the corresponding part of the LdMNPV IAP-3 gene and L.dispar IAP-1 mRNA for an inhibitor of apoptosis protein with the high cover by query, allows assuming that we cloned a part of gypsy moth anti-apoptosis gene. This finding gives the grounding that proposed here DNA insecticides might act through the blocking of the mechanisms involved in post transcriptional expression of insect anti-apoptosis genes. The results show the insecticidal potential of the viral genome fragments that can be used to create safe and relatively fast-acting DNA insecticides to control the quantity of gypsy moth populations, important task for forestry and agriculture.

  2. Single-stranded DNA fragments of insect-specific nuclear polyhedrosis virus act as selective DNA insecticides for gypsy moth control.

    PubMed

    Oberemok, Volodymyr V; Skorokhod, Oleksii A

    2014-07-01

    This paper focuses on the DNA insecticides as a novel preparation against gypsy moth (Lymantria dispar) based on DNA fragments of the anti-apoptotic gene of its nuclear polyhedrosis virus. It was found that the external application of a solution with two single-stranded DNA fragments from BIR and RING domains of LdMNPV (L.dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene induces a significantly higher mortality of gypsy moth caterpillars in comparison with the application of the control solutions. This effect does not depend on the infection of caterpillars with LdMNPV. The results also show that DNA insecticides based on LdMNPV IAP-3 gene fragments can be selective in action, and at least are not harmful to tobacco hornworm (Manduca sexta) and black cutworm (Agrotis ipsilon). Part of the gypsy moth genome cloned with the fragments of BIR and RING domains of LdMNPV IAP-3 gene as primers, has an overlap with the corresponding part of the LdMNPV IAP-3 gene and L.dispar IAP-1 mRNA for an inhibitor of apoptosis protein with the high cover by query, allows assuming that we cloned a part of gypsy moth anti-apoptosis gene. This finding gives the grounding that proposed here DNA insecticides might act through the blocking of the mechanisms involved in post transcriptional expression of insect anti-apoptosis genes. The results show the insecticidal potential of the viral genome fragments that can be used to create safe and relatively fast-acting DNA insecticides to control the quantity of gypsy moth populations, important task for forestry and agriculture. PMID:25052520

  3. Cytoplasmic hydrogen ion diffusion coefficient.

    PubMed Central

    al-Baldawi, N F; Abercrombie, R F

    1992-01-01

    The apparent cytoplasmic proton diffusion coefficient was measured using pH electrodes and samples of cytoplasm extracted from the giant neuron of a marine invertebrate. By suddenly changing the pH at one surface of the sample and recording the relaxation of pH within the sample, an apparent diffusion coefficient of 1.4 +/- 0.5 x 10(-6) cm2/s (N = 7) was measured in the acidic or neutral range of pH (6.0-7.2). This value is approximately 5x lower than the diffusion coefficient of the mobile pH buffers (approximately 8 x 10(-6) cm2/s) and approximately 68x lower than the diffusion coefficient of the hydronium ion (93 x 10(-6) cm2/s). A mobile pH buffer (approximately 15% of the buffering power) and an immobile buffer (approximately 85% of the buffering power) could quantitatively account for the results at acidic or neutral pH. At alkaline pH (8.2-8.6), the apparent proton diffusion coefficient increased to 4.1 +/- 0.8 x 10(-6) cm2/s (N = 7). This larger diffusion coefficient at alkaline pH could be explained quantitatively by the enhanced buffering power of the mobile amino acids. Under the conditions of these experiments, it is unlikely that hydroxide movement influences the apparent hydrogen ion diffusion coefficient. PMID:1617134

  4. Measurement of Cytoplasmic Streaming in Drosophila Melanogaster

    NASA Astrophysics Data System (ADS)

    Ganguly, Sujoy; Williams, Lucy; Palacios, Isabel; Goldstein, Raymond

    2010-11-01

    During stage 9 of Drosophila melanogastor oogenesis flow of the oocyte cytoplasm, driven by kinesin 1 motor protein is observed. This cytoplasmic streaming is analyzed by PIV in both wild type and kinesin light chain mutants, revealing striking statistical differences. Further measurements of the rheology of the oocyte allow for estimations of the mechanical energy needed to generate the observed flows.

  5. Cytoplasmic Streaming - Skylab Student Experiment ED-63

    NASA Technical Reports Server (NTRS)

    1973-01-01

    This chart describes the Skylab student experiment (ED-63), Cytoplasmic Streaming, proposed by Cheryl A. Peitz of Arapahoe High School, Littleton, Colorado. Experiment ED-63 was to observe the effect of zero-gravity on cytoplasmic streaming in the aquatic plant named Elodea, commonly called water weed or water thyme. The phenomenon of cytoplasmic streaming is not well understood, but it is recognized as the circulation mechanism of the internal materials or cytoplasm of a cell. Cytoplasm is a gelatinous substance that has the ability to change its viscosity and flow, carrying various cell materials with it. The activity can be stimulated by sunlight or heat. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

  6. Deep cytoplasmic rearrangements in ventralized Xenopus embryos

    NASA Technical Reports Server (NTRS)

    Brown, E. E.; Denegre, J. M.; Danilchik, M. V.

    1993-01-01

    Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos

  7. Baculovirus replication: characterization of DNA and proteins synthesized by a nuclear polyhedrosis virus of Lymantria dispar, the gypsy moth, in a homologous cell line

    SciTech Connect

    McClintock, J.T.

    1985-01-01

    A multiple-embedded nuclear polyhedrosis virus (NPV) of the gypsy moth, Lymantria dispar (LdMNPV), is used for biological control. However, LdMNPV has low natural virulence and a long infection cycle in relation to other NPVs. Therefore, the replicative cycle of LdMNPV was investigated using a homologous cell line, IPLB-LD-652Y. Based on analyses of virus growth curves LdMNPV nonoccluded virus and polyhedral inclusion bodies appeared approximately 20 and 50 hr postinfection (p.i.), respectively. LdMNPV polypeptides, identified by autoradiography of (/sup 35/S)-methionine labeled fractions in SDS-PAGE, were synthesized in sequential phases: (1) an early ..cap alpha.. phase of replication (4 polypeptides from 4 to 12 hr p.i.), (2) an intermediate ..beta.. phase (20 polypeptides from 12 to 24 hr p.i.), and a late ..gamma.. phase (4 polypeptides from 24 to 28 hr p.i.). In infected cells at least four polypeptides were post-translational cleaved and/or modified. Pulse-labeling with (/sup 3/H)-mannose, (/sup 3/H)-N-acetyl-glucosamine or (/sup 32/P)-monosodium phosphate revealed several viral polypeptides which were glycosylated and/or phosphorylated. DNA:DNA dot hybridization experiments suggested that LdMNPV DNA synthesis was initiated between 12 to 16 hr p.i., increasing significantly thereafter.

  8. The RING for gypsy moth control: Topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide.

    PubMed

    Oberemok, Volodymyr V; Laikova, Kateryna V; Zaitsev, Aleksei S; Gushchin, Vladimir A; Skorokhod, Oleksii A

    2016-07-01

    Numerous studies suggest a cellular origin for the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) anti-apoptosis genes IAPs, thus opening a possibility to use the fragments of these genes for modulation of host metabolism. We report here the strong insecticidal and metabolic effect of single-stranded antisense DNA fragment from RING (really interesting new gene) domain of gypsy moth LdMNPV IAP-3 gene: specifically, on reduction of biomass (by 35%) and survival of L. dispar caterpillars. The treatment with this DNA fragment leads to a significantly higher mortality rates of female insects (1.7 fold) accompanied with the signs of apoptosis. Additionally, we show increased expression of host IAP-1, caspase-4 and gelsolin genes in eggs laid by survived females treated with RING DNA fragment accompanied with calcium and magnesium imbalance, indicating that the strong stress reactions and metabolic effects are not confined to treated insects but likely led to apoptosis in eggs too. The proposed new approach for insect pest management, which can be considered as advancement of "microbial pesticides", is based on the application of the specific virus DNA, exploiting the knowledge about virus-pest interactions and putting it to the benefit of mankind.

  9. The RING for gypsy moth control: Topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide.

    PubMed

    Oberemok, Volodymyr V; Laikova, Kateryna V; Zaitsev, Aleksei S; Gushchin, Vladimir A; Skorokhod, Oleksii A

    2016-07-01

    Numerous studies suggest a cellular origin for the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) anti-apoptosis genes IAPs, thus opening a possibility to use the fragments of these genes for modulation of host metabolism. We report here the strong insecticidal and metabolic effect of single-stranded antisense DNA fragment from RING (really interesting new gene) domain of gypsy moth LdMNPV IAP-3 gene: specifically, on reduction of biomass (by 35%) and survival of L. dispar caterpillars. The treatment with this DNA fragment leads to a significantly higher mortality rates of female insects (1.7 fold) accompanied with the signs of apoptosis. Additionally, we show increased expression of host IAP-1, caspase-4 and gelsolin genes in eggs laid by survived females treated with RING DNA fragment accompanied with calcium and magnesium imbalance, indicating that the strong stress reactions and metabolic effects are not confined to treated insects but likely led to apoptosis in eggs too. The proposed new approach for insect pest management, which can be considered as advancement of "microbial pesticides", is based on the application of the specific virus DNA, exploiting the knowledge about virus-pest interactions and putting it to the benefit of mankind. PMID:27265824

  10. Cytoplasmic rearrangements associated with amphibian egg symmetrization

    NASA Technical Reports Server (NTRS)

    Malacinski, G. M.

    1984-01-01

    Cytoplasmic rearrangements which follow fertilization were mentioned in normal and inverted eggs. A set of yolk compartments was resolved by cytological analyses of both normally oriented and inverted eggs. Those compartments were characterized by their yolk platelet compositions and movement during egg inversion. It is found that during egg inversion the yolk compartments shift minor cytoplasmic compartments which line the egg cortex. Those yolk mass shifts occurred only after the inverted egg was activated. The direction of shift of the major yolk components, rather than the sperm entrance site, determines the dorsal/ventral polarity of the inverted egg. Among different spawnings the rate of shift varied. Eggs that displayed the fastest rate of shift exhibited the highest frequency of developmental abnormalities during organogenesis. Interpretation of novel observations on cytoplasmic organization provide criticism of some earlier models. A new density compartment model is presented as a coherent way to view the organization of the egg cytoplasm and the development of bilateral symmetry.

  11. Reorganization of cytoplasmic structures during cell fusion.

    PubMed

    Zheng, Q A; Chang, D C

    1991-11-01

    In order to provide a better understanding of the dynamic process of cell fusion, we studied the reorganization of cytoplasmic structures in electro-fused CV-1 cells. Using fluorescence microscopy and double staining methods, we examined correlations between the structural patterns of the major cytoskeletal proteins (microtubules, actin and vimentin intermediate filaments) and the distribution of various organelles (endoplasmic reticulum, mitochondria and nuclei) at different stages of cell fusion. Our results suggest that microtubules appear to play a primary role in the process of cytoplasmic reorganization. At the early stage of cell fusion, microtubules were observed to infiltrate rapidly into the newly formed cytoplasmic bridges and establish a connection between the cytoskeletal networks of fusing cells. The reorganization of microtubules was found to be correlated with the redistribution of endoplasmic reticulum (ER), vimentin intermediate filaments, mitochondria, and the aggregation of nuclei. The F-actin system, on the other hand, appeared to be independent of the reorganization of the other cytoplasmic structures. The principal function of F-actin during cell fusion is probably to widen the cytoplasmic bridges by lamellipodial extension.

  12. Rotavirus disrupts cytoplasmic P bodies during infection.

    PubMed

    Bhowmick, Rahul; Mukherjee, Arpita; Patra, Upayan; Chawla-Sarkar, Mamta

    2015-12-01

    Cytoplasmic Processing bodies (P bodies), the RNA-protein aggregation foci of translationally stalled and potentially decaying mRNA, have been reported to be differentially modulated by viruses. Rotavirus, the causative agent of acute infantile gastroenteritis is a double stranded RNA virus which completes its entire life-cycle exclusively in host cell cytoplasm. In this study, the fate of P bodies was investigated upon rotavirus infection. It was found that P bodies get disrupted during rotavirus infection. The disruption occurred by more than one different mechanism where deadenylating P body component Pan3 was degraded by rotavirus NSP1 and exonuclease XRN1 along with the decapping enzyme hDCP1a were relocalized from cytoplasm to nucleus. Overall the study highlights decay and subcellular relocalization of P body components as novel mechanisms by which rotavirus subverts cellular antiviral responses.

  13. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  14. Cytoplasmic Sterility Factors in VICIA FABA L

    PubMed Central

    Edwardson, J. R.; Bond, D. A.; Christie, R. G.

    1976-01-01

    Tissues of cytoplasmic male sterile, maintainer, restorer, and restored lines, and sterile plants which reverted to fertility in Vicia faba were examined in ultrathin sections. Cytoplasmic spherical bodies (CSB), ca. 70 nm in diameter, were observed in tissues of all sterile plants but not in tissues of maintainer, restorer or restored sterile plants. No CSB were observed in a reverted fertile branch of a tiller-sterile plant, nor in 5 of 6 reverted fertile plants. One reverted fertile plant contained CSB in ovules. It is proposed that the CSB are the sites of, or possibly, products of, sterility factors in Vicia faba. PMID:17248701

  15. Subunit organization in cytoplasmic dynein subcomplexes

    PubMed Central

    King, Stephen J.; Bonilla, Myriam; Rodgers, Michael E.; Schroer, Trina A.

    2002-01-01

    Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain–binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo. PMID:11967380

  16. Detection of cytoplasmic glycosylation associated with hydroxyproline.

    PubMed

    West, Christopher M; van der Wel, Hanke; Blader, Ira J

    2006-01-01

    A special class of glycosylation occurs on a proline residue of the cytoplasmic/nuclear protein Skp1 in the social amoeba Dictyostelium. For this glycosylation to occur, the proline must first be hydroxylated by the action of a soluble prolyl 4-hydroxylase acting on the protein. Cytoplasmic prolyl 4-hydroxylases are dioxygen-dependent enzymes that have low affinity for their O2 substrate and, therefore, have been implicated in O2-sensing in Dictyostelium, as well as in vertebrates and invertebrates. The sugar-hydroxyproline linkage has low abundance, is resistant to alkali cleavage and known glycosidases, and does not bind known lectins. However, initial screens for this modification can be made by assessing changes in electrophoretic mobility of candidate proteins after treatment of cells with prolyl hydroxylase inhibitors, and/or by metabolic labeling with [3H]sugar precursors. In addition, cytoplasmic hydroxylation/glycosylation can be assessed by assaying for cytoplasmic glycosyltransferases. Here we describe these methods and examples of their use in analyzing Skp1 glycosylation in Dictyostelium and the apicomplexan Toxoplasma gondii, the causative agent of toxoplasmosis in humans. PMID:17132515

  17. Reconstitution of the human cytoplasmic dynein complex.

    PubMed

    Trokter, Martina; Mücke, Norbert; Surrey, Thomas

    2012-12-18

    Cytoplasmic dynein is the major motor protein responsible for microtubule minus-end-directed movements in most eukaryotic cells. It transports a variety of cargoes and has numerous functions during spindle assembly and chromosome segregation. It is a large complex of about 1.4 MDa composed of six different subunits, interacting with a multitude of different partners. Most biochemical studies have been performed either with the native mammalian cytoplasmic dynein complex purified from tissue or, more recently, with recombinant dynein fragments from budding yeast and Dictyostelium. Hardly any information exists about the properties of human dynein. Moreover, experiments with an entire human dynein complex prepared from recombinant subunits with a well-defined composition are lacking. Here, we reconstitute a complete cytoplasmic dynein complex using recombinant human subunits and characterize its biochemical and motile properties. Using analytical gel filtration, sedimentation-velocity ultracentrifugation, and negative-stain electron microscopy, we demonstrate that the smaller subunits of the complex have an important structural function for complex integrity. Fluorescence microscopy experiments reveal that while engaged in collective microtubule transport, the recombinant human cytoplasmic dynein complex is an active, microtubule minus-end-directed motor, as expected. However, in contrast to recombinant dynein of nonmetazoans, individual reconstituted human dynein complexes did not show robust processive motility, suggesting a more intricate mechanism of processivity regulation for the human dynein complex. In the future, the comparison of reconstituted dynein complexes from different species promises to provide molecular insight into the mechanisms regulating the various functions of these large molecular machines.

  18. Hybridization using cytoplasmic male sterility, cytoplasmic herbicide tolerance, and herbicide tolerance from nuclear genes

    SciTech Connect

    Beversdorf, W.D.; Erickson, L.R.; Grant, I.

    1987-04-14

    An improved process is described for producing a substantially homogeneous population of plants of a predetermined hybrid variety of crop which is capable of undergoing self-pollination and cross-pollination. The process comprises: growing in a first planting area a substantially random population of cytoplasmic male sterile plants which exhibit cytoplasmic herbicide tolerance to at least one Type A herbicide and exhibit tolerance to at least one Type B herbicide which is attributable solely to homozygous dominant nuclear genes and male fertile plants which are homozygous recessive maintainer plants for the cytoplasmic male sterile plants and which lack the cytoplasmic herbicide tolerance to at least one Type A herbicide and exhibit tolerance to at least one Type B herbicide attributable solely to the homozygous dominant nuclear genes.

  19. Nuclear and cytoplasmic actin in dinoflagellates.

    PubMed

    Soyer-Gobillard, M O; Ausseil, J; Géraud, M L

    1996-01-01

    Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the

  20. The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.

    PubMed Central

    Zhang, Z; Tanaka, Y; Nonaka, S; Aizawa, H; Kawasaki, H; Nakata, T; Hirokawa, N

    1993-01-01

    Overlapping cDNA clones encoding the heavy chain of rat brain cytoplasmic dynein have been isolated. The isolated cDNA clones contain an open reading frame of 13,932 bp encoding 4644 aa (M(r), 532,213). The deduced protein sequence of the heavy chain of rat brain dynein shows significant similarity to sea urchin flagellar beta-dynein (27.0% identical) and to Dictyostelium cytoplasmic dynein (53.5% identical) throughout the entire sequence. The heavy chain of rat brain (cytoplasmic) dynein contains four putative nucleotide-binding consensus sequences [GX4GK(T/S)] in the central one-third region that are highly similar to those of sea urchin and Dictyostelium dyneins. The N-terminal one-third of the heavy chain of rat brain (cytoplasmic) dynein shows high similarity (43.8% identical) to that of Dictyostelium cytoplasmic dynein but poor similarity (19.4% identical) to that of sea urchin flagellar dynein. These results suggested that the C-terminal two-thirds of the dynein molecule is conserved and plays an essential role in microtubule-dependent motility activity, whereas the N-terminal regions are different between cytoplasmic and flagellar dyneins. Images Fig. 1 PMID:7690137

  1. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  2. Cytoplasmic male sterility in Brassicaceae crops.

    PubMed

    Yamagishi, Hiroshi; Bhat, Shripad R

    2014-05-01

    Brassicaceae crops display strong hybrid vigor, and have long been subject to F1 hybrid breeding. Because the most reliable system of F1 seed production is based on cytoplasmic male sterility (CMS), various types of CMS have been developed and adopted in practice to breed Brassicaceae oil seed and vegetable crops. CMS is a maternally inherited trait encoded in the mitochondrial genome, and the male sterile phenotype arises as a result of interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene. Therefore, CMS has been intensively investigated for gaining basic insights into molecular aspects of nuclear-mitochondrial genome interactions and for practical applications in plant breeding. Several CMS genes have been identified by molecular genetic studies, including Ogura CMS from Japanese radish, which is the most extensively studied and most widely used. In this review, we discuss Ogura CMS, and other CMS systems, and the causal mitochondrial genes for CMS. Studies on nuclear Rf genes and the cytoplasmic effects of alien cytoplasm on general crop performance are also reviewed. Finally, some of the unresolved questions about CMS are highlighted. PMID:24987289

  3. Cytoplasmic male sterility in Brassicaceae crops

    PubMed Central

    Yamagishi, Hiroshi; Bhat, Shripad R.

    2014-01-01

    Brassicaceae crops display strong hybrid vigor, and have long been subject to F1 hybrid breeding. Because the most reliable system of F1 seed production is based on cytoplasmic male sterility (CMS), various types of CMS have been developed and adopted in practice to breed Brassicaceae oil seed and vegetable crops. CMS is a maternally inherited trait encoded in the mitochondrial genome, and the male sterile phenotype arises as a result of interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene. Therefore, CMS has been intensively investigated for gaining basic insights into molecular aspects of nuclear-mitochondrial genome interactions and for practical applications in plant breeding. Several CMS genes have been identified by molecular genetic studies, including Ogura CMS from Japanese radish, which is the most extensively studied and most widely used. In this review, we discuss Ogura CMS, and other CMS systems, and the causal mitochondrial genes for CMS. Studies on nuclear Rf genes and the cytoplasmic effects of alien cytoplasm on general crop performance are also reviewed. Finally, some of the unresolved questions about CMS are highlighted. PMID:24987289

  4. Cytoplasm-to-myonucleus ratios following microgravity

    NASA Technical Reports Server (NTRS)

    Kasper, C. E.; Xun, L.

    1996-01-01

    The cytoplasmic volume-to-myonucleus ratio in the tibialis anterior and gastrocnemius muscles of juvenile rats after 5.4 days of microgravity was studied. Three groups of rats (n = 8 each) were used. The experimental group (space rats) was flown aboard the space shuttle Discovery (NASA, STS-48), while two ground-based groups, one hindlimb suspended (suspended rats), one non-suspended (control), served as controls. Single fibre analysis revealed a significant decrease in cross-sectional area (microns2) in the gastrocnemius for both the space and the suspended rats; in the tibialis anterior only the suspended rats showed a significant decrease. Myonuclei counts (myonuclei per mm) in both the tibialis anterior and gastrocnemius were significantly increased in the space rats but not in the suspended rats. The mean myonuclear volume (individual nuclei: microns3) in tibialis anterior fibres from the space rats, and in gastrocnemius fibres from both the space and the suspended rats, was significantly lower than that in the respective control group. Estimation of the total myonuclear volume (microns3 per.mm), however, revealed no significant differences between the three groups in either the tibialis anterior or gastrocnemius. The described changes in the cross-sectional area and myonuclei numbers resulted in significant decreases in the cytoplasmic volume-to-myonucleus ratio (microns3 x 10(3)) in both muscles and for both space and suspended rats (tibialis anterior; 15.6 +/- 0.6 (space), 17.2 +/- 1.0 (suspended), 20.8 +/- 0.9 (control): gastrocnemius; 13.4 +/- 0.4 (space) and 14.9 +/- 1.1 (suspended) versus 18.1 +/- 1.1 (control)). These results indicate that even short periods of unweighting due to microgravity or limb suspension result in changes in skeletal muscle fibres which lead to significant decreases in the cytoplasmic volume-to-myonucleus ratio.

  5. Mitochondria and cytoplasmic male sterility in plants.

    PubMed

    Hu, Jun; Huang, Wenchao; Huang, Qi; Qin, Xiaojian; Yu, Changchun; Wang, Lili; Li, Shaoqing; Zhu, Renshan; Zhu, Yingguo

    2014-11-01

    Mitochondria are essential organelles in cells not only because they supply over 90% of the cell's energy but also because their dysfunction is associated with disease. Owing to the importance of mitochondria, there are many questions about mitochondria that must be answered. Cytoplasmic male sterility (CMS) is a mysterious natural phenomenon, and the mechanism of the origin of CMS is unknown. Despite successful utilization of CMS and restoration of fertility (Rf) in practice, the underlying mechanisms of these processes remain elusive. This review summarizes the genes involved in CMS and Rf, with a special focus on recent studies reporting the mechanisms of the CMS and Rf pathways, and concludes with potential working models.

  6. Structural responses of amoebae to the injection of heterologous cytoplasm.

    PubMed

    Flickinger, C J; Read, G A; Kabana, E M

    1980-10-01

    Responses to the introduction of heterologous cytoplasm and the fate of foreign organelles were investigated in amoebae. Heterologous cytoplasm was transferred by microinjection from Pelomyxa carolinensis to Amoeba discoides. In control experiments, homologous cytoplasm was transferred from one A. discoides to another. Recipient cells were observed by light microscopy, and samples were prepared for ultrastructural study at intervals between 15 min nad 3 days after operation. Recipients of heterologous cytoplasm showed two main responses. First, about 40% recipients expelled small amounts of cytoplasm by a blebbing process within 30 min after injection. Second, organelles were segregated and broken down in membrane-bounded cytoplasmic vacuoles between 6 h and 2 days after operation. Acid phosphatase reaction product was observed in these vacuoles along with altered organelles. Use of electron-dense thoria particles to mark donor cells demonstrated the presence of injected cytoplasm in the vacuoles. In contrast, when amoebae were injected with homologous cytoplasm, none was expelled and vacuoles containing degenerating organelles were uncommon. The survival rate and general appearance of recipients of heterologous cytoplasm were much poorer than those of homologous recipients, and most of the former died by I week after operation. It is postulated that amoeba are capable of recognizing heterologous organelles introduced into the cytoplasm and that they respond by expulsion and/or destruction of the foreign cellular components. The previously described lethal effect of heterologous cytoplasm was confirmed. PMID:7462341

  7. PTEN functions by recruitment to cytoplasmic vesicles.

    PubMed

    Naguib, Adam; Bencze, Gyula; Cho, Hyejin; Zheng, Wu; Tocilj, Ante; Elkayam, Elad; Faehnle, Christopher R; Jaber, Nadia; Pratt, Christopher P; Chen, Muhan; Zong, Wei-Xing; Marks, Michael S; Joshua-Tor, Leemor; Pappin, Darryl J; Trotman, Lloyd C

    2015-04-16

    PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles. PMID:25866245

  8. The epididymis, cytoplasmic droplets and male fertility

    PubMed Central

    Cooper, Trevor G

    2011-01-01

    The potential of spermatozoa to become motile during post-testicular maturation, and the relationship between the cytoplasmic droplet and fertilizing capacity are reviewed. Post-testicular maturation of spermatozoa involves the autonomous induction of motility, which can occur in vivo in testes with occluded excurrent ducts and in vitro in testicular explants, and artefactual changes in morphology that appear to occur in the testis in vitro. Both modifications may reflect time-dependent oxidation of disulphide bonds of head and tail proteins. Regulatory volume decrease (RVD), which counters sperm swelling at ejaculation, is discussed in relation to loss of cytoplasmic droplets and consequences for fertility. It is postulated that: (i) fertile males possess spermatozoa with sufficient osmolytes to drive RVD at ejaculation, permitting the droplet to round up and pinch off without membrane rupture; and (ii) infertile males possess spermatozoa with insufficient osmolytes so that RVD is inadequate, the droplet swells and the resulting flagellar angulation prevents droplet loss. Droplet retention at ejaculation is a harbinger of infertility caused by failure of the spermatozoon to negotiate the uterotubal junction or mucous and reach the egg. In this hypothesis, the epididymis regulates fertility indirectly by the extent of osmolyte provision to spermatozoa, which influences RVD and therefore droplet loss. Man is an exception, because ejaculated human spermatozoa retain their droplets. This may reflect their short midpiece, approximating head length, permitting a swollen droplet to extend along the entire midpiece; this not only obviates droplet migration and flagellar angulation but also hampers droplet loss. PMID:21076437

  9. Cytoplasmic RNA Granules and Viral Infection

    PubMed Central

    Tsai, Wei-Chih; Lloyd, Richard E.

    2016-01-01

    RNA granules are dynamic cellular structures essential for proper gene expression and homeostasis. The two principle types of cytoplasmic RNA granules are stress granules (SGs), which contain stalled translation initiation complexes, and processing bodies (P-bodies, PBs), which concentrate factors involved in mRNA degradation. RNA granules are associated with gene silencing of transcripts, thus, viruses repress RNA granule functions to favor replication. This review discusses the breadth of viral interactions with cytoplasmic RNA granules, focusing on mechanisms that modulate the functions of RNA granules and that typically promote viral replication. Currently mechanisms for virus manipulation of RNA granules can be loosely grouped into three non-exclusive categories; i) cleavage of key RNA granule factors, ii) regulation of PKR activation and iii) co-opting RNA granule factors for new roles in viral replication. Viral repression of RNA granules supports productive infection by inhibiting their gene silencing functions and counteracting their role in linking stress sensing with innate immune activation. PMID:26958719

  10. Cytoplasmic sensing of viral nucleic acids.

    PubMed

    Habjan, Matthias; Pichlmair, Andreas

    2015-04-01

    Viruses are the most abundant pathogens on earth. A fine-tuned framework of intervening pathways is in place in mammalian cells to orchestrate the cellular defence against these pathogens. Key for this system is sensor proteins that recognise specific features associated with nucleic acids of incoming viruses. Here we review the current knowledge on cytoplasmic sensors for viral nucleic acids. These sensors induce expression of cytokines, affect cellular functions required for virus replication and directly target viral nucleic acids through degradation or sequestration. Their ability to respond to a given nucleic acid is based on both the differential specificity of the individual proteins and the downstream signalling or adaptor proteins. The cooperation of these multiple proteins and pathways plays a key role in inducing successful immunity against virus infections.

  11. Quantifying intermittent transport in cell cytoplasm

    NASA Astrophysics Data System (ADS)

    Lagache, Thibault; Holcman, David

    2008-03-01

    Active cellular transport is a fundamental mechanism for protein and vesicle delivery, cell cycle, and molecular degradation. Viruses can hijack the transport system and use it to reach the nucleus. Most transport processes consist of intermittent dynamics, where the motion of a particle, such as a virus, alternates between pure Brownian and directed movement along microtubules. In this Rapid Communication, we estimate the mean time for a particle to attach to a microtubule network. This computation leads to a coarse grained equation of the intermittent motion in radial and cylindrical geometries. Finally, by using the degradation activity inside the cytoplasm, we obtain refined asymptotic estimations for the probability and the mean time a virus reaches a small nuclear pore.

  12. Physical properties of cytoplasmic intermediate filaments.

    PubMed

    Block, Johanna; Schroeder, Viktor; Pawelzyk, Paul; Willenbacher, Norbert; Köster, Sarah

    2015-11-01

    Intermediate filaments (IFs) constitute a sophisticated filament system in the cytoplasm of eukaryotes. They form bundles and networks with adapted viscoelastic properties and are strongly interconnected with the other filament types, microfilaments and microtubules. IFs are cell type specific and apart from biochemical functions, they act as mechanical entities to provide stability and resilience to cells and tissues. We review the physical properties of these abundant structural proteins including both in vitro studies and cell experiments. IFs are hierarchical structures and their physical properties seem to a large part be encoded in the very specific architecture of the biopolymers. Thus, we begin our review by presenting the assembly mechanism, followed by the mechanical properties of individual filaments, network and structure formation due to electrostatic interactions, and eventually the mechanics of in vitro and cellular networks. This article is part of a Special Issue entitled: Mechanobiology.

  13. Inborn errors of cytoplasmic triglyceride metabolism.

    PubMed

    Wu, Jiang Wei; Yang, Hao; Wang, Shu Pei; Soni, Krishnakant G; Brunel-Guitton, Catherine; Mitchell, Grant A

    2015-01-01

    Triglyceride (TG) synthesis, storage, and degradation together constitute cytoplasmic TG metabolism (CTGM). CTGM is mostly studied in adipocytes, where starting from glycerol-3-phosphate and fatty acyl (FA)-coenzyme A (CoA), TGs are synthesized then stored in cytoplasmic lipid droplets. TG hydrolysis proceeds sequentially, producing FAs and glycerol. Several reactions of CTGM can be catalyzed by more than one enzyme, creating great potential for complex tissue-specific physiology. In adipose tissue, CTGM provides FA as a systemic energy source during fasting and is related to obesity. Inborn errors and mouse models have demonstrated the importance of CTGM for non-adipose tissues, including skeletal muscle, myocardium and liver, because steatosis and dysfunction can occur. We discuss known inborn errors of CTGM, including deficiencies of: AGPAT2 (a form of generalized lipodystrophy), LPIN1 (childhood rhabdomyolysis), LPIN2 (an inflammatory condition, Majeed syndrome, described elsewhere in this issue), DGAT1 (protein loosing enteropathy), perilipin 1 (partial lipodystrophy), CGI-58 (gene ABHD5, neutral lipid storage disease (NLSD) with ichthyosis and "Jordan's anomaly" of vacuolated polymorphonuclear leukocytes), adipose triglyceride lipase (ATGL, gene PNPLA2, NLSD with myopathy, cardiomyopathy and Jordan's anomaly), hormone-sensitive lipase (HSL, gene LIPE, hypertriglyceridemia, and insulin resistance). Two inborn errors of glycerol metabolism are known: glycerol kinase (GK, causing pseudohypertriglyceridemia) and glycerol-3-phosphate dehydrogenase (GPD1, childhood hepatic steatosis). Mouse models often resemble human phenotypes but may diverge markedly. Inborn errors have been described for less than one-third of CTGM enzymes, and new phenotypes may yet be identified. PMID:25300978

  14. Inborn errors of cytoplasmic triglyceride metabolism.

    PubMed

    Wu, Jiang Wei; Yang, Hao; Wang, Shu Pei; Soni, Krishnakant G; Brunel-Guitton, Catherine; Mitchell, Grant A

    2015-01-01

    Triglyceride (TG) synthesis, storage, and degradation together constitute cytoplasmic TG metabolism (CTGM). CTGM is mostly studied in adipocytes, where starting from glycerol-3-phosphate and fatty acyl (FA)-coenzyme A (CoA), TGs are synthesized then stored in cytoplasmic lipid droplets. TG hydrolysis proceeds sequentially, producing FAs and glycerol. Several reactions of CTGM can be catalyzed by more than one enzyme, creating great potential for complex tissue-specific physiology. In adipose tissue, CTGM provides FA as a systemic energy source during fasting and is related to obesity. Inborn errors and mouse models have demonstrated the importance of CTGM for non-adipose tissues, including skeletal muscle, myocardium and liver, because steatosis and dysfunction can occur. We discuss known inborn errors of CTGM, including deficiencies of: AGPAT2 (a form of generalized lipodystrophy), LPIN1 (childhood rhabdomyolysis), LPIN2 (an inflammatory condition, Majeed syndrome, described elsewhere in this issue), DGAT1 (protein loosing enteropathy), perilipin 1 (partial lipodystrophy), CGI-58 (gene ABHD5, neutral lipid storage disease (NLSD) with ichthyosis and "Jordan's anomaly" of vacuolated polymorphonuclear leukocytes), adipose triglyceride lipase (ATGL, gene PNPLA2, NLSD with myopathy, cardiomyopathy and Jordan's anomaly), hormone-sensitive lipase (HSL, gene LIPE, hypertriglyceridemia, and insulin resistance). Two inborn errors of glycerol metabolism are known: glycerol kinase (GK, causing pseudohypertriglyceridemia) and glycerol-3-phosphate dehydrogenase (GPD1, childhood hepatic steatosis). Mouse models often resemble human phenotypes but may diverge markedly. Inborn errors have been described for less than one-third of CTGM enzymes, and new phenotypes may yet be identified.

  15. Cytoplasmic peptidoglycan intermediate levels in Staphylococcus aureus.

    PubMed

    Vemula, Harika; Ayon, Navid J; Gutheil, William G

    2016-02-01

    Intracellular cytoplasmic peptidoglycan (PG) intermediate levels were determined in Staphylococcus aureus during log-phase growth in enriched media. Levels of UDP-linked intermediates were quantitatively determined using ion pairing LC-MS/MS in negative mode, and amine intermediates were quantitatively determined stereospecifically as their Marfey's reagent derivatives in positive mode. Levels of UDP-linked intermediates in S. aureus varied from 1.4 μM for UDP-GlcNAc-Enolpyruvyate to 1200 μM for UDP-MurNAc. Levels of amine intermediates (L-Ala, D-Ala, D-Ala-D-Ala, L-Glu, D-Glu, and L-Lys) varied over a range of from 860 μM for D-Ala-D-Ala to 30-260 mM for the others. Total PG was determined from the D-Glu content of isolated PG, and used to estimate the rate of PG synthesis (in terms of cytoplasmic metabolite flux) as 690 μM/min. The total UDP-linked intermediates pool (2490 μM) is therefore sufficient to sustain growth for 3.6 min. Comparison of UDP-linked metabolite levels with published pathway enzyme characteristics demonstrates that enzymes on the UDP-branch range from >80% saturation for MurA, Z, and C, to <5% saturation for MurB. Metabolite levels were compared with literature values for Escherichia coli, with the major difference in UDP-intermediates being the level of UDP-MurNAc, which was high in S. aureus (1200 μM) and low in E. coli (45 μM). PMID:26612730

  16. The molecular mechanism and physiological role of cytoplasmic streaming.

    PubMed

    Tominaga, Motoki; Ito, Kohji

    2015-10-01

    Cytoplasmic streaming occurs widely in plants ranging from algae to angiosperms. However, the molecular mechanism and physiological role of cytoplasmic streaming have long remained unelucidated. Recent molecular genetic approaches have identified specific myosin members (XI-2 and XI-K as major and XI-1, XI-B, and XI-I as minor motive forces) for the generation of cytoplasmic streaming among 13 myosin XIs in Arabidopsis thaliana. Simultaneous knockout of these myosin XI members led to a reduced velocity of cytoplasmic streaming and marked defects of plant development. Furthermore, the artificial modifications of myosin XI-2 velocity changed plant and cell sizes along with the velocity of cytoplasmic streaming. Therefore, we assume that cytoplasmic streaming is one of the key regulators in determining plant size.

  17. Internal Sense of Direction: Sensing and Signaling from Cytoplasmic Chemoreceptors

    PubMed Central

    Collins, Kieran D.; Lacal, Jesus

    2014-01-01

    SUMMARY Chemoreceptors sense environmental signals and drive chemotactic responses in Bacteria and Archaea. There are two main classes of chemoreceptors: integral inner membrane and soluble cytoplasmic proteins. The latter were identified more recently than integral membrane chemoreceptors and have been studied much less thoroughly. These cytoplasmic chemoreceptors are the subject of this review. Our analysis determined that 14% of bacterial and 43% of archaeal chemoreceptors are cytoplasmic, based on currently sequenced genomes. Cytoplasmic chemoreceptors appear to share the same key structural features as integral membrane chemoreceptors, including the formations of homodimers, trimers of dimers, and 12-nm hexagonal arrays within the cell. Cytoplasmic chemoreceptors exhibit varied subcellular locations, with some localizing to the poles and others appearing both cytoplasmic and polar. Some cytoplasmic chemoreceptors adopt more exotic locations, including the formations of exclusively internal clusters or moving dynamic clusters that coalesce at points of contact with other cells. Cytoplasmic chemoreceptors presumably sense signals within the cytoplasm and bear diverse signal input domains that are mostly N terminal to the domain that defines chemoreceptors, the so-called MA domain. Similar to the case for transmembrane receptors, our analysis suggests that the most common signal input domain is the PAS (Per-Arnt-Sim) domain, but a variety of other N-terminal domains exist. It is also common, however, for cytoplasmic chemoreceptors to have C-terminal domains that may function for signal input. The most common of these is the recently identified chemoreceptor zinc binding (CZB) domain, found in 8% of all cytoplasmic chemoreceptors. The widespread nature and diverse signal input domains suggest that these chemoreceptors can monitor a variety of cytoplasmically based signals, most of which remain to be determined. PMID:25428939

  18. Cytoplasmic incorporation of a ribonucleic acid precursor in Amoeba proteus.

    PubMed

    PLAUT, W; RUSTAD, R C

    1957-07-25

    The question of RNA synthesis in enucleate cytoplasm of Amoeba has been approached experimentally by incubating enucleate amoebae in a labelled RNA precursor and determining the incorporation into RNA autoradiographically. The results indicate that there is a cytoplasmic incorporation mechanism which can operate in the absence of the nucleus. A comparison is made between Acetabularia and Amoeba with respect to the origins of cytoplasmic RNA. It is concluded that the existing data are consistent with the assumption that some cytoplasmic RNA is of nuclear origin in both organisms.

  19. Infective organisms in the cytoplasm of Amoeba proteus.

    PubMed

    ROTH, L E; DANIELS, E W

    1961-02-01

    Evidence from electron and phase microscopy is given which shows that infective organisms are present in the cytoplasm of Amoeba proteus. Vesicles containing living organisms have been observed after repeated washing and starvation of the amebae for a period of 2 weeks. Exposure to gamma-radiation in conjunction with starvation, repeated washing, isolation of single amebae, refeeding with contaminant-free Tetrahymena, and clone selection has produced clones with reduced cytoplasmic infection. These findings are discussed in regard to the autoradiographic studies of other investigators on Amoeba proteus. The controversies over whether DNA and RNA are synthesized in the cytoplasm may be resolved by the finding of cytoplasmic infection.

  20. Molecular analysis of cytoplasmic male sterility

    SciTech Connect

    Hanson, M.R.

    1990-01-01

    The ultimate aims of the project are to understand the molecular mechanism of the disruption in pollen development which occurs in cytoplasmic male sterile plants and to understand the control of respiratory energy flow in the higher plant cell. A mitochondrial locus termed S-pcf segregates with sterility and with an alteration in respiration in Petunia. This cloned locus contains three genes, an abnormal fused gene termed pcf, a gene for a subunit of an NADH dehydrogenase complex, and a small ribosomal subunit protein. The pcf gene is comprised of partial sequences of ATPase subunit 9, cytochrome oxidase subunit II, and an unidentified reading frame. Components of the S-Pcf locus will be introduced into the nuclear of a fertile genotype under the control of appropriate regulatory signals, and polypeptide products of introduced genes will be directed to the mitochondrion with a transit peptide. By examining transgenic plants, we can determine what elements of the locus are critical for altered respiration or sterility. Such knowledge could explain how mitochondrial DNA affects pollen development in the large number of plant species which exhibit the agronomically important trait of male sterility. 10 refs., 3 figs.

  1. Evolution of Wolbachia cytoplasmic incompatibility types.

    PubMed

    Dobson, Stephen L

    2004-10-01

    The success of obligate endosymbiotic Wolbachia infections in insects is due in part to cytoplasmic incompatibility (CI), whereby Wolbachia bacteria manipulate host reproduction to promote their invasion and persistence within insect populations. The observed diversity of CI types raises the question of what the evolutionary pathways are by which a new CI type can evolve from an ancestral type. Prior evolutionary models assume that Wolbachia exists within a host individual as a clonal infection. While endosymbiotic theory predicts a general trend toward clonality, Wolbachia provides an exception in which there is selection to maintain diversity. Here, evolutionary trajectories are discussed that assume that a novel Wolbachia variant will co-exist with the original infection type within a host individual as a superinfection. Relative to prior models, this assumption relaxes requirements and allows additional pathways for the evolution of novel CI types. In addition to describing changes in the Wolbachia infection frequency associated with the hypothesized evolutionary events, the predicted impact of novel CI variants on the host population is also described. This impact, resulting from discordant evolutionary interests of symbiont and host, is discussed as a possible cause of Wolbachia loss from the host population or host population extinction. The latter is also discussed as the basis for an applied strategy for the suppression of insect pest populations. Model predictions are discussed relative to a recently published Wolbachia genome sequence and prior characterization of CI in naturally and artificially infected insects.

  2. Cytoplasmic dynein and early endosome transport

    PubMed Central

    Xiang, Xin; Qiu, Rongde; Yao, Xuanli; Arst, Herbert N.; Peñalva, Miguel A.; Zhang, Jun

    2015-01-01

    Microtubule-based distribution of organelles/vesicles is crucial for the function of many types of eukaryotic cells and the molecular motor cytoplasmic dynein is required for transporting a variety of cellular cargos toward the microtubule minus ends. Early endosomes represent a major cargo of dynein in filamentous fungi, and dynein regulators such as LIS1 and the dynactin complex are both required for early endosome movement. In fungal hyphae, kinesin-3 and dynein drive bi-directional movements of early endosomes. Dynein accumulates at microtubule plus ends; this accumulation depends on kinesin-1 and dynactin, and it is important for early endosome movements towards the microtubule minus ends. The physical interaction between dynein and early endosome requires the dynactin complex, and in particular, its p25 component. The FTS-Hook-FHIP (FHF) complex links dynein-dynactin to early endosomes, and within the FHF complex, Hook interacts with dynein-dynactin, and Hook-early endosome interaction depends on FHIP and FTS. PMID:26001903

  3. Wolbachia and cytoplasmic incompatibility in mosquitoes.

    PubMed

    Sinkins, Steven P

    2004-07-01

    Wolbachia are maternally inherited bacteria that induce cytoplasmic incompatibility in mosquitoes, and are able to use these patterns of sterility to spread themselves through populations. For this reason they have been proposed as a gene drive system for mosquito genetic replacement, as well as for the reduction of population size or for modulating population age structure in order to reduce disease transmission. Here, recent progress in the study of mosquito Wolbachia is reviewed. We now have much more comprehensive estimates of the parameters that can affect the spread of Wolbachia through natural populations from low starting frequencies, and for waves of spread to be maintained in the face of partial barriers to gene flow. In Aedes albopictus these dynamics are extremely favourable, with very high maternal transmission fidelity and levels of incompatibility recorded. Correspondence between measurements taken in the lab and field is much better than in the Drosophila simulans model system. Important research goals are also discussed, including Wolbachia transformation, interspecific transfer and the elucidation of the mechanisms of incompatibility and rescue; all will be aided by a wealth of new Wolbachia genome information.

  4. Epidermal cytoplasmic antibodies. (Incidence and clinical significance).

    PubMed

    Betterle, C; Peserico, A; Bersani, G; Rigon, F; Del Prete, G

    1977-01-01

    Antibodies to epidermal cytoplasmic antigens were detected by the indirect immunofluorescence (IF) technique in 36% of 100 adult healthy subjects and in 17.6% of 17 normal newborn infants. This type of autoantibody occurred in 33% of 100 cases with vitiligo, in 32.5% of 40 cases with psoriasis, in 55.3% of patients with malignant tumours and in 72.7% of subjects with malignant melanoma. The frequency of the autoimmune reactions was statistically significant only in patients with malignant neoplasms. In the majority of positive cases the IF pattern involved the upper layers of the epidermal cells (U-CYT). The basal layer was generally negative. Only a few cases showed a pattern involving both the upper and the basal layers (G-CYT). However, a wide variation in staining was noted when sera were tested on different skin specimens or different sections of the same skin. To identify the nature of the target antigen(s), absorption experiments of sera were attempted with lyophilized and particulate antigens. Animal and human blood cells and lyophilized homogenates of malignant tumours failed to absorb the autoimmune activity of positive sera. Only a powder preparation of keratin induced a decrease in antibody titres. It is postulated that they are the result of an antigenic stimulation by exogenous substances commonly present in the environment.

  5. A physical perspective on cytoplasmic streaming

    PubMed Central

    Goldstein, Raymond E.; van de Meent, Jan-Willem

    2015-01-01

    Organisms show a remarkable range of sizes, yet the dimensions of a single cell rarely exceed 100 µm. While the physical and biological origins of this constraint remain poorly understood, exceptions to this rule give valuable insights. A well-known counterexample is the aquatic plant Chara, whose cells can exceed 10 cm in length and 1 mm in diameter. Two spiralling bands of molecular motors at the cell periphery drive the cellular fluid up and down at speeds up to 100 µm s−1, motion that has been hypothesized to mitigate the slowness of metabolite transport on these scales and to aid in homeostasis. This is the most organized instance of a broad class of continuous motions known as ‘cytoplasmic streaming’, found in a wide range of eukaryotic organisms—algae, plants, amoebae, nematodes and flies—often in unusually large cells. In this overview of the physics of this phenomenon, we examine the interplay between streaming, transport and cell size and discuss the possible role of self-organization phenomena in establishing the observed patterns of streaming. PMID:26464789

  6. Single molecule analysis of cytoplasmic dynein motility

    NASA Astrophysics Data System (ADS)

    Yildiz, Ahmet

    2014-03-01

    Cytoplasmic dynein is a homodimeric AAA + motor that transports a multitude of cargos towards the microtubule (MT) minus end. The mechanism of dynein motility remains unclear, due to its large size (2.6 MDa) and the complexity of its structure. By tracking the stepping motion of both heads at nanometer resolution, we observed that dynein heads move independently along the MT, in contrast to hand over hand movement of kinesins and myosin. Stepping behavior of the heads varies as a function of interhead separation and establishing the basis of high variability in dynein step size. By engineering the mechanical and catalytic properties of the dynein motor domain, we show that a rigid linkage between monomers and dimerization between N-terminal tail domains are not essential for processive movement. Instead, dynein processivity minimally requires the linker domain of one active monomer to be attached to an inert MT tether retaining only the MT-binding domain. The release of a dynein monomer from the MT can be mediated either by nucleotide binding or external load. Nucleotide dependent release is inhibited by the tension on the linker domain at high interhead separations. Tension dependent release is highly asymmetric, with faster release towards the minus-end. Reversing the asymmetry of the MT binding interface results in plus end directed motility, even though the force was generated by the dynein motor activity. On the basis of these measurements, we propose a model that describes the basis of dynein processivity, directionality and force generation.

  7. Model for bidirectional movement of cytoplasmic dynein.

    PubMed

    Sumathy, S; Satyanarayana, S V M

    2015-09-01

    Cytoplasmic dynein exhibits a directional processive movement on microtubule filaments and is known to move in steps of varying length based on the number of ATP molecules bound to it and the load that it carries. It is experimentally observed that dynein takes occasional backward steps and the frequency of such backward steps increases as the load approaches the stall force. Using a stochastic process model, we investigate the bidirectional movement of single head of a dynein motor. The probability for backward step is implemented based on fluctuation theorem of non-equilibrium statistical mechanics. We find that the movement of dynein motor is characterized with negative velocity implying backward motion beyond stall force. We observe that the motor moves backward for super stall forces by hydrolyzing the ATP exactly the same way as it does while moving forward for sub-stall forces. Movement of dynein is also simulated using a kinetic Monte Carlo method and the simulated velocities are in good agreement with velocities obtained using a stochastic rate equation model.

  8. Cytoplasmic dynein and early endosome transport.

    PubMed

    Xiang, Xin; Qiu, Rongde; Yao, Xuanli; Arst, Herbert N; Peñalva, Miguel A; Zhang, Jun

    2015-09-01

    Microtubule-based distribution of organelles/vesicles is crucial for the function of many types of eukaryotic cells and the molecular motor cytoplasmic dynein is required for transporting a variety of cellular cargos toward the microtubule minus ends. Early endosomes represent a major cargo of dynein in filamentous fungi, and dynein regulators such as LIS1 and the dynactin complex are both required for early endosome movement. In fungal hyphae, kinesin-3 and dynein drive bi-directional movements of early endosomes. Dynein accumulates at microtubule plus ends; this accumulation depends on kinesin-1 and dynactin, and it is important for early endosome movements towards the microtubule minus ends. The physical interaction between dynein and early endosome requires the dynactin complex, and in particular, its p25 component. The FTS-Hook-FHIP (FHF) complex links dynein-dynactin to early endosomes, and within the FHF complex, Hook interacts with dynein-dynactin, and Hook-early endosome interaction depends on FHIP and FTS.

  9. Tropomyosin-Mediated Regulation of Cytoplasmic Myosins.

    PubMed

    Manstein, Dietmar J; Mulvihill, Daniel P

    2016-08-01

    The ability of the actin-based cytoskeleton to rapidly reorganize is critical for maintaining cell organization and viability. The plethora of activities in which actin polymers participate require different biophysical properties, which can vary significantly between the different events that often occur simultaneously at separate cellular locations. In order to modify the biophysical properties of an actin polymer for a particular function, the cell contains diverse actin-binding proteins that modulate the growth, regulation and molecular interactions of actin-based structures according to functional requirements. In metazoan and yeast cells, tropomyosin is a key regulator of actin-based structures. Cells have the capacity to produce multiple tropomyosin isoforms, each capable of specifically associating as copolymers with actin at distinct cellular locations to fine-tune the functional properties of discrete actin structures. Here, we present a unifying theory in which tropomyosin isoforms critically define the surface landscape of copolymers with cytoplasmic β- or γ-actin. Decoration of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins. Conversely, changes in the nucleotide state of actin and posttranslational modifications affect the composition, morphology, subcellular localization and allosteric coupling of the associated actin-based superstructures. PMID:27060364

  10. A physical perspective on cytoplasmic streaming.

    PubMed

    Goldstein, Raymond E; van de Meent, Jan-Willem

    2015-08-01

    Organisms show a remarkable range of sizes, yet the dimensions of a single cell rarely exceed 100 µm. While the physical and biological origins of this constraint remain poorly understood, exceptions to this rule give valuable insights. A well-known counterexample is the aquatic plant Chara, whose cells can exceed 10 cm in length and 1 mm in diameter. Two spiralling bands of molecular motors at the cell periphery drive the cellular fluid up and down at speeds up to 100 µm s(-1), motion that has been hypothesized to mitigate the slowness of metabolite transport on these scales and to aid in homeostasis. This is the most organized instance of a broad class of continuous motions known as 'cytoplasmic streaming', found in a wide range of eukaryotic organisms-algae, plants, amoebae, nematodes and flies-often in unusually large cells. In this overview of the physics of this phenomenon, we examine the interplay between streaming, transport and cell size and discuss the possible role of self-organization phenomena in establishing the observed patterns of streaming.

  11. Developing improved durum wheat germplasm by altering the cytoplasmic genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotic organisms, nuclear and cytoplasmic genomes interact to drive cellular functions. These genomes have co-evolved to form specific nuclear-cytoplasmic interactions that are essential to the origin, success, and evolution of diploid and polyploid species. Hundreds of genetic diseases in h...

  12. Dexamethasone and Acetate Modulate Cytoplasmic Leptin in Bovine Preadipocytes

    PubMed Central

    Yonekura, Shinichi; Hirota, Shohei; Tokutake, Yukako; Rose, Michael T.; Katoh, Kazuo; Aso, Hisashi

    2014-01-01

    Hormonal and nutrient signals regulate leptin synthesis and secretion. In rodents, leptin is stored in cytosolic pools of adipocytes. However, not much information is available regarding the regulation of intracellular leptin in ruminants. Recently, we demonstrated that leptin mRNA was expressed in bovine intramuscular preadipocyte cells (BIP cells) and that a cytoplasmic leptin pool may be present in preadipocytes. In the present study, we investigated the expression of cytoplasmic leptin protein in BIP cells during differentiation as well as the effects of various factors added to the differentiation medium on its expression in BIP cells. Leptin mRNA expression was observed only at 6 and 8 days after adipogenic induction, whereas the cytoplasmic leptin concentration was the highest on day 0 and decreased gradually thereafter. Cytoplasmic leptin was detected at 6 and 8 days after adipogenic induction, but not at 4 days after adipogenic induction. The cytoplasmic leptin concentration was reduced in BIP cells at 4 days after treatment with dexamethasone, whereas cytoplasmic leptin was not observed at 8 days after treatment. In contrast, acetate significantly enhanced the cytoplasmic leptin concentration in BIP cells at 8 days after treatment, although acetate alone did not induce adipocyte differentiation in BIP cells. These results suggest that dexamethasone and acetate modulate the cytoplasmic leptin concentration in bovine preadipocytes. PMID:25049989

  13. Translation of tobacco mosaic virus RNA In Acetabularia cell cytoplasm.

    PubMed

    Cairns, E; Sarkar, S; Schweiger, H G

    1978-11-01

    Isolated Acetabularia nuclei were microinjected with Tobacco Mosaic Virus RNA and then implanted into an anucleate posterior fragment of an Acetabularia cell. Injected RNA was translated in the Acetabularia cytoplasm from the first to twelfth day after implantation of the nuclei. The production of specific virus proteins was detected and localized in the Acetabularia cytoplasm by an immunofluorescence precipitation technique.

  14. Can paternal leakage maintain sexually antagonistic polymorphism in the cytoplasm?

    PubMed Central

    Kuijper, B; Lane, N; Pomiankowski, A

    2015-01-01

    A growing number of studies in multicellular organisms highlight low or moderate frequencies of paternal transmission of cytoplasmic organelles, including both mitochondria and chloroplasts. It is well established that strict maternal inheritance is selectively blind to cytoplasmic elements that are deleterious to males – ’mother's curse’. But it is not known how sensitive this conclusion is to slight levels of paternal cytoplasmic leakage. We assess the scope for polymorphism when individuals bear multiple cytoplasmic alleles in the presence of paternal leakage, bottlenecks and recurrent mutation. When fitness interactions among cytoplasmic elements within an individual are additive, we find that sexually antagonistic polymorphism is restricted to cases of strong selection on males. However, when fitness interactions among cytoplasmic elements are nonlinear, much more extensive polymorphism can be supported in the cytoplasm. In particular, mitochondrial mutants that have strong beneficial fitness effects in males and weak deleterious fitness effects in females when rare (i.e. ’reverse dominance’) are strongly favoured under paternal leakage. We discuss how such epistasis could arise through preferential segregation of mitochondria in sex-specific somatic tissues. Our analysis shows how paternal leakage can dampen the evolution of deleterious male effects associated with predominant maternal inheritance of cytoplasm, potentially explaining why ’mother's curse’ is less pervasive than predicted by earlier work. PMID:25653025

  15. The cytoplasm of living cells behaves as a poroelastic material

    PubMed Central

    Moeendarbary, Emad; Valon, Léo; Fritzsche, Marco; Harris, Andrew R.; Moulding, Dale A.; Thrasher, Adrian J.; Stride, Eleanor; Mahadevan, L.; Charras, Guillaume T.

    2014-01-01

    The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at physiologically relevant timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology. PMID:23291707

  16. The cytoplasm of living cells behaves as a poroelastic material

    NASA Astrophysics Data System (ADS)

    Moeendarbary, Emad; Valon, Léo; Fritzsche, Marco; Harris, Andrew R.; Moulding, Dale A.; Thrasher, Adrian J.; Stride, Eleanor; Mahadevan, L.; Charras, Guillaume T.

    2013-03-01

    The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at short timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology at short timescales.

  17. Cytoplasmic streaming velocity as a plant size determinant.

    PubMed

    Tominaga, Motoki; Kimura, Atsushi; Yokota, Etsuo; Haraguchi, Takeshi; Shimmen, Teruo; Yamamoto, Keiichi; Nakano, Akihiko; Ito, Kohji

    2013-11-11

    Cytoplasmic streaming is active transport widely occurring in plant cells ranging from algae to angiosperms. Although it has been revealed that cytoplasmic streaming is generated by organelle-associated myosin XI moving along actin bundles, the fundamental function in plants remains unclear. We generated high- and low-speed chimeric myosin XI by replacing the motor domains of Arabidopsis thaliana myosin XI-2 with those of Chara corallina myosin XI and Homo sapiens myosin Vb, respectively. Surprisingly, the plant sizes of the transgenic Arabidopsis expressing high- and low-speed chimeric myosin XI-2 were larger and smaller, respectively, than that of the wild-type plant. This size change correlated with acceleration and deceleration, respectively, of cytoplasmic streaming. Our results strongly suggest that cytoplasmic streaming is a key determinant of plant size. Furthermore, because cytoplasmic streaming is a common system for intracellular transport in plants, our system could have applications in artificial size control in plants.

  18. Antineutrophil Cytoplasmic Antibodies Associated With Infective Endocarditis

    PubMed Central

    Langlois, Vincent; Lesourd, Anais; Girszyn, Nicolas; Ménard, Jean-Francois; Levesque, Hervé; Caron, Francois; Marie, Isabelle

    2016-01-01

    Abstract To determine the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in patients with infective endocarditis (IE) in internal medicine; and to compare clinical and biochemical features and outcome between patients exhibiting IE with and without ANCA. Fifty consecutive patients with IE underwent ANCA testing. The medical records of these patients were reviewed. Of the 50 patients with IE, 12 exhibited ANCA (24%). ANCA-positive patients with IE exhibited: longer duration between the onset of first symptoms and IE diagnosis (P = 0.02); and more frequently: weight loss (P = 0.017) and renal impairment (P = 0.08), lower levels of C-reactive protein (P = 0.0009) and serum albumin (P = 0.0032), involvement of both aortic and mitral valves (P = 0.009), and longer hospital stay (P = 0.016). Under multivariate analysis, significant factors for ANCA-associated IE were: longer hospital stay (P = 0.004), lower level of serum albumin (P = 0.02), and multiple valve involvement (P = 0.04). Mortality rate was 25% in ANCA patients; death was because of IE complications in all these patients. Our study identifies a high prevalence of ANCA in unselected patients with IE in internal medicine (24%). Our findings further underscore that ANCA may be associated with a subacute form of IE leading to multiple valve involvement and more frequent renal impairment. Because death was due to IE complications in all patients, our data suggest that aggressive therapy may be required to improve such patients’ outcome. PMID:26817911

  19. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    SciTech Connect

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  20. Differences in catalytic properties between cerebral cytoplasmic and mitochondrial hexokinases.

    PubMed

    Thompson, M F; Bachelard, H S

    1977-03-01

    1. Clear kinetic differences between cytoplasmic and mitochondrial forms of type-I cerebral hexokinase were demonstrated from experiments performed under identical conditions on three (cytoplasmic, bound mitochondrial and solubilized mitochondrial) preparations of the enzyme. 2. Whereas the Michaelis constant for glucose (KmGlc) was consistent, that for MgATP2- (KmATP) was lower in the cytoplasmic than in the two mitochondrial preparations. The substrate dissociation constants (KsGlc and KsATP) were both higher in the cytoplasmic than in the mitochondrial preparations. A further difference in the substrate kinetic patterns was that KmATP=KmATP for the cytoplasmic enzyme, in contrast with the mitochondrial enzyme, where KmATP was clearly not equal to KsATP [Bachelard et al. (1971) Biochem. J. 123, 707-715]. 3. Dead-end inhibition produced by N-acetyl-glucosamine and by AMP also exhibited different quantitative kinetic patterns for the two enzyme sources. Both inhibitions gave Ki values similar or equal to those of Ki' for the cytoplasmic activity, whereas Ki was clearly not equal to Ki' for the mitochondrial activity. 4. All of these studies demonstrated the similarity of the two mitochondrial activities (particulate and solubilized), which were both clearly different from the cytoplasmic activity. 5. The analysis gives a practical example of our previous theoretical treatment on the derivation of true inhibition constants. 6. The results are discussed in terms of the function of cerebral hexokinases.

  1. Single cytoplasmic dynein molecule movements: characterization and comparison with kinesin.

    PubMed Central

    Wang, Z; Khan, S; Sheetz, M P

    1995-01-01

    Cytoplasmic dynein is a major microtubule motor for minus-end directed movements including retrograde axonal transport. To better understand the mechanism by which cytoplasmic dynein converts ATP energy into motility, we have analyzed the nanometer-level displacements of latex beads coated with low numbers of cytoplasmic dynein molecules. Cytoplasmic dynein-coated beads exhibited greater lateral movements among microtubule protofilaments (ave. 5.1 times/microns of displacement) compared with kinesin (ave. 0.9 times/micron). In addition, dynein moved rearward up to 100 nm over several hundred milliseconds, often in correlation with off-axis movements from one protofilament to another. We suggest that single molecules of cytoplasmic dynein move the beads because 1) there is a linear dependence of bead motility on dynein/bead ratio, 2) the binding of beads to microtubules studied by laser tweezers is best fit by a first-order Poisson, and 3) the run length histogram of dynein beads follows a first-order decay. At the cellular level, the greater disorder of cytoplasmic dynein movements may facilitate transport by decreasing the duration of collisions between kinesin and cytoplasmic dynein-powered vesicles. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 9 PMID:8580344

  2. Cross-species cloning: influence of cytoplasmic factors on development.

    PubMed

    Sun, Yong-Hua; Zhu, Zuo-Yan

    2014-06-01

    It is widely accepted that the crosstalk between naive nucleus and maternal factors deposited in the egg cytoplasm before zygotic genome activation is crucial for early development. This crosstalk may also exert some influence on later development. It is interesting to clarify the relative roles of the zygotic genome and the cytoplasmic factors in development. Cross-species nuclear transfer (NT) between two distantly related species provides a unique system to study the relative role and crosstalk between egg cytoplasm and zygotic nucleus in development. In this review, we will summarize the recent progress of cross-species NT, with emphasis on the cross-species NT in fish and the influence of cytoplasmic factors on development. Finally, we conclude that the developmental process and its evolution should be interpreted in a systemic way, rather than in a way that solely focuses on the role of the nuclear genome.

  3. Stabilization and Degradation Mechanisms of Cytoplasmic Ataxin-1

    PubMed Central

    Kohiyama, Mayumi F.; Lagalwar, Sarita

    2015-01-01

    Aggregation-prone proteins in neurodegenerative disease disrupt cellular protein stabilization and degradation pathways. The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is caused by a coding polyglutamine expansion in the Ataxin-1 gene (ATXN1), which gives rise to the aggregation-prone mutant form of ATXN1 protein. Cerebellar Purkinje neurons, preferentially vulnerable in SCA1, produce ATXN1 protein in both cytoplasmic and nuclear compartments. Cytoplasmic stabilization of ATXN1 by phosphorylation and 14-3-3-mediated mechanisms ultimately drive translocation of the protein to the nucleus where aggregation may occur. However, experimental inhibition of phosphorylation and 14-3-3 binding results in rapid degradation of ATXN1, thus preventing nuclear translocation and cellular toxicity. The exact mechanism of cytoplasmic ATXN1 degradation is currently unknown; further investigation of degradation may provide future therapeutic targets. This review examines the present understanding of cytoplasmic ATXN1 stabilization and potential degradation mechanisms during normal and pathogenic states. PMID:27168726

  4. Nuclear Proteins Hijacked by Mammalian Cytoplasmic Plus Strand RNA Viruses

    PubMed Central

    Lloyd, Richard E.

    2015-01-01

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. PMID:25818028

  5. Regulation of cytoplasmic streaming in Vallisneria mesophyll cells.

    PubMed

    Takagi, S; Nagai, R

    1983-07-01

    Induction and cessation of the rotational cytoplasmic streaming in Vallisneria mesophyll cells could be controlled by external stimuli. In cells that had been kept in darkness the cytoplasm remained quiescent. However, when the cells were treated in the dark with EGTA solution (10 mM or 20 mM buffered with 10 mM-Tris-maleate at pH 7.0), rotational cytoplasmic streaming was induced. When the cells were transferred again to artificial pond water in the dark, the induced streaming was inhibited; that is, only 50% of the observed cells exhibited active streaming after 2 h. When the cells were irradiated continuously with far-red light (lambda max = 750 nm, 0.4 W/m2) in the same external medium, the induced streaming was inhibited almost completely within 2 h. The relative quantum effectiveness of monochromatic light (450--800 nm) in producing cessation of streaming was also investigated. Irradiation with light of 450, 550 and 600 nm was almost as effective as darkness. Light of 500 and 650 nm was less effective than dark exposure. Only irradiation at 750 nm stopped streaming in almost all cells. But when calcium was excluded from the external medium, the effect of far-red light decreased to almost the dark control level. Light of 800 nm also inhibited the streaming but the effect was much less than that of far-red light. Microfilaments in bundles with the long axis parallel to the streaming direction were localized in the vicinity of the cell membrane. Their configuration, localization and distribution were the same in the present experimental system irrespective of whether the cytoplasm was streaming or quiescent. Intracellular calcium was examined by electron microscopic cytochemistry and X-ray microanalysis. In cells with streaming induced by EGTA, only a small amount of calcium-containing precipitates formed in the cytoplasm in the presence of antimony. A few precipitates were found in the chloroplasts, the middle lamella of the cell wall and at the border between

  6. Immunohistochemical expression of nuclear and cytoplasmic survivin in gastrointestinal carcinoma.

    PubMed

    Shintani, Michiko; Sangawa, Akiko; Yamao, Naoki; Kamoshida, Shingo

    2013-01-01

    Survivin is a protein that is highly expressed in many embryonic tissues, as well as most human tumors. Prior studies have reported both positive and negative correlations between survivin expression and cancer prognosis, but these associations remain controversial. In the present study, we assessed the expression of nuclear and cytoplasmic survivin in gastrointestinal carcinomas. Using these data, we determined the correlation between nuclear and cytoplasmic survivin and, further, investigated correlations between survivin expression and clinicopathological parameters. Seventy-two advanced gastric adenocarcinomas and 78 colorectal adenocarcinomas were analyzed for survivin expression by immunohistochemistry. Expression of both nuclear and cytoplasmic survivin was significantly higher in colorectal carcinomas than in gastric carcinomas (P < 0.01). There was a positive correlation between nuclear and cytoplasmic expression of survivin (r = 0.42, P < 0.001). In gastric carcinomas, the level of survivin protein expression was associated with tumor differentiation, patient age, and lymphatic invasion (P < 0.05, 0.01, and 0.01, respectively). In colorectal carcinomas, the level of nuclear survivin expression was significantly higher in females than in males (P < 0.05). There were no significant associations between survivin expression and most of the clinicopathological parameters. Nevertheless, there was a trend towards an inverse correlation between nuclear survivin expression and tumor aggressiveness in gastric carcinoma; there was a similar trend for cytoplasmic survivin expression. In summary, our results suggest that levels of nuclear and cytoplasmic survivin expression differ between gastric carcinoma and colorectal carcinoma.

  7. Immunohistochemical expression of nuclear and cytoplasmic survivin in gastrointestinal carcinoma

    PubMed Central

    Shintani, Michiko; Sangawa, Akiko; Yamao, Naoki; Kamoshida, Shingo

    2013-01-01

    Survivin is a protein that is highly expressed in many embryonic tissues, as well as most human tumors. Prior studies have reported both positive and negative correlations between survivin expression and cancer prognosis, but these associations remain controversial. In the present study, we assessed the expression of nuclear and cytoplasmic survivin in gastrointestinal carcinomas. Using these data, we determined the correlation between nuclear and cytoplasmic survivin and, further, investigated correlations between survivin expression and clinicopathological parameters. Seventy-two advanced gastric adenocarcinomas and 78 colorectal adenocarcinomas were analyzed for survivin expression by immunohistochemistry. Expression of both nuclear and cytoplasmic survivin was significantly higher in colorectal carcinomas than in gastric carcinomas (P < 0.01). There was a positive correlation between nuclear and cytoplasmic expression of survivin (r = 0.42, P < 0.001). In gastric carcinomas, the level of survivin protein expression was associated with tumor differentiation, patient age, and lymphatic invasion (P < 0.05, 0.01, and 0.01, respectively). In colorectal carcinomas, the level of nuclear survivin expression was significantly higher in females than in males (P < 0.05). There were no significant associations between survivin expression and most of the clinicopathological parameters. Nevertheless, there was a trend towards an inverse correlation between nuclear survivin expression and tumor aggressiveness in gastric carcinoma; there was a similar trend for cytoplasmic survivin expression. In summary, our results suggest that levels of nuclear and cytoplasmic survivin expression differ between gastric carcinoma and colorectal carcinoma. PMID:24294379

  8. tRNA travels from the cytoplasm to organelles

    PubMed Central

    Rubio, Mary Anne T.; Hopper, Anita K.

    2011-01-01

    Transfer RNAs (tRNAs) encoded by the nuclear genome are surprisingly dynamic. Although tRNAs function in protein synthesis occurring on cytoplasmic ribosomes, tRNAs can transit from the cytoplasm to the nucleus and then again return to the cytoplasm by a process known as the tRNA retrograde process. Subsets of the cytoplasmic tRNAs are also imported into mitochondria and function in mitochondrial protein synthesis. The numbers of tRNA species that are imported into mitchondria differ among organisms, ranging from just a few to the entire set needed to decode mitochondrially encoded mRNAs. For some tRNAs, import is dependent on the mitochondrial protein import machinery, whereas the majority of tRNA mitochondrial import is independent of this machinery. Although cytoplasmic proteins and proteins located on the mitochondrial surface participating in the tRNA import process have been described for several organisms, the identity of these proteins differ among organisms. Likewise, the tRNA determinants required for mitochondrial import differ among tRNA species and organisms. Here, we present an overview and discuss the current state of knowledge regarding the mechanisms involved in the tRNA retrograde process and continue with an overview of tRNA import into mitochondria. Finally, we highlight areas of future research to understand the function and regulation of movement of tRNAs between the cytoplasm and organelles. PMID:21976284

  9. Mutualistic Wolbachia infection in Aedes albopictus: accelerating cytoplasmic drive.

    PubMed Central

    Dobson, Stephen L; Marsland, Eric J; Rattanadechakul, Wanchai

    2002-01-01

    Maternally inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods, often behaving as reproductive parasites by manipulating host reproduction to enhance the vertical transmission of infections. One manipulation is cytoplasmic incompatibility (CI), which causes a significant reduction in brood hatch and promotes the spread of the maternally inherited Wolbachia infection into the host population (i.e., cytoplasmic drive). Here, we have examined a Wolbachia superinfection in the mosquito Aedes albopictus and found the infection to be associated with both cytoplasmic incompatibility and increased host fecundity. Relative to uninfected females, infected females live longer, produce more eggs, and have higher hatching rates in compatible crosses. A model describing Wolbachia infection dynamics predicts that increased fecundity will accelerate cytoplasmic drive rates. To test this hypothesis, we used population cages to examine the rate at which Wolbachia invades an uninfected Ae. albopictus population. The observed cytoplasmic drive rates were consistent with model predictions for a CI-inducing Wolbachia infection that increases host fecundity. We discuss the relevance of these results to both the evolution of Wolbachia symbioses and proposed applied strategies for the use of Wolbachia infections to drive desired transgenes through natural populations (i.e., population replacement strategies). PMID:11901124

  10. Evidence for a cytoplasmic microprocessor of pri-miRNAs.

    PubMed

    Shapiro, Jillian S; Langlois, Ryan A; Pham, Alissa M; Tenoever, Benjamin R

    2012-07-01

    microRNAs (miRNAs) represent a class of noncoding RNAs that fine-tune gene expression through post-transcriptional silencing. While miRNA biogenesis occurs in a stepwise fashion, initiated by the nuclear microprocessor, rare noncanonical miRNAs have also been identified. Here we characterize the molecular components and unique attributes associated with the processing of virus-derived cytoplasmic primary miRNAs (c-pri-miRNAs). RNA in situ hybridization and inhibition of cellular division demonstrated a complete lack of nuclear involvement in c-pri-miRNA cleavage while genetic studies revealed that maturation still relied on the canonical nuclear RNase III enzyme, Drosha. The involvement of Drosha was mediated by a dramatic relocalization to the cytoplasm following virus infection. Deep sequencing analyses revealed that the cytoplasmic localization of Drosha does not impact the endogenous miRNA landscape during infection, despite allowing for robust synthesis of virus-derived miRNAs in the cytoplasm. Taken together, this research describes a unique function for Drosha in the processing of highly structured cytoplasmic RNAs in the context of virus infection.

  11. Assessment of cytoplasm conductivity by nanosecond pulsed electric fields.

    PubMed

    Denzi, Agnese; Merla, Caterina; Palego, Cristiano; Paffi, Alessandra; Ning, Yaqing; Multari, Caroline R; Cheng, Xuanhong; Apollonio, Francesca; Hwang, James C M; Liberti, Micaela

    2015-06-01

    The aim of this paper is to propose a new method for the better assessment of cytoplasm conductivity, which is critical to the development of electroporation protocols as well as insight into fundamental mechanisms underlying electroporation. For this goal, we propose to use nanosecond electrical pulses to bypass the complication of membrane polarization and a single cell to avoid the complication of the application of the "mixing formulas." Further, by suspending the cell in a low-conductivity medium, it is possible to force most of the sensing current through the cytoplasm for a more direct assessment of its conductivity. For proof of principle, the proposed technique was successfully demonstrated on a Jurkat cell by comparing the measured and modeled currents. The cytoplasm conductivity was best assessed at 0.32 S/m and it is in line with the literature. The cytoplasm conductivity plays a key role in the understanding of the basis mechanism of the electroporation phenomenon, and in particular, a large error in the cytoplasm conductivity determination could result in a correspondingly large error in predicting electroporation. Methods for a good estimation of such parameter become fundamental.

  12. Biotoxicity assays for fruiting body lectins and other cytoplasmic proteins.

    PubMed

    Künzler, Markus; Bleuler-Martinez, Silvia; Butschi, Alex; Garbani, Mattia; Lüthy, Peter; Hengartner, Michael O; Aebi, Markus

    2010-01-01

    Recent studies suggest that a specific class of fungal lectins, commonly referred to as fruiting body lectins, play a role as effector molecules in the defense of fungi against predators and parasites. Hallmarks of these fungal lectins are their specific expression in reproductive structures, fruiting bodies, and/or sclerotia and their synthesis on free ribosomes in the cytoplasm. Fruiting body lectins are released upon damage of the fungal cell and bind to specific carbohydrate structures of predators and parasites, which leads to deterrence, inhibition of growth, and development or even killing of these organisms. Here, we describe assays to assess the toxicity of such lectins and other cytoplasmic proteins toward three different model organisms: the insect Aedes aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on heterologous expression of the examined proteins in the cytoplasm of Escherichia coli and feeding of these recombinant bacteria to omnivorous and bacterivorous organisms. PMID:20816208

  13. Ionizing Radiation Induces HMGB1 Cytoplasmic Translocation and Extracellular Release

    PubMed Central

    Wang, Lili; He, Li; Bao, Guoqiang; He, Xin; Fan, Saijun; Wang, Haichao

    2016-01-01

    Objective A nucleosomal protein, HMGB1, can be secreted by activated immune cells or passively released by dying cells, thereby amplifying rigorous inflammatory responses. In this study we aimed to test the possibility that ionizing radiation similarly induces cytoplasmic HMGB1 translocation and extracellular release. Method Human skin fibroblast (GM0639) and bronchial epithelial (16HBE) cells and animals (rats) were exposed to X-ray radiation, and HMGB1 translocation and release were assessed by immunocytochemistry and immunoassay, respectively. Results At a wide dose range (4.0 – 12.0 Gy), X-ray radiation induced a dramatic cytoplasmic HMGB1 translocation, and triggered a time- and dose-dependent HMGB1 release both in vitro and in vivo. The radiation-mediated HMGB1 release was associated with noticeable chromosomal DNA damage and loss of cell viability. Conclusion radiation induces HMGB1 cytoplasmic translocation and extracellular release through active secretion and passive leakage processes. PMID:27331198

  14. Mitochondrial DNA phylogeny of cultivated and wild beets: relationships among cytoplasmic male-sterility-inducing and nonsterilizing cytoplasms.

    PubMed

    Nishizawa, Satsuki; Mikami, Tetsuo; Kubo, Tomohiko

    2007-11-01

    Cytoplasmic male sterility (CMS), the maternally inherited failure to produce functional pollen, has been used in the breeding of sugar beet (Beta vulgaris ssp. vulgaris). At least three different sources of CMS can be distinguished from one another as well as from normal fertile cytoplasm by polymorphisms in their mitochondrial genomes. Here we analyzed 50 accessions of cultivated and wild beets to investigate the phylogenetic relationships among male-sterility-inducing and normal cytoplasms. The haplotypes were characterized by the nucleotide sequence of the mitochondrial cox2-cox1 spacer region and mitochondrial minisatellite loci. The results indicated that (1) a normal cytoplasm line, cv. TK81-O, was situated at the major core node of the haplotype network, and (2) the three sterilizing cytoplasms in question derived independently from the core haplotype. The evolutionary pathway was investigated by physical mapping study of the mitochondrial genome of a wild beet (B. vulgaris ssp. orientalis) accession BGRC56777 which shared the same mitochondrial haplotype with TK81-O, but was not identical to TK81-O for the RFLP profiles of mitochondrial DNA. Interestingly, three sets of inverted repeated sequences appeared to have been involved in a series of recombination events during the course of evolution between the BGRC56777 and the TK81-O mitochondrial genomes.

  15. Colicin S8 export: extracellular and cytoplasmic colicin are different.

    PubMed

    Garcia Diaz, Maria-Elena; Concepción Curbelo, Juan Luis

    2003-12-01

    The properties of colicin S8 are different for the cytoplasmic, periplasmic and extracellular protein. Interactions with its specific receptors reflect this. Active cell extracts separate into a non-anionic along with an anionic fraction by DEAE-Sephacell chromatography. Previously, we have purified cell-associated colicin S8 as an aggregation of highly related polypeptides; cytoplasmic colicin S8 seems to be post-translationally processed into an aggregation of polypeptides of molecular mass ranging from 45,000 Da to 60,000 Da. We suggest that a conformational change to colicin S8 may occur related to the export process.

  16. Cytoplasmic transduction peptide (CTP): New approach for the delivery of biomolecules into cytoplasm in vitro and in vivo

    SciTech Connect

    Kim, Daeyou; Jeon, Choonju; Kim, Jeong-Hwan; Kim, Mi-Seon; Yoon, Cheol-Hee; Choi, In-Soo; Kim, Sung-Hoon; Bae, Yong-Soo . E-mail: ysbae04@skku.edu

    2006-05-01

    The protein transduction domain (PTD) of HIV-1 TAT has been extensively documented with regard to its membrane transduction potential, as well as its efficient delivery of biomolecules in vivo. However, the majority of PTD and PTD-conjugated molecules translocate to the nucleus rather than to the cytoplasm after transduction, due to the functional nuclear localization sequence (NLS). Here, we report a cytoplasmic transduction peptide (CTP), which was deliberately designed to ensure the efficient cytoplasmic delivery of the CTP-fused biomolecules. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential. Unlike the mechanism underlying PTD-mediated transduction, CTP-mediated transduction occurs independently of the lipid raft-dependent macropinocytosis pathway. The CTP-conjugated Smac/DIABLO peptide (Smac-CTP) was also shown to be much more efficient than Smac-PTD in the blockage of the antiapoptotic properties of XIAP, suggesting that cytoplasmic functional molecules can be more efficiently targeted by CTP-mediated delivery. In in vivo trafficking studies, CTP-fused {beta}-gal exhibited unique organ tropisms to the liver and lymph nodes when systemically injected into mice, whereas PTD-{beta}-gal exhibited no such tropisms. Taken together, our findings implicate CTP as a novel delivery peptide appropriate for (i) molecular targeting to cytoplasmic compartments in vitro, (ii) the development of class I-associated CTL vaccines, and (iii) special drug delivery in vivo, without causing any untoward effects on nuclear genetic material.

  17. Further analysis of cytoplasmic polyadenylation in Xenopus embryos and identification of embryonic cytoplasmic polyadenylation element-binding proteins.

    PubMed

    Simon, R; Richter, J D

    1994-12-01

    Early development in Xenopus laevis is programmed in part by maternally inherited mRNAs that are synthesized and stored in the growing oocyte. During oocyte maturation, several of these messages are translationally activated by poly(A) elongation, which in turn is regulated by two cis elements in the 3' untranslated region, the hexanucleotide AAUAAA and a cytoplasmic polyadenylation element (CPE) consisting of UUUUUAU or similar sequence. In the early embryo, a different set of maternal mRNAs is translationally activated. We have shown previously that one of these, C12, requires a CPE consisting of at least 12 uridine residues, in addition to the hexanucleotide, for its cytoplasmic polyadenylation and subsequent translation (R. Simon, J.-P. Tassan, and J.D. Richter, Genes Dev. 6:2580-2591, 1992). To assess whether this embryonic CPE functions in other maternal mRNAs, we have chosen Cl1 RNA, which is known to be polyadenylated during early embryogenesis (J. Paris, B. Osborne, A. Couturier, R. LeGuellec, and M. Philippe, Gene 72:169-176, 1988). Wild-type as well as mutated versions of Cl1 RNA were injected into fertilized eggs and were analyzed for cytoplasmic polyadenylation at times up to the gastrula stage. This RNA also required a poly(U) CPE for cytoplasmic polyadenylation in embryos, but in this case the CPE consisted of 18 uridine residues. In addition, the timing and extent of cytoplasmic poly(A) elongation during early embryogenesis were dependent upon the distance between the CPE and the hexanucleotide. Further, as was the case with Cl2 RNA, Cl1 RNA contains a large masking element that prevents premature cytoplasmic polyadenylation during oocyte maturation. To examine the factors that may be involved in the cytoplasmic polyadenylation of both C12 and C11 RNAs, we performed UV cross-linking experiments in egg extracts. Two proteins with sizes of ~36 and ~45 kDa interacted specifically with the CPEs of both RNAs, although they bound preferentially to the C12

  18. Experimental Analysis of Cell Function Using Cytoplasmic Streaming

    ERIC Educational Resources Information Center

    Janssens, Peter; Waldhuber, Megan

    2012-01-01

    This laboratory exercise investigates the phenomenon of cytoplasmic streaming in the fresh water alga "Nitella". Students use the fungal toxin cytochalasin D, an inhibitor of actin polymerization, to investigate the mechanism of streaming. Students use simple statistical methods to analyze their data. Typical student data are provided. (Contains 3…

  19. Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

    PubMed Central

    Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

    2012-01-01

    The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

  20. Nuclear repulsion enables division autonomy in a single cytoplasm

    PubMed Central

    Anderson, Cori A.; Eser, Umut; Korndorf, Therese; Borsuk, Mark E.; Skotheim, Jan M.; Gladfelter, Amy S.

    2014-01-01

    Summary Background Current models of cell cycle control, based on classic studies of fused cells, predict that nuclei in a shared cytoplasm respond to the same CDK activities to undergo synchronous cycling. However, synchrony is rarely observed in naturally occurring syncytia, such as the multinucleate fungus Ashbya gossypii. In this system, nuclei divide asynchronously raising the question of how nuclear timing differences are maintained despite sharing a common milieu. Results We observe that neighboring nuclei are highly variable in division cycle duration and neighbors repel one another to space apart and demarcate their own cytoplasmic territories. The size of these territories increases as a nucleus approaches mitosis and can influence cycling rates. This non-random nuclear spacing is regulated by microtubules and is required for nuclear asynchrony, as nuclei that transiently come in very close proximity will partially synchronize. Sister nuclei born of the same mitosis are generally not persistent neighbors over their lifetimes yet remarkably retain similar division cycle times. This indicates that nuclei carry a memory of their birth state that influences their division timing and supports that nuclei subdivide a common cytosol into functionally distinct yet mobile compartments. Conclusions These findings support that nuclei use cytoplasmic microtubules to establish “cells within cells.” Individual compartments appear to push against one another to compete for cytoplasmic territory and insulate the division cycle. This provides a mechanism by which syncytial nuclei can spatially organize cell cycle signaling and suggests size control can act in a system without physical boundaries. PMID:24094857

  1. Optomechatronic System For Automated Intra Cytoplasmic Sperm Injection

    NASA Astrophysics Data System (ADS)

    Shulev, Assen; Tiankov, Tihomir; Ignatova, Detelina; Kostadinov, Kostadin; Roussev, Ilia; Trifonov, Dimitar; Penchev, Valentin

    2015-12-01

    This paper presents a complex optomechatronic system for In-Vitro Fertilization (IVF), offering almost complete automation of the Intra Cytoplasmic Sperm Injection (ICSI) procedure. The compound parts and sub-systems, as well as some of the computer vision algorithms, are described below. System capabilities for ICSI have been demonstrated on infertile oocyte cells.

  2. Structure of human cytoplasmic dynein-2 primed for its powerstroke

    PubMed Central

    Urnavicius, Linas; Carter, Andrew P.

    2014-01-01

    Members of the dynein family, consisting of cytoplasmic and axonemal isoforms, are motors that move towards the minus ends of microtubules. Cytoplasmic dynein-1 (dynein-1) plays roles in mitosis and cellular cargo transport1, and is implicated in viral infections2 and neurodegenerative diseases3. Cytoplasmic dynein-2 (dynein-2) carries out intraflagellar transport4 and is associated with human skeletal ciliopathies5. Dyneins share a conserved motor domain that couples cycles of ATP hydrolysis with conformational changes to produce movement6-9. Here we present the crystal structure of the human cytoplasmic dynein-2 motor bound to the ATP-hydrolysis transition state analogue ADP.vanadate (ADP.Vi)10. The structure reveals a closure of the motor’s ring of six AAA+ domains (ATPases associated with various cellular activites: AAA1-AAA6). This induces a steric clash with the linker, the key element for the generation of movement, driving it into a conformation that is primed to produce force. Ring closure also changes the interface between the stalk and buttress coiled-coil extensions of the motor domain. This drives helix sliding in the stalk that causes the microtubule binding domain (MTBD) at its tip to release from the microtubule. Our structure answers the key questions of how ATP hydrolysis leads to linker remodelling and microtubule affinity regulation. PMID:25470043

  3. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

    SciTech Connect

    Lloyd, Richard E.

    2015-05-15

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

  4. Membrane binding of human phospholipid scramblase 1 cytoplasmic domain.

    PubMed

    Posada, Itziar M D; Sánchez-Magraner, Lissete; Hervás, Javier H; Alonso, Alicia; Monaco, Hugo L; Goñi, Félix M

    2014-07-01

    Human phospholipid scramblase 1 (SCR) consists of a large cytoplasmic domain and a small presumed transmembrane domain near the C-terminal end of the protein. Previous studies with the SCRΔ mutant lacking the C-terminal portion (last 28 aa) revealed the importance of this C-terminal moiety for protein function and calcium-binding affinity. The present contribution is intended to elucidate the effect of the transmembrane domain suppression on SCRΔ binding to model membranes (lipid monolayers and bilayers) and on SCRΔ reconstitution in proteoliposomes. In all cases the protein cytoplasmic domain showed a great affinity for lipid membranes, and behaved in most aspects as an intrinsic membrane protein. Assays have been performed in the presence of phosphatidylserine, presumably important for the SCR cytoplasmic domain to be electrostatically anchored to the plasma membrane inner surface. The fusion protein maltose binding protein-SCR has also been studied as an intermediate case of a molecule that can insert into the bilayer hydrophobic core, yet it is stable in detergent-free buffers. Although the intracellular location of SCR has been the object of debate, the present data support the view of SCR as an integral membrane protein, in which not only the transmembrane domain but also the cytoplasmic moiety play a role in membrane docking of the protein.

  5. Cytoplasmic mechanisms of axonal and dendritic growth in neurons.

    PubMed

    Heidemann, S R

    1996-01-01

    The structural mechanisms responsible for the gradual elaboration of the cytoplasmic elongation of neurons are reviewed. In addition to discussing recent work, important older work is included to inform newcomers to the field how the current perspective arose. The highly specialized axon and the less exaggerated dendrite both result from the advance of the motile growth cone. In the area of physiology, studies in the last decade have directly confirmed the classic model of the growth cone pulling forward and the axon elongating from this tension. Particularly in the case of the axon, cytoplasmic elongation is closely linked to the formation of an axial microtubule bundle from behind the advancing growth cone. Substantial progress has been made in understanding the expression of microtubule-associated proteins during neuronal differentiation to stiffen and stabilize axonal microtubules, providing specialized structural support. Studies of membrane organelle transport along the axonal microtubules produced an explosion of knowledge about ATPase molecules serving as motors driving material along microtubule rails. However, most aspects of the cytoplasmic mechanisms responsible for neurogenesis remain poorly understood. There is little agreement on mechanisms for the addition of new plasma membrane or the addition of new cytoskeletal filaments in the growing axon. Also poorly understood are the mechanisms that couple the promiscuous motility of the growth cone to the addition of cytoplasmic elements.

  6. Gravity-dependent polarity of cytoplasmic streaming in Nitellopsis

    NASA Technical Reports Server (NTRS)

    Wayne, R.; Staves, M. P.; Leopold, A. C.

    1990-01-01

    The internodal cells of the characean alga Nitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downward-streaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR < 1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K+ channels. The K+ channel blocker TEA Cl- inhibits the NR effect. External Ca2+ is required for normal graviresponsiveness. The [Ca2+] of the medium determines the polarity of cytoplasmic streaming. Less than 1 micromole Ca2+ resulted in a PR < 1 while greater than 1 micromole Ca2+ resulted in the normal gravity response. The voltage-dependent Ca(2+)-channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl3 resulted in a PR < 1, indicating the presence of two types of Ca2+ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR of Nitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.

  7. Mechanodelivery of nanoparticles to the cytoplasm of living cells

    NASA Astrophysics Data System (ADS)

    Emerson, Nyssa T.; Hsia, Chih-Hao; Rafalska-Metcalf, Ilona U.; Yang, Haw

    2014-04-01

    Nanotechnology has opened up the opportunity to probe, sense, and manipulate the chemical environment of biological systems with an unprecedented level of spatiotemporal control. A major obstacle to the full realization of these novel technologies is the lack of a general, robust, and simple method for the delivery of arbitrary nanostructures to the cytoplasm of intact live cells. Here, we identify a new delivery modality, based on mechanical disruption of the plasma membrane, which efficiently mediates the delivery of nanoparticles to the cytoplasm of mammalian cells. We use two distinct execution modes, two adherent cell lines, and three sizes of semiconducting nanocrystals, or quantum dots, to demonstrate its applicability and effectiveness. As the underlying mechanism is purely physical, we anticipate that such ``mechanodelivery'' can be generalized to other modes of execution as well as to the cytoplasmic introduction of a structurally diverse array of functional nanomaterials.Nanotechnology has opened up the opportunity to probe, sense, and manipulate the chemical environment of biological systems with an unprecedented level of spatiotemporal control. A major obstacle to the full realization of these novel technologies is the lack of a general, robust, and simple method for the delivery of arbitrary nanostructures to the cytoplasm of intact live cells. Here, we identify a new delivery modality, based on mechanical disruption of the plasma membrane, which efficiently mediates the delivery of nanoparticles to the cytoplasm of mammalian cells. We use two distinct execution modes, two adherent cell lines, and three sizes of semiconducting nanocrystals, or quantum dots, to demonstrate its applicability and effectiveness. As the underlying mechanism is purely physical, we anticipate that such ``mechanodelivery'' can be generalized to other modes of execution as well as to the cytoplasmic introduction of a structurally diverse array of functional nanomaterials

  8. Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis.

    PubMed Central

    Lam, E; Green, P J; Wong, M; Chua, N H

    1989-01-01

    We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organellar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes. Images PMID:2583082

  9. Chara myosin and the energy of cytoplasmic streaming.

    PubMed

    Yamamoto, Keiichi; Shimada, Kiyo; Ito, Khoji; Hamada, Saeko; Ishijima, Akio; Tsuchiya, Takayoshi; Tazawa, Masashi

    2006-10-01

    Recently, it was found that myosin generating very fast cytoplasmic streaming in Chara corallina has very high ATPase activity. To estimate the energy consumed by this myosin, its concentration in the internodal cells of C. corallina was determined by quantitative immunoblot. It was found that the concentration of Chara myosin was considerably high (200 nM) and the amount of ATP consumed by this myosin would exceed that supplied by dark respiration if all myosin molecules were fully activated by the interaction with actin. These results and model calculations suggested that the energy required to generate cytoplasmic streaming is very small and only one-hundredth of the existing myosin is enough to maintain the force for the streaming in the Chara cell.

  10. Automated nucleus and cytoplasm segmentation of overlapping cervical cells.

    PubMed

    Lu, Zhi; Carneiro, Gustavo; Bradley, Andrew P

    2013-01-01

    In this paper we describe an algorithm for accurately segmenting the individual cytoplasm and nuclei from a clump of overlapping cervical cells. Current methods cannot undertake such a complete segmentation due to the challenges involved in delineating cells with severe overlap and poor contrast. Our approach initially performs a scene segmentation to highlight both free-lying cells, cell clumps and their nuclei. Then cell segmentation is performed using a joint level set optimization on all detected nuclei and cytoplasm pairs. This optimisation is constrained by the length and area of each cell, a prior on cell shape, the amount of cell overlap and the expected gray values within the overlapping regions. We present quantitative nuclei detection and cell segmentation results on a database of synthetically overlapped cell images constructed from real images of free-lying cervical cells. We also perform a qualitative assessment of complete fields of view containing multiple cells and cell clumps.

  11. Genetic analysis of the cytoplasmic dynein subunit families.

    PubMed

    Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  12. Genetic Analysis of the Cytoplasmic Dynein Subunit Families

    PubMed Central

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles. PMID:16440056

  13. Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity.

    PubMed

    See, Stephanie K; Hoogendoorn, Sascha; Chung, Andrew H; Ye, Fan; Steinman, Jonathan B; Sakata-Kato, Tomoyo; Miller, Rand M; Cupido, Tommaso; Zalyte, Ruta; Carter, Andrew P; Nachury, Maxence V; Kapoor, Tarun M; Chen, James K

    2016-01-15

    Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.

  14. Bidirectional helical motility of cytoplasmic dynein around microtubules.

    PubMed

    Can, Sinan; Dewitt, Mark A; Yildiz, Ahmet

    2014-07-28

    Cytoplasmic dynein is a molecular motor responsible for minus-end-directed cargo transport along microtubules (MTs). Dynein motility has previously been studied on surface-immobilized MTs in vitro, which constrains the motors to move in two dimensions. In this study, we explored dynein motility in three dimensions using an MT bridge assay. We found that dynein moves in a helical trajectory around the MT, demonstrating that it generates torque during cargo transport. Unlike other cytoskeletal motors that produce torque in a specific direction, dynein generates torque in either direction, resulting in bidirectional helical motility. Dynein has a net preference to move along a right-handed helical path, suggesting that the heads tend to bind to the closest tubulin binding site in the forward direction when taking sideways steps. This bidirectional helical motility may allow dynein to avoid roadblocks in dense cytoplasmic environments during cargo transport.

  15. Cytoplasmic streaming in plant cells: the role of wall slip.

    PubMed

    Wolff, K; Marenduzzo, D; Cates, M E

    2012-06-01

    We present a computer simulation study, via lattice Boltzmann simulations, of a microscopic model for cytoplasmic streaming in algal cells such as those of Chara corallina. We modelled myosin motors tracking along actin lanes as spheres undergoing directed motion along fixed lines. The sphere dimension takes into account the fact that motors drag vesicles or other organelles, and, unlike previous work, we model the boundary close to which the motors move as walls with a finite slip layer. By using realistic parameter values for actin lane and myosin density, as well as for endoplasmic and vacuole viscosity and the slip layer close to the wall, we find that this simplified view, which does not rely on any coupling between motors, cytoplasm and vacuole other than that provided by viscous Stokes flow, is enough to account for the observed magnitude of streaming velocities in intracellular fluid in living plant cells.

  16. Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

    PubMed Central

    Lakdawala, Seema S.; Wu, Yicong; Wawrzusin, Peter; Kabat, Juraj; Broadbent, Andrew J.; Lamirande, Elaine W.; Fodor, Ervin; Altan-Bonnet, Nihal; Shroff, Hari; Subbarao, Kanta

    2014-01-01

    Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm. PMID:24603687

  17. Influenza a virus assembly intermediates fuse in the cytoplasm.

    PubMed

    Lakdawala, Seema S; Wu, Yicong; Wawrzusin, Peter; Kabat, Juraj; Broadbent, Andrew J; Lamirande, Elaine W; Fodor, Ervin; Altan-Bonnet, Nihal; Shroff, Hari; Subbarao, Kanta

    2014-03-01

    Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm. PMID:24603687

  18. Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking

    SciTech Connect

    Funasaka, Tatsuyoshi; Balan, Vitaly; Raz, Avraham; Wong, Richard W.

    2013-04-26

    Highlights: •Nuclear pore protein Nup98 is a novel binding partner of galectin-3. •Nup98 transports galectin-3 into cytoplasm. •Nup98 depletion leads to galectin-3 nuclear transport and induces growth retardation. •Nup98 may involve in ß-catenin pathway through interaction with galectin-3. -- Abstract: Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus–cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3–nucleus translocation rate. Overall, the results show Nup98’s involvement in nuclear–cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.

  19. Toxicological studies of pesticides on cytoplasmic streaming in Nitella.

    PubMed

    Pandey, Sudhir Kumar; Fatma, Tasneem

    2004-07-01

    In the present study, changes in velocity of cytoplasmic streaming in the giant internodal cells of Nitella for varying concentration of the pesticides, 2,4-D, dieldrin, malathion, methyl parathion and endosulfan, were measured. Marked decrease in the velocity of cytoplasmic streaming was found at the concentrations of 0.01, 0.1, 1, 10 and 100mM. Dieldrin was the most toxic to all the pesticides investigated, followed by methyl parathion, endosulfan, malathion and 2,4-D. Threshold values for dieldrin, methylparathion, endosulfan, malathion and 2,4-D as indicated by the onset of decrease in the normal cytoplasmic streaming velocity were less than 6.25 x 10(-6), 2.5 x 10(-5), 5 x 10(-5), 5 x 10(-5) and 1.25 x 10(-5)M respectively. Cessation of streaming was noticed above 1mM in dieldrin and above 10mM when exposed to methylparathion and endosulfan. Cessation of streaming was not seen up to 100mM concentration of 2,4-D and malathion.

  20. Female breast carcinomas: nuclear and cytoplasmic proteins versus steroid receptors.

    PubMed

    Bryś, M; Romanowicz-Makowska, H; Nawrocka, A; Krajewska, W M

    2000-01-01

    Nuclear and cytoplasmic proteins of human female breast cancer were analysed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Oestrogen receptor and progesterone receptor expression was determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. The electropherograms were developed by silver nitrate staining and quantitative analysis was carried out by video densitometer using the software Gel-Pro Analyzer. Nuclear and cytoplasmic proteins of breast carcinomas and normal tissue differed both qualitatively and quantitatively. Nuclear polypeptides of 108, 53 and 48 kD as well as the 36 kD cytoplasmic polypeptide were specific for tumour samples, while the 51 kD nuclear polypeptide was detected only in normal tissue. Quantitative differences in band density were noted in the 32 kD nuclear polypeptide. This polypeptide was expressed in greatest concentration in infiltrating ductal carcinomas which also indicated the greatest oestrogen receptor gene expression. This relationship appeared to be statistically significant (p < 0.005). No correlations were evident between the 32 kD protein expression and the progesterone receptor gene expression in any of the tissue types examined, nor between the 32 kD protein and the patient's age or tumour grade. PMID:10756981

  1. Sequences determining the cytoplasmic localization of a chemoreceptor domain.

    PubMed Central

    Seligman, L; Bailey, J; Manoil, C

    1995-01-01

    The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure. PMID:7730259

  2. Genetic studies on cytoplasmic male sterility in maize

    SciTech Connect

    Laughnan, J.R.

    1992-01-01

    Our research concerns the basic mechanisms of cytoplasmic male sterility (CMS) and fertility restoration in maize. The molecular determination of CMS is in the DNA of the mitochondria (mtDNA) but specific nuclear restorer-of-fertility (Rf) genes can overrule the male-sterile effect of the cytoplasm. Our approach to the study of the Rf genes is threefold. We are attempting to tag the cms-S Rf genes and the cms-T Rf2 gene with controlling elements (CEs). Since we have identified a number of spontaneous Rf genes for cms-S and have demonstrated that they are themselves transposable, we are also searching for cases in which an Rf gene is inserted into a wild-type gene. The other aspect of our research involves the nuclear control over the organization of the mitochondrial genome. We found that the changes in mtDNA organization upon cytoplasmic reversion to fertility were characteristic of the nuclear background in which the reversion event occurred. We have investigated whether these differences are a reflection of differences in the organization of the mtDNA genome before reversion.

  3. Alterations in hepatic pericanalicular cytoplasm during enhanced bile secretory activity.

    PubMed

    Jones, A L; Schmucker, D L; Mooney, J S; Ockner, R K; Adler, R D

    1979-04-01

    In an attempt to demonstrate the morphology of the bile secretory apparatus, male rats were restrained and maintained on an isocaloric diet with (experimental) and without (control) taurocholate, which was continuously infused via a duodenal cannula. This method of taurocholate administration promotes a 2-fold increase in the bile acid pool size and bile secretory rate and increases the transport maximum of taurocholate by approximately 50%. After 48 hours, the livers from both the control and experimental animals were perfusion-fixed and whole hepatocytes as well as pericanalicular cytoplasm (defined as a 1-micron. wide zone of cytoplasm adjacent to the bile canaliculus) in both centrolobular and periportal cells were subjected to a stereologic analysis. Although taurocholate infusion produced relatively few changes in the amounts of organelles or inclusionswithin hepatocytes, it caused highly significant increases in the amount ofGolgi-rich area, Golgi membranes, and the number of vesicles with diameters greater than 1000 A in the pericanalicular area of cytoplasm. In addition to these changes, which occurred in both central and periportal zones, decreases in the volume of lysosomes and the surface area of smooth surfaced endoplasmic reticulum were observed. These data provide new evidence that the "bile secretory apparatus" may encompass several hepatocellular components which include the Golgi complex and a vesicular transport system.

  4. Nuclear versus perinuclear and cytoplasmic calcium in osteoclasts.

    PubMed

    Ferrier, J; Yu, H

    1996-11-01

    We measured fluorescence from the calcium indicator Fluo-3 in multinucleated osteoclasts. In the initial state, each nucleus is surrounded by a ring of bright fluorescence. Following activation of purinergic receptors by 100 microM ATP there is a pulse of cellular fluorescence increase, and nuclear fluorescence intensity becomes greater than that of the cytoplasm. This is followed by a period during which the fluorescence of the cell decreases below that of the initial state. During the pulsed increase following purinergic receptor activation, the perinuclear fluorescence intensity does not increase as much as that in the nuclear centers and, following this pulse, the perinuclear fluorescence intensity decreases more than that in the nuclear centers, relative to the initial state. Measurements in which Mn2+ was introduced into the cell show that the number of Fluo-3 molecules per unit horizontal area in the nuclear centers is slightly greater than that in the perinuclear regions, and more than twice that in the surrounding cytoplasm. These results show that there is a much higher free calcium concentration in the perinuclear regions than in the nuclear centers in the initial state, with a release of free calcium from the perinuclear regions following activation of the purinergic receptors. These data also provide evidence that the free calcium concentration in the nuclear centers is lower than in the cytoplasm in the initial state.

  5. Cytoplasmic Control of Sense-Antisense mRNA Pairs.

    PubMed

    Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

    2015-09-22

    Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. PMID:26344770

  6. Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking

    PubMed Central

    Funasaka, Tatsuyoshi; Balan, Vitaly; Raz, Avraham; Wong, Richard W.

    2013-01-01

    Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus–cytoplasm transport of galectin-3, which is a β-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the β-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the β-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to β-catenin to inhibit transcriptional activity. Reduced expression of β-catenin target genes is consistent with the Nup98 reduction and the galectin-3–nucleus translocation rate. Overall, the results show Nup98’s involvement in nuclear–cytoplasm translocation of galectin-3 and β-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers. PMID:23541576

  7. Increased cytoplasm viscosity hampers aggregate polar segregation in Escherichia coli.

    PubMed

    Oliveira, Samuel M D; Neeli-Venkata, Ramakanth; Goncalves, Nadia S M; Santinha, João A; Martins, Leonardo; Tran, Huy; Mäkelä, Jarno; Gupta, Abhishekh; Barandas, Marilia; Häkkinen, Antti; Lloyd-Price, Jason; Fonseca, José M; Ribeiro, Andre S

    2016-02-01

    In Escherichia coli, under optimal conditions, protein aggregates associated with cellular aging are excluded from midcell by the nucleoid. We study the functionality of this process under sub-optimal temperatures from population and time lapse images of individual cells and aggregates and nucleoids within. We show that, as temperature decreases, aggregates become homogeneously distributed and uncorrelated with nucleoid size and location. We present evidence that this is due to increased cytoplasm viscosity, which weakens the anisotropy in aggregate displacements at the nucleoid borders that is responsible for their preference for polar localisation. Next, we show that in plasmolysed cells, which have increased cytoplasm viscosity, aggregates are also not preferentially located at the poles. Finally, we show that the inability of cells with increased viscosity to exclude aggregates from midcell results in enhanced aggregate concentration in between the nucleoids in cells close to dividing. This weakens the asymmetries in aggregate numbers between sister cells of subsequent generations required for rejuvenating cell lineages. We conclude that the process of exclusion of protein aggregates from midcell is not immune to stress conditions affecting the cytoplasm viscosity. The findings contribute to our understanding of E. coli's internal organisation and functioning, and its fragility to stressful conditions. PMID:26507787

  8. Mitochondrial and Cytoplasmic ROS Have Opposing Effects on Lifespan

    PubMed Central

    Schaar, Claire E.; Dues, Dylan J.; Spielbauer, Katie K.; Machiela, Emily; Cooper, Jason F.; Senchuk, Megan; Hekimi, Siegfried; Van Raamsdonk, Jeremy M.

    2015-01-01

    Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the clk-1 mitochondrial mutant, but not other long-lived mitochondrial mutants such as isp-1 or nuo-6. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (sod) genes in clk-1 mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in clk-1 worms. In contrast, deletion of either of the two cytoplasmic sod genes, sod-1 or sod-5, significantly decreases the lifespan of clk-1 worms. Further, we show that increasing mitochondrial superoxide levels through deletion of sod-2 or treatment with paraquat can still increase lifespan in clk-1;sod-1 double mutants, which live shorter than clk-1 worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan – elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of clk-1 worms. PMID:25671321

  9. Mitochondrial and cytoplasmic ROS have opposing effects on lifespan.

    PubMed

    Schaar, Claire E; Dues, Dylan J; Spielbauer, Katie K; Machiela, Emily; Cooper, Jason F; Senchuk, Megan; Hekimi, Siegfried; Van Raamsdonk, Jeremy M

    2015-02-01

    Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the clk-1 mitochondrial mutant, but not other long-lived mitochondrial mutants such as isp-1 or nuo-6. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (sod) genes in clk-1 mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in clk-1 worms. In contrast, deletion of either of the two cytoplasmic sod genes, sod-1 or sod-5, significantly decreases the lifespan of clk-1 worms. Further, we show that increasing mitochondrial superoxide levels through deletion of sod-2 or treatment with paraquat can still increase lifespan in clk-1;sod-1 double mutants, which live shorter than clk-1 worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan - elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of clk-1 worms.

  10. Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

    PubMed Central

    Pietrocola, Federico; Mariño, Guillermo; Lissa, Delphine; Vacchelli, Erika; Malik, Shoaib Ahmad; Niso-Santano, Mireia; Zamzami, Naoufal; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Kroemer, Guido

    2012-01-01

    Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components. PMID:23070521

  11. Symmetrical infantile thalamic degeneration with focal cytoplasmic calcification.

    PubMed

    Ambler, M; O'Neil, W

    1975-10-27

    Infantile thalamic degeneration is a rare clinico-pathological entity. Restricted location of the lesion and peculiar cytopathological changes serve to distinguish this disorder from other common encephalopathies. Optical and ultrastructural studies demonstrate cytoplasmic calcopherules in previously viable cells. According to current concepts of acute cellular reactions to injury and mechanism of intracellular calcification, the cytological changes cannot be attributed to either hypoxic ischemic cell change or dystrophic calcification. By analogy to other human and pathological material, the most likely basis for nondystrophic calcopherule formation is toxic or infectious injury with local synthesis, or autophagic or phagolysosomal degradation of cellular debris of specific chemical composition favoring calcium deposition.

  12. NUCLEAR DNA AND CYTOPLASMIC DNA FROM TISSUES OF HIGHER PLANTS

    PubMed Central

    Hotta, Yasuo; Bassel, Alix; Stern, Herbert

    1965-01-01

    Young wheat roots were labeled with 32P-inorganic phosphate. Following the labeling period, roots were homogenized in a sucrose medium and fractionated into nuclei, cytoplasmic particles (including proplastids and mitochondria), and a soluble fraction containing most of the microsomes. DNA prepared from the particles had a higher buoyant density than that from the nuclei and showed a marked loss in total label if the roots were exposed to non-radioactive medium for 48 hours prior to fractionation of the cells. PMID:5885425

  13. Antineutrophil cytoplasmic antibodies (ANCA) testing: detection methods and clinical application.

    PubMed

    Sinico, Renato Alberto; Radice, Antonella

    2014-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are considered the diagnostic biomarker of some necrotising vasculitis such as granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and, to a lesser extent, eosinophilic granulomatosis with polyangiitis (EGPA). According to the current recommendations, combining indirect immunofluorescence and proteinase 3 (PR3) and myeloperoxidase (MPO) antigen specific immunometric assays, in the proper clinical setting, assures the best diagnostic specificity. When such conditions are satisfied, ANCA are detected in up to 90% of patients with active generalised GPA and MPA and in about 40% of patients with EGPA. Cytoplasmic ANCA (C-ANCA) with specificity for PR3 are usually found in patients with GPA whereas perinuclear ANCA (P-ANCA) in patients with MPA and EGPA. However, ANCA antigen specificity is more closely associated with disease phenotype and prognosis than clinical diagnosis. The clinical value of serial ANCA testing in monitoring disease activity is still debated. Recently, new promising developments in methodology and techniques (computer-based image analysis of immunofluorescence patterns, novel generation of PR3-/MPO-ANCA immunometric assays and multiplex technology) have been proposed but studies comparing the performances of the different assays are scarce. PMID:24854381

  14. Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro

    PubMed Central

    Jin, Yong-Xun; Kwon, Jeong-Woo

    2016-01-01

    Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.

  15. Microfluidics of cytoplasmic streaming and its implications for intracellular transport.

    PubMed

    Goldstein, Raymond E; Tuval, Idan; van de Meent, Jan-Willem

    2008-03-11

    Found in many large eukaryotic cells, particularly in plants, cytoplasmic streaming is the circulation of their contents driven by fluid entrainment from particles carried by molecular motors at the cell periphery. In the more than two centuries since its discovery, streaming has frequently been conjectured to aid in transport and mixing of molecular species in the cytoplasm and, by implication, in cellular homeostasis, yet no theoretical analysis has been presented to quantify these processes. We show by a solution to the coupled dynamics of fluid flow and diffusion appropriate to the archetypal "rotational streaming" of algal species such as Chara and Nitella that internal mixing and the transient dynamical response to changing external conditions can indeed be enhanced by streaming, but to an extent that depends strongly on the pitch of the helical flow. The possibility that this may have a developmental consequence is illustrated by the coincidence of the exponential growth phase of Nitella and the point of maximum enhancement of those processes.

  16. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  17. Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro

    PubMed Central

    Jin, Yong-Xun; Kwon, Jeong-Woo

    2016-01-01

    Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression. PMID:27672508

  18. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein

    PubMed Central

    Much, Christian; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O’Carroll, Dónal

    2016-01-01

    Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse. PMID:27254021

  19. Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity

    PubMed Central

    2015-01-01

    Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure–activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes. PMID:26555042

  20. Subunit composition of the human cytoplasmic dynein-2 complex

    PubMed Central

    Asante, David; Stevenson, Nicola L.; Stephens, David J.

    2014-01-01

    ABSTRACT Cytoplasmic dynein-2 is the motor for retrograde intraflagellar transport (IFT), and mutations in dynein-2 are known to cause skeletal ciliopathies. Here, we define for the first time the composition of the human cytoplasmic dynein-2 complex. We show that the proteins encoded by the ciliopathy genes WDR34 and WDR60 are bona fide dynein-2 intermediate chains and are both required for dynein-2 function. In addition, we identify TCTEX1D2 as a unique dynein-2 light chain that is itself required for cilia function. We define several subunits common to both dynein-1 and dynein-2, including TCTEX-1 (also known as DYNLT1) and TCTEX-3 (also known as DYNLT3), roadblock-1 (also known as DYNLRB1) and roadblock-2 (also known as DYNLRB2), and LC8-1 and LC8-2 light chains (DYNLL1 and DYNLL2, respectively). We also find that NudCD3 associates with dynein-2 as it does with dynein-1. By contrast, the common dynein-1 regulators dynactin, LIS1 (also known as PAFAH1B1) and BICD2 are not found in association with dynein-2. These data explain why mutations in either WDR34 or WDR60 cause disease, as well as identifying TCTEX1D2 as a candidate ciliopathy gene. PMID:25205765

  1. Mitochondrion role in molecular basis of cytoplasmic male sterility.

    PubMed

    Horn, Renate; Gupta, Kapuganti J; Colombo, Noemi

    2014-11-01

    Cytoplasmic male sterility and its fertility restoration via nuclear genes offer the possibility to understand the role of mitochondria during microsporogenesis. In most cases rearrangements in the mitochondrial DNA involving known mitochondrial genes as well as unknown sequences result in the creation of new chimeric open reading frames, which encode proteins containing transmembrane domains. So far, most of the CMS systems have been characterized via restriction fragment polymorphisms followed by transcript analysis. However, whole mitochondrial genome sequence analyses comparing male sterile and fertile cytoplasm open options for deeper insights into mitochondrial genome rearrangements. We more and more start to unravel how mitochondria are involved in triggering death of the male reproductive organs. Reduced levels of ATP accompanied by increased concentrations of reactive oxygen species, which are produced more under conditions of mitochondrial dysfunction, seem to play a major role in the fate of pollen production. Nuclear genes, so called restorer-of-fertility are able to restore the male fertility. Fertility restoration can occur via pentatricopeptide repeat (PPR) proteins or via different mechanisms involving non-PPR proteins.

  2. A Balance between Nuclear and Cytoplasmic Volumes Controls Spindle Length

    PubMed Central

    Novakova, Lucia; Kovacovicova, Kristina; Dang-Nguyen, Thanh Quang; Sodek, Martin; Skultety, Michal; Anger, Martin

    2016-01-01

    Proper assembly of the spindle apparatus is crucially important for faithful chromosome segregation during anaphase. Thanks to the effort over the last decades, we have very detailed information about many events leading to spindle assembly and chromosome segregation, however we still do not understand certain aspects, including, for example, spindle length control. When tight regulation of spindle size is lost, chromosome segregation errors emerge. Currently, there are several hypotheses trying to explain the molecular mechanism of spindle length control. The number of kinetochores, activity of molecular rulers, intracellular gradients, cell size, limiting spindle components, and the balance of the spindle forces seem to contribute to spindle size regulation, however some of these mechanisms are likely specific to a particular cell type. In search for a general regulatory mechanism, in our study we focused on the role of cell size and nuclear to cytoplasmic ratio in this process. To this end, we used relatively large cells isolated from 2-cell mouse embryos. Our results showed that the spindle size upper limit is not reached in these cells and suggest that accurate control of spindle length requires balanced ratio between nuclear and cytoplasmic volumes. PMID:26886125

  3. Cytoplasmic Incompatibility and Bacterial Density in Nasonia Vitripennis

    PubMed Central

    Breeuwer, JAJ.; Werren, J. H.

    1993-01-01

    Cytoplasmically (maternally) inherited bacteria that cause reproductive incompatibility between strains are widespread among insects. In the parasitoid wasp Nasonia, incompatibility results in improper condensation and fragmentation of the paternal chromosomes in fertilized eggs. Some form of genome imprinting may be involved. Because of haplodiploidy, incompatibility results in conversion of (diploid) female eggs into (haploid) males. Experiments show that bacterial density is correlated with compatibility differences between male and female Nasonia. Males from strains with high bacterial numbers are incompatible with females from strains with lower numbers. Temporal changes in compatibility of females after tetracycline treatment are generally correlated with decreases in bacterial levels in eggs. However, complete loss of bacteria in mature eggs precedes conversion of eggs to the ``asymbiont'' compatibility type by 3-4 days. This result is consistent with a critical ``imprinting'' period during egg maturation, when cytoplasmic bacteria determine compatibility. Consequent inheritance of reduced bacterial numbers in F(1) progeny has different effects on compatibility type of subsequent male vs. female progeny. In some cases, partial incompatibility occurs which results in reduced offspring numbers, apparently due to incomplete paternal chromosome elimination resulting in aneuploidy. PMID:8244014

  4. Cytoplasm dynamics and cell motion: two-phase flow models.

    PubMed

    Alt, W; Dembo, M

    1999-03-01

    The motion of amoeboid cells is characterized by cytoplasmic streaming and by membrane protrusions and retractions which occur even in the absence of interactions with a substratum. Cell translocation requires, in addition, a transmission mechanism wherein the power produced by the cytoplasmic engine is applied to the substratum in a highly controlled fashion through specific adhesion proteins. Here we present a simple mechano-chemical model that tries to capture the physical essence of these complex biomolecular processes. Our model is based on the continuum equations for a viscous and reactive two-phase fluid model with moving boundaries, and on force balance equations that average the stochastic interactions between actin polymers and membrane proteins. In this paper we present a new derivation and analysis of these equations based on minimization of a power functional. This derivation also leads to a clear formulation and classification of the kinds of boundary conditions that should be specified at free surfaces and at the sites of interaction of the cell and the substratum. Numerical simulations of a one-dimensional lamella reveal that even this extremely simplified model is capable of producing several typical features of cell motility. These include periodic 'ruffle' formation, protrusion-retraction cycles, centripetal flow and cell-substratum traction forces. PMID:10204394

  5. A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis.

    PubMed

    Boutte, Cara C; Baer, Christina E; Papavinasasundaram, Kadamba; Liu, Weiru; Chase, Michael R; Meniche, Xavier; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R; Rubin, Eric J

    2016-01-01

    Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance. PMID:27304077

  6. Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro.

    PubMed

    Yuan, Bao; Liang, Shuang; Jin, Yong-Xun; Kwon, Jeong-Woo; Zhang, Jia-Bao; Kim, Nam-Hyung

    2016-01-01

    Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression. PMID:27672508

  7. CYTOPLASMIC INCLUSIONS RESEMBLING NUCLEOLI IN SYMPATHETIC NEURONS OF ADULT RATS

    PubMed Central

    Grillo, Mary A.

    1970-01-01

    A distinctive cytoplasmic inclusion consisting of a convoluted network of electron-opaque strands embedded in a less dense matrix was identified in the neurons, but not in the supporting cells, of rat sympathetic ganglia. This ball-like structure, designated "nematosome," measures ∼ 0.9 µ and lacks a limiting membrane. Its strands (diameter = 400–600 A) appear to be made of an entanglement of tightly packed filaments and particles ∼ 25–50 A thick. Cytochemical studies carried out with the light microscope suggest the presence of nonhistone proteins and some RNA. Usually only one such structure is present in a cell, and it appears to occur in most ganglion cells. Although they can be seen anywhere in the cell body, nematosomes are typically located in the perinuclear cytoplasm, where they are often associated with smooth-surfaced and coated vesicles. In fine structure and stainability, they bear a resemblance to the fibrous component of the nucleolus. Subsynaptic formations, which are a special feature of some of the synapses in sympathetic ganglia, appear similar to the threadlike elements in the nematosomes. The possibility that these three structures—nucleolus, nematosome, and subsynaptic formation—may be interrelated in origin and function is discussed. PMID:5458990

  8. RNA TRANSPORT FROM NUCLEUS TO CYTOPLASM IN CHIRONOMUS SALIVARY GLANDS

    PubMed Central

    Stevens, Barbara J.; Swift, Hewson

    1966-01-01

    The fine structure and cytochemistry of the extremely large RNA puffs, or Balbiani rings, in salivary gland nuclei of midge, Chironomus thummi, larvae have been investigated. The Balbiani rings are composed of a diffuse mass of electron-opaque 400 to 500 A granules, short threads about 180 to 220 A in diameter and associated fine chromatin fibrils. These components appear to be organized into brushlike elements which form the ring. Electron microscope cytochemistry has shown that the granules and short threads contain RNA. After ribonuclease digestion, only 50 to 100 A chromatin fibrils were apparent in the Balbiani ring, and the granules were no longer demonstrable. Deoxyribonuclease digestion had no apparent effect on these structures. Observations indicate that the granules are formed from the short threads and released into the nucleoplasm in which they are evenly distributed. At the nuclear envelope, many granules have been observed partially or completely within the nuclear pores. These granules become elongated and are shown to penetrate the center of the pore in a rodlike form, about 200 A in diameter. The Balbiani ring granules are not normally visible within the cytoplasm adjacent to the nuclear envelope, but have been rarely found in this region. It is suggested that the granules represent the product of the Balbiani ring, possibly a messenger RNA bound to protein, and that they regularly pass into the cytoplasm through a narrow central channel in the pores of the nuclear envelope. PMID:5971975

  9. Sodium channels in the cytoplasm of Schwann cells

    SciTech Connect

    Ritchie, J.M. ); Black, J.A.; Waxman, S.G. Veterans Affairs Medical Center, West Haven, CT ); Angelides, K.J. )

    1990-12-01

    Immunoblotting, ultrastructural immunocytochemistry, and tritiated saxitoxin (({sup 3}H)STX) binding experiments were used to study sodium channel localization in Schwann cells. Polyclonal antibody 7493, which is directed against purifed sodium channels from rat brain, specifically recognized a 260-kDa protein corresponding to the {alpha} subunit of the sodium channel in immunoblots of crude glycoproteins from rat sciatic nerve. Electron microscopic localization of sodium channel immunoreactivity within adult rat sciatic nerves reveals heavy staining of the axon membrane at the node of Ranvier, in contrast to the internodal axon membrane, which does not stain. Schwann cells including perinodal processes also exhibit antibody 7493 immunoreactivity, localized within both the cytoplasm and the plasmalemma of the Schwann cell. To examine further the possibility that sodium channels are localized within Schwann cell cytoplasm, ({sup 3}H)STX binding was studied in cultured rabbit Schwann cells, both intact and after homogenization. Saturable binding of STX was singificantly higher in homogenized Schwann cells than in intact Schwann cells. Moreover, the equilibrium dissociation constant was higher for homogenized preparations (1.77 {plus minus} 0.37 nM) than for intact Schwann cells (1.06 {plus minus} 0.29 nM). These data suggest the presence of an intracellular pool of sodium channels or channel presursors in Schwann cells.

  10. Subunit composition of the human cytoplasmic dynein-2 complex.

    PubMed

    Asante, David; Stevenson, Nicola L; Stephens, David J

    2014-11-01

    Cytoplasmic dynein-2 is the motor for retrograde intraflagellar transport (IFT), and mutations in dynein-2 are known to cause skeletal ciliopathies. Here, we define for the first time the composition of the human cytoplasmic dynein-2 complex. We show that the proteins encoded by the ciliopathy genes WDR34 and WDR60 are bona fide dynein-2 intermediate chains and are both required for dynein-2 function. In addition, we identify TCTEX1D2 as a unique dynein-2 light chain that is itself required for cilia function. We define several subunits common to both dynein-1 and dynein-2, including TCTEX-1 (also known as DYNLT1) and TCTEX-3 (also known as DYNLT3), roadblock-1 (also known as DYNLRB1) and roadblock-2 (also known as DYNLRB2), and LC8-1 and LC8-2 light chains (DYNLL1 and DYNLL2, respectively). We also find that NudCD3 associates with dynein-2 as it does with dynein-1. By contrast, the common dynein-1 regulators dynactin, LIS1 (also known as PAFAH1B1) and BICD2 are not found in association with dynein-2. These data explain why mutations in either WDR34 or WDR60 cause disease, as well as identifying TCTEX1D2 as a candidate ciliopathy gene.

  11. Cytoplasmic protein methylation is essential for neural crest migration

    PubMed Central

    Vermillion, Katie L.; Lidberg, Kevin A.

    2014-01-01

    As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration. PMID:24379414

  12. QTL analysis of fertility restoration in cytoplasmic male sterile pepper.

    PubMed

    Wang, L H; Zhang, B X; Lefebvre, V; Huang, S W; Daubèze, A M; Palloix, A

    2004-09-01

    Fertility restoration of Peterson's cytoplasmic male-sterility in pepper (Capsicum annuum L.) is quantitative and environment-dependent. QTL analysis of fertility restoration was performed based on the test-cross progeny of 77013A (a strict cytoplasmic-genetic male sterile line) and a doubled haploid population of 114 lines obtained from an F1 hybrid between Yolo wonder (a sterility maintainer line) and Perennial (a fertility-restorer line). The fertility of the test-crossed lines was assessed under greenhouse and open field conditions using three criteria related to pollen or seed production. One major QTL for fertility restoration was mapped to chromosome P6. It was significant in all the environments and for all the traits, accounting for 20-69% of the phenotypic variation, depending on the trait. Four additional minor QTLs were also detected on chromosomes P5, P2, and linkage groups PY3 and PY1, accounting for 7-17% of the phenotypic variation. Most of the alleles increasing fertility originated from the restorer parent, except for two alleles at minor QTLs. Phenotypic analysis and genetic dissection indicated that breeding pepper for complete sterility of female lines and high hybrid fertility requires complex combinations of alleles from both parents and a strict control of the environment. PMID:15173931

  13. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

    PubMed

    Much, Christian; Auchynnikava, Tania; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O'Carroll, Dónal

    2016-06-01

    Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse. PMID:27254021

  14. A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis

    PubMed Central

    Boutte, Cara C; Baer, Christina E; Papavinasasundaram, Kadamba; Liu, Weiru; Chase, Michael R; Meniche, Xavier; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R; Rubin, Eric J

    2016-01-01

    Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance. DOI: http://dx.doi.org/10.7554/eLife.14590.001 PMID:27304077

  15. Multivalent niacin nanoconjugates for delivery to cytoplasmic lipid droplets.

    PubMed

    Sharma, Anjali; Khatchadourian, Armen; Khanna, Kunal; Sharma, Rishi; Kakkar, Ashok; Maysinger, Dusica

    2011-02-01

    We report here the design, synthesis, and properties, of multifunctional niacin nanoconjugates based on dendritic, miktoarm and linear backbone nanocarriers, using "click" chemistry. The conjugates were in this instance used to deliver the therapeutic agent niacin to lipid droplets. The desired combination of niacin, a lipophilic fluorescent dye (BODIPY), and polyethylene glycol (PEG), was achieved by covalently linking the desired agents to the selected carrier. The nanocarriers containing niacin and BODIPY were found almost exclusively within cytoplasmic lipid droplets in the cells used in this study (living hepatocytes and microglia), whereas the trifunctional carrier containing niacin, BODIPY and PEG was partially localized within these organelles but also elsewhere in the cytoplasmic compartment. Spectrofluorometric analyses, confocal microscopy and fluorescence cell sorting revealed different rates and extent of multifunctional conjugate(s) internalization in the two cell types. Even micromolar concentrations of the internalized multifunctional conjugates did not cause significant cell death or mitochondrial functional impairment, suggesting that they are suitable candidate nanostructures for lipid droplet imaging and for targeting drugs to these cellular organelles. These studies provide an efficient and easy way to synthesize multifunctional nanocarriers by click chemistry, applicable to the synthesis of related multifunctional nanostructures and to their use in the targeting of cellular organelles, including lipid droplets.

  16. The partitioning of cytoplasmic organelles at cell division.

    PubMed

    Birky, C W

    1983-01-01

    When an organism has only one or two mitochondria or chloroplasts per cell, it is probable that their partitioning is always stringently controlled so that each daughter cell always receives half the organelles in the parent cell. When there are more copies of an organelle, the available data suggest that partitioning is stochastic but far from random, with a strong tendency toward equality. The molecular mechanisms that promote equal partitioning are not known in any case, but the great variety of organelle behavior suggests that many different mechanisms are involved in different organisms. As Wilson (1925) pointed out, the precision of partitioning of cytoplasmic organelles rarely if ever equals that of mitosis, but it is still an expression of selection for mechanisms that will ensure the hereditary continuity of the organelles. How cells compensate for unequal partitioning by controlling organelle replication is known for only one case. But when one considers that Tetrahymena and Paramecium use different methods to compensate for unequal partitioning of macronuclear DNA, it would not be surprising if organisms use a variety of different compensating replication modes for organelles as well. What is surprising is that so little attention has been paid to these problems. Nothing could be simpler than counting organelles in dividing cells, but this has been done on a large scale in only two systems. Quantitative techniques in cell biology have been developed to the point where such studies could be done even on cells that have too many organelles for direct counting. Molecular mechanisms of partitioning have scarcely been touched on. Much more has been done on the role of the cytoskeleton in determining cell shape, and some observations have been made on its role in positioning organelles in interphase cells, but these kinds of studies have not been extended to dividing cells. Some experiments and observations have been made on the role of microtubules and

  17. Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry

    PubMed Central

    1991-01-01

    Information about the rheological characteristics of the aqueous cytoplasm can be provided by analysis of the rotational motion of small polar molecules introduced into the cell. To determine fluid-phase cytoplasmic viscosity in intact cells, a polarization microscope was constructed for measurement of picosecond anisotropy decay of fluorescent probes in the cell cytoplasm. We found that the rotational correlation time (tc) of the probes, 2,7-bis-(2-carboxyethyl)-5-(and-6- )carboxyfluorescein (BCECF), 6-carboxyfluorescein, and 8-hydroxypyrene- 1,3,6-trisulfonic acid (HPTS) provided a direct measure of fluid-phase cytoplasmic viscosity that was independent of probe binding. In quiescent Swiss 3T3 fibroblasts, tc values were 20-40% longer than those in water, indicating that the fluid-phase cytoplasm is only 1.2- 1.4 times as viscous as water. The activation energy of fluid-phase cytoplasmic viscosity was 4 kcal/mol, which is similar to that of water. Fluid-phase cytoplasmic viscosity was altered by less than 10% upon addition of sucrose to decrease cell volume, cytochalasin B to disrupt cell cytoskeleton, and vasopressin to activate phospholipase C. Nucleoplasmic and peripheral cytoplasmic viscosities were not different. Our results establish a novel method to measure fluid-phase cytoplasmic viscosity, and indicate that fluid-phase cytoplasmic viscosity in fibroblasts is similar to that of free water. PMID:1993739

  18. Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers.

    PubMed

    van der Honing, Hannie S; de Ruijter, Norbert C A; Emons, Anne Mie C; Ketelaar, Tijs

    2010-01-01

    Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

  19. Geologic Map of the Mylitta Fluctus Quadrangle (V-61), Venus

    USGS Publications Warehouse

    Ivanov, Mikhail A.; Head, James W.

    2006-01-01

    INTRODUCTION The Magellan Mission The Magellan spacecraft orbited Venus from August 10, 1990, until it plunged into the Venusian atmosphere on October 12, 1994. Magellan Mission objectives included: (1) improving knowledge of the geological processes, surface properties, and geologic history of Venus by analysis of surface radar characteristics, topography, and morphology, and (2) improving the knowledge of the geophysics of Venus by analysis of Venusian gravity. The Magellan spacecraft carried a 12.6-cm radar system to map the surface of Venus. The transmitter and receiver systems were used to collect three data sets: (1) synthetic aperture radar (SAR) images of the surface, (2) passive microwave thermal emission observations, and (3) measurements of the backscattered power at small angles of incidence, which were processed to yield altimetric data. Radar imaging, altimetric, and radiometric mapping of the Venusian surface was done in mission cycles 1, 2, and 3 from September 1990 until September 1992. Ninety-eight percent of the surface was mapped with radar resolution on the order of 120 meters. The SAR observations were projected to a 75-m nominal horizontal resolution, and these full-resolution data compose the image base used in geologic mapping. The primary polarization mode was horizontal-transmit, horizontal-receive (HH), but additional data for selected areas were collected for the vertical polarization sense. Incidence angles varied between about 20? and 45?. High resolution Doppler tracking of the spacecraft took place from September 1992 through October 1994 (mission cycles 4, 5, 6). Approximately 950 orbits of high-resolution gravity observations were obtained between September 1992 and May 1993 while Magellan was in an elliptical orbit with a periapsis near 175 km and an apoapsis near 8,000 km. An additional 1,500 orbits were obtained following orbit-circularization in mid-1993. These data exist as a 75? by 75? harmonic field.

  20. Cytoplasmic sulfur trafficking in sulfur-oxidizing prokaryotes.

    PubMed

    Dahl, Christiane

    2015-04-01

    Persulfide groups are chemically versatile and participate in a wide array of biochemical pathways. Although it is well documented that persulfurated proteins supply a number of important and elaborate biosynthetic pathways with sulfane sulfur, it is far less acknowledged that the enzymatic generation of persulfidic sulfur, the successive transfer of sulfur as a persulfide between multiple proteins, and the oxidation of sulfane sulfur in protein-bound form are also essential steps during dissimilatory sulfur oxidation in bacteria and archaea. Here, the currently available information on sulfur trafficking in sulfur oxidizing prokaryotes is reviewed, and the idea is discussed that sulfur is always presented to cytoplasmic oxidizing enzymes in a protein-bound form, thus preventing the occurrence of free sulfide inside of the prokaryotic cell. Thus, sulfur trafficking emerges as a central element in sulfur-oxidizing pathways, and TusA homologous proteins appear to be central and common elements in these processes.

  1. Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation.

    PubMed

    Boren, J; Brindle, K M

    2012-09-01

    A characteristic of apoptosis is the rapid accumulation of cytoplasmic lipid droplets, which are composed largely of neutral lipids. The proton signals from these lipids have been used for the non-invasive detection of cell death using magnetic resonance spectroscopy. We show here that despite an apoptosis-induced decrease in the levels and activities of enzymes involved in lipogenesis, which occurs downstream of p53 activation and inhibition of the mTOR signaling pathway, the increase in lipid accumulation is due to increased de novo lipid synthesis. This results from inhibition of mitochondrial fatty acid β-oxidation, which coupled with an increase in acyl-CoA synthetase activity, diverts fatty acids away from oxidation and into lipid synthesis. The inhibition of fatty acid oxidation can be explained by a rapid rise in mitochondrial membrane potential and an attendant increase in the levels of reactive oxygen species. PMID:22460322

  2. The Cytoplasm-to-Vacuole Targeting Pathway: A Historical Perspective

    PubMed Central

    Umekawa, Midori; Klionsky, Daniel J.

    2012-01-01

    From today's perspective, it is obvious that macroautophagy (hereafter autophagy) is an important pathway that is connected to a range of developmental and physiological processes. This viewpoint, however, is relatively recent, coinciding with the molecular identification of autophagy-related (Atg) components that function as the protein machinery that drives the dynamic membrane events of autophagy. It may be difficult, especially for scientists new to this area of research, to appreciate that the field of autophagy long existed as a “backwater” topic that attracted little interest or attention. Paralleling the development of the autophagy field was the identification and analysis of the cytoplasm-to-vacuole targeting (Cvt) pathway, the only characterized biosynthetic route that utilizes the Atg proteins. Here, we relate some of the initial history, including some never-before-revealed facts, of the analysis of the Cvt pathway and the convergence of those studies with autophagy. PMID:22481942

  3. Minocycline-induced cutaneous polyarteritis nodosa with antineutrophil cytoplasmic antibodies.

    PubMed

    Pelletier, Fabien; Puzenat, Eve; Blanc, Dominique; Faivre, Brigitte; Humbert, Philippe; Aubin, François

    2003-01-01

    Minocycline is an antibiotic widely used in the treatment of acne. Among the induced auto-immune disorders, cutaneous polyarteritis nodosa (PAN) is very rare. A new case is reported below. A 23-year-old female patient treated with minocycline for acne for 24 months developed sub-cutaneous nodules, livedo reticularis and pigmented lesions of the lower limbs. Antineutrophil cytoplasmic antibodies (ANCA) were positive at 1/320. Skin biopsy showed vasculitis of a medium-sized artery. The role of minocycline was suspected using the imputability criteria. The diagnosis of minocycline-induced cutaneous PAN with ANCA was sustained. After withdrawal of the treatment, the nodular lesions decreased spontaneously, whereas livedo disappeared and inflammatory parameters were normalized after oral corticosteroid therapy. Minocycline is a tetracycline which is efficient for treating acne. Auto-immune disorders are frequently observed. Among them, it is very rare to observe cutaneous PAN associated with positive ANCA. The pathophysiological mechanisms are discussed.

  4. Cytoplasmic effect on gene function in Xenopus laevis.

    PubMed

    Yu, H J; Shi, C P; Niu, M C

    1987-05-01

    The pigmentation gene of Xenopus laevis is dominant and that of albino aP mutant recessive. Heterologous haploid hybrids are produced by UV inactivation of the egg nuclei during second polar body formation in the mutant sperm-fertilized Xenopus eggs. During development of these hybrids, melanin appeared in the eye and melanophores in the skin at stages comparable to those of the wild type, but much earlier than in the albino mutant. The number and intensity of pigment cells are intermediate between the black Xenopus and albino mutant. While a number of pigment cells remain in the hybrids, those in the albino eventually degenerate. Therefore, the development and maintenance of pigmentation in heterologous hybrids are contributed by Xenopus cytoplasm. Tadpole tail-tips were squashed and stained for chromosome counting. The results show that Xenopus and mutants are diploid (36 chromosomes) and heterologous haploid hybrids have 18 chromosomes. PMID:3672098

  5. A cytoplasm to vacuole targeting pathway in P. pastoris.

    PubMed

    Farré, Jean-Claude; Vidal, Jason; Subramani, Suresh

    2007-01-01

    The cytoplasm-to-vacuole targeting (Cvt) pathway of Saccharomyces cerevisiae delivers aminopeptidase I (Ape1) from the cytosol to the vacuole, bypassing the normal secretory route. The Cvt pathway, although well-studied, was known only in S. cerevisiae. We demonstrate its existence in the methylotrophic yeast, Pichia pastoris, where it also delivers P. pastoris Ape1 (PpApe1) to the vacuole. Most proteins known to be required for the Cvt pathway in S. cerevisiae were, to the extent we found orthologs, also required in P. pastoris. The P. pastoris Cvt pathway differs, however, from that in S. cerevisiae, in that new proteins, such as PpAtg28 and PpAtg26, are involved. The discovery of a Cvt pathway in P. pastoris makes it an excellent model system for the dissection of autophagy-related pathways in a single organism and for the discovery of new Cvt pathway components.

  6. The kinase domain of mitochondrial PINK1 faces the cytoplasm

    PubMed Central

    Zhou, Chun; Huang, Yong; Shao, Yufang; May, Jessica; Prou, Delphine; Perier, Celine; Dauer, William; Schon, Eric A.; Przedborski, Serge

    2008-01-01

    Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinson's disease (PD). Efforts in deducing the PINK1 signaling pathway have been hindered by controversy around its subcellular and submitochondrial localization and the authenticity of its reported substrates. We show here that this mitochondrial protein exhibits a topology in which the kinase domain faces the cytoplasm and the N-terminal tail is inside the mitochondria. Although deletion of the transmembrane domain disrupts this topology, common PD-linked PINK1 mutations do not. These results are critical in rectifying the location and orientation of PINK1 in mitochondria, and they should help decipher its normal physiological function and potential pathogenic role in PD. PMID:18687899

  7. A Cytoplasmic Dynein Tail Mutation Impairs Motor Processivity

    PubMed Central

    Ori-McKenney, Kassandra M.; Xu, Jing; Gross, Steven P.; Vallee, Richard B.

    2012-01-01

    Mutations in the tail of the cytoplasmic dynein molecule have been reported to cause neurodegenerative disease in mice. The mutant mouse strain Legs at Odd Angles (Loa) exhibits impaired retrograde axonal transport, but the molecular deficiencies in the mutant dynein molecule, and how they contribute to neurodegeneration, are unknown. To address these questions, we purified wild-type and mutant mouse dynein. Using biochemical, single molecule, and live cell imaging techniques, we find a strong inhibition of motor run-length in vitro and in vivo, and significantly altered motor domain coordination in the mutant dynein. These results suggest a potential role for the dynein tail in motor function, and provide the first direct evidence for a link between single-motor processivity and disease. PMID:21102439

  8. The Whereabouts of microRNA Actions: Cytoplasm and Beyond.

    PubMed

    Leung, Anthony K L

    2015-10-01

    MicroRNAs (miRNAs) are a conserved class of approximately 22 nucleotide (nt) short noncoding RNAs that normally silence gene expression via translational repression and/or degradation of targeted mRNAs in plants and animals. Identifying the whereabouts of miRNAs potentially informs miRNA functions, some of which are perhaps specialized to specific cellular compartments. In this review, the significance of miRNA localizations in the cytoplasm, including those at RNA granules and endomembranes, and the export of miRNAs to extracellular space will be discussed. How miRNA localizations and functions are regulated by protein modifications on the core miRNA-binding protein Argonaute (AGO) during normal and stress conditions will be explored, and in conclusion new AGO partners, non-AGO miRNA-binding proteins, and the emergent understanding of miRNAs found in the nucleoplasm, nucleoli, and mitochondria will be discussed. PMID:26410406

  9. Lateral Movements of Membrane Glycoproteins Restricted by Dynamic Cytoplasmic Barriers

    NASA Astrophysics Data System (ADS)

    Edidin, Michael; Kuo, Scot C.; Sheetz, Michael P.

    1991-11-01

    Cell membranes often are patchy, composed of lateral domains. These domains may be formed by barriers within or on either side of the membrane bilayer. Major histocompatibility complex (MHC) class 1 molecules that were either transmembrane- (H-2D^b) or glycosylphosphatidylinositol (GPI)-anchored (Qa2) were labeled with antibody-coated gold particles and moved across the cell surface with a laser optical tweezers until they encountered a barrier, the barrier-free path length (BFP). At room temperature, the BFPs of Qa2 and H-2D^b were 1.7 ± 0.2 and 0.6 ± 0.1 (micrometers ± SEM), respectively. Barriers persisted at 34^circC, although the BFP for both MHC molecules was fivefold greater at 34^circC than at 23^circC. This indicates that barriers to lateral movement are primarily on the cytoplasmic half of the membrane and are dynamic.

  10. Myeloperoxidase-antineutrophil cytoplasmic antibody-associated sensorineural hearing loss.

    PubMed

    Maguchi, S; Fukuda, S; Chida, E; Terayama, Y

    2001-05-01

    A 36-year-old female with hyperthyroidism that had been treated with propilthiouracil (PTU) complained of tinnitus and hearing loss in both ears. She was treated with steroid administration by an otolaryngologist; however, hearing continued to fluctuate when the steroids were tapered. Laboratory evaluation revealed a decreased complement level and elevated levels of immunoglobulin M (IgM) and myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA). With the withdrawal of PTU and high-dose methylprednisolone, she had excellent return of right-sided hearing. In recent years, there have been many reports about MPO-ANCA-associated small vessel vasculitis. Although any organ may be affected by this disease, there are no reports about MPO-ANCA-associated progressive hearing loss without any other organ involvement. The present case suggests the possibility that inner ear blood flow impairment due to ANCA-associated small vessel vasculitis induces the so-called autoimmune sensorineural hearing loss.

  11. Deep cytoplasmic rearrangements in axis-respecified Xenopus embryos

    NASA Technical Reports Server (NTRS)

    Denegre, J. M.; Danilchik, M. V.

    1993-01-01

    In fertilized eggs of the frog Xenopus, the vegetal yolk mass rotates away from the future dorsal side (J. P. Vincent and J. Gerhart, 1987, Dev. Biol. 123, 526-539), and a major rearrangement of the deep animal hemisphere cytoplasm produces a characteristic swirl in the prospective dorsal side (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). The relationship between this swirl and determination of the dorsal-ventral axis was further investigated by attempting to experimentally separate the positions of the swirl and the dorsal-ventral axis. Eggs were obliquely oriented in the gravity field to respecify the direction of yolk mass rotation and the position of the dorsal-ventral axis. When yolk mass rotation occurred in the absence of a sperm, as in activated eggs, a swirl pattern formed on the side away from which the yolk mass had rotated. In fertilized eggs tipped with the sperm entry point (SEP) down or to the side, swirl patterns were always found to form on the side away from which the yolk mass was displaced. However, in eggs tipped SEP up, in which the yolk mass was forced to rotate away from the SEP, more complicated rearrangements were observed in addition to the rotation-oriented swirl. Because the direction of yolk mass rotation was found to be influenced by both gravity and the actual position of the SEP in obliquely oriented eggs (SEP to the side), such complicated rearrangement patterns may result from opposing forces generated by both yolk mass rotation and the expanding sperm aster. Thus, except in cases in which the influences of SEP position and unit gravity opposed each other, it was not possible to experimentally separate the position of the deep cytoplasmic swirl from the direction of yolk mass rotation, and therefore the position of the prospective dorsal side.

  12. Regulation of Cytoplasmic Dynein ATPase by Lis1

    PubMed Central

    Mesngon, Mariano T.; Tarricone, Cataldo; Hebbar, Sachin; Guillotte, Aimee M.; Schmitt, E. William; Lanier, Lorene; Musacchio, Andrea; King, Stephen J.; Smith, Deanna S.

    2015-01-01

    Mutations in Lis1 cause classical lissencephaly, a developmental brain abnormality characterized by defects in neuronal positioning. Over the last decade, a clear link has been forged between Lis1 and the microtubule motor cytoplasmic dynein. Substantial evidence indicates that Lis1 functions in a highly conserved pathway with dynein to regulate neuronal migration and other motile events. Yeast two-hybrid studies predict that Lis1 binds directly to dynein heavy chains (Sasaki et al., 2000; Tai et al., 2002), but the mechanistic significance of this interaction is not well understood. We now report that recombinant Lis1 binds to native brain dynein and significantly increases the microtubule-stimulated enzymatic activity of dynein in vitro. Lis1 does this without increasing the proportion of dynein that binds to microtubules, indicating that Lis1 influences enzymatic activity rather than microtubule association. Dynein stimulation in vitro is not a generic feature of microtubule-associated proteins, because tau did not stimulate dynein. To our knowledge, this is the first indication that Lis1 or any other factor directly modulates the enzymatic activity of cytoplasmic dynein. Lis1 must be able to homodimerize to stimulate dynein, because a C-terminal fragment (containing the dynein interaction site but missing the self-association domain) was unable to stimulate dynein. Binding and colocalization studies indicate that Lis1 does not interact with all dynein complexes found in the brain. We propose a model in which Lis1 stimulates the activity of a subset of motors, which could be particularly important during neuronal migration and long-distance axonal transport. PMID:16481446

  13. Antineutrophil Cytoplasmic Antibody Frequency in Chronic Hepatitis B Patients

    PubMed Central

    Calhan, Turan; Kahraman, Resul; Altunoz, Mustafa Erhan; Ozbakır, Fatma; Ozdil, Kamil; Sokmen, Hacı Mehmet

    2014-01-01

    Background. Chronic hepatitis B (CHB) is a viral disease, common across the world, and associated with several extraintestinal manifestations including vasculitis. Antineutrophil cytoplasmic antibodies (ANCAs) are sensitive and specific markers for vasculitides. There is limited data available in the literature on whether ANCA formation is stimulated by CHB infection. In the present study we aimed to identify the frequency of ANCA in CHB patients. Methods. A total of 174 subjects were included in the study (87 CHB patients, 87 control subjects). Perinuclear-ANCA (P-ANCA), cytoplasmic-ANCA (C-ANCA), myeloperoxidase ANCA (MPO-ANCA), and proteinase 3-ANCA (PR3-ANCA) were studied. IFA was used for P-ANCA and C-ANCA assays, and ELISA was used for MPO-ANCA and PR3-ANCA assays. Results. ANCA positivity was high in both groups (31% in the CHB group and 26% among controls). There were no significant differences between the groups for P-ANCA and MPO-ANCA (P = 0.6 and P = 0.6, resp.). Frequency of borderline positive C-ANCA and all positive PR3-ANCA (positive + borderline positive) was significantly higher in the CHB group (P = 0.009 and P = 0.005, resp.). Conclusions. In the present study, the frequency of ANCA was high in both groups. The CHB group had a relatively higher frequency of ANCA positivity compared to controls. Borderline positive C-ANCA and positive PR3-ANCA were significantly higher in the CHB group. These results suggest that ANCA may have a high prevalence in Turkey. Patients with CHB should be evaluated particularly for C-ANCA and PR3-ANCA in the presence of vasculitic complaints and lesions. PMID:25161336

  14. Novel origin of lamin-derived cytoplasmic intermediate filaments in tardigrades.

    PubMed

    Hering, Lars; Bouameur, Jamal-Eddine; Reichelt, Julian; Magin, Thomas M; Mayer, Georg

    2016-02-03

    Intermediate filament (IF) proteins, including nuclear lamins and cytoplasmic IF proteins, are essential cytoskeletal components of bilaterian cells. Despite their important role in protecting tissues against mechanical force, no cytoplasmic IF proteins have been convincingly identified in arthropods. Here we show that the ancestral cytoplasmic IF protein gene was lost in the entire panarthropod (onychophoran + tardigrade + arthropod) rather than arthropod lineage and that nuclear, lamin-derived proteins instead acquired new cytoplasmic roles at least three times independently in collembolans, copepods, and tardigrades. Transcriptomic and genomic data revealed three IF protein genes in the tardigrade Hypsibius dujardini, one of which (cytotardin) occurs exclusively in the cytoplasm of epidermal and foregut epithelia, where it forms belt-like filaments around each epithelial cell. These results suggest that a lamin derivative has been co-opted to enhance tissue stability in tardigrades, a function otherwise served by cytoplasmic IF proteins in all other bilaterians.

  15. [Possible role of the cytoplasm in the course of morphogenesis, namely, in the case of twinning].

    PubMed

    Giroud, A

    1975-01-01

    A number of facts that cannot be interpreted in terms of nuclear genes would seem to be interpretable in terms of cytoplasmic heredity. The hereditary role of the mitochondria has already been demonstrated in molds. The role of the cytoplasm (matrilineal heredity) has also been shown in some phanerogams, and analogous facts have been noted in insects and molluscs. In amphibians, the influence of an alteration of the egg cortical cytoplasm has been shown to reappear in the following generations. This cortical cytoplasm includes the morphological plan of the organism with its bilateral symmetry. In Tatusia novemcincta the twins may be morphologically or chemically different, which may only be explained by an unequal subdivision of a heterogenic cytoplasm. Similar facts are observed in human twinning. Monozygotic twins are usually discordant with respect to congenital malformations (especially symmelia and anencephaly), which may only be interpreted in terms of unequal distribution of cytoplasmic properties.

  16. [Possible role of the cytoplasm in the course of morphogenesis, namely, in the case of twinning].

    PubMed

    Giroud, A

    1975-01-01

    A number of facts that cannot be interpreted in terms of nuclear genes would seem to be interpretable in terms of cytoplasmic heredity. The hereditary role of the mitochondria has already been demonstrated in molds. The role of the cytoplasm (matrilineal heredity) has also been shown in some phanerogams, and analogous facts have been noted in insects and molluscs. In amphibians, the influence of an alteration of the egg cortical cytoplasm has been shown to reappear in the following generations. This cortical cytoplasm includes the morphological plan of the organism with its bilateral symmetry. In Tatusia novemcincta the twins may be morphologically or chemically different, which may only be explained by an unequal subdivision of a heterogenic cytoplasm. Similar facts are observed in human twinning. Monozygotic twins are usually discordant with respect to congenital malformations (especially symmelia and anencephaly), which may only be interpreted in terms of unequal distribution of cytoplasmic properties. PMID:1242075

  17. Novel origin of lamin-derived cytoplasmic intermediate filaments in tardigrades.

    PubMed

    Hering, Lars; Bouameur, Jamal-Eddine; Reichelt, Julian; Magin, Thomas M; Mayer, Georg

    2016-01-01

    Intermediate filament (IF) proteins, including nuclear lamins and cytoplasmic IF proteins, are essential cytoskeletal components of bilaterian cells. Despite their important role in protecting tissues against mechanical force, no cytoplasmic IF proteins have been convincingly identified in arthropods. Here we show that the ancestral cytoplasmic IF protein gene was lost in the entire panarthropod (onychophoran + tardigrade + arthropod) rather than arthropod lineage and that nuclear, lamin-derived proteins instead acquired new cytoplasmic roles at least three times independently in collembolans, copepods, and tardigrades. Transcriptomic and genomic data revealed three IF protein genes in the tardigrade Hypsibius dujardini, one of which (cytotardin) occurs exclusively in the cytoplasm of epidermal and foregut epithelia, where it forms belt-like filaments around each epithelial cell. These results suggest that a lamin derivative has been co-opted to enhance tissue stability in tardigrades, a function otherwise served by cytoplasmic IF proteins in all other bilaterians. PMID:26840051

  18. A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

    PubMed Central

    Eystathioy, Theophany; Chan, Edward K. L.; Tenenbaum, Scott A.; Keene, Jack D.; Griffith, Kevin; Fritzler, Marvin J.

    2002-01-01

    A novel human cellular structure has been identified that contains a unique autoimmune antigen and multiple messenger RNAs. This complex was discovered using an autoimmune serum from a patient with motor and sensory neuropathy and contains a protein of 182 kDa. The gene and cDNA encoding the protein indicated an open reading frame with glycine-tryptophan (GW) repeats and a single RNA recognition motif. Both the patient's serum and a rabbit serum raised against the recombinant GW protein costained discrete cytoplasmic speckles designated as GW bodies (GWBs) that do not overlap with the Golgi complex, endosomes, lysosomes, or peroxisomes. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within the GW complexes. We propose that the GW ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis. PMID:11950943

  19. Distribution of cytoplasmic vacuoles in blood T and B lymphocytes in two lysosomal disorders.

    PubMed

    Aula, P; Rapola, J; Andersson, L C

    1975-09-11

    Distribution of cytoplasmic vacuoles in purified T and B lymphocytes was analyzed in four cases of aspartylglucosaminuria (AGU) and in one case of neuronal ceroid lipofuscinosis (Spielmeyer-Sjörgren type). In all cases T cells were significantly more vacuolized than B cells. The ultrastructure of the cytoplasmic vacuoles was consistent with the concept of storage lysosomes. The cytoplasmic vacuoles both in T and B cells similar to abnormal lysosomes seen in the visceral organs in these diseases. PMID:170734

  20. Maternal transmission of cytoplasmic DNA in interspecific hybrids of peat mosses, Sphagnum (Bryophyta).

    PubMed

    Natcheva, R; Cronberg, N

    2007-07-01

    The progeny of spontaneous interspecific hybrid sporophytes of Sphagnum were used to analyse the inheritance of cytoplasmic DNA. The analysis showed that only the female parent donated chloroplasts and mitochondria in Sphagnum hybrids. Thus, this is the first study demonstrating maternal cytoplasmic inheritance in a nonvascular land plant. This finding has important implications for phylogenetic reconstructions utilizing chloroplast and mitochondrial DNA sequences as well as for the evolution of cytoplasmic inheritance in relation to the life cycle of land plants.

  1. Amphibian egg cytoplasm response to altered g-forces and gravity orientation

    NASA Technical Reports Server (NTRS)

    Neff, A. W.; Smith, R. C.; Malacinski, G. M.

    1986-01-01

    Elucidation of dorsal/ventral polarity and primary embryonic axis development in amphibian embryos requires an understanding of cytoplasmic rearrangements in fertile eggs at the biophysical, physiological, and biochemical levels. Evidence is presented that amphibian egg cytoplasmic components are compartmentalized. The effects of altered orientation to the gravitational vector (i.e., egg inversion) and alterations in gravity force ranging from hypergravity (centrifugation) to simulated microgravity (i.e., horizontal clinostat rotation) on cytoplasmic compartment rearrangements are reviewed. The behavior of yolk compartments as well as a newly defined (with monoclonal antibody) nonyolk cytoplasmic compartment, in inverted eggs and in eggs rotated on horizontal clinostats at their buoyant density, is discussed.

  2. Ectopic mesodermal expression promoted by the eight-cell stage Bufo arenarum subequatorial cytoplasm.

    PubMed

    Casal, F C; Manes, M E

    1999-07-01

    Mesodermal determinants were investigated by cytoplasmic transfer and blastomere isolation in the eight-cell stage of Bufo arenarum. Their existence was confirmed by assaying the subequatorial cytoplasm's ability to respecify the developmental potency of animal quartets. The gray subequatorial cytoplasm, but not animal cytoplasm, is able to divert the ectodermal fate of animal quartets to several mesodermal components. The source of the transplanted cytoplasm was important in determining the category of the resulting structures. Ventral subequatorial cytoplasm from ventrovegetal blastomeres generated ventral derivatives, namely erythrocytes and mesenchyma. Dorsal subequatorial cytoplasm from dorsovegetal blastomeres produced dorsolateral derivatives, such as notochord, muscle, nephric tubules, and coelomic epithelium, including mesenchyma. On the other hand, transfer of vegetal pole cytoplasm to animal quartets resulted in the formation of groups of endoderm-like cells dispersed among epidermal cells. However, the presence of such cells did not cause any mesodermal induction. The present findings suggest the existence of cytoplasmic information responsible for mesodermal specification. The alternative hypothesis that animal blastomeres become mesoderm due to vegetal induction is questioned.

  3. Identification of cytoplasm types in rapeseed (Brassica napus L.) accessions by a multiplex PCR assay.

    PubMed

    Zhao, H X; Li, Z J; Hu, S W; Sun, G L; Chang, J J; Zhang, Z H

    2010-08-01

    Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F(1) hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F(1) hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS "three-line" breeding, selection and validation of hybrid rapeseed. PMID:20401459

  4. A computational approach to detect and segment cytoplasm in muscle fiber images.

    PubMed

    Guo, Yanen; Xu, Xiaoyin; Wang, Yuanyuan; Yang, Zhong; Wang, Yaming; Xia, Shunren

    2015-06-01

    We developed a computational approach to detect and segment cytoplasm in microscopic images of skeletal muscle fibers. The computational approach provides computer-aided analysis of cytoplasm objects in muscle fiber images to facilitate biomedical research. Cytoplasm in muscle fibers plays an important role in maintaining the functioning and health of muscular tissues. Therefore, cytoplasm is often used as a marker in broad applications of musculoskeletal research, including our search on treatment of muscular disorders such as Duchenne muscular dystrophy, a disease that has no available treatment. However, it is often challenging to analyze cytoplasm and quantify it given the large number of images typically generated in experiments and the large number of muscle fibers contained in each image. Manual analysis is not only time consuming but also prone to human errors. In this work we developed a computational approach to detect and segment the longitudinal sections of cytoplasm based on a modified graph cuts technique and iterative splitting method to extract cytoplasm objects from the background. First, cytoplasm objects are extracted from the background using the modified graph cuts technique which is designed to optimize an energy function. Second, an iterative splitting method is designed to separate the touching or adjacent cytoplasm objects from the results of graph cuts. We tested the computational approach on real data from in vitro experiments and found that it can achieve satisfactory performance in terms of precision and recall rates.

  5. Aluminum toxicity studies in Vaucheria longicaulis var. macounii (Xanthophyta, Tribophyceae). I. Effects on cytoplasmic organization.

    PubMed

    Alessa, L; Oliveira, L

    2001-06-01

    Using differential interference contrast (DIC) and epifluorescence microscopy, we tested the hypothesis that exposure to environmentally significant levels of aluminum (Al) would cause rapid changes in cytoplasmic organization in vegetative filaments of the coenocytic alga, Vaucheria longicaulis Hoppaugh var. macounii Blum resulting in the loss of cytoplasmic streaming. In untreated cells, DIC microscopy revealed the presence of cortical cytoplasmic strands that were oriented longitudinally to the cell axis as well as sub-cortical cytoplasmic strands that exhibited a reticulate morphology. Organelles such as chloroplasts and mitochondria translocated throughout the cell in close association with the cortical longitudinal cytoplasmic strands. Staining with the lipophilic dye, 3,3-dihexyloloxacarbocyanine, revealed structures that appeared to be endoplasmic reticulum (ER). This organelle closely resembled, in location and appearance, the cytoplasmic strands visualized using DIC microscopy. The addition of Al (80 µM) resulted in the inhibition of cytoplasmic streaming as well as the dissipation of the putative cortical longitudinal ER within one minute. Subsequently, the DIC-visible cortical cytoplasmic strands exhibited progressive degrees of disorganization. Throughout these changes, chloroplasts and mitochondria remained visibly associated with the cortical cytoplasmic strands.

  6. Nucleo-cytoplasmic interaction during macronuclear differentiation in ciliate protists: genetic basis for cytoplasmic control of SerH expression during macronuclear development in Tetrahymena thermophila.

    PubMed

    Doerder, F P; Berkowitz, M S

    1987-09-01

    A novel class of mutations affecting the developmental expression of SerH cell surface antigen genes of Tetrahymena thermophila is described. Unlike previous categories of mutation, the four independently isolated mutations of this class act through the cytoplasm to affect SerH genes during macronuclear development. That is, macronuclei which develop under the influence of mutant cytoplasm do not subsequently express H, most likely because the developmental processing of SerH genes is affected. The cytoplasmic effect is specific for the SerH locus and is independent of which SerH allele is present. In place of H, hitherto unknown antigens are expressed. Expression of SerH can be rescued during development either by wild-type cytoplasm exchanged between conjugants or by the homozygous wild-type genotype. The mutations segregate independently of the SerH genes and identify one, possibly two, bistable genes. Possible models to explain these results are discussed.

  7. Isolation of Cytoplasmic Pituitary Granules with Gonadotropic Activity

    PubMed Central

    Hartley, Marshall W.; McShan, W. H.; Ris, Hans

    1960-01-01

    A fraction isolated from the anterior pituitary glands of rats castrate for 8 weeks contained essentially a single cytoplasmic constituent with which the major portion of the gonadotropic hormone activity was associated. The glands were homogenized in an 0.25 M sucrose + 7.3 per cent polyvinylpyrrolidone (PVP) solution and fractionated by differential centrifugation to give a heterogeneous small granule fraction which contained almost all the gonadotropic hormone activity. The active supernatant containing this small granule fraction was separated into layers by isopycnic gradient centrifugation on a continuous 6 to 45 per cent sucrose + 17.5 per cent "diodrast" + 5 x 10-4 M "versene" gradient at 100,000 g for 2 hours. Three layers were obtained and the pellet from the active bottom layer was sectioned, examined with the electron microscope, and found to contain 200 mµ granules, mitochondria, ergastoplasm, and other cellular debris. This layer was fractionated further by isopycnic and differential centrifugation to obtain a pellet which contained the major portion of the gonadotropic hormone activity. Because of the heterogeneity of this fraction, due to the contamination of the 200 mµ granules with mitochondria and other cellular debris, the active layer and the resuspended active pellet, obtained by centrifuging this layer first at 17,000 g then diluting the supernatant and centrifuging at 30,000 g for 1 hour, were filtered through Millipore HA paper with a pore size of 0.45 µ. The cytoplasmic material containing the gonadotropic hormone activity passed through the filter paper and this activity was recovered in the pellets obtained by centrifuging at 100,000 g for 1 hour. These active pellets consisted almost entirely of 200 mµ granules with a minimum amount of contamination, and they contained the major portion of the gonadotropic hormone activity with practically none remaining in the supernatant fraction. These results are discussed in view of their

  8. Effects of alien and intraspecies cytoplasms on manifestation of nuclear genes for wheat resistance to brown rust: II. Specificity of cytoplasm influence on different Lr genes

    SciTech Connect

    Voluevich, E.A.; Buloichik, A.A.; Palilova, A.N.

    1995-04-01

    Specificity of expression of the major nuclear genes Lr to two brown rust clones in hybrids with the same maternal cytoplasm was analyzed. It was evaluated by a resistant: susceptible ratio in the F{sub 2}. Reciprocal hybrids were obtained from the cross between the progeny of homozygous susceptible plants of the cultivar Penjamo 62 and its alloplasmatic lines carrying cytoplasms of Triticum dicoccoides var. fulvovillosum, Aegilops squarrosa var. typical, Agropyron trichophorum, and isogenic lines of the cultivar Thatcher (Th) with the Lr1, Lr9, Lr15, and Lr19 genes. It was shown that the effect of the Lr1 gene in the cytoplasm of cultivar Thatcher and in eu-, and alloplasmatic forms of Penjamo 62 was less expressed than that of other Lr genes. Cytoplasm of the alloplasmatic line (dicoccoides)-Penjamo 62 was the only exception: in the F{sub 2}, hybrids with Th (Lr1) had a higher yield of resistant forms than those with Th (Lr15). In the hybrid combinations studied, expression and/or transmission of the Lr19 gene was more significant than that of other genes. This gene had no advantages over Lr15 and Lr19 only in cytoplasm of the alloplasmatic line (squarrosa)-Penjamo 62. In certain hybrid cytoplasms, the display of the Lr1, Lr15, and Lr19 genes, in contrast to Lr9, varied with the virulence of the pathogen clones. 15 refs., 5 tabs.

  9. Ubiquitin-proteasome-rich cytoplasmic structures in neutrophils of patients with Shwachman-Diamond syndrome

    PubMed Central

    Necchi, Vittorio; Minelli, Antonella; Sommi, Patrizia; Vitali, Agostina; Caruso, Roberta; Longoni, Daniela; Frau, Maria Rita; Nasi, Cristina; De Gregorio, Fabiola; Zecca, Marco; Ricci, Vittorio; Danesino, Cesare; Solcia, Enrico

    2012-01-01

    Background Shwachman–Diamond syndrome is an autosomal recessive disorder in which severe bone marrow dysfunction causes neutropenia and an increased risk of leukemia. Recently, novel particulate cytoplasmic structures, rich in ubiquitinated and proteasomal proteins, have been detected in epithelial cells and neutrophils from patients with Helicobacter pylori gastritis and several epithelial neoplasms. Design and Methods Blood neutrophils from 13 cases of Shwachman–Diamond syndrome – ten with and three without SBDS gene mutation – and ten controls were investigated by confocal microscopy and ultrastructural immunocytochemistry using antibodies against ubiquitinated proteins, proteasomes, p62 protein, and Helicobacter pylori VacA, urease and outer membrane proteins. Results Many extensively disseminated particulate cytoplasmic structures, accounting for 22.78±5.57% (mean ± standard deviation) of the total cytoplasm, were found in blood neutrophils from mutated Shwachman–Diamond syndrome patients. The particulate cytoplasmic structures showed immunoreactivity for polyubiquitinated proteins and proteasomes, but no reactivity for Helicobacter pylori products, which are present in particulate cytoplasmic structures of Helicobacter pylori-positive gastritis. Neutrophils from patients with Shwachman–Diamond syndrome frequently showed p62-positive autophagic vacuoles and apoptotic changes in 5% of cells. No particulate cytoplasmic structures were observed in most control neutrophils; however, in a few cells from two cases we noted focal development of minute particulate cytoplasmic structures, accounting for 0.74±0.56% of the total cytoplasm (P<0.001 versus particulate cytoplasmic structures from mutated Shwachman–Diamond syndrome patients). Neutrophils from non-mutated Shwachman–Diamond-syndrome-like patients resembled controls in two cases, and a third case showed particulate cytoplasmic structure patterns intermediate between those in controls and

  10. Novel clinical and diagnostic aspects of antineutrophil cytoplasmic antibodies.

    PubMed

    Schulte-Pelkum, Johannes; Radice, Antonella; Norman, Gary L; Lόpez Hoyos, Marcos; Lakos, Gabriella; Buchner, Carol; Musset, Lucile; Miyara, Makoto; Stinton, Laura; Mahler, Michael

    2014-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of some idiopathic systemic vasculitides. Besides the investigation of ANCA-associated vasculitis (AAV) and constant effort for a standardized nomenclature and classification of the AAV, a main focus of research during the last few years has been to constantly improve the performance of enzyme immunoassays. With the latest so called third generation ELISA, this goal seemed to be fulfilled. The International Consensus Statement on Testing and Reporting of ANCA gave recommendations for standardized strategies for the serological diagnosis of ANCA. New developments now target the system immanent drawbacks of the respective diagnostic methods, be it the need for batching and the long time to result for ELISA, or the high likelihood of error and subjectivity of indirect immunofluorescence (IIF). Random access technology and multiplexing for solid phase assays as well as digital imaging for IIF are tools which may help to expedite and simplify routine diagnostics in the lab and in emergency settings. Recent findings indicate that PR3-ANCA have clinical utility beyond the diagnosis of AAV. PR3-ANCA can also serve as an aid for the differentiation between ulcerative colitis (UC) and Crohn's disease (CrD) and the stratification of UC patients. This review provides a detailed review of what is known about ANCA and highlights the latest research and state-of-the-art developments in this area. PMID:24995343

  11. Cytoplasmic dynein functions as a gear in response to load

    NASA Astrophysics Data System (ADS)

    Mallik, Roop; Carter, Brian C.; Lex, Stephanie A.; King, Stephen J.; Gross, Steven P.

    2004-02-01

    Cytoskeletal molecular motors belonging to the kinesin and dynein families transport cargos (for example, messenger RNA, endosomes, virus) on polymerized linear structures called microtubules in the cell. These `nanomachines' use energy obtained from ATP hydrolysis to generate force, and move in a step-like manner on microtubules. Dynein has a complex and fundamentally different structure from other motor families. Thus, understanding dynein's force generation can yield new insight into the architecture and function of nanomachines. Here, we use an optical trap to quantify motion of polystyrene beads driven along microtubules by single cytoplasmic dynein motors. Under no load, dynein moves predominantly with a mixture of 24-nm and 32-nm steps. When moving against load applied by an optical trap, dynein can decrease step size to 8nm and produce force up to 1.1pN. This correlation between step size and force production is consistent with a molecular gear mechanism. The ability to take smaller but more powerful strokes under load-that is, to shift gears-depends on the availability of ATP. We propose a model whereby the gear is downshifted through load-induced binding of ATP at secondary sites in the dynein head.

  12. Gram's Stain Does Not Cross the Bacterial Cytoplasmic Membrane.

    PubMed

    Wilhelm, Michael J; Sheffield, Joel B; Sharifian Gh, Mohammad; Wu, Yajing; Spahr, Christian; Gonella, Grazia; Xu, Bolei; Dai, Hai-Lung

    2015-07-17

    For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.

  13. In Silico Classification of Proteins from Acidic and Neutral Cytoplasms

    PubMed Central

    Fang, Yaping; Middaugh, C. Russell; Fang, Jianwen

    2012-01-01

    Protein acidostability is a common problem in biopharmaceutical and other industries. However, it remains a great challenge to engineer proteins for enhanced acidostability because our knowledge of protein acidostabilization is still very limited. In this paper, we present a comparative study of proteins from bacteria with acidic (AP) and neutral cytoplasms (NP) using an integrated statistical and machine learning approach. We construct a set of 393 non-redundant AP-NP ortholog pairs and calculate a total of 889 sequence based features for these proteins. The pairwise alignments of these ortholog pairs are used to build a residue substitution propensity matrix between APs and NPs. We use Gini importance provided by the Random Forest algorithm to rank the relative importance of these features. A scoring function using the 10 most significant features is developed and optimized using a hill climbing algorithm. The accuracy of the score function is 86.01% in predicting AP-NP ortholog pairs and is 76.65% in predicting non-ortholog AP-NP pairs, suggesting that there are significant differences between APs and NPs which can be used to predict relative acidostability of proteins. The overall trends uncovered in the study can be used as general guidelines for designing acidostable proteins. To best of our knowledge, this work represents the first systematic comparative study of the acidostable proteins and their non-acidostable orthologs. PMID:23049817

  14. Sexy sons: a dead end for cytoplasmic genes.

    PubMed

    Zeh, Jeanne A

    2004-08-01

    Critics of sexual conflict theory argue that females may gain a net reproductive benefit from mating with manipulative males because the direct costs that they suffer may be offset by the production of sexy, i.e. manipulative, sons. However, this exclusive focus on nuclear gene effects represents an incomplete view of female fitness. Females differ fundamentally from males in transmitting not only nuclear genes but also a wide range of cytoplasmic genetic elements (CGEs) that can have profound effects, from male killing to influencing development of the nervous system and cognitive ability. Maternal transmission of CGEs has two major implications for sexual selection. First, the evolution of male fitness traits, such as sperm competitive ability, may be constrained because response to selection on mitochondrial genomes can occur only through the female line. Second, CGEs bear the direct costs of male manipulation but gain no indirect benefits when females produce sexy sons. This should result in perpetual antagonistic coevolution between nuclear genes involved in male manipulation and CGEs that promote female resistance to male sexually selected traits. Explicit consideration of the consequences of selection acting on CGEs is therefore necessary for a better understanding of the relationship between sexual selection and sexual conflict.

  15. Association of Endophilin B1 with Cytoplasmic Vesicles.

    PubMed

    Li, Jinhui; Barylko, Barbara; Eichorst, John P; Mueller, Joachim D; Albanesi, Joseph P; Chen, Yan

    2016-08-01

    Endophilins are SH3- and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2 promote the budding of endocytic vesicles from the plasma membrane, whereas endophilin B1 has been implicated in vesicle budding from intracellular organelles, including the trans-Golgi network and late endosomes. We previously reported that endophilins A1 and A2 exist almost exclusively as soluble dimers in the cytosol. Here, we present results of fluorescence fluctuation spectroscopy analyses indicating that, in contrast, the majority of endophilin B1 is present in multiple copies on small, highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. Using dual-color heterospecies partition analysis, we identified the epidermal growth factor receptor on endophilin B1 vesicles. Moreover, a proportion of endophilin B1 vesicles also contained caveolin, whereas clathrin was almost undetectable on those vesicles. These results raise the possibility that endophilin B1 participates in dynamin 2-dependent formation of a population of transport vesicles distinct from those generated by A-type endophilins. PMID:27508440

  16. Cytoplasmic dynein: a key player in neurodegenerative and neurodevelopmental diseases.

    PubMed

    Chen, Xiang-Jun; Xu, Huan; Cooper, Helen M; Liu, Yaobo

    2014-04-01

    Cytoplasmic dynein is the most important molecular motor driving the movement of a wide range of cargoes towards the minus ends of microtubules. As a molecular motor protein, dynein performs a variety of basic cellular functions including organelle transport and centrosome assembly. In the nervous system, dynein has been demonstrated to be responsible for axonal retrograde transport. Many studies have revealed direct or indirect evidence of dynein in neurodegenerative diseases such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Alzheimer's disease, Parkinson's disease and Huntington's disease. Among them, a number of mutant proteins involved in various neurodegenerative diseases interact with dynein. Axonal transport disruption is presented as a common feature occurring in neurodegenerative diseases. Dynein heavy chain mutant mice also show features of neurodegenerative diseases. Moreover, defects of dynein-dependent processes such as autophagy or clearance of aggregation-prone proteins are found in most of these diseases. Lines of evidence have also shown that dynein is associated with neurodevelopmental diseases. In this review, we focus on dynein involvement in different neurological diseases and discuss potential underlying mechanisms.

  17. Negative Regulation of Cytoplasmic RNA-Mediated Antiviral Signaling

    PubMed Central

    Komuro, Akihiko; Bamming, Darja

    2008-01-01

    The recent, rapid progress in our understanding of cytoplasmic RNA-mediated antiviral innate immune signaling was initiated by the discovery of retinoic acid-inducible gene I (RIG-I) as a sensor of viral RNA [1]. It is now widely recognized that RIG-I and related RNA helicases, melanoma differentiated-associated gene-5 (MDA5) and laboratory of genetics and physiology-2 (LGP2), can initiate and/or regulate RNA and virus -mediated type I IFN production and antiviral responses. As with other cytokine systems, production of type I IFN is a transient process, and can be hazardous to the host if unregulated, resulting in chronic cellular toxicity or inflammatory and autoimmune diseases [2-9]. In addition, the RIG-I-like receptor (RLR) system is a fundamental target for virus-encoded immune suppression, with many indirect and direct examples of interference described. In this article, we review the current understanding of endogenous negative regulation in RLR signaling and explore direct inhibition of RLR signaling by viruses as a host immune evasion strategy. PMID:18703349

  18. Cytoplasmic sphingosine-1-phosphate pathway modulates neuronal autophagy

    PubMed Central

    Moruno Manchon, Jose Felix; Uzor, Ndidi-Ese; Dabaghian, Yuri; Furr-Stimming, Erin E.; Finkbeiner, Steven; Tsvetkov, Andrey S.

    2015-01-01

    Autophagy is an important homeostatic mechanism that eliminates long-lived proteins, protein aggregates and damaged organelles. Its dysregulation is involved in many neurodegenerative disorders. Autophagy is therefore a promising target for blunting neurodegeneration. We searched for novel autophagic pathways in primary neurons and identified the cytosolic sphingosine-1-phosphate (S1P) pathway as a regulator of neuronal autophagy. S1P, a bioactive lipid generated by sphingosine kinase 1 (SK1) in the cytoplasm, is implicated in cell survival. We found that SK1 enhances flux through autophagy and that S1P-metabolizing enzymes decrease this flux. When autophagy is stimulated, SK1 relocalizes to endosomes/autophagosomes in neurons. Expression of a dominant-negative form of SK1 inhibits autophagosome synthesis. In a neuron model of Huntington’s disease, pharmacologically inhibiting S1P-lyase protected neurons from mutant huntingtin-induced neurotoxicity. These results identify the S1P pathway as a novel regulator of neuronal autophagy and provide a new target for developing therapies for neurodegenerative disorders. PMID:26477494

  19. Does a parthenogenesis-inducing Wolbachia induce vestigial cytoplasmic incompatibility?

    NASA Astrophysics Data System (ADS)

    Kraaijeveld, Ken; Reumer, Barbara M.; Mouton, Laurence; Kremer, Natacha; Vavre, Fabrice; van Alphen, Jacques J. M.

    2011-03-01

    Wolbachia is a maternally inherited bacterium that manipulates the reproduction of its host. Recent studies have shown that male-killing strains can induce cytoplasmic incompatibility (CI) when introgressed into a resistant host. Phylogenetic studies suggest that transitions between CI and other Wolbachia phenotypes have also occurred frequently, raising the possibility that latent CI may be widespread among Wolbachia. Here, we investigate whether a parthenogenesis-inducing Wolbachia strain can also induce CI. Parthenogenetic females of the parasitoid wasp Asobara japonica regularly produce a small number of males that may be either infected or not. Uninfected males were further obtained through removal of the Wolbachia using antibiotics and from a naturally uninfected strain. Uninfected females that had mated with infected males produced a slightly, but significantly more male-biased sex ratio than uninfected females that had mated with uninfected males. This effect was strongest in females that mated with males that had a relatively high Wolbachia titer. Quantitative PCR indicated that infected males did not show higher ratios of nuclear versus mitochondrial DNA content. Wolbachia therefore does not cause diploidization of cells in infected males. While these results are consistent with CI, other alternatives such as production of abnormal sperm by infected males cannot be completely ruled out. Overall, the effect was very small (9%), suggesting that if CI is involved it may have degenerated through the accumulation of mutations.

  20. Cytoplasmic pathway followed by chloride ions to enter the CFTR channel pore.

    PubMed

    El Hiani, Yassine; Negoda, Alexander; Linsdell, Paul

    2016-05-01

    Most ATP-binding cassette (ABC) proteins function as ATP-dependent membrane pumps. One exception is the cystic fibrosis transmembrane conductance regulator (CFTR), an ABC protein that functions as a Cl(-) ion channel. As such, the CFTR protein must form a continuous pathway for the movement of Cl(-) ions from the cytoplasm to the extracellular solution when in its open channel state. Extensive functional investigations have characterized most parts of this Cl(-) permeation pathway. However, one region remains unexplored-the pathway connecting the cytoplasm to the membrane-spanning pore. We used patch clamp recording and extensive substituted cysteine accessibility mutagenesis to identify amino acid side-chains in cytoplasmic regions of CFTR that lie close to the pathway taken by Cl(-) ions as they pass from the cytoplasm through this pathway. Our results suggest that Cl(-) ions enter the permeation pathway via a single lateral tunnel formed by the cytoplasmic parts of the protein, and then follow a fairly direct central pathway towards the membrane-spanning parts of the protein. However, this pathway is not lined continuously by any particular part of the protein; instead, the contributions of different cytoplasmic regions of the protein appear to change as the permeation pathway approaches the membrane, which appears to reflect the ways in which different cytoplasmic regions of the protein are oriented towards its central axis. Our results allow us to define for the first time the complete Cl(-) permeation pathway in CFTR, from the cytoplasm to the extracellular solution.

  1. Response of amphibian egg non-yolk cytoplasm to gravity orientation

    NASA Technical Reports Server (NTRS)

    Smith, R. C.; Neff, A. W.; Malacinski, G. M.

    1985-01-01

    In order to study amphibian egg cytoplasmic organization and egg symmetrization at the molecular level, a library of seventeen monoclonal antibodies (MoAbs) against Xenopus laevis non-yolk egg proteins was produced. Several of these MoAbs react with non-yolk cytoplasmic antigens which are unevenly distributed in the fertile Xenopus egg.

  2. TRIM5{alpha} association with cytoplasmic bodies is not required for antiretroviral activity

    SciTech Connect

    Song, Byeongwoon; Diaz-Griffero, Felipe; Park, Do Hyun; Rogers, Thomas; Stremlau, Matthew; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-12-20

    The tripartite motif (TRIM) protein, TRIM5{alpha}, restricts infection by particular retroviruses. Many TRIM proteins form cytoplasmic bodies of unknown function. We investigated the relationship between cytoplasmic body formation and the structure and antiretroviral activity of TRIM5{alpha}. In addition to diffuse cytoplasmic staining, the TRIM5{alpha} proteins from several primate species were located in cytoplasmic bodies of different sizes; by contrast, TRIM5{alpha} from spider monkeys did not form cytoplasmic bodies. Despite these differences, all of the TRIM5{alpha} proteins exhibited the ability to restrict infection by particular retroviruses. Treatment of cells with geldanamycin, an Hsp90 inhibitor, resulted in disappearance or reduction of the TRIM5{alpha}-associated cytoplasmic bodies, yet exerted little effect on the restriction of retroviral infection. Studies of green fluorescent protein-TRIM5{alpha} fusion proteins indicated that no TRIM5{alpha} domain is specifically required for association with cytoplasmic bodies. Apparently, the formation of cytoplasmic bodies is not required for the antiretroviral activity of TRIM5{alpha}.

  3. Cytoplasmic Rbfox1 Regulates the Expression of Synaptic and Autism-Related Genes.

    PubMed

    Lee, Ji-Ann; Damianov, Andrey; Lin, Chia-Ho; Fontes, Mariana; Parikshak, Neelroop N; Anderson, Erik S; Geschwind, Daniel H; Black, Douglas L; Martin, Kelsey C

    2016-01-01

    Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is also alternatively spliced to produce cytoplasmic isoforms. To investigate the function of cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that Rbfox1 bound predominantly to introns in nascent RNA, while cytoplasmic Rbox1 bound to 3' UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and Rbfox1 and miRNA binding sites overlapped significantly. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease. PMID:26687839

  4. Effect of wild Helianthus cytoplasms on agronomic and oil characteristics of cultivated sunflower (H. annuus L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sunflower (Helianthus annuus L.) productions reliance on a single source of cytoplasmic male-sterility, PET1, derived from H. petiolaris Nutt., makes the crop genetically vulnerable. Twenty diverse cytoplasmic substitution lines from annual and perennial wild species were compared with the inbred li...

  5. Cytoplasmic pathway followed by chloride ions to enter the CFTR channel pore.

    PubMed

    El Hiani, Yassine; Negoda, Alexander; Linsdell, Paul

    2016-05-01

    Most ATP-binding cassette (ABC) proteins function as ATP-dependent membrane pumps. One exception is the cystic fibrosis transmembrane conductance regulator (CFTR), an ABC protein that functions as a Cl(-) ion channel. As such, the CFTR protein must form a continuous pathway for the movement of Cl(-) ions from the cytoplasm to the extracellular solution when in its open channel state. Extensive functional investigations have characterized most parts of this Cl(-) permeation pathway. However, one region remains unexplored-the pathway connecting the cytoplasm to the membrane-spanning pore. We used patch clamp recording and extensive substituted cysteine accessibility mutagenesis to identify amino acid side-chains in cytoplasmic regions of CFTR that lie close to the pathway taken by Cl(-) ions as they pass from the cytoplasm through this pathway. Our results suggest that Cl(-) ions enter the permeation pathway via a single lateral tunnel formed by the cytoplasmic parts of the protein, and then follow a fairly direct central pathway towards the membrane-spanning parts of the protein. However, this pathway is not lined continuously by any particular part of the protein; instead, the contributions of different cytoplasmic regions of the protein appear to change as the permeation pathway approaches the membrane, which appears to reflect the ways in which different cytoplasmic regions of the protein are oriented towards its central axis. Our results allow us to define for the first time the complete Cl(-) permeation pathway in CFTR, from the cytoplasm to the extracellular solution. PMID:26659082

  6. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

    PubMed Central

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J.; Tuckey, Corinna; Riggs, Paul D.; Colussi, Paul A.; Noren, Christopher J.; Taron, Christopher H.; DeLisa, Matthew P.; Berkmen, Mehmet

    2015-01-01

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. PMID:26311203

  7. Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles.

    PubMed

    Vaisberg, E A; Grissom, P M; McIntosh, J R

    1996-05-01

    We describe two dynein heavy chain (DHC)-like polypeptides (DHCs 2 and 3) that are distinct from the heavy chain of conventional cytoplasmic dynein (DHC1) but are expressed in a variety of mammalian cells that lack axonemes. DHC2 is a distant member of the "cytoplasmic" branch of the dynein phylogenetic tree, while DHC3 shares more sequence similarity with dynein-like polypeptides that have been thought to be axonemal. Each cytoplasmic dynein is associated with distinct cellular organelles. DHC2 is localized predominantly to the Golgi apparatus. Moreover, the Golgi disperses upon microinjection of antibodies to DHC2, suggesting that this motor is involved in establishing proper Golgi organization. DCH3 is associated with as yet unidentified structures that may represent transport intermediates between two or more cytoplasmic compartments. Apparently, specific cytoplasmic dyneins, like individual members of the kinesin superfamily, play unique roles in the traffic of cytomembranes.

  8. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.

    PubMed

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J; Tuckey, Corinna; Riggs, Paul D; Colussi, Paul A; Noren, Christopher J; Taron, Christopher H; DeLisa, Matthew P; Berkmen, Mehmet

    2015-01-01

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named 'cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. PMID:26311203

  9. Cytoplasmic dynein is a minus end-directed motor for membranous organelles

    SciTech Connect

    Schroer, T.A.; Steuer, E.R.; Sheetz, M.P.

    1989-03-24

    The role of cytoplasmic dynein in microtubule-based organelle transport was examined using a reconstituted assay developed from chick embryo fibroblasts. Factors present in a high-speed cytosol caused the movement of purified organelles on microtubules predominantly in the minus end direction. Inactivation of cytoplasmic dynein in the high-speed cytosol by vanadate-mediated UV photocleavage inhibited minus end-directed organelle motility by over 90%. Addition of purified cytoplasmic dynein to the inactive cytosol restored minus end-directed organelle motility, although purified cytoplasmic dynein by itself did not support organelle movement. We propose that cytoplasmic dynein is the motor for minus end-directed organelle movement, but that additional cytosolic factors are also required to produce organelle motility.

  10. A cryptic cytoplasmic male sterility unveils a possible gynodioecious past for Arabidopsis thaliana.

    PubMed

    Gobron, Nicolas; Waszczak, Cezary; Simon, Matthieu; Hiard, Sophie; Boivin, Stéphane; Charif, Delphine; Ducamp, Aloïse; Wenes, Estelle; Budar, Françoise

    2013-01-01

    Gynodioecy, the coexistence of hermaphrodites and females (i.e. male-sterile plants) in natural plant populations, most often results from polymorphism at genetic loci involved in a particular interaction between the nuclear and cytoplasmic genetic compartments (cytonuclear epistasis): cytoplasmic male sterility (CMS). Although CMS clearly contributes to the coevolution of involved nuclear loci and cytoplasmic genomes in gynodioecious species, the occurrence of CMS genetic factors in the absence of sexual polymorphism (cryptic CMS) is not easily detected and rarely taken in consideration. We found cryptic CMS in the model plant Arabidopsis thaliana after crossing distantly related accessions, Sha and Mr-0. Male sterility resulted from an interaction between the Sha cytoplasm and two Mr-0 genomic regions located on chromosome 1 and chromosome 3. Additional accessions with either nuclear sterility maintainers or sterilizing cytoplasms were identified from crosses with either Sha or Mr-0. By comparing two very closely related cytoplasms with different male-sterility inducing abilities, we identified a novel mitochondrial ORF, named orf117Sha, that is most likely the sterilizing factor of the Sha cytoplasm. The presence of orf117Sha was investigated in worldwide natural accessions. It was found mainly associated with a single chlorotype in accessions belonging to a clade predominantly originating from Central Asia. More than one-third of accessions from this clade carried orf117Sha, indicating that the sterilizing-inducing cytoplasm had spread in this lineage. We also report the coexistence of the sterilizing cytoplasm with a non-sterilizing cytoplasm at a small, local scale in a natural population; in addition a correlation between cytotype and nuclear haplotype was detected in this population. Our results suggest that this CMS system induced sexual polymorphism in A. thaliana populations, at the time when the species was mainly outcrossing.

  11. Studying Genome Heterogeneity within the Arbuscular Mycorrhizal Fungal Cytoplasm

    PubMed Central

    Halary, Sébastien; Bapteste, Eric; Hijri, Mohamed

    2015-01-01

    Although heterokaryons have been reported in nature, multicellular organisms are generally assumed genetically homogeneous. Here, we investigate the case of arbuscular mycorrhizal fungi (AMF) that form symbiosis with plant roots. The growth advantages they confer to their hosts are of great potential benefit to sustainable agricultural practices. However, measuring genetic diversity for these coenocytes is a major challenge: Within the same cytoplasm, AMF contain thousands of nuclei and show extremely high levels of genetic variation for some loci. The extent and physical location of polymorphism within and between AMF genomes is unclear. We used two complementary strategies to estimate genetic diversity in AMF, investigating polymorphism both on a genome scale and in putative single copy loci. First, we used data from whole-genome pyrosequencing of four AMF isolates to describe genetic diversity, based on a conservative network-based clustering approach. AMF isolates showed marked differences in genome-wide diversity patterns in comparison to a panel of control fungal genomes. This clustering approach further allowed us to provide conservative estimates of Rhizophagus spp. genomes sizes. Second, we designed new putative single copy genomic markers, which we investigated by massive parallel amplicon sequencing for two Rhizophagus irregularis and one Rhizophagus sp. isolates. Most loci showed high polymorphism, with up to 103 alleles per marker. This polymorphism could be distributed within or between nuclei. However, we argue that the Rhizophagus isolates under study might be heterokaryotic, at least for the putative single copy markers we studied. Considering that genetic information is the main resource for identification of AMF, we suggest that special attention is warranted for the study of these ecologically important organisms. PMID:25573960

  12. Cytoplasmic molecular delivery with shock waves: importance of impulse.

    PubMed Central

    Kodama, T; Hamblin, M R; Doukas, A G

    2000-01-01

    Cell permeabilization using shock waves may be a way of introducing macromolecules and small polar molecules into the cytoplasm, and may have applications in gene therapy and anticancer drug delivery. The pressure profile of a shock wave indicates its energy content, and shock-wave propagation in tissue is associated with cellular displacement, leading to the development of cell deformation. In the present study, three different shock-wave sources were investigated; argon fluoride excimer laser, ruby laser, and shock tube. The duration of the pressure pulse of the shock tube was 100 times longer than the lasers. The uptake of two fluorophores, calcein (molecular weight: 622) and fluorescein isothiocyanate-dextran (molecular weight: 71,600), into HL-60 human promyelocytic leukemia cells was investigated. The intracellular fluorescence was measured by a spectrofluorometer, and the cells were examined by confocal fluorescence microscopy. A single shock wave generated by the shock tube delivered both fluorophores into approximately 50% of the cells (p < 0.01), whereas shock waves from the lasers did not. The cell survival fraction was >0.95. Confocal microscopy showed that, in the case of calcein, there was a uniform fluorescence throughout the cell, whereas, in the case of FITC-dextran, the fluorescence was sometimes in the nucleus and at other times not. We conclude that the impulse of the shock wave (i.e., the pressure integrated over time), rather than the peak pressure, was a dominant factor for causing fluorophore uptake into living cells, and that shock waves might have changed the permeability of the nuclear membrane and transferred molecules directly into the nucleus. PMID:11023888

  13. Studying genome heterogeneity within the arbuscular mycorrhizal fungal cytoplasm.

    PubMed

    Boon, Eva; Halary, Sébastien; Bapteste, Eric; Hijri, Mohamed

    2015-01-07

    Although heterokaryons have been reported in nature, multicellular organisms are generally assumed genetically homogeneous. Here, we investigate the case of arbuscular mycorrhizal fungi (AMF) that form symbiosis with plant roots. The growth advantages they confer to their hosts are of great potential benefit to sustainable agricultural practices. However, measuring genetic diversity for these coenocytes is a major challenge: Within the same cytoplasm, AMF contain thousands of nuclei and show extremely high levels of genetic variation for some loci. The extent and physical location of polymorphism within and between AMF genomes is unclear. We used two complementary strategies to estimate genetic diversity in AMF, investigating polymorphism both on a genome scale and in putative single copy loci. First, we used data from whole-genome pyrosequencing of four AMF isolates to describe genetic diversity, based on a conservative network-based clustering approach. AMF isolates showed marked differences in genome-wide diversity patterns in comparison to a panel of control fungal genomes. This clustering approach further allowed us to provide conservative estimates of Rhizophagus spp. genomes sizes. Second, we designed new putative single copy genomic markers, which we investigated by massive parallel amplicon sequencing for two Rhizophagus irregularis and one Rhizophagus sp. isolates. Most loci showed high polymorphism, with up to 103 alleles per marker. This polymorphism could be distributed within or between nuclei. However, we argue that the Rhizophagus isolates under study might be heterokaryotic, at least for the putative single copy markers we studied. Considering that genetic information is the main resource for identification of AMF, we suggest that special attention is warranted for the study of these ecologically important organisms.

  14. Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring

    PubMed Central

    Szalay, Beata; Hagel, Christian; Hohenberg, Heinrich; Deppert, Wolfgang; Bohn, Wolfgang

    2009-01-01

    Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner. PMID:19562035

  15. Formation of antiviral cytoplasmic granules during orthopoxvirus infection.

    PubMed

    Simpson-Holley, M; Kedersha, N; Dower, K; Rubins, K H; Anderson, P; Hensley, L E; Connor, J H

    2011-02-01

    Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (ΔE3L mutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2α phosphorylation and impaired translation initiation. Here we show that cells infected with ΔE3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in >80% of cells infected with ΔE3L mutant VV but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2α, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2α showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased ΔE3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of ΔE3L mutant VV, despite increased eIF2α phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of ΔE3L mutant VV replication requires AVG formation subsequent to eIF2α phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an important component of the successful cellular antiviral response.

  16. Nuclear synthesis of cytoplasmic ribonucleic acid in Amoeba proteus.

    PubMed

    PRESCOTT, D M

    1959-10-01

    The enucleation technique has been applied to Amoeba proteus by several laboratories in attempts to determine whether the cytoplasm is capable of nucleus-independent ribonucleic acid synthesis. This cell is very convenient for micrurgy, but its use requires a thorough starvation period to eliminate the possibility of metabolic influence by food vacuoles and frequent washings and medium renewal to maintain asepsis. In the experiments described here, amoebae were starved for periods of 24 to 96 hours, cut into nucleated and enucleated halves, and exposed to either C-14 uracil, C-14 adenine, C-14 orotic acid, or a mixture of all three. When the starvation period was short (less than 72 hours), organisms (especially yeast cells) contained within amoeba food vacuoles frequently showed RNA synthesis in both nucleated and enucleated amoebae. When the preperiod of starvation was longer than 72 hours, food vacuole influence was apparently negligible, and a more meaningful comparison between enucleated and nucleated amoebae was possible. Nucleated cells incorporated all three precursors into RNA; enucleated cells were incapable of such incorporation. The experiments indicate a complete dependence on the nucleus for RNA synthesis. The conflict with the experimental results of others on this problem could possibly stem from differences in culture conditions, starvation treatment, or experimental conditions. For an unequivocal answer in experiments of this design, ideally the cells should be capable of growth on an entirely synthetic medium under aseptic conditions. The use of a synthetic medium (experiments with A. proteus are done under starvation conditions) would permit, moreover, a more realistic comparison of metabolic capacities of nucleated and enucleated cells.

  17. Molecular mechanism of motion and force generation by cytoplasmic dynein

    NASA Astrophysics Data System (ADS)

    Gennerich, Arne

    2013-03-01

    Cytoplasmic dynein is an intricate microtubule (MT) motor with four AAA (ATPase associated with various cellular activities) ATPases per head domain. Dynein homodimers take hundreds of consecutive steps, during which the leading and trailing heads experience intramolecular tension in opposite directions. We hypothesize that this asymmetry may differentially regulate the MT-binding and ATPase functions in each head, thereby facilitating processive movement. Here, we elucidate the function of tension in regulating dynein-MT interactions, and dissect the roles of its multiple AAA subunits in effecting and modulating this behavior. Using optical tweezers to measure unbinding forces of single S. cerevisiae dynein heads in the absence of nucleotide, we show that intrinsic dynein-MT binding is significantly weaker under forward (MT-minus-end directed) tension than under rearward tension. Thus, forward tension likely promotes rear head detachment in the dimeric motor. The nucleotide states of specific AAA sites modify this intrinsic behavior. Mutational analysis shows that ATP binding to AAA1 substantially weakens MT binding. Moreover, ADP binding to AAA3 `locks' dynein in a previously undescribed, weak MT-binding state with a relatively symmetric response to tension. Interestingly, tension also affects nucleotide affinity: ADP affinity is lower under rearward than under forward load, suggesting that the front head preferentially releases ADP (likely from AAA3), perhaps driving a transition from an ADP state with relatively weak MT attachment to a strongly MT-attached, nucleotide-free state. Our analysis suggests that intramolecular tension is key to dynein motility, and highlights the importance of including multiple AAA ATPases in models for dynein mechanochemistry. NIH R01GM098469

  18. Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells.

    PubMed

    Kalantari, Roya; Hicks, Jessica A; Li, Liande; Gagnon, Keith T; Sridhara, Viswanadham; Lemoff, Andrew; Mirzaei, Hamid; Corey, David R

    2016-07-01

    Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners.

  19. Targeted Cytoplasmic Irradiation with Alpha Particles Induces Mutations in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Wu, Li-Jun; Randers-Pehrson, Gerhard; Xu, An; Waldren, Charles A.; Geard, Charles R.; Yu, Zengliang; Hei, Tom K.

    1999-04-01

    Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

  20. Novel nuclear-cytoplasmic interaction in wheat (Triticum aestivum) induces vigorous plants.

    PubMed

    Soltani, Ali; Kumar, Ajay; Mergoum, Mohamed; Pirseyedi, Seyed Mostafa; Hegstad, Justin B; Mazaheri, Mona; Kianian, Shahryar F

    2016-03-01

    Interspecific hybridization can be considered an accelerator of evolution, otherwise a slow process, solely dependent on mutation and recombination. Upon interspecific hybridization, several novel interactions between nuclear and cytoplasmic genomes emerge which provide additional sources of diversity. The magnitude and essence of intergenomic interactions between nuclear and cytoplasmic genomes remain unknown due to the direction of many crosses. This study was conducted to address the role of nuclear-cytoplasmic interactions as a source of variation upon hybridization. Wheat (Triticum aestivum) alloplasmic lines carrying the cytoplasm of Aegilops mutica along with an integrated approach utilizing comparative quantitative trait locus (QTL) and epigenome analysis were used to dissect this interaction. The results indicate that cytoplasmic genomes can modify the magnitude of QTL controlling certain physiological traits such as dry matter weight. Furthermore, methylation profiling analysis detected eight polymorphic regions affected by the cytoplasm type. In general, these results indicate that novel nuclear-cytoplasmic interactions can potentially trigger an epigenetic modification cascade in nuclear genes which eventually change the genetic network controlling physiological traits. These modified genetic networks can serve as new sources of variation to accelerate the evolutionary process. Furthermore, this variation can synthetically be produced by breeders in their programs to develop epigenomic-segregating lines. PMID:26860316

  1. Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

    PubMed Central

    Kalantari, Roya; Hicks, Jessica A.; Li, Liande; Gagnon, Keith T.; Sridhara, Viswanadham; Lemoff, Andrew; Mirzaei, Hamid; Corey, David R.

    2016-01-01

    Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners. PMID:27198507

  2. Pulmonary Fibrosis in Antineutrophil Cytoplasmic Antibodies (ANCA)-Associated Vasculitis

    PubMed Central

    Comarmond, Cloé; Crestani, Bruno; Tazi, Abdellatif; Hervier, Baptiste; Adam-Marchand, Sylvain; Nunes, Hilario; Cohen-Aubart, Fleur; Wislez, Marie; Cadranel, Jacques; Housset, Bruno; Lloret-Linares, Célia; Sève, Pascal; Pagnoux, Christian; Abad, Sébastien; Camuset, Juliette; Bienvenu, Boris; Duruisseaux, Michaël; Hachulla, Eric; Arlet, Jean-Benoît; Hamidou, Mohammed; Mahr, Alfred; Resche-Rigon, Matthieu; Brun, Anne-Laure; Grenier, Philippe; Cacoub, Patrice; Saadoun, David

    2014-01-01

    Abstract Pulmonary fibrosis (PF) is an uncommon manifestation observed in patients with antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV), particularly microscopic polyangiitis (MPA). While patients with PF associated with AAV seem to have a worse prognosis, these patients have been described only in case reports or small retrospective case series. In this retrospective multicenter study, we report the main features and long-term outcomes of patients with PF associated with AAV, fulfilling the American College of Rheumatology criteria and/or Chapel Hill definitions. Forty-nine patients (30 men [61%]; median age at diagnosis of AAV, 68 [interquartile range, 58–73] years) with PF associated with AAV were identified. Forty (81.6%) patients had MPA and 9 (18.4%) had granulomatosis with polyangiitis. The diagnosis of PF preceded the onset of vasculitis in 22 (45%) patients. Usual interstitial pneumonia was the main radiologic pattern (n = 18, 43%). ANCA were mostly of antimyeloperoxidase specificity (88%). All patients were treated with glucocorticoids as induction therapy, combined with cyclophosphamide (CYC) (n = 36, 73.5%) or rituximab (RTX) (n = 1, 2%). Factors associated with mortality included occurrence of chronic respiratory insufficiency (hazard ratio [HR], 7.44; 95% confidence interval [CI], 1.6–34.5; p = 0.003), induction therapy with glucocorticoids alone (HR, 2.94; CI, 1.05–8.33; p = 0.04), and initial weigh loss (HR, 2.83; CI, 1.05–7.65; p = 0.041). The 3-year survival rate in patients treated with glucocorticoids alone or combined with an immunosuppressant (CYC or RTX) as induction therapy was 64% (95% CI, 41–99) and 94% (95% CI, 86–100), respectively (p = 0.03). After a median follow-up of 48 months [interquartile range, 14–88 mo], 18 (37%) patients died, including 11 related to respiratory insufficiency. PF is a rare manifestation of AAV with a very poor prognosis. Induction therapy with

  3. Staphylococcal Major Autolysin (Atl) Is Involved in Excretion of Cytoplasmic Proteins*

    PubMed Central

    Pasztor, Linda; Ziebandt, Anne-Kathrin; Nega, Mulugeta; Schlag, Martin; Haase, Sabine; Franz-Wachtel, Mirita; Madlung, Johannes; Nordheim, Alfred; Heinrichs, David E.; Götz, Friedrich

    2010-01-01

    Many microorganisms excrete typical cytoplasmic proteins into the culture supernatant. As none of the classical secretion systems appears to be involved, this type of secretion was referred to as “nonclassical protein secretion.” Here, we demonstrate that in Staphylococcus aureus the major autolysin plays a crucial role in release of cytoplasmic proteins. Comparative secretome analysis revealed that in the wild type S. aureus strain, 22 typical cytoplasmic proteins were excreted into the culture supernatant, although in the atl mutant they were significantly decreased. The presence or absence of prophages had little influence on the secretome pattern. In the atl mutant, secondary peptidoglycan hydrolases were increased in the secretome; the corresponding genes were transcriptionally up-regulated suggesting a compensatory mechanism for the atl mutation. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic indicator enzyme, we showed that all clinical isolates tested excreted this protein. In the wall teichoic acid-deficient tagO mutant with its increased autolysis activity, GAPDH was excreted in even higher amounts than in the WT, confirming the importance of autolysis in excretion of cytoplasmic proteins. To answer the question of how discriminatory the excretion of cytoplasmic proteins is, we performed a two-dimensional PAGE of cytoplasmic proteins isolated from WT. Surprisingly, the most abundant proteins in the cytoplasm were not found in the secretome of the WT, suggesting that there exists a selection mechanism in the excretion of cytoplasmic proteins. As the major autolysin binds at the septum site, we assume that the proteins are preferentially released at and during septum formation. PMID:20847047

  4. Cytoplasm segmentation on cervical cell images using graph cut-based approach.

    PubMed

    Zhang, Ling; Kong, Hui; Chin, Chien Ting; Wang, Tianfu; Chen, Siping

    2014-01-01

    This paper proposes a method to segment the cytoplasm in cervical cell images using graph cut-based algorithm. First, the A* channel in CIE LAB color space is extracted for contrast enhancement. Then, in order to effectively extract cytoplasm boundaries when image histograms present non-bimodal distribution, Otsu multiple thresholding is performed on the contrast enhanced image to generate initial segments, based on which the segments are refined by the multi-way graph cut method. We use 21 cervical cell images with non-ideal imaging condition to evaluate cytoplasm segmentation performance. The proposed method achieved a 93% accuracy which outperformed state-of-the-art works.

  5. Cytoplasmic RNA modulators of an inside-out signal-transduction cascade

    PubMed Central

    Blind, Michael; Kolanus, Waldemar; Famulok, Michael

    1999-01-01

    A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the β2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathways in vivo could potentially be applied wherever targeted modulation of a signal-transduction cascade is desired. PMID:10097084

  6. Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy

    NASA Astrophysics Data System (ADS)

    Choma, Michael A.; Ellerbee, Audrey K.; Yazdanfar, Siavash; Izatt, Joseph A.

    2006-03-01

    Spectral domain phase microscopy (SDPM) is a function extension of spectral domain optical coherence tomography. SDPM achieves exquisite levels of phase stability by employing common-path interferometry. We discuss the theory and limitations of Doppler flow imaging using SDPM, demonstrate monitoring the thermal contraction of a glass sample with nanometer per second velocity sensitivity, and apply this technique to measurement of cytoplasmic streaming in an Amoeba proteus pseudopod. We observe reversal of cytoplasmic flow induced by extracellular CaCl2, and report results that suggest parabolic flow of cytoplasm in the A. proteus pseudopod.

  7. Life as a moving fluid: fate of cytoplasmic macromolecules in dynamic fungal syncytia

    PubMed Central

    Roper, Marcus; Lee, ChangHwan; Hickey, Patrick C.; Gladfelter, Amy S.

    2015-01-01

    In fungal syncytia dozens, or even millions of nuclei may coexist in a single connected cytoplasm. Recent discoveries have exposed some of the adaptations that enable fungi to marshall these nuclei to produce complex coordinated behaviors, including cell growth, nuclear division, secretion and communication. In addition to shedding light on the principles by which syncytia (including embryos and osteoplasts) are organized, fungal adaptations for dealing with internal genetic diversity and physically dynamic cytoplasm may provide mechanistic insights into how cells generally are carved into different functional compartments. In this review we focus on enumerating the physical constraints associated with maintaining macromolecular distributions within a fluctuating and often flowing cytoplasmic interior. PMID:26226449

  8. Cytoplasmic streaming affects gravity-induced amyloplast sedimentation in maize coleoptiles

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Leopold, A. C.

    1985-01-01

    Living maize (Zea mays L.) coleoptile cells were observed using a horizontal microscope to determine the interaction between cytoplasmic streaming and gravity-induced amyloplast sedimentation. Sedimentation is heavily influenced by streaming which may (1) hasten or slow the velocity of amyloplast movement and (2) displace the plastid laterally or even upwards before or after sedimentation. Amyloplasts may move through transvacuolar strands or through the peripheral cytoplasm which may be divided into fine cytoplasmic strands of much smaller diameter than the plastids. The results indicate that streaming may contribute to the dynamics of graviperception by influencing amyloplast movement.

  9. Evolutionary genomics of nucleo-cytoplasmic large DNA viruses.

    PubMed

    Iyer, Lakshminarayan M; Balaji, S; Koonin, Eugene V; Aravind, L

    2006-04-01

    A previous comparative-genomic study of large nuclear and cytoplasmic DNA viruses (NCLDVs) of eukaryotes revealed the monophyletic origin of four viral families: poxviruses, asfarviruses, iridoviruses, and phycodnaviruses [Iyer, L.M., Aravind, L., Koonin, E.V., 2001. Common origin of four diverse families of large eukaryotic DNA viruses. J. Virol. 75 (23), 11720-11734]. Here we update this analysis by including the recently sequenced giant genome of the mimiviruses and several additional genomes of iridoviruses, phycodnaviruses, and poxviruses. The parsimonious reconstruction of the gene complement of the ancestral NCLDV shows that it was a complex virus with at least 41 genes that encoded the replication machinery, up to four RNA polymerase subunits, at least three transcription factors, capping and polyadenylation enzymes, the DNA packaging apparatus, and structural components of an icosahedral capsid and the viral membrane. The phylogeny of the NCLDVs is reconstructed by cladistic analysis of the viral gene complements, and it is shown that the two principal lineages of NCLDVs are comprised of poxviruses grouped with asfarviruses and iridoviruses grouped with phycodnaviruses-mimiviruses. The phycodna-mimivirus grouping was strongly supported by several derived shared characters, which seemed to rule out the previously suggested basal position of the mimivirus [Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M., Claverie, J.M. 2004. The 1.2-megabase genome sequence of Mimivirus. Science 306 (5700), 1344-1350]. These results indicate that the divergence of the major NCLDV families occurred at an early stage of evolution, prior to the divergence of the major eukaryotic lineages. It is shown that subsequent evolution of the NCLDV genomes involved lineage-specific expansion of paralogous gene families and acquisition of numerous genes via horizontal gene transfer from the eukaryotic hosts, other viruses, and bacteria

  10. Nonlinearity in cytoplasm viscosity can generate an essential symmetry breaking in cellular behaviors.

    PubMed

    Tachikawa, Masashi; Mochizuki, Atsushi

    2015-01-01

    The cytoplasms of ameboid cells are nonlinearly viscous. The cell controls this viscosity by modulating the amount, localization and interactions of bio-polymers. Here we investigated how the nonlinearity infers the cellular behaviors and whether nonlinearity-specific behaviors exist. We modeled the developed plasmodium of the slime mold Physarum polycephalum as a network of branching tubes and examined the linear and nonlinear viscous cytoplasm flows in the tubes. We found that the nonlinearity in the cytoplasm׳s viscosity induces a novel type of symmetry breaking in the protoplasmic flow. We also show that symmetry breaking can play an important role in adaptive behaviors, namely, connection of behavioral modes implemented on different time scales and transportation of molecular signals from the front to the rear of the cell during cellular locomotion. PMID:25261729

  11. The microtubule nucleation activity of centrobin in both the centrosome and cytoplasm

    PubMed Central

    Shin, Wonjung; Yu, Nam-Kyung; Kaang, Bong-Kiun; Rhee, Kunsoo

    2015-01-01

    Centrobin resides in daughter centriole and play a critical role in centriole duplication. Nucleation and stabilization of microtubules are known biological activities of centrobin. Here, we report a specific localization of centrobin outside the centrosome. Centrobin was associated with the stable microtubules. In hippocampal cells, centrobin formed cytoplasmic dots in addition to the localization at both centrosomes with the mother and daughter centrioles. Such specific localization pattern suggests that cytoplasmic centrobin is not just a reserved pool for centrosomal localization but also has a specific role in the cytoplasm. In fact, centrobin enhanced microtubule formation outside as well as inside the centrosome. These results propose specific roles of the cytoplasmic centrobin for noncentrosomal microtubule formation in specific cell types and during the cell cycle. PMID:26083938

  12. Reactive oxygen species (ROS) is not a promotor of taxol-induced cytoplasmic vacuolization

    NASA Astrophysics Data System (ADS)

    Sun, Qingrui; Chen, Tongsheng

    2009-02-01

    we have previously reported that taxol, a potent anticancer agent, induces caspase-independent cell death and cytoplasmic vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. However, the mechanisms of taxol-induced cytoplasmic vacuolization are poorly understood. Reactive oxygen species (ROS) has been reported to be involved in the taxol-induced cell death. Here, we employed confocal fluorescence microscopy imaging to explore the role of ROS in taxol-induced cytoplasmic vacuolization. We found that ROS inhibition by addition of N-acetycysteine (NAC), a total ROS scavenger, did not suppress these vacuolization but instead increased vacuolization. Take together, our results showed that ROS is not a promotor of the taxol-induced cytoplasmic vacuolization.

  13. Cytoplasm enhancement operator of peripheral blood smear images that are instable-stained and overexposed

    NASA Astrophysics Data System (ADS)

    Zheng, Xin; Wang, Guoyou; Liu, Jianguo

    2015-12-01

    Nucleus and cytoplasm are both essential for white blood cell recognition but the edges of cytoplasm are too blurry to be detected because of instable staining and overexposure. This paper aims at proposing a cytoplasm enhancement operator (CEO) to achieve accurate convergence of the active contour model. The CEO contains two parts. First, a nonlinear over-exposure enhancer map is yielded to correct over-exposure, which suppresses background noise while preserving details and improving contrast. Second, the over-exposed regions of cytoplasm in particular is further enhanced by a tri- modal histogram specification based on the scale-space filtering. The experimental results show that the proposed CEO and its corresponding GVF snake is superior to other unsupervised segmentation approaches.

  14. Hybridization using cytoplasmic male sterility and herbicide tolerance from nuclear genes

    SciTech Connect

    Beversdorf, W.D.; Erickson, L.R.; Grant, I.

    1987-04-14

    An improved process is described for producing a substantially homogeneous population of plants of a predetermined hybrid variety of a crop which is capable of undergoing both self-pollination and cross-pollination. This process comprises: growing in a first planting area a substantially random population of cytoplasmic male sterile plants which exhibit tolerance to at least one herbicide attributable solely to homozygous dominant nuclear genes, and male fertile plants which are capable of pollinating the cytoplasmic male sterile plants and which lack the herbicide tolerance because the presence of homozygous recessive nuclear genes for such trait. The cytoplasmic male sterile plants and the male fertile plants are pollinated with pollen derived from the male fertile plants. Seed is formed on the cytoplasmic male sterile plants and on the male fertile plants. Harvesting in bulk the seed is formed on the plants of the first planting area.

  15. Relation Between Basophilia and Fine Structure of Cytoplasm in the Fungus Allomyces macrogynus Em

    PubMed Central

    Blondel, Benigna; Turian, Gilbert

    1960-01-01

    In a fungus, Allomyces macrogynus Em., staining tests have revealed changes in the location of cytoplasmic basophilia following different phases of the developmental cycle. These variations in location were used to observe which fine structures correspond to basophile and non-basophile areas of the cytoplasm. Hyphae, gametangia, zygotes, and plants were fixed at various developmental stages in OsO4, pH 6.1, and embedded in vestopal. Sections were examined in the electron microscope. Comparison of basophile and non-basophile cytoplasms leads to the conclusion that cytoplasmic particles of 150 to 200 A in diameter are responsible for basophilia. The possibility of these particles being ribosomes is discussed and confirmed. The present paper also describes some observations on the fine structure of other cellular components of this fungus, such as nuclei, mitochondria, various granules, and flagella. PMID:13801597

  16. Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells.

    PubMed

    Mhlanga, Musa M; Tyagi, Sanjay

    2006-01-01

    Imaging products of gene expression in live cells will provide unique insights into the biology of cells. Molecular beacons make attractive probes for imaging mRNA in live cells as they can report the presence of an RNA target by turning on the fluorescence of a quenched fluorophore. However, when oligonucleotide probes are introduced into cells, they are rapidly sequestered in the nucleus, making the detection of cytoplasmic mRNAs difficult. We have shown that if a molecular beacon is linked to a tRNA, it stays in the cytoplasm and permits detection of cytoplasmic mRNAs. Here we describe two methods of linking molecular beacons to tRNA and show how the joint molecules can be used for imaging an mRNA that is normally present in the cytoplasm in live cultured cells. This protocol should take a total of 4 d to complete.

  17. Novel nuclear-cytoplasmic interaction in wheat (Triticum aestivum) induces vigorous plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interspecific hybridization can be considered an accelerator of evolution, otherwise a slow process, solely dependent on mutation and recombination. Upon interspecific hybridization, several novel interactions between nuclear and cytoplasmic genomes emerge which provide additional sources of diversi...

  18. Sequential processing of lysosomal acid phosphatase by a cytoplasmic thiol proteinase and a lysosomal aspartyl proteinase.

    PubMed Central

    Gottschalk, S; Waheed, A; Schmidt, B; Laidler, P; von Figura, K

    1989-01-01

    BHK cells expressing human lysosomal acid phosphatase (LAP) transport LAP to lysosomes as an integral membrane protein. In lysosomes LAP is released from the membrane by proteolytic processing, which involves at least two cleavages at the C terminus of LAP. The first cleavage is catalysed by a thiol proteinase at the outside of the lysosomal membrane and removes the bulk of the cytoplasmic tail of LAP. The second cleavage is catalysed by an aspartyl proteinase inside the lysosomes and releases the luminal part of LAP from the membrane-spanning domain. The first cleavage at the cytoplasmic side of the lysosomal membrane depends on acidification of lysosomes and the second cleavage inside the lysosomes depends on prior processing of the cytoplasmic tail. These results suggest that the cytoplasmic tail controls the conformation of the luminal portion of LAP and vice versa. Images PMID:2684640

  19. Beyond Histone and Deacetylase: An Overview of Cytoplasmic Histone Deacetylases and Their Nonhistone Substrates

    PubMed Central

    Yao, Ya-Li; Yang, Wen-Ming

    2011-01-01

    Acetylation of lysines is a prominent form of modification in mammalian proteins. Deacetylation of proteins is catalyzed by histone deacetylases, traditionally named after their role in histone deacetylation, transcriptional modulation, and epigenetic regulation. Despite the link between histone deacetylases and chromatin structure, some of the histone deacetylases reside in various compartments in the cytoplasm. Here, we review how these cytoplasmic histone deacetylases are regulated, the identification of nonhistone substrates, and the functional implications of their nondeacetylase enzymatic activities. PMID:21234400

  20. Natural selection with nuclear and cytoplasmic transmission. II. Tests with Drosophila from diverse populations.

    PubMed

    Clark, A G

    1985-09-01

    Observations of intraspecific variation in organelle DNA have prompted a renewed interest in the evolutionary consequences of cytoplasmically transmitted factors. Attempts to quantify the significance of cytoplasmic effects are frequently limited by the difficulty in partitioning the cause of reciprocal cross differences among a series of possibilities. In the experiment reported here the nuclear genomes of a set of six lines of Drosophila melanogaster from diverse geographic locations were replaced in a series of cytoplasms. The segregation of the SM5 balancer chromosome was scored in a factorial design, and the data allowed a partitioning of variance such that cytoplasmic effects were distinguished from maternal effects and meiotic drive. An attempt was made to avoid the confounding problem of hybrid dysgenesis by performing the entire experiment (including chromosomal extractions) in a P cytotype. Results indicated a significant contribution of cytoplasm to the variance in SM5 segregation. Error variance showed an increasing trend as the experiment proceeded, and additional tests indicated that this was due to an accumulation of chromosomal mutations. These findings are interpreted in light of the population genetic theory that addresses the maintenance of cytoplasmic polymorphism.

  1. Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network

    PubMed Central

    Luo, Weiwei; Lieu, Zi Zhao; Manser, Ed; Bershadsky, Alexander D.; Sheetz, Michael P.

    2016-01-01

    A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. PMID:27760153

  2. Exploration of cytoplasmic inheritance as a contributor to maternal effects in Welsh Mountain sheep.

    PubMed

    Pritchard, Tracey; Cahalan, Christine; Ap Dewi, Ioan

    2008-01-01

    Cytoplasmic effects were investigated using a dataset comprising three breeding groups of Welsh Mountain sheep. The influences of cytoplasmic effects were investigated by comparing animal models with and without a random term representing cytoplasmic effects. The models were applied to the eight-week weight, scan weight (mean 152 days) and ultrasonically scanned muscle and fat depth. The animal model included the random effects of animals and the maternal additive genetic, maternal permanent environmental and maternal common environmental effects. In total there were 24 569, 10 509, 8389, 8369 records for the eight-week weight, scan weight, muscle depth and fat depth respectively. Four subsets were further analysed containing maternal lines with at least five, ten, fifteen and twenty animals/line. There was no evidence of cytoplasmic effects on eight-week weight and muscle depth. Cytoplasmic effects contributed 1-2% of phenotypic variance for scan-weight and fat depth, but the effect was generally non-significant (P >0.05). As the number of animals per maternal line increased, the magnitude of cytoplasmic effects also increased for these traits. Direct heritability estimates for the eight-week weight, scan weight, muscle depth and fat depth using the full dataset were 0.18, 0.25, 0.24, and 0.21 respectively.

  3. Cell fusion to study nuclear-cytoplasmic interactions in endothelial cell apoptosis.

    PubMed Central

    Polunovsky, V. A.; Ingbar, D. H.; Peterson, M.; Bitterman, P. B.

    1996-01-01

    Studies examining the regulation of nuclear rearrangements during apoptosis have led to conflicting results. Cytoplasmic control of nuclear events has been strongly suggested by cell-free experimental systems. In contrast, strict cytoplasmic control cannot account for the results of fibroblast-thymocyte fusion experiments in which dexamethasone induction of polykaryons led only to thymocyte nuclear apoptosis. Unresolved by these fusion studies was whether fibroblast nuclei were indifferent to heterologous cytoplasmic signals. Our objective was to resolve this discrepancy using cell fusion in a homologous system. Our strategy was to fuse endothelial cells with high levels of susceptibility to the induction of apoptosis (log phase cells arrested in G1 for 48 hours by isoleucine deprivation) with those manifesting low levels of susceptibility (serum-deprived, G0). Resultant fused and unfused cells were induced to undergo apoptosis by incubation with tumor necrosis factor-alpha and cycloheximide. Depending on the parental cell of origin, between 14 and 30% of dikaryons contained one apoptotic and one intact nucleus, indicating that strict cytoplasmic control was not occurring. In accord with this, the total frequency of nuclear apoptosis was unchanged after fusion. However, the distribution of apoptotic nuclei revealed a pronounced cytoplasmic influence, with a two- to fivefold increase in coordinate nuclear behavior. This pattern of nuclear apoptosis was consistent with a model of control in which both the state of nuclear susceptibility to apoptosis and expression of cytoplasmic pro-apoptotic regulators determined whether nuclear apoptosis would eventuate. Images Figure 3 Figure 4 PMID:8686735

  4. Anti-neutrophil cytoplasmic antibodies in rheumatoid arthritis: two case reports and review of literature

    PubMed Central

    2012-01-01

    Background Anti-neutrophil cytoplasmic antibodies are typically detected in anti-neutrophil cytoplasmic antibody associated vasculitis, but are also present in a number of chronic inflammatory non-vasculitic conditions like rheumatoid arthritis. Rare cases of granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis, a vasculitic disorder frequently associated with the presence of anti-neutrophil cytoplasmic antibodies) in patients with rheumatoid arthritis have been described in literature. Case presentation We report two middle-aged female patients with rheumatoid arthritis who developed anti-neutrophil cytoplasmic antibodies and symptoms reminiscent of granulomatosis with polyangiitis. Despite the lack of antibodies specific for proteinase 3 and the absence of a classical histology, we report a probable case of granulomatosis with polyangiitis in the first patient, and consider rheumatoid vasculitis in the second patient. Conclusion Taken together with previous reports, these cases highlight that anti-neutrophil cytoplasmic antibodies have to be evaluated very carefully in patients with rheumatoid arthritis. In this context, anti-neutrophil cytoplasmic antibodies detected by indirect immunofluorescence appear to have a low diagnostic value for granulomatosis with polyangiitis. Instead they may have prognostic value for assessing the course of rheumatoid arthritis. PMID:23253567

  5. Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

    SciTech Connect

    Liu, Yan; Zheng, Bin; Zhang, Xin-hua; Nie, Chan-juan; Li, Yong-hui; Wen, Jin-kun

    2013-06-28

    Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs.

  6. Phage Display Based Cloning of Proteins Interacting with the Cytoplasmic Tail of Membrane Immunoglobulins

    PubMed Central

    Geisberger, Roland; Prlic, Martin; Achatz-Straussberger, Gertrude; Oberndorfer, Iris; Luger, Elke; Lamers, Marinus; Crameri, Reto; Appenzeller, Ulrich; Wienands, Jürgen; Breitenbach, Michael; Ferreira, Fatima; Achatz, Gernot

    2002-01-01

    The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Igα/Igβ heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation. PMID:12885153

  7. Cytoplasmic long noncoding RNAs are frequently bound to and degraded at ribosomes in human cells.

    PubMed

    Carlevaro-Fita, Joana; Rahim, Anisa; Guigó, Roderic; Vardy, Leah A; Johnson, Rory

    2016-06-01

    Recent footprinting studies have made the surprising observation that long noncoding RNAs (lncRNAs) physically interact with ribosomes. However, these findings remain controversial, and the overall proportion of cytoplasmic lncRNAs involved is unknown. Here we make a global, absolute estimate of the cytoplasmic and ribosome-associated population of stringently filtered lncRNAs in a human cell line using polysome profiling coupled to spike-in normalized microarray analysis. Fifty-four percent of expressed lncRNAs are detected in the cytoplasm. The majority of these (70%) have >50% of their cytoplasmic copies associated with polysomal fractions. These interactions are lost upon disruption of ribosomes by puromycin. Polysomal lncRNAs are distinguished by a number of 5' mRNA-like features, including capping and 5'UTR length. On the other hand, nonpolysomal "free cytoplasmic" lncRNAs have more conserved promoters and a wider range of expression across cell types. Exons of polysomal lncRNAs are depleted of endogenous retroviral insertions, suggesting a role for repetitive elements in lncRNA localization. Finally, we show that blocking of ribosomal elongation results in stabilization of many associated lncRNAs. Together these findings suggest that the ribosome is the default destination for the majority of cytoplasmic long noncoding RNAs and may play a role in their degradation.

  8. Novel origin of lamin-derived cytoplasmic intermediate filaments in tardigrades

    PubMed Central

    Hering, Lars; Bouameur, Jamal-Eddine; Reichelt, Julian; Magin, Thomas M; Mayer, Georg

    2016-01-01

    Intermediate filament (IF) proteins, including nuclear lamins and cytoplasmic IF proteins, are essential cytoskeletal components of bilaterian cells. Despite their important role in protecting tissues against mechanical force, no cytoplasmic IF proteins have been convincingly identified in arthropods. Here we show that the ancestral cytoplasmic IF protein gene was lost in the entire panarthropod (onychophoran + tardigrade + arthropod) rather than arthropod lineage and that nuclear, lamin-derived proteins instead acquired new cytoplasmic roles at least three times independently in collembolans, copepods, and tardigrades. Transcriptomic and genomic data revealed three IF protein genes in the tardigrade Hypsibius dujardini, one of which (cytotardin) occurs exclusively in the cytoplasm of epidermal and foregut epithelia, where it forms belt-like filaments around each epithelial cell. These results suggest that a lamin derivative has been co-opted to enhance tissue stability in tardigrades, a function otherwise served by cytoplasmic IF proteins in all other bilaterians. DOI: http://dx.doi.org/10.7554/eLife.11117.001 PMID:26840051

  9. Structure of bacterial cytoplasmic chemoreceptor arrays and implications for chemotactic signaling.

    PubMed

    Briegel, Ariane; Ladinsky, Mark S; Oikonomou, Catherine; Jones, Christopher W; Harris, Michael J; Fowler, Daniel J; Chang, Yi-Wei; Thompson, Lynmarie K; Armitage, Judith P; Jensen, Grant J

    2014-01-01

    Most motile bacteria sense and respond to their environment through a transmembrane chemoreceptor array whose structure and function have been well-studied, but many species also contain an additional cluster of chemoreceptors in their cytoplasm. Although the cytoplasmic cluster is essential for normal chemotaxis in some organisms, its structure and function remain unknown. Here we use electron cryotomography to image the cytoplasmic chemoreceptor cluster in Rhodobacter sphaeroides and Vibrio cholerae. We show that just like transmembrane arrays, cytoplasmic clusters contain trimers-of-receptor-dimers organized in 12-nm hexagonal arrays. In contrast to transmembrane arrays, however, cytoplasmic clusters comprise two CheA/CheW baseplates sandwiching two opposed receptor arrays. We further show that cytoplasmic fragments of normally transmembrane E. coli chemoreceptors form similar sandwiched structures in the presence of molecular crowding agents. Together these results suggest that the 12-nm hexagonal architecture is fundamentally important and that sandwiching and crowding can replace the stabilizing effect of the membrane. DOI: http://dx.doi.org/10.7554/eLife.02151.001.

  10. The cytoplasm as a radiation target: an in silico study of microbeam cell irradiation.

    PubMed

    Byrne, H L; Domanova, W; McNamara, A L; Incerti, S; Kuncic, Z

    2015-03-21

    We performed in silico microbeam cell irradiation modelling to quantitatively investigate ionisations resulting from soft x-ray and alpha particle microbeams targeting the cytoplasm of a realistic cell model. Our results on the spatial distribution of ionisations show that as x-rays are susceptible to scatter within a cell that can lead to ionisations in the nucleus, soft x-ray microbeams may not be suitable for investigating the DNA damage response to radiation targeting the cytoplasm alone. In contrast, ionisations from an ideal alpha microbeam are tightly confined to the cytoplasm, but a realistic alpha microbeam degrades upon interaction with components upstream of the cellular target. Thus it is difficult to completely rule out a contribution from alpha particle hits to the nucleus when investigating DNA damage response to cytoplasmic irradiation. We find that although the cytoplasm targeting efficiency of an alpha microbeam is better than that of a soft x-ray microbeam (the probability of stray alphas hitting the nucleus is 0.2% compared to 3.6% for x-rays), stray alphas produce more ionisations in the nucleus and thus have greater potential for initiating damage responses therein. Our results suggest that observed biological responses to cytoplasmic irradiation include a small component that can be attributed to stray ionisations in the nucleus resulting from the stochastic nature of particle interactions that cause out-of-beam scatter. This contribution is difficult to isolate experimentally, thus demonstrating the value of the in silico approach.

  11. The internalization signal in the cytoplasmic tail of lysosomal acid phosphatase consists of the hexapeptide PGYRHV.

    PubMed Central

    Lehmann, L E; Eberle, W; Krull, S; Prill, V; Schmidt, B; Sander, C; von Figura, K; Peters, C

    1992-01-01

    Lysosomal acid phosphatase (LAP) is rapidly internalized from the cell surface due to a tyrosine-containing internalization signal in its 19 amino acid cytoplasmic tail. Measuring the internalization of a series of LAP cytoplasmic tail truncation and substitution mutants revealed that the N-terminal 12 amino acids of the cytoplasmic tail are sufficient for rapid endocytosis and that the hexapeptide 411-PGYRHV-416 is the tyrosine-containing internalization signal. Truncation and substitution mutants of amino acid residues following Val416 can prevent internalization even though these residues do not belong to the internalization signal. It was shown recently that part of the LAP cytoplasmic tail peptide corresponding to 410-PPGY-413 forms a well-ordered beta turn structure in solution. Two-dimensional NMR spectroscopy of two modified LAP tail peptides, in which the single tyrosine was substituted either by phenylalanine or by alanine, revealed that the tendency to form a beta turn is reduced by 25% in the phenylalanine-containing peptide and by approximately 50% in the alanine-containing mutant peptide. Our results suggest, that in the short cytoplasmic tail of LAP tyrosine is required for stabilization of the right turn and that the aromatic ring system of the tyrosine residue is a contact point to the putative cytoplasmic receptor. Images PMID:1425575

  12. The bacterial cytoplasm has glass-like properties and is fluidized by metabolic activity

    NASA Astrophysics Data System (ADS)

    Parry, Brad; Surovtsev, Ivan; Cabeen, Matthew; O'Hern, Corey; Dufresne, Eric; Jacobs-Wagner, Christine

    2014-03-01

    In eukaryotes, active transport involves motor proteins and cytoskeletal filaments. In contrast, bacteria (which lack cytoskeletal motor proteins) are thought to rely on diffusion for molecular transport, though the physical properties of the bacterial cytoplasm are poorly understood. Through single particle tracking of foreign particles of different sizes, we have found that the bacterial cytoplasm exhibits striking similarities to glass-forming liquids. Glass-forming liquids are noted for their metastability near the glass transition where their behavior changes from liquid-like to amorphous solid with even small perturbations. Particles of different sizes exhibit distinct dynamics and their mobility changes from fluid-like to glassy with increasing size. This size dependency provides an explanation for previous reports of both normal and anomalous diffusion in the bacterial cytoplasm. Moreover, we find that cellular metabolism attenuates the glassy properties of the bacterial cytoplasm. As a result, components that would otherwise be caged in narrow regions of confinement are able to explore the cytoplasmic space under metabolically active conditions. These findings have broad implications for our understanding of bacterial physiology as the glassy behavior of the cytoplasm impacts all intracellular processes involving large cellular components. Supported by the Howard Hughes Medical Institute.

  13. The cytoplasm as a radiation target: an in silico study of microbeam cell irradiation

    NASA Astrophysics Data System (ADS)

    Byrne, H. L.; Domanova, W.; McNamara, A. L.; Incerti, S.; Kuncic, Z.

    2015-03-01

    We performed in silico microbeam cell irradiation modelling to quantitatively investigate ionisations resulting from soft x-ray and alpha particle microbeams targeting the cytoplasm of a realistic cell model. Our results on the spatial distribution of ionisations show that as x-rays are susceptible to scatter within a cell that can lead to ionisations in the nucleus, soft x-ray microbeams may not be suitable for investigating the DNA damage response to radiation targeting the cytoplasm alone. In contrast, ionisations from an ideal alpha microbeam are tightly confined to the cytoplasm, but a realistic alpha microbeam degrades upon interaction with components upstream of the cellular target. Thus it is difficult to completely rule out a contribution from alpha particle hits to the nucleus when investigating DNA damage response to cytoplasmic irradiation. We find that although the cytoplasm targeting efficiency of an alpha microbeam is better than that of a soft x-ray microbeam (the probability of stray alphas hitting the nucleus is 0.2% compared to 3.6% for x-rays), stray alphas produce more ionisations in the nucleus and thus have greater potential for initiating damage responses therein. Our results suggest that observed biological responses to cytoplasmic irradiation include a small component that can be attributed to stray ionisations in the nucleus resulting from the stochastic nature of particle interactions that cause out-of-beam scatter. This contribution is difficult to isolate experimentally, thus demonstrating the value of the in silico approach.

  14. Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer

    PubMed Central

    Santacana, Maria; Fernández-Hernández, Rita; Gatius, Sònia; Pedraza, Neus; Pallarés, Judit; Cemeli, Tània; Valls, Joan; Tarres, Marc; Ferrezuelo, Francisco; Dolcet, Xavier; Matias-Guiu, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) is a proto-oncogen amplified in many different cancers and nuclear accumulation of Ccnd1 is a characteristic of tumor cells. Ccnd1 activates the transcription of a large set of genes involved in cell cycle progress and proliferation. However, Ccnd1 also targets cytoplasmic proteins involved in the regulation of cell migration and invasion. In this work, we have analyzed by immunohistochemistry the localization of Ccnd1 in endometrial, breast, prostate and colon carcinomas with different types of invasion. The number of cells displaying membranous or cytoplasmic Ccnd1 was significantly higher in peripheral cells than in inner cells in both collective and pushing invasion patterns of endometrial carcinoma, and in collective invasion pattern of colon carcinoma. Also, the cytoplasmic localization of Ccnd1 was higher when tumors infiltrated as single cells, budding or small clusters of cells. To evaluate cytoplasmic function of cyclin D1, we have built a variant (Ccnd1-CAAX) that remains attached to the cell membrane therefore sequestering this cyclin in the cytoplasm. Tumor cells harboring Ccnd1-CAAX showed high levels of invasiveness and metastatic potential compared to those containing the wild type allele of Ccnd1. However, Ccnd1-CAAX expression did not alter proliferative rates of tumor cells. We hypothesize that the role of Ccnd1 in the cytoplasm is mainly associated with the invasive capability of tumor cells. Moreover, we propose that subcellular localization of Ccnd1 is an interesting guideline to measure cancer outcome. PMID:27105504

  15. Evaluation of line and breed of cytoplasm effects on performance of purebred Brangus cattle.

    PubMed

    Rohrer, G A; Taylor, J F; Sanders, J O; Thallman, R M

    1994-11-01

    Substantial differences between reciprocally crossed Bos taurus x Bos indicus calves for birth, weaning, and yearling weights have been reported. To determine whether cytoplasmic inheritance is responsible for a portion of these differences, field records for birth and weaning weight (n = 7,353) and postweaning average daily gain (n = 2,746) from registered Brangus calves were analyzed. An animal model that included maternal effects was fit for each trait. Breed of cytoplasmic origin was fit as a fixed effect and coded as Angus, Brahman, or unknown. Cytoplasmic line within each breed of origin was treated as a random effect. Variance components for random effects were estimated using derivative-free REML procedures. Line of cytoplasm accounted for less than .002% of the phenotypic variance in all three traits. Estimates for cytoplasmic breed of origin effects were small in magnitude, and contrasts tested (Angus vs Brahman and Angus vs Unknown) were not significant (P > .10). Estimates of heritability of direct (maternal) effects were .36 (.20), .41 (.27), and .21 (.08) for birth weight, weaning weight, and postweaning average daily gain, respectively. Estimates of genetic correlations between direct and maternal effects ranged from -.27 for postweaning average daily gain to -.58 for birth weight. No evidence for breed or line within breed of cytoplasmic origin effects was detected in these data. PMID:7730171

  16. Microtubule-microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes.

    PubMed

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill; Gelfand, Vladimir I

    2016-08-23

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

  17. Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm.

    PubMed

    Steed, P Ryan; Kraft, Kaitlin A; Fillingame, Robert H

    2014-11-25

    H(+)-transporting F1F0 ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F0 and F1. H(+) transport at the subunit a-c interface in transmembranous F0 drives rotation of a cylindrical c10 oligomer within the membrane, which is coupled to rotation of subunit γ within the α3β3 sector of F1 to mechanically drive ATP synthesis. F1F0 functions in a reversible manner, with ATP hydrolysis driving H(+) transport. ATP-driven H(+) transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag(+) and/or Cd(+2) with the substituted sulfhydryl groups. The H(+) transport pathway mapped via these Ag(+)(Cd(+2))-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H(+) release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd(+2) directly inhibits passive H(+) transport mediated by F0 reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H(+) transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H(+) transport likely interact in a single structural domain, which then functions in gating H(+) release to the cytoplasm.

  18. Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm.

    PubMed

    Hasan, Mahadi; Nishimoto, Akinori; Ohgita, Takashi; Hama, Susumu; Kashida, Hiromu; Asanuma, Hiroyuki; Kogure, Kentaro

    2016-04-28

    Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules. PMID:26944781

  19. Cytoplasmic SET induces tau hyperphosphorylation through a decrease of methylated phosphatase 2A

    PubMed Central

    2014-01-01

    Background The neuronal cytoplasmic localization of SET, an inhibitor of the phosphatase 2A (PP2A), results in tau hyperphosphorylation in the brains of Alzheimer patients through mechanisms that are still not well defined. Results We used primary neurons and mouse brain slices to show that SET is translocated to the cytoplasm in a manner independent of both its cleavage and over-expression. The localization of SET in the cytoplasm, either by the translocation of endogenous SET or by internalization of the recombinant full-length SET protein, induced tau hyperphosphorylation. Cytoplasmic recombinant full-length SET in mouse brain slices induced a decrease of PP2A activity through a decrease of methylated PP2A levels. The levels of methylated PP2A were negatively correlated with tau hyperphosphorylation at Ser-202 but not with the abnormal phosphorylation of tau at Ser-422. Conclusions The presence of full-length SET in the neuronal cytoplasm is sufficient to impair PP2A methylation and activity, leading to tau hyperphosphorylation. In addition, our data suggest that tau hyperphosphorylation is regulated by different mechanisms at distinct sites. The translocation of SET to the neuronal cytoplasm, the low activity of PP2A, and tau hyperphosphorylation are associated in the brains of Alzheimer patients. Our data show a link between the translocation of SET in the cytoplasm and the decrease of methylated PP2A levels leading to a decrease of PP2A activity and tau hyperphosphorylation. This chain of events may contribute to the pathogenesis of Alzheimer disease. PMID:24981783

  20. Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles

    PubMed Central

    1994-01-01

    Two microtubule-stimulated ATPases, cytoplasmic dynein, and kinesin, are believed to be responsible for the intracellular movement of membrane-bound organelles in opposite directions along microtubules. An unresolved component of this model is the mechanism by which cells regulate these two motors to direct various membrane-bound organelles to their proper locations. To determine if phosphorylation may play a role in the regulation of cytoplasmic dynein, the in vivo phosphorylation state of cytoplasmic dynein from two cellular pools was examined. The entire cellular pool of brain cytoplasmic dynein was metabolically labeled by the infusion of [32P]orthophosphate into the cerebrospinal fluid of rat brain ventricles. To characterize the phosphorylation of dynein associated with anterograde membrane-bound organelles, the optic nerve fast axonal transport system was used. Using a monoclonal antibody to the 74-kD polypeptide of brain cytoplasmic dynein, the native dynein complex was immunoprecipitated from the radiolabled tissue extracts. Autoradiographs of one and two dimensional gels showed labeling of nearly all of the polypeptide isoforms of cytoplasmic dynein from rat brain. These polypeptides are phosphorylated on serine residues. Comparison of the amount of 32P incorporated into the dynein polypeptides revealed differences in the phosphorylation of dynein polypeptides from the anterograde and the cellular pools. Most interestingly, the 530-kD heavy chain of dynein appears to be phosphorylated to a lesser extent in the anterograde pool than in the cellular pool. Since the anterograde pool contains inactive dynein, while the entire cellular pool contains both inactive and active dynein, these results are consistent with the hypothesis that phosphorylation regulates the functional activity of cytoplasmic dynein. PMID:7528220

  1. Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2015-06-19

    As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm. PMID:25944907

  2. Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm.

    PubMed

    Hasan, Mahadi; Nishimoto, Akinori; Ohgita, Takashi; Hama, Susumu; Kashida, Hiromu; Asanuma, Hiroyuki; Kogure, Kentaro

    2016-04-28

    Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules.

  3. Detection of beta-tubulin in the cytoplasm of the interphasic Entamoeba histolytica trophozoites.

    PubMed

    Gómez-Conde, Eduardo; Vargas-Mejía, Miguel Ángel; Díaz-Orea, María Alicia; Hernández-Rivas, Rosaura; Cárdenas-Perea, María Elena; Guerrero-González, Tayde; González-Barrios, Juan Antonio; Montiel-Jarquín, Álvaro José

    2016-08-01

    It is known that the microtubules (MT) of Entamoeba histolytica trophozoites form an intranuclear mitotic spindle. However, electron microscopy studies and the employment of anti-beta-tubulin (β-tubulin) antibodies have not exhibited these cytoskeletal structures in the cytoplasm of these parasites. The purpose of this work was to detect β-tubulin in the cytoplasm of interphasic E. histolytica trophozoites. Activated or non-activated HMI-IMSS-strain E. histolytica trophozoites were used and cultured for 72 h at 37 °C in TYI-S-33 medium, and then these were incubated with the anti-β-tubulin antibody of E. histolytica. The anti-β-tubulin antibody reacted with the intranuclear mitotic spindle of E. histolytica-activated trophozoites as control. In contrast, in non-activated interphasic parasites, anti-β-tubulin antibody reacted with diverse puntiform structures in the cytoplasm and with ring-shaped structures localized in the cytoplasm, cellular membrane and endocytic stomas. In this work, for the first time, the presence of β-tubulin is shown in the cytoplasm of E. histolytica trophozoites.

  4. Cytoplasmic Sequestration of Rel Proteins by IκBα Requires CRM1-Dependent Nuclear Export

    PubMed Central

    Tam, Winnie F.; Lee, Linda H.; Davis, Laura; Sen, Ranjan

    2000-01-01

    Rel and IκB protein families form a complex cellular regulatory network. A major regulatory function of IκB proteins is to retain Rel proteins in the cell cytoplasm. In addition, IκB proteins have also been postulated to serve nuclear functions. These include the maintenance of inducible NF-κB-dependent gene transcription, as well as termination of inducible transcription. We show that IκBα shuttles between the nucleus and the cytoplasm, utilizing the nuclear export receptor CRM1. A CRM1-binding export sequence was identified in the N-terminal domain of IκBα but not in that of IκBβ or IκBɛ. By reconstituting major aspects of NF-κB–IκB sequestration in yeast, we demonstrate that cytoplasmic retention of p65 (also called RelA) by IκBα requires Crm1p-dependent nuclear export. In mammalian cells, inhibition of CRM1 by leptomycin B resulted in nuclear localization of cotransfected p65 and IκBα in COS cells and enhanced nuclear relocation of endogenous p65 in T cells. These observations suggest that the main function of IκBα is that of a nuclear export chaperone rather than a cytoplasmic tether. We propose that the nucleus is the major site of p65-IκBα association, from where these complexes must be exported in order to create the cytoplasmic pool. PMID:10688673

  5. Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte

    PubMed Central

    1994-01-01

    The unidirectional movements of the microtubule-associated motors, dyneins, and kinesins, provide an important mechanism for the positioning of cellular organelles and molecules. An intriguing possibility is that this mechanism may underlie the directed transport and asymmetric positioning of morphogens that influence the development of multicellular embryos. In this report, we characterize the Drosophila gene, Dhc64C, that encodes a cytoplasmic dynein heavy chain polypeptide. The primary structure of the Drosophila cytoplasmic dynein heavy chain polypeptide has been determined by the isolation and sequence analysis of overlapping cDNA clones. Drosophila cytoplasmic dynein is highly similar in sequence and structure to cytoplasmic dynein isoforms reported for other organisms. The Dhc64C dynein transcript is differentially expressed during development with the highest levels being detected in the ovaries of adult females. Within the developing egg chambers of the ovary, the dynein gene is predominantly transcribed in the nurse cell complex. In contrast, the encoded dynein motor protein displays a striking accumulation in the single cell that will develop as the oocyte. The temporal and spatial pattern of dynein accumulation in the oocyte is remarkably similar to that of several maternal effect gene products that are essential for oocyte differentiation and axis specification. This distribution and its disruption by specific maternal effect mutations lends support to recent models suggesting that microtubule motors participate in the transport of these morphogens from the nurse cell cytoplasm to the oocyte. PMID:8089180

  6. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes

    PubMed Central

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S.; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming. PMID:27472658

  7. The Role of Preassembled Cytoplasmic Complexes in Assembly of Flagellar Dynein Subunits

    PubMed Central

    Fowkes, Mary Elizabeth; Mitchell, David Rees

    1998-01-01

    Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCβ. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete Mr ≃ 2 × 106 dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment. PMID:9725897

  8. Detection of beta-tubulin in the cytoplasm of the interphasic Entamoeba histolytica trophozoites.

    PubMed

    Gómez-Conde, Eduardo; Vargas-Mejía, Miguel Ángel; Díaz-Orea, María Alicia; Hernández-Rivas, Rosaura; Cárdenas-Perea, María Elena; Guerrero-González, Tayde; González-Barrios, Juan Antonio; Montiel-Jarquín, Álvaro José

    2016-08-01

    It is known that the microtubules (MT) of Entamoeba histolytica trophozoites form an intranuclear mitotic spindle. However, electron microscopy studies and the employment of anti-beta-tubulin (β-tubulin) antibodies have not exhibited these cytoskeletal structures in the cytoplasm of these parasites. The purpose of this work was to detect β-tubulin in the cytoplasm of interphasic E. histolytica trophozoites. Activated or non-activated HMI-IMSS-strain E. histolytica trophozoites were used and cultured for 72 h at 37 °C in TYI-S-33 medium, and then these were incubated with the anti-β-tubulin antibody of E. histolytica. The anti-β-tubulin antibody reacted with the intranuclear mitotic spindle of E. histolytica-activated trophozoites as control. In contrast, in non-activated interphasic parasites, anti-β-tubulin antibody reacted with diverse puntiform structures in the cytoplasm and with ring-shaped structures localized in the cytoplasm, cellular membrane and endocytic stomas. In this work, for the first time, the presence of β-tubulin is shown in the cytoplasm of E. histolytica trophozoites. PMID:27156446

  9. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.

    PubMed

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji; Kimura, Akatsuki

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming. PMID:27472658

  10. Sumoylation is Required for the Cytoplasmic Accumulation of a Subset of mRNAs

    PubMed Central

    Zhang, Hui; Mahadevan, Kohila; Palazzo, Alexander F.

    2014-01-01

    In order to discover novel proteins that promote the nuclear export of newly synthesized mRNAs in mammalian cells, we carried out a limited RNAi screen for proteins required for the proper cytoplasmic distribution of a model intronless mRNA. From this screen we obtained two hits, Ubc9 (SUMO-conjugating E2 enzyme) and GANP (germinal center-associated nuclear protein). Depletion of Ubc9 inhibited the proper cytoplasmic distribution of certain overexpressed intronless mRNAs, while depletion of GANP affected all tested mRNAs. Depletion of Sae1, which is also required for sumoylation, partially inhibited the cytoplasmic distribution of our model mRNA. Interestingly, the block in cytoplasmic accumulation in Ubc9-depleted cells could be overcome if an intron was incorporated into the mRNA. Surprisingly, Ubc9-depleted cells had normal nuclear export of newly synthesized intronless mRNAs, indicating that the observed accumulation of the model mRNA in the nuclei of transfected cells was likely due to some more general perturbation. Indeed, depletion of Ubc9, coupled with the overexpression of the intronless mRNAs, caused the redistribution of the nuclear speckle protein SC35 to cytoplasmic foci. Our results suggest that sumoylation may play a role in the proper assembly of mRNPs and/or the distribution of key RNA binding proteins, and may thus contribute to general protein expression patterns. PMID:25333844

  11. Detection of gravity-induced polarity of cytoplasmic streaming in Chara

    NASA Technical Reports Server (NTRS)

    Staves, M. P.; Wayne, R.; Leopold, A. C.

    1995-01-01

    Gravity induces a polarity of cytoplasmic streaming in vertically-oriented internodal cells of characean algae. The motive force that powers cytoplasmic streaming is generated at the ectoplasmic/endoplasmic interface. The velocity of streaming, which is about 100 micrometers/s at this interface, decreases with distance from the interface on either side of the cell to 0 micrometers/s near the middle. Therefore, when discussing streaming velocity it is necessary to specify the tangential plane through the cell in which streaming is being measured. This is easily done with a moderate resolution light microscope (which has a lateral resolution of 0.6 micrometers and a depth of field of 1.4 micrometers), but is obscured when using any low resolution technique, such as low magnification light microscopy or laser Doppler spectroscopy. In addition, the effect of gravity on the polarity of cytoplasmic streaming declines with increasing physiological age of isolated cells. Using a classical mechanical analysis, we show that the effect of gravity on the polarity of cytoplasmic streaming cannot result from the effect of gravity acting directly on individual cytoplasmic particles. We suggest that gravity may best be perceived by the entire cell at the plasma membrane-extracellular matrix junction.

  12. Cytoplasmic flows as signatures for the mechanics of mitotic spindle positioning

    NASA Astrophysics Data System (ADS)

    Nazockdast, Ehssan; Rahimian, Abtin; Needleman, Daniel; Shelley, Michael

    2015-11-01

    The proper positioning of the mitotic spindle is crucial for asymmetric cell division and generating cell diversity during development. We use dynamic simulations to study the cytoplasmic flows generated by three possible active forcing mechanisms involved in positioning of the mitotic spindle in the first cell division of C.elegans embryo namely cortical pulling, cortical pushing, and cytoplasmic pulling mechanisms. The numerical platform we have developed for simulating cytoskeletal assemblies is the first to incorporate the interactions between the fibers and other intracellular bodies with the cytoplasmic fluid, while also accounting for their polymerization, and interactions with motor proteins. The hydrodynamic interactions are computed using boundary integral methods in Stokes flow coupled with highly efficient fast summation techniques that reduce the computational cost to scale linearly with the number of fibers and other bodies. We show that although all three force transduction mechanisms predict proper positioning and orientation of the mitotic spindle, each model produces a different signature in its induced cytoplasmic flow and MT conformation. We suggest that cytoplasmic flows and MT conformation can be used to differentiate between these mechanisms.

  13. Cytoplasmic calcium levels in protoplasts from the cap and elongation zone of maize roots

    NASA Technical Reports Server (NTRS)

    Kiss, H. G.; Evans, M. L.; Johnson, J. D.

    1991-01-01

    Calcium has been implicated as a key component in the signal transduction process of root gravitropism. We measured cytoplasmic free calcium in protoplasts isolated from the elongation zone and cap of primary roots of light-grown, vertically oriented seedlings of Zea mays L. Protoplasts were loaded with the penta-potassium salts of fura-2 and indo-1 by incubation in acidic solutions of these calcium indicators. Loading increased with decreasing pH but the pH dependence was stronger for indo-1 than for fura-2. In the case of fura-2, loading was enhanced only at the lowest pH (4.5) tested. Dyes loaded in this manner were distributed predominantly in the cytoplasm as indicated by fluorescence patterns. As an alternative method of loading, protoplasts were incubated with the acetoxymethylesters of fura-2 and indo-1. Protoplasts loaded by this method exhibited fluorescence both in the cytoplasm and in association with various organelles. Cytoplasmic calcium levels measured using spectrofluorometry, were found to be 160 +/- 40 nM and 257 +/- 27 nM, respectively, in populations of protoplasts from the root cap and elongation zone. Cytoplasmic free calcium did not increase upon addition of calcium to the incubation medium, indicating that the passive permeability to calcium was low.

  14. Interactions of cations with the cytoplasmic pores of inward rectifier K(+) channels in the closed state.

    PubMed

    Inanobe, Atsushi; Nakagawa, Atsushi; Kurachi, Yoshihisa

    2011-12-01

    Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K(+) (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg(2+) by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg(2+) when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues by Mg(2+) indicates that the mode of binding of Mg(2+) is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore. PMID:21982822

  15. Tubulin dynamics during the cytoplasmic cohesiveness cycle in artificially activated sea urchin eggs.

    PubMed

    Coffe, G; Foucault, G; Raymond, M N; Pudles, J

    1983-12-01

    Sedimentation studies and [3H]colchicine-binding assays have demonstrated a relationship between the cytoplasmic cohesiveness cycles and the changes in tubulin organization in Paracentrotus lividus eggs activated by 2.5 mM procaine. The same amount of tubulin (20-25% of the total egg tubulin) is involved in these cyclic process and appears to undergo polymerization and depolymerization cycles. Electron microscopy studies reveal that the microtubules formed during these cytoplasmic cohesiveness cycles are under a particulate form which is sedimentable at low speed. Activation experiments carried out in the presence of cytochalasin B (CB) show that the increase in the cytoplasmic cohesiveness is highly reduced while tubulin polymerization and depolymerization cycles and pronuclear centration are not affected. Although tubulin or actin polymerization can be independently triggered in procaine-activated eggs, the increase in cytoplasmic cohesiveness requires the polymerization of both proteins. However, the cytoplasmic cohesiveness cycles appear to be regulated by tubulin polymerization and depolymerization cycles. PMID:6641809

  16. Cytoplasmic long noncoding RNAs are frequently bound to and degraded at ribosomes in human cells

    PubMed Central

    Carlevaro-Fita, Joana; Rahim, Anisa; Guigó, Roderic; Vardy, Leah A.; Johnson, Rory

    2016-01-01

    Recent footprinting studies have made the surprising observation that long noncoding RNAs (lncRNAs) physically interact with ribosomes. However, these findings remain controversial, and the overall proportion of cytoplasmic lncRNAs involved is unknown. Here we make a global, absolute estimate of the cytoplasmic and ribosome-associated population of stringently filtered lncRNAs in a human cell line using polysome profiling coupled to spike-in normalized microarray analysis. Fifty-four percent of expressed lncRNAs are detected in the cytoplasm. The majority of these (70%) have >50% of their cytoplasmic copies associated with polysomal fractions. These interactions are lost upon disruption of ribosomes by puromycin. Polysomal lncRNAs are distinguished by a number of 5′ mRNA-like features, including capping and 5′UTR length. On the other hand, nonpolysomal “free cytoplasmic” lncRNAs have more conserved promoters and a wider range of expression across cell types. Exons of polysomal lncRNAs are depleted of endogenous retroviral insertions, suggesting a role for repetitive elements in lncRNA localization. Finally, we show that blocking of ribosomal elongation results in stabilization of many associated lncRNAs. Together these findings suggest that the ribosome is the default destination for the majority of cytoplasmic long noncoding RNAs and may play a role in their degradation. PMID:27090285

  17. Export of Precursor tRNAIle from the Nucleus to the Cytoplasm in Human Cells.

    PubMed

    Wei, Min; Zhao, Xia; Liu, Mi; Niu, Meijuan; Seif, Elias; Kleiman, Lawrence

    2016-01-01

    In the current concept, tRNA maturation in vertebrate cells, including splicing of introns, trimming of 5' leader and 3' trailer, and adding of CCA, is thought to occur exclusively in the nucleus. Here we provide evidence to challenge this concept. Unspliced intron-containing precursor tRNAIle was identified in Human Immunodeficiency Virus type 1 (HIV-1) virions, which are synthesized in the cytoplasm. Northern blot, confocal microscopy and quantitative RT-PCR further verified enrichment of this unspliced tRNAIle within the cytoplasm in human cells. In addition to containing an intron, the cytoplasmic precursor tRNAIle also contains a short incompletely processed 5´ leader and a 3´ trailer, which abundance is around 1000 fold higher than the nuclear precursor tRNAIle with long 5' leader and long 3' trailer. In vitro data also suggest that the cytoplasmic unspliced end-immature precursor tRNAIle could be processed by short isoform of RNase Z, but not long isoform of RNase Z. These data suggest that precursor tRNAs could export from the nucleus to the cytoplasm in human cells, instead of be processed only in the nucleus.

  18. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.

    PubMed

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji; Kimura, Akatsuki

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.

  19. Do cytoplasmic lipid droplets accumulate in immature oocytes from over-conditioned repeat breeder dairy heifers?

    PubMed

    Awasthi, H; Saravia, F; Rodríguez-Martínez, H; Båge, R

    2010-10-01

    One of the main sources of repeat breeding in dairy cattle, caused by fertilization failure or early embryonic death, is metabolic stress during lactation. Nutrition seems also to play a role when the condition is seen in heifers, where oocyte cytoplasmic maturation is impaired. To determine whether over conditioning affects oocyte morphology, immature oocytes were collected by ovum pick-up (OPU) twice weekly during 5 weeks from three over-conditioned repeat breeder dairy heifers (RBH) and two normal virgin heifers (VH, controls) of the Swedish Red breed, monitored by body weight and condition. Oocyte quality was assessed under stereomicroscope and further examined by transmission electron microscope for accumulation of cytoplasmic lipid deposits. After OPU, the RBH yielded more low quality oocytes (60% vs 52% for VH, p = 0.14). The relative occupancy of osmophilic lipid droplets in the cytoplasm was higher in oocytes of bad quality compared with good ones, especially in RBH (p = 0.08) but also in VH (p = 0.11). Moreover, the oocytes from over-conditioned RBH showed higher amounts of cytoplasmic lipid deposits both in good (p = 0.14) and, even more prominent, in bad quality oocytes (p = 0.06). Such accumulation of lipid droplets may imply increased sensitivity to oxidative stress, hinder cytoplasmic maturation and lead to subfertility, as accounted in over-conditioned repeat breeders of the Swedish Red breed. PMID:19735437

  20. Molecular analyses of nuclear-cytoplasmic interactions affecting plant growth and yield. Final technical report

    SciTech Connect

    Newton, K.J.

    1998-11-01

    Mitochondria have a central role in the production of cellular energy. The biogenesis and functioning of mitochondria depends on the expression of both mitochondrial and nuclear genes. One approach to investigating the role of nuclear-mitochondrial cooperation in plant growth and development is to identify combinations of nuclear and mitochondrial genomes that result in altered but sublethal phenotypes. Plants that have certain maize nuclear genotypes in combination with cytoplasmic genomes from more distantly-related teosintes can exhibit incompatible phenotypes, such as reduced plant growth and yield and cytoplasmic male sterility, as well as altered mitochondrial gene expression. The characterization of these nuclear-cytoplasmic interactions was the focus of this grant. The authors were investigating the effects of two maize nuclear genes, RcmI and Mct, on mitochondrial function and gene expression. Plants with the teosinte cytoplasms and homozygous for the recessive rcm allele are small (miniature) and-slow-growing and the kernels are reduced in size. The authors mapped this locus to molecular markers on chromosome 7 and attempted to clone this locus by transposon tagging. The effects of the nuclear-cytoplasmic interaction on mitochondrial function and mitochondrial protein profiles were also studied.

  1. Regulation of actin-dependent cytoplasmic motility by type II phytochrome occurs within seconds in Vallisneria gigantea epidermal cells.

    PubMed

    Takagi, Shingo; Kong, Sam-Geun; Mineyuki, Yoshinobu; Furuya, Masaki

    2003-02-01

    The effects of light on actin-dependent cytoplasmic motility in epidermal cells of green leaves of the aquatic angiosperm Vallisneria gigantea were investigated quantitatively using a custom-made dynamic image analyzer. Cytoplasmic motility was measured by monitoring changes in the brightness of individual pixels on digitized images taken sequentially under infrared light. Acceleration and deceleration of cytoplasmic motility were regulated photoreversibly by type II phytochrome(s). This phytochrome-dependent induction of cytoplasmic motility did not occur uniformly in cytoplasm but took place as scattered patches in which no particular organelles, including nucleus, existed. The induction became detectable at 2.5 s after the start of irradiation with pulsed red light. In cells exposed to microbeam irradiation, cytoplasmic motility was induced only in sites in the cytoplasm that were irradiated directly, whereas nonirradiated neighboring areas were unaffected. The effect was short-lived, disappearing within a few minutes, and no signal was transmitted from an irradiated cell to its neighbors. Anti-phytochrome antibody-responsive protein(s) was detectable in the leaf extract by immunoblot and zinc blot analyses and in cryosections of the epidermis by immunocytochemistry. Although the phytochrome-dependent cytoplasmic motility was blocked by exogenously applied latrunculin B or cytochalasins, treatment of the dark-adapted cells with Ca(2+)-chelating reagents induced the cytoplasmic motility. We have proposed a model for the phytochrome regulation of cytoplasmic motility as one of the earliest responses to a light stimulus. PMID:12566576

  2. Regulation of Actin-Dependent Cytoplasmic Motility by Type II Phytochrome Occurs within Seconds in Vallisneria gigantea Epidermal Cells

    PubMed Central

    Takagi, Shingo; Kong, Sam-Geun; Mineyuki, Yoshinobu; Furuya, Masaki

    2003-01-01

    The effects of light on actin-dependent cytoplasmic motility in epidermal cells of green leaves of the aquatic angiosperm Vallisneria gigantea were investigated quantitatively using a custom-made dynamic image analyzer. Cytoplasmic motility was measured by monitoring changes in the brightness of individual pixels on digitized images taken sequentially under infrared light. Acceleration and deceleration of cytoplasmic motility were regulated photoreversibly by type II phytochrome(s). This phytochrome-dependent induction of cytoplasmic motility did not occur uniformly in cytoplasm but took place as scattered patches in which no particular organelles, including nucleus, existed. The induction became detectable at 2.5 s after the start of irradiation with pulsed red light. In cells exposed to microbeam irradiation, cytoplasmic motility was induced only in sites in the cytoplasm that were irradiated directly, whereas nonirradiated neighboring areas were unaffected. The effect was short-lived, disappearing within a few minutes, and no signal was transmitted from an irradiated cell to its neighbors. Anti-phytochrome antibody–responsive protein(s) was detectable in the leaf extract by immunoblot and zinc blot analyses and in cryosections of the epidermis by immunocytochemistry. Although the phytochrome-dependent cytoplasmic motility was blocked by exogenously applied latrunculin B or cytochalasins, treatment of the dark-adapted cells with Ca2+-chelating reagents induced the cytoplasmic motility. We have proposed a model for the phytochrome regulation of cytoplasmic motility as one of the earliest responses to a light stimulus. PMID:12566576

  3. Complete mitochondrial genome sequence and identification of a candidate gene responsible for cytoplasmic male sterility in radish (Raphanus sativus L.) containing DCGMS cytoplasm.

    PubMed

    Park, Jee Young; Lee, Young-Pyo; Lee, Jonghoon; Choi, Beom-Soon; Kim, Sunggil; Yang, Tae-Jin

    2013-07-01

    A novel cytoplasmic male sterility (CMS) conferred by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its restorer-of-fertility gene (Rfd1) was previously reported in radish (Raphanus sativus L.). Its inheritance of fertility restoration and profiles of mitochondrial DNA (mtDNA)-based molecular markers were reported to be different from those of Ogura CMS, the first reported CMS in radish. The complete mitochondrial genome sequence (239,186 bp; GenBank accession No. KC193578) of DCGMS mitotype is reported in this study. Thirty-four protein-coding genes and three ribosomal RNA genes were identified. Comparative analysis of a mitochondrial genome sequence of DCGMS and previously reported complete sequences of normal and Ogura CMS mitotypes revealed various recombined structures of seventeen syntenic sequence blocks. Short-repeat sequences were identified in almost all junctions between syntenic sequence blocks. Phylogenetic analysis of three radish mitotypes showed that DCGMS was more closely related to the normal mitotype than to the Ogura mitotype. A single 1,551-bp unique region was identified in DCGMS mtDNA sequences and a novel chimeric gene, designated orf463, consisting of 128-bp partial sequences of cox1 gene and 1,261-bp unidentified sequences were found in the unique region. No other genes with a chimeric structure, a major feature of most characterized CMS-associated genes in other plant species, were found in rearranged junctions of syntenic sequence blocks. Like other known CMS-associated mitochondrial genes, the predicted gene product of orf463 contained 12 transmembrane domains. Thus, this gene product might be integrated into the mitochondrial membrane. In total, the results indicate that orf463 is likely to be a casual factor for CMS induction in radish containing the DCGMS cytoplasm.

  4. Alloplasmic male-sterile Brassica juncea with Enarthrocarpus lyratus cytoplasm and the introgression of gene(s) for fertility restoration from cytoplasm donor species.

    PubMed

    Banga, S S; Deol, J S; Banga, S K

    2003-05-01

    A new cytoplasmic male sterility (CMS) source in Brassica juncea (2n = 36; AABB) was developed by substituting its nucleus into the cytoplasm of Enarthrocarpus lyratus (2n = 20; E(l)E(l)). Male sterility was complete, stable and manifested in either petaloid- or rudimentary-anthers which were devoid of fertile pollen grains. Male sterile plants resembled the euplasmic B. juncea except for slight leaf yellowing and delayed maturity. Leaf yellowing was due mainly to higher level of carotenoids rather than a reduction in chlorophyll pigments. Female fertility in male-sterile plants varied; it was normal in lines having rudimentary anthers but poor in those with petaloid anthers. Each of the 62 evaluated germplasm lines of B. juncea was a functional maintainer of male sterility. The gene(s) for male-fertility restoration ( Rf) were introgressed from the cytoplasm donor species through homoeologous pairing between A and E(l) chromosomes in monosomic addition plants (2n = 18II+1E(l)). The percent pollen fertility of restored F(1) ( lyr CMS x putative restorer) plants ranged from 60 to 80%. This, however, was sufficient to ensure complete seed set upon by bag selfing. The CMS ( lyr) B. juncea compared favourably with the existing CMS systems for various productivity related characteristics. However, the reduced transmission frequency of the Rf gene(s) through pollen grains, which was evident from the sporadic occurrence of male-sterile plants in restored F(1) hybrids, remains a limitation.

  5. Agonist binding to the NMDA receptor drives movement of its cytoplasmic domain without ion flow

    PubMed Central

    Dore, Kim; Aow, Jonathan; Malinow, Roberto

    2015-01-01

    The NMDA receptor (R) plays important roles in brain physiology and pathology as an ion channel. Here we examine the ion flow-independent coupling of agonist to the NMDAR cytoplasmic domain (cd). We measure FRET between fluorescently tagged cytoplasmic domains of GluN1 subunits of NMDARs expressed in neurons. Different neuronal compartments display varying levels of FRET, consistent with different NMDARcd conformations. Agonist binding drives a rapid and transient ion flow-independent reduction in FRET between GluN1 subunits within individual NMDARs. Intracellular infusion of an antibody targeting the GluN1 cytoplasmic domain blocks agonist-driven FRET changes in the absence of ion flow, supporting agonist-driven movement of the NMDARcd. These studies indicate that extracellular ligand binding to the NMDAR can transmit conformational information into the cell in the absence of ion flow. PMID:26553997

  6. The Effect of Light and Inhibitors on Chloroplast and Cytoplasmic RNA Synthesis

    PubMed Central

    Ingle, J.

    1968-01-01

    Chloroplast RNA is synthesized in dark-grown radish cotyledons at about one-third the rate of that in the light. The synthesis, however, continues for longer in the dark and the percentage of chloroplast RNA can approach that in light-grown tissue. Light stimulates the synthesis and accumulation of both cytoplasmic and chloroplast RNA, but shows a 4-fold greater stimulation of the chloroplast RNA. Chloramphenicol, streptomycin and cycloheximide inhibit the synthesis of chloroplast RNA with little effect on cytoplasmic RNA. 5-Fluorouracil inhibits the synthesis of cytoplasmic more than chloroplast RNA. Synthesis of the 0.56 × 106 mol wt chloroplast RNA is inhibited much less than the other ribosomal RNA components by actinomycin D. PMID:5699149

  7. Identification of a Sterility-Inducing Cytoplasm in a Fertile Accession Line of Phaseolus Vulgaris L

    PubMed Central

    Mackenzie, S. A.

    1991-01-01

    Previous investigations into the genetic mechanism of fertility restoration in cytoplasmic male sterile Phaseolus vulgaris suggested that this is a particularly interesting system for the study of nuclear-mitochondrial interactions. This study was conducted to investigate the nature of nuclear-mitochondrial compatibility in fertile accession line G08063, the reported progenitor to the cytoplasmic male sterile line. Results from genetic analysis indicated that fertile line G08063 carried a sterility-inducing cytoplasm with a fertility restoring nuclear genotype. Mitochondrial DNA analysis indicated that the mechanism of fertility restoration by line G08063 was different from that conditioned by Fr, a previously described restorer gene. A mitochondrial DNA sequence associated with sterility and lost upon fertility restoration by nuclear gene Fr was present in the mitochondrial genome of fertile line G08063; this sequence was not carried within the mitochondrial genome of any other P. vulgaris accession line tested. PMID:1706293

  8. Complete atomistic model of a bacterial cytoplasm for integrating physics, biochemistry, and systems biology.

    PubMed

    Feig, Michael; Harada, Ryuhei; Mori, Takaharu; Yu, Isseki; Takahashi, Koichi; Sugita, Yuji

    2015-05-01

    A model for the cytoplasm of Mycoplasma genitalium is presented that integrates data from a variety of sources into a physically and biochemically consistent model. Based on gene annotations, core genes expected to be present in the cytoplasm were determined and a metabolic reaction network was reconstructed. The set of cytoplasmic genes and metabolites from the predicted reactions were assembled into a comprehensive atomistic model consisting of proteins with predicted structures, RNA, protein/RNA complexes, metabolites, ions, and solvent. The resulting model bridges between atomistic and cellular scales, between physical and biochemical aspects, and between structural and systems views of cellular systems and is meant as a starting point for a variety of simulation studies.

  9. Complete Atomistic Model of a Bacterial Cytoplasm for Integrating Physics, Biochemistry, and Systems Biology

    PubMed Central

    Feig, Michael; Harada, Ryuhei; Mori, Takaharu; Yu, Isseki; Takahashi, Koichi; Sugita, Yuji

    2015-01-01

    A model for the cytoplasm of Mycoplasma genitalium is presented that integrates data from a variety of sources into a physically and biochemically consistent model. Based on gene annotations, core genes expected to be present in the cytoplasm were determined and a metabolic reaction network was reconstructed. The set of cytoplasmic genes and metabolites from the predicted reactions were assembled into a comprehensive atomistic model consisting of proteins with predicted structures, RNA, protein/RNA complexes, metabolites, ions, and solvent. The resulting model bridges between atomistic and cellular scales, between physical and biochemical aspects, and between structural and systems views of cellular systems and is meant as a starting point for a variety of simulation studies. PMID:25765281

  10. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells

    PubMed Central

    Tichon, Ailone; Gil, Noa; Lubelsky, Yoav; Havkin Solomon, Tal; Lemze, Doron; Itzkovitz, Shalev; Stern-Ginossar, Noam; Ulitsky, Igor

    2016-01-01

    Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionarily conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD—an abundant and highly conserved cytoplasmic lncRNA. Here we show that most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two mammalian Pumilio homologues. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA-binding proteins, an activity which positions them at key junctions of cellular signalling pathways. PMID:27406171

  11. Vesicular Nucleo-Cytoplasmic Transport—Herpesviruses as Pioneers in Cell Biology

    PubMed Central

    Mettenleiter, Thomas C.

    2016-01-01

    Herpesviruses use a vesicle-mediated transfer of intranuclearly assembled nucleocapsids through the nuclear envelope (NE) for final maturation in the cytoplasm. The molecular basis for this novel vesicular nucleo-cytoplasmic transport is beginning to be elucidated in detail. The heterodimeric viral nuclear egress complex (NEC), conserved within the classical herpesviruses, mediates vesicle formation from the inner nuclear membrane (INM) by polymerization into a hexagonal lattice followed by fusion of the vesicle membrane with the outer nuclear membrane (ONM). Mechanisms of capsid inclusion as well as vesicle-membrane fusion, however, are largely unclear. Interestingly, a similar transport mechanism through the NE has been demonstrated in nuclear export of large ribonucleoprotein complexes during Drosophila neuromuscular junction formation, indicating a widespread presence of a novel concept of cellular nucleo-cytoplasmic transport. PMID:27690080

  12. Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm.

    PubMed

    Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

    2016-08-01

    There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (D_{tot}) and the displacement distribution functions (P(r,t)) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ, which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ=0.65, while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ. We also investigate the distribution (P(θ,t)) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system. PMID:27627367

  13. Analysis of Cytoplasmic Effects and Fine-Mapping of a Genic Male Sterile Line in Rice

    PubMed Central

    Li, Yuanyuan; Ma, Bingtian; Li, Shigui

    2013-01-01

    Cytoplasm has substantial genetic effects on progeny and is important for yield improvement in rice breeding. Studies on the cytoplasmic effects of cytoplasmic male sterility (CMS) show that most types of CMS have negative effects on yield-related traits and that these negative effects vary among CMS. Some types of genic male sterility (GMS), including photo-thermo sensitive male sterility (PTMS), have been widely used in rice breeding, but the cytoplasmic effects of GMS remain unknown. Here, we identified a GMS mutant line, h2s, which exhibited small, white anthers and failed to produce mature pollen. Unlike CMS, the h2s had significant positive cytoplasmic effects on the seed set rate, weight per panicle, yield, and general combining ability (GCA) for plant height, seed set rate, weight per panicle, and yield. These effects indicated that h2s cytoplasm may show promise for the improvement of rice yield. Genetic analysis suggested that the phenotype of h2s was controlled by a single recessive locus. We mapped h2s to a 152 kb region on chromosome 6, where 22 candidate genes were predicted. None of the 22 genes had previously been reported to be responsible for the phenotypes of h2s. Sequencing analysis showed a 12 bp deletion in the sixth exon of Loc_Os06g40550 in h2s in comparison to wild type, suggesting that Loc_Os06g40550 is the best candidate gene. These results lay a strong foundation for cloning of the H2S gene to elucidate the molecular mechanism of male reproduction. PMID:23613915

  14. A Mechanism for Cytoplasmic Streaming: Kinesin-Driven Alignment of Microtubules and Fast Fluid Flows.

    PubMed

    Monteith, Corey E; Brunner, Matthew E; Djagaeva, Inna; Bielecki, Anthony M; Deutsch, Joshua M; Saxton, William M

    2016-05-10

    The transport of cytoplasmic components can be profoundly affected by hydrodynamics. Cytoplasmic streaming in Drosophila oocytes offers a striking example. Forces on fluid from kinesin-1 are initially directed by a disordered meshwork of microtubules, generating minor slow cytoplasmic flows. Subsequently, to mix incoming nurse cell cytoplasm with ooplasm, a subcortical layer of microtubules forms parallel arrays that support long-range, fast flows. To analyze the streaming mechanism, we combined observations of microtubule and organelle motions with detailed mathematical modeling. In the fast state, microtubules tethered to the cortex form a thin subcortical layer and undergo correlated sinusoidal bending. Organelles moving in flows along the arrays show velocities that are slow near the cortex and fast on the inward side of the subcortical microtubule layer. Starting with fundamental physical principles suggested by qualitative hypotheses, and with published values for microtubule stiffness, kinesin velocity, and cytoplasmic viscosity, we developed a quantitative coupled hydrodynamic model for streaming. The fully detailed mathematical model and its simulations identify key variables that can shift the system between disordered (slow) and ordered (fast) states. Measurements of array curvature, wave period, and the effects of diminished kinesin velocity on flow rates, as well as prior observations on f-actin perturbation, support the model. This establishes a concrete mechanistic framework for the ooplasmic streaming process. The self-organizing fast phase is a result of viscous drag on kinesin-driven cargoes that mediates equal and opposite forces on cytoplasmic fluid and on microtubules whose minus ends are tethered to the cortex. Fluid moves toward plus ends and microtubules are forced backward toward their minus ends, resulting in buckling. Under certain conditions, the buckling microtubules self-organize into parallel bending arrays, guiding varying directions

  15. Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm

    NASA Astrophysics Data System (ADS)

    Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

    2016-08-01

    There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (Dtot) and the displacement distribution functions (P (r ,t ) ) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ , which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ =0.65 , while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ . We also investigate the distribution (P (θ ,t ) ) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

  16. PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae.

    PubMed Central

    Anderson, J T; Paddy, M R; Swanson, M S

    1993-01-01

    Proteins that directly associate with nuclear polyadenylated RNAs, or heterogeneous nuclear RNA-binding proteins (hnRNPs), and those that associate with cytoplasmic mRNAs, or mRNA-binding proteins (mRNPs), play important roles in regulating gene expression at the posttranscriptional level. Previous work with a variety of eukaryotic cells has demonstrated that hnRNPs are localized predominantly within the nucleus whereas mRNPs are cytoplasmic. While studying proteins associated with polyadenylated RNAs in Saccharomyces cerevisiae, we discovered an abundant polyuridylate-binding protein, PUB1, which appears to be both an hnRNP and an mRNP. PUB1 and PAB1, the polyadenylate tail-binding protein, are the two major proteins cross-linked by UV light to polyadenylated RNAs in vivo. The deduced primary structure of PUB1 indicates that it is a member of the ribonucleoprotein consensus sequence family of RNA-binding proteins and is structurally related to the human hnRNP M proteins. Even though the PUB1 protein is a major cellular polyadenylated RNA-binding protein, it is nonessential for cell growth. Indirect cellular immunofluorescence combined with digital image processing allowed a detailed comparison of the intracellular distributions of PUB1 and PAB1. While PAB1 is predominantly, and relatively uniformly, distributed within the cytoplasm, PUB1 is localized in a nonuniform pattern throughout both the nucleus and the cytoplasm. The cytoplasmic distribution of PUB1 is considerably more discontinuous than that of PAB1. Furthermore, sucrose gradient sedimentation analysis demonstrates that PAB1 cofractionates with polyribosomes whereas PUB1 does not. These results suggest that PUB1 is both an hnRNP and an mRNP and that it may be stably bound to a translationally inactive subpopulation of mRNAs within the cytoplasm. Images PMID:8413212

  17. Comparative assessment of genetic diversity in cytoplasmic and nuclear genome of upland cotton.

    PubMed

    Egamberdiev, Sharof S; Saha, Sukumar; Salakhutdinov, Ilkhom; Jenkins, Johnie N; Deng, Dewayne; Y Abdurakhmonov, Ibrokhim

    2016-06-01

    The importance of the cytoplasmic genome for many economically important traits is well documented in several crop species, including cotton. There is no report on application of cotton chloroplast specific SSR markers as a diagnostic tool to study genetic diversity among improved Upland cotton lines. The complete plastome sequence information in GenBank provided us an opportunity to report on 17 chloroplast specific SSR markers using a cost-effective data mining strategy. Here we report the comparative analysis of genetic diversity among a set of 42 improved Upland cotton lines using SSR markers specific to chloroplast and nuclear genome, respectively. Our results revealed that low to moderate level of genetic diversity existed in both nuclear and cytoplasm genome among this set of cotton lines. However, the specific estimation suggested that genetic diversity is lower in cytoplasmic genome compared to the nuclear genome among this set of Upland cotton lines. In summary, this research is important from several perspectives. We detected a set of cytoplasm genome specific SSR primer pairs by using a cost-effective data mining strategy. We reported for the first time the genetic diversity in the cytoplasmic genome within a set of improved Upland cotton accessions. Results revealed that the genetic diversity in cytoplasmic genome is narrow, compared to the nuclear genome within this set of Upland cotton accessions. Our results suggested that most of these polymorphic chloroplast SSRs would be a valuable complementary tool in addition to the nuclear SSR in the study of evolution, gene flow and genetic diversity in Upland cotton.

  18. Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

    PubMed

    Ganguly, Abira; Bhattacharjee, Chumki; Bhave, Madhura; Kailaje, Vaishali; Jain, Bhawik K; Sengupta, Isha; Rangarajan, Annapoorni; Bhattacharyya, Dibyendu

    2016-03-01

    Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation.

  19. Speciation of mercury in salmon egg cell cytoplasm in relation with metallomics research.

    PubMed

    Hasegawa, Takuya; Asano, Motoki; Takatani, Kohei; Matsuura, Hirotaka; Umemura, Tomonari; Haraguchi, Hiroki

    2005-12-15

    Speciation of mercury in salmon egg cell cytoplasm was investigated by surfactant-mediated high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS), where an ODS (octadecylsilica) column coated with a bile acid derivative, CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), was used for species separation. Prior to the speciation analysis, total Hg in the cell cytoplasm was determined by ICP-MS at m/z 202 in a flow injection mode. For the precise measurement, salmon egg cell cytoplasm was diluted five-fold with 0.1M Tris (Tris(hydroxymethyl)aminomethane)-HNO(3) buffer solution, and the standard addition method was employed. Thus, the total concentration of Hg in cell cytoplasm was estimated to be 12.4ngg(-1) on the wet weight basis. Next, the cell cytoplasm diluted five-fold with 0.1M Tris-HNO(3) buffer solution was analyzed by surfactant-mediated HPLC with the dual detection system of a UV absorption detector and an ICP-MS instrument. Two peaks corresponding to some proteins and small molecules were mainly observed in those chromatograms. When salmon egg cell cytoplasm was diluted five-fold with 0.01M Tris buffer solution or pure water, some precipitates appeared probably because of precipitation of hydrophobic proteins in cytoplasm. After the precipitates were eliminated with a membrane filter, the filtrate was subjected to the analysis by surfactant-mediated HPLC/UV/ICP-MS. As a result, the peaks for small molecular species of Hg were clearly observed at the retention time near 4.0min (corresponding to low-molecular weight zone) in the chromatograms with UV absorption detection as well as with Hg- and S-specific ICP-MS detections. The small molecule bound with Hg was identified as cysteine through the cysteine-spiked experiment. In addition, the protein fraction on the chromatogram obtained by using the CHAPS-coated ODS column was further analyzed by SEC (size exclusion chromatography). Consequently

  20. Cytoplasmic intermediate filaments mediate actin-driven positioning of the nucleus.

    PubMed

    Dupin, Isabelle; Sakamoto, Yasuhisa; Etienne-Manneville, Sandrine

    2011-03-15

    The localization of the nucleus is precisely regulated, and defects in nuclear positioning are observed in diseases such as lissencephaly, cerebellar ataxia and dysplasia. We show here that cytoplasmic intermediate filaments are essential players in actin-dependent positioning of the nucleus. The actin retrograde flow is relayed by a flow of intermediate filaments that accumulate asymmetrically around the nuclear envelope. Perturbations of the intermediate filament network alter positioning of the nucleus in both migrating and immobile astrocytes. This function of intermediate filaments might be crucial for regulating cell motility, in particular in tumor cells expressing high levels of cytoplasmic intermediate filaments.

  1. Beyond the cytoplasm of Escherichia coli: localizing recombinant proteins where you want them.

    PubMed

    Boock, Jason T; Waraho-Zhmayev, Dujduan; Mizrachi, Dario; DeLisa, Matthew P

    2015-01-01

    Recombinant protein expression in Escherichia coli represents a cornerstone of the biotechnology enterprise. While cytoplasmic expression in this host has received the most attention, achieving substantial yields of correctly folded proteins in this compartment can sometimes be met with difficulties. These issues can often be overcome by targeting protein expression to extracytoplasmic compartments (e.g., membrane, periplasm) or to the culture medium. This chapter discusses various strategies for exporting proteins out of the cytoplasm as well as tools for monitoring and optimizing these different export mechanisms. PMID:25447860

  2. A comparison of nuclear and cytoplasmic genetic effects on sperm competitiveness and female remating in a seed beetle.

    PubMed

    Dowling, D K; Friberg, U; Arnqvist, G

    2007-11-01

    It is widely assumed that male sperm competitiveness evolves adaptively. However, recent studies have found a cytoplasmic genetic component to phenotypic variation in some sperm traits presumed important in sperm competition. As cytoplasmic genes are maternally transmitted, they cannot respond to selection on sperm and this constraint may affect the scope in which sperm competitiveness can evolve adaptively. We examined nuclear and cytoplasmic genetic contributions to sperm competitiveness, using populations of Callosobruchus maculatus carrying orthogonal combinations of nuclear and cytoplasmic lineages. Our design also enabled us to examine genetic contributions to female remating. We found that sperm competitiveness and remating are primarily encoded by nuclear genes. In particular, a male's sperm competitiveness phenotype was contingent on an interaction between the competing male genotypes. Furthermore, cytoplasmic effects were detected on remating but not sperm competitiveness, suggesting that cytoplasmic genes do not generally play a profound evolutionary role in sperm competition.

  3. Relative Roles of Gap Junction Channels and Cytoplasm in Cell-to-Cell Diffusion of Fluorescent Tracers

    NASA Astrophysics Data System (ADS)

    Safranyos, Richard G. A.; Caveney, Stanley; Miller, James G.; Petersen, Nils O.

    1987-04-01

    Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic diffusion, which may limit intercellular diffusion.

  4. Effect of Paramecium biaurelia cytoplasm transplantation on the duration of the interautogamous interval (IAI) in Paramecium tetraurelia.

    PubMed

    Prajer, M

    1994-01-01

    The cytoplasm of Paramecium biaurelia in various stages of IAI was transplanted to Paramecium tetraurelia cells of the same clonal age. Such donor cytoplasm was effective in retardation of the expression of autogamy in the recipient clones. The results suggest that the cytoplasm of Paramecium biaurelia may contain the autogamous immaturity factor whose level changes during the run of IAI and whose specificity is the same as in Paramecium tetraurelia. PMID:7982503

  5. Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Kuang, A.; Musgrave, M. E.

    1996-01-01

    Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultrastructure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyosomes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.

  6. Detection of cytoplasmic proteins from Helicobacter pylori in Colony Lift Immunoassay.

    PubMed

    Rojas-Rengifo, Diana F; Jaramillo, Carlos A; Haas, Rainer; Jiménez-Soto, Luisa F

    2015-12-01

    Use of the Colony Lift Immunoassay has been described for several Gram negative bacteria of medical interest. In all cases detection was limited to the use of antibodies against outer membrane proteins. Here we describe the adaptation of this method for detection of the cytoplasmic CagA toxin from Helicobacter pylori.

  7. Identifying cytoplasmic input to the cell wall of growing Chara corallina.

    PubMed

    Proseus, Timothy E; Boyer, John S

    2006-01-01

    Plants enlarge mostly because the walls of certain cells enlarge, with accompanying input of wall constituents and other factors from the cytoplasm. However, the enlargement can occur without input, suggesting an uncertain relationship between cytoplasmic input and plant growth. Therefore, the role of the input was investigated by quantitatively comparing growth in isolated walls (no input) with that in living cells (input occurring). Cell walls were isolated from growing internodes of Chara corallina and filled with pressurized oil to control turgor pressure while elongation was monitored. Turgor pressure in living cells was similarly controlled and monitored by adding/removing cell solution. Temperature was varied in some experiments. At all pressures and temperatures, isolated walls displayed turgor-driven growth indistinguishable in every respect from that in living cells, except the rate decelerated in the isolated walls while the living cells grew rapidly. The growth in the isolated walls was highly responsive to temperature, in contrast to the elastic extension that has been shown to be insensitive to similar temperatures. Consequently, strong intermolecular bonds were responsible for growth and weak bonds for elastic extension. Boiling the walls gave the same results, indicating that enzyme activities were not controlling these bonds. However, pectin added to isolated walls reversed their growth deceleration and returned the rate to that in the living cells. The pectin was similar to that normally produced by the cytoplasm and deposited in the wall, suggesting that continued cytoplasmic input of pectin may play a role in sustaining turgor-driven growth in Chara.

  8. Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans

    PubMed Central

    1993-01-01

    We have examined the cortex of Caenorhabditis elegans eggs during pseudocleavage (PC), a period of the first cell cycle which is important for the generation of asymmetry at first cleavage (Strome, S. 1989. Int. Rev. Cytol. 114: 81-123). We have found that directed, actin dependent, cytoplasmic, and cortical flow occurs during this period coincident with a rearrangement of the cortical actin cytoskeleton (Strome, S. 1986. J. Cell Biol. 103: 2241-2252). The flow velocity (4-7 microns/min) is similar to previously determined particle movements driven by cortical actin flows in motile cells. We show that directed flows occur in one of the daughters of the first division that itself divides asymmetrically, but not in its sister that divides symmetrically. The cortical and cytoplasmic events of PC can be mimicked in other cells during cytokinesis by displacing the mitotic apparatus with the microtubule polymerization inhibitor nocodazole. In all cases, the polarity of the resulting cortical and cytoplasmic flows correlates with the position of the attenuated mitotic spindle formed. These cortical flows are also accompanied by a change in the distribution of the cortical actin network. The polarity of this redistribution is similarly correlated with the location of the attenuated spindle. These observations suggest a mechanism for generating polarized flows of cytoplasmic and cortical material during embryonic cleavages. We present a model for the events of PC and suggest how the poles of the mitotic spindle mediate the formation of the contractile ring during cytokinesis in C. elegans. PMID:8509454

  9. Cytoplasmic Streaming and Ion Transport: A Laboratory Exercise which Tests a Longstanding Botanical Concept.

    ERIC Educational Resources Information Center

    Perley, James E.; Glass, A. D. M.

    1979-01-01

    Presents a method for microscopically examining cytoplasmic streaming in root cells, and physiologically examining the delivery of ions to the xylem exudate. The expected result does not occur and the authors believe that students learn to re-examine the assumptions, thereby increasing their skills in scientific inquiry. (Authors/SA)

  10. Strategies for investigating nuclear-cytoplasmic tRNA dynamics in yeast and mammalian cells.

    PubMed

    Pierce, Jacqueline B; Chafe, Shawn C; Eswara, Manoja B K; van der Merwe, George; Mangroo, Dev

    2014-01-01

    Nuclear-cytoplasmic tRNA transport involves multiple pathways that are segregated by the involvement of distinct proteins. The tRNA export process begins in the nucleolus, where the functionality of newly produced tRNAs are tested by aminoacylation, and ends with the delivery of the exported aminoacyl tRNAs to the eukaryotic elongation factor eEF-1A for utilization in protein synthesis in the cytoplasm. Recent studies have identified a number of proteins that participate in nuclear tRNA export in both yeast and mammals. However, genetic and biochemical evidence suggest that additional components, which have yet to be identified, also participate in nuclear-cytoplasmic tRNA trafficking. Here we review key strategies that have led to the identification and characterization of proteins that are involved in the nuclear tRNA export process in yeasts and mammals. The approaches described will greatly facilitate the identification and delineation of the roles of new proteins involved in nuclear export of tRNAs to the cytoplasm.

  11. [Antibody adsorption to tetrodotoxin-sensitive cytoplasmic proteins by murine neuroblastoma cells].

    PubMed

    Pinchuk, G V; Pinchuk, L N; Gerasimenko, O V

    1988-01-01

    The study was aimed to examine properties of polyclonal antibodies elicited after immunization by cytoplasmic nerve cell glycoproteins forming sodium channels in liposomes. It was shown that intact murine neuroblastoma cells can absorb these antibodies. Absorbing cell dose-effect curves were found to have a characteristic form able to shift when the cell culture time or serum concentration in the growth medium varied.

  12. Transport of the outer dynein arm complex to cilia requires a cytoplasmic protein Lrrc6.

    PubMed

    Inaba, Yasuko; Shinohara, Kyosuke; Botilde, Yanick; Nabeshima, Ryo; Takaoka, Katsuyoshi; Ajima, Rieko; Lamri, Lynda; Takeda, Hiroyuki; Saga, Yumiko; Nakamura, Tetsuya; Hamada, Hiroshi

    2016-07-01

    Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism.

  13. ERAD of proteins containing aberrant transmembrane domains requires ubiquitylation of cytoplasmic lysine residues

    PubMed Central

    Briant, Kit; Koay, Yee-Hui; Otsuka, Yuka; Swanton, Eileithyia

    2015-01-01

    ABSTRACT Clearance of misfolded proteins from the endoplasmic reticulum (ER) is mediated by the ubiquitin-proteasome system in a process known as ER-associated degradation (ERAD). The mechanisms through which proteins containing aberrant transmembrane domains are degraded by ERAD are poorly understood. To address this question, we generated model ERAD substrates based on CD8 with either a non-native transmembrane domain but a folded ER luminal domain (CD8TMD*), or the native transmembrane domain but a misfolded luminal domain (CD8LUM*). Although both chimeras were degraded by ERAD, we found that the location of the folding defect determined the initial site of ubiquitylation. Ubiquitylation of cytoplasmic lysine residues was required for the extraction of CD8TMD* from the ER membrane during ERAD, whereas CD8LUM* continued to be degraded in the absence of cytoplasmic lysine residues. Cytoplasmic lysine residues were also required for degradation of an additional ERAD substrate containing an unassembled transmembrane domain and when a non-native transmembrane domain was introduced into CD8LUM*. Our results suggest that proteins with defective transmembrane domains are removed from the ER through a specific ERAD mechanism that depends upon ubiquitylation of cytoplasmic lysine residues. PMID:26446255

  14. A new nomenclature for the cytoplasmic ribosomal proteins of Saccharomyces cerevisiae.

    PubMed Central

    Mager, W H; Planta, R J; Ballesta, J G; Lee, J C; Mizuta, K; Suzuki, K; Warner, J R; Woolford, J

    1997-01-01

    The availability of the complete sequence of the Saccharomyces cerevisiae genome has allowed a comprehensive analysis of the genes encoding cytoplasmic ribosomal proteins in this organism. On the basis of this complete inventory a new nomenclature for the yeast ribosomal proteins is presented. PMID:9396790

  15. Cytoplasmic Membrane Changes during Adaptation of the Fresh Water Cyanobacterium Synechococcus 6311 to Salinity 1

    PubMed Central

    Lefort-Tran, Marcelle; Pouphile, Monique; Spath, Susan; Packer, Lester

    1988-01-01

    In this investigation, changes were characterized in cell structure and cytoplasmic membrane organization that occur when the freshwater cyanobacterium Synechococcus 6311 is transferred from `low salt' (0.03 molar NaCl) to `high salt' (0.5 molar NaCl) media (i.e. sea water concentration). Cells were examined at several time points after the imposition of the salt stress and compared to control cells, in thin sections and freeze fracture electron microscopy, and by flow cytometry. One minute after exposure to high salt, i.e. `salt shock,' virtually all intracellular granules disappeared, the density of the cytoplasm decreased, and the appearance of DNA material was changed. Glycogen and other granules, however, reappeared by 4 hours after salt exposure. The organization of the cytoplasmic membrane undergoes major reorganization following salt shock. Freeze-fracture electron microscopy showed that small intramembrane particles (diameters 7.5 and 8.5 nanometers) are reduced in number by two- to fivefold, whereas large particles, (diameters 14.5 and 17.5 nanometers) increase two- to fourfold in frequency, compared to control cells grown in low salt medium. The changes in particle size distribution suggest synthesis of new membrane proteins, in agreement with the known increases in respiration, cytochrome oxidase, and sodium proton exchange activity of the cytoplasmic membrane. Images Fig. 2 Fig. 1 Fig. 5 PMID:11537874

  16. Diffusive Promotion by Velocity Gradient of Cytoplasmic Streaming (CPS) in Nitella Internodal Cells.

    PubMed

    Kikuchi, Kenji; Mochizuki, Osamu

    2015-01-01

    Cytoplasmic streaming (CPS) is well known to assist the movement of nutrients, organelles and genetic material by transporting all of the cytoplasmic contents of a cell. CPS is generated by motility organelles that are driven by motor proteins near a membrane surface, where the CPS has been found to have a flat velocity profile in the flow field according to the sliding theory. There is a consistent mixing of contents inside the cell by CPS if the velocity gradient profile is flattened, which is not assisted by advection diffusion but is only supported by Brownian diffusion. Although the precise flow structure of the cytoplasm has an important role for cellular metabolism, the hydrodynamic mechanism of its convection has not been clarified. We conducted an experiment to visualise the flow of cytoplasm in Nitella cells by injecting tracer fluorescent nanoparticles and using a flow visualisation system in order to understand how the flow profile affects their metabolic system. We determined that the velocity field in the cytosol has an obvious velocity gradient, not a flattened gradient, which suggests that the gradient assists cytosolic mixing by Taylor-Aris dispersion more than by Brownian diffusion. PMID:26694322

  17. The nuclear protein Sam68 is recruited to the cytoplasmic stress granules during enterovirus 71 infection.

    PubMed

    Zhang, Hua; Chen, Ning; Li, Pengfei; Pan, Ziye; Ding, Yun; Zou, Dehua; Li, Liyang; Xiao, Lijie; Shen, Binglei; Liu, Shuxia; Cao, Hongwei; Cui, Yudong

    2016-07-01

    Our previous study found that the nuclear protein, 68-kDa Src-associated in mitosis protein (Sam68), is translocated to the cytoplasm and forms punctate pattern during enterovirus 71 (EV71) infection [Virus Research, 180 (2014), 1-11]. However, the exact function of this punctate pattern in cytoplasm during EV71 infection remains unknown. In this study, we firstly have examined this punctate pattern of Sam68 re-localization in the cytoplasm, and observed the obvious recruitments of Sam68 to the EV71-induced stress granules (SGs). Sam68, belongs to the KH domain family of RNA binding proteins (RBPs), was then confirmed that its KH domain was essential for this recruitment. Nevertheless, Knockdown of Sam68 expression using ShRNA had no effects on SGs assembly, indicating that Sam68 is not a constitutive component of the SGs during EV71 infection. Lastly, we investigated the importance of microtubulin transport to SGs aggregation, and revealed that microtubule depolymerization inhibited SGs formation, suggesting that EV71-induced SGs move throughout the cytoplasm in a microtubule-dependent manner. Taken together, these results illuminated that EV71 infections can induce SGs formation, and Sam68, as a SGs component, migrates alone with SGs dependent on intact microtubule upon the viral infections. These findings may provide novel underlying mechanism for delineating the role of SGs during EV71 infection. PMID:27057671

  18. Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.

    PubMed

    Gatla, Himavanth R; Singha, Bipradeb; Persaud, Valerie; Vancurova, Ivana

    2014-01-01

    Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types. PMID:24908314

  19. Cytoplasmic domains of the reduced folate carrier are essential for trafficking, but not function.

    PubMed Central

    Sadlish, Heather; Williams, Frederick M R; Flintoff, Wayne F

    2002-01-01

    The reduced folate carrier (RFC) protein has a secondary structure consistent with the predicted 12 transmembrane (TM) domains, intracellular N- and C-termini and a large cytoplasmic loop between TM6 and TM7. In the present study, the role of the cytoplasmic domains in substrate transport and protein biogenesis were examined using an array of hamster RFC deletion mutants fused to enhanced green fluorescent protein and expressed in Chinese hamster ovary cells. The N- and C-terminal tails were removed both individually and together, or the large cytoplasmic loop was modified such that the domain size and role of conserved sequences could be examined. The loss of the N- or C-terminal tails did not appear to significantly disrupt protein function, although both termini appeared to have a role in the efficiency with which molecules exited the endoplasmic reticulum to localize at the plasma membrane. There appeared to be both size and sequence requirements for the intracellular loop, which are able to drastically affect protein stability and function unless met. Furthermore, there might be an indirect role for the loop in substrate translocation, since even moderate changes significantly reduced the V(max) for methotrexate transport. Although these cytoplasmic domains do not appear to be absolutely essential for substrate transport, each one is important for biogenesis and localization. PMID:12049642

  20. Localization and topogenesis studies of cytoplasmic and vacuolar homologs of the Galanthus nivalis agglutinin.

    PubMed

    Fouquaert, Elke; Hanton, Sally L; Brandizzi, Federica; Peumans, Willy J; Van Damme, Els J M

    2007-07-01

    The Galanthus nivalis agglutinin (GNA) is synthesized as a preproprotein. To corroborate the role of the different targeting peptides in the topogenesis of GNA and related proteins, different constructs were made whereby both the complete original GNA gene and different truncated sequences were coupled to the enhanced green fluorescent protein (EGFP). In addition, a GNA ortholog from rice that lacks the signal peptide and C-terminal propeptide sequence was fused to EGFP. These fusion constructs were expressed in tobacco BY-2 cells and their localization analyzed by confocal fluorescence microscopy. We observed that the processed preproprotein of GNA was directed towards the vacuolar compartment, whereas both the truncated forms of GNA corresponding to the mature lectin polypeptide and the rice ortholog of GNA were located in the nucleus and the cytoplasm. It can be concluded, therefore, that removal of the C-terminal propeptide and the signal peptide is sufficient to change the subcellular targeting of a normally vacuolar protein to the nuclear/cytoplasmic compartment of the BY-2 cells. These findings support the proposed hypothesis that cytoplasmic/nuclear GNA-like proteins and their vacuolar homologs are evolutionarily related and that the classical GNA-related lectins might have evolved from cytoplasmic orthologs through an evolutionary event involving the insertion of a signal peptide and a C-terminal propeptide.

  1. The Effect of Temperature on Microtubule-Based Transport by Cytoplasmic Dynein and Kinesin-1 Motors.

    PubMed

    Hong, Weili; Takshak, Anjneya; Osunbayo, Olaolu; Kunwar, Ambarish; Vershinin, Michael

    2016-09-20

    Cytoplasmic dynein and kinesin are both microtubule-based molecular motors but are structurally and evolutionarily unrelated. Under standard conditions, both move with comparable unloaded velocities toward either the microtubule minus (dynein) or plus (most kinesins) end. This similarity is important because it is often implicitly incorporated into models that examine the balance of cargo fluxes in cells and into models of the bidirectional motility of individual cargos. We examined whether this similarity is a robust feature, and specifically whether it persists across the biologically relevant temperature range. The velocity of mammalian cytoplasmic dynein, but not of mammalian kinesin-1, exhibited a break from simple Arrhenius behavior below 15°C-just above the restrictive temperature of mammalian fast axonal transport. In contrast, the velocity of yeast cytoplasmic dynein showed a break from Arrhenius behavior at a lower temperature (∼8°C). Our studies implicate cytoplasmic dynein as a more thermally tunable motor and therefore a potential thermal regulator of microtubule-based transport. Our theoretical analysis further suggests that motor velocity changes can lead to qualitative changes in individual cargo motion and hence net intracellular cargo fluxes. We propose that temperature can potentially be used as a noninvasive probe of intracellular transport. PMID:27653487

  2. NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5.

    PubMed

    Baker, Paul J; Boucher, Dave; Bierschenk, Damien; Tebartz, Christina; Whitney, Paul G; D'Silva, Damian B; Tanzer, Maria C; Monteleone, Mercedes; Robertson, Avril A B; Cooper, Matthew A; Alvarez-Diaz, Silvia; Herold, Marco J; Bedoui, Sammy; Schroder, Kate; Masters, Seth L

    2015-10-01

    Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase-4 and caspase-5. When activated, these trigger pyroptotic cell death and caspase-1-dependent IL-1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase-4/5-dependent IL-1β production elicited by transfected LPS. Given that both caspase-4 and caspase-5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase-4 and caspase-5 were genetically deleted either individually or together. We found that the deletion of caspase-4 suppressed cell death and IL-1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase-5 did not confer protection against transfected LPS, cell death and IL-1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase-4 and caspase-5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL-1β maturation, downstream of cytoplasmic LPS detection by caspase-4/5. We also show that both caspase-4 and caspase-5 are functionally important for appropriate responses to intracellular Gram-negative bacteria.

  3. Diffusive Promotion by Velocity Gradient of Cytoplasmic Streaming (CPS) in Nitella Internodal Cells

    PubMed Central

    Kikuchi, Kenji; Mochizuki, Osamu

    2015-01-01

    Cytoplasmic streaming (CPS) is well known to assist the movement of nutrients, organelles and genetic material by transporting all of the cytoplasmic contents of a cell. CPS is generated by motility organelles that are driven by motor proteins near a membrane surface, where the CPS has been found to have a flat velocity profile in the flow field according to the sliding theory. There is a consistent mixing of contents inside the cell by CPS if the velocity gradient profile is flattened, which is not assisted by advection diffusion but is only supported by Brownian diffusion. Although the precise flow structure of the cytoplasm has an important role for cellular metabolism, the hydrodynamic mechanism of its convection has not been clarified. We conducted an experiment to visualise the flow of cytoplasm in Nitella cells by injecting tracer fluorescent nanoparticles and using a flow visualisation system in order to understand how the flow profile affects their metabolic system. We determined that the velocity field in the cytosol has an obvious velocity gradient, not a flattened gradient, which suggests that the gradient assists cytosolic mixing by Taylor–Aris dispersion more than by Brownian diffusion. PMID:26694322

  4. Transport of the outer dynein arm complex to cilia requires a cytoplasmic protein Lrrc6.

    PubMed

    Inaba, Yasuko; Shinohara, Kyosuke; Botilde, Yanick; Nabeshima, Ryo; Takaoka, Katsuyoshi; Ajima, Rieko; Lamri, Lynda; Takeda, Hiroyuki; Saga, Yumiko; Nakamura, Tetsuya; Hamada, Hiroshi

    2016-07-01

    Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism. PMID:27353389

  5. Cell type-specific loss of atp6 RNA editing in cytoplasmic male sterile Sorghum bicolor.

    PubMed

    Howad, W; Kempken, F

    1997-09-30

    RNA editing and cytoplasmic male sterility are two important phenomena in higher plant mitochondria. To determine whether correlations might exist between the two, RNA editing in different tissues of Sorghum bicolor was compared employing reverse transcription-PCR and subsequent sequence analysis. In etiolated shoots, RNA editing of transcripts of plant mitochondrial atp6, atp9, nad3, nad4, and rps12 genes was identical among fertile or cytoplasmic male sterile plants. We then established a protocol for mitochondrial RNA isolation from plant anthers and pollen to include in these studies. Whereas RNA editing of atp9, nad3, nad4, and rps12 transcripts in anthers was similar to etiolated shoots, mitochondrial atp6 RNA editing was strongly reduced in anthers of the A3Tx398 male sterile line of S. bicolor. atp6 transcripts of wheat and selected plastid transcripts in S. bicolor showed normal RNA editing, indicating that loss of atp6 RNA editing is specific for cytoplasmic male sterility S. bicolor mitochondria. Restoration of fertility in F1 and F2 lines correlated with an increase in RNA editing of atp6 transcripts. Our data suggest that loss of atp6 RNA editing contributes to or causes cytoplasmic male sterility in S. bicolor. Further analysis of the mechanism of cell type-specific loss of atp6 RNA editing activity may advance our understanding of the mechanism of RNA editing.

  6. The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections

    PubMed Central

    1985-01-01

    High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2. PMID:4039327

  7. Gene knockouts reveal separate functions for two cytoplasmic dyneins in Tetrahymena thermophila.

    PubMed

    Lee, S; Wisniewski, J C; Dentler, W L; Asai, D J

    1999-03-01

    In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

  8. Live cell imaging of cytoplasmic Ca2+ dynamics in Arabidopsis guard cells.

    PubMed

    Behera, Smrutisanjita; Kudla, Jörg

    2013-07-01

    This protocol describes a classical method for measuring cytoplasmic Ca(2+) dynamics in Arabidopsis thaliana guard cells. The Förster (fluorescence) resonance energy transfer (FRET)-based genetically modified Ca(2+) indicator Yellow Cameleon YC3.6, under the control of the guard cell-specific promoter GC1, is used for Ca(2+) measurements.

  9. The border-to-border distribution method for analysis of cytoplasmic particles and organelles.

    PubMed

    Yacovone, Shalane K; Ornelles, David A; Lyles, Douglas S

    2016-02-01

    Comparing the distribution of cytoplasmic particles and organelles between different experimental conditions can be challenging due to the heterogeneous nature of cell morphologies. The border-to-border distribution method was created to enable the quantitative analysis of fluorescently labeled cytoplasmic particles and organelles of multiple cells from images obtained by confocal microscopy. The method consists of four steps: (1) imaging of fluorescently labeled cells, (2) division of the image of the cytoplasm into radial segments, (3) selection of segments of interest, and (4) population analysis of fluorescence intensities at the pixel level either as a function of distance along the selected radial segments or as a function of angle around an annulus. The method was validated using the well-characterized effect of brefeldin A (BFA) on the distribution of the vesicular stomatitis virus G protein, in which intensely labeled Golgi membranes are redistributed within the cytoplasm. Surprisingly, in untreated cells, the distribution of fluorescence in Golgi membrane-containing radial segments was similar to the distribution of fluorescence in other G protein-containing segments, indicating that the presence of Golgi membranes did not shift the distribution of G protein towards the nucleus compared to the distribution of G protein in other regions of the cell. Treatment with BFA caused only a slight shift in the distribution of the brightest G protein-containing segments which had a distribution similar to that in untreated cells. Instead, the major effect of BFA was to alter the annular distribution of G protein in the perinuclear region.

  10. Cycloheximide- and puromycin-induced heat resistance: different effects on cytoplasmic and nuclear luciferases.

    PubMed

    Michels, A A; Kanon, B; Konings, A W; Bensaude, O; Kampinga, H H

    2000-07-01

    Inhibition of translation can result in cytoprotection against heat shock. The mechanism of this protection has remained elusive so far. Here, the thermoprotective effects of the translation inhibitor cycloheximide (CHX) and puromycin were investigated, using as reporter firefly luciferase localized either in the nucleus or in the cytoplasm. A short preincubation of O23 cells with either translation inhibitor was found to attenuate the heat inactivation of a luciferase directed into the cytoplasm, whereas the heat sensitivity of a nuclear-targeted luciferase remained unaffected. After a long-term CHX pretreatment, both luciferases were more heat resistant. Both the cytoplasmic and the nuclear luciferase are protected against heat-induced inactivation in thermotolerant cells and in cells overexpressing heat shock protein (Hsp)70. CHX incubations further attenuated cytoplasmic luciferase inactivation in thermotolerant and in Hsp70 overexpressing cells, even when Hsp70-mediated protection was saturated. It is concluded that protection by translation inhibition is unlikely due to an increase in the pool of free Hsps normally engaged in translation and released from the nascent polypeptide chains on the ribosomes. Rather, a decrease in nascent chains and thermolabile polypeptides may account for the heat resistance promoted by inhibitors of translation.

  11. Towards the elucidation of the cytoplasmic diversity of North American Grape Breeding Programs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants have an intriguing tripartite genetic system: Nuclear genome × Mitochondria × Plastids, and their interactions may impact germplasm breeding. In grapevine, the study of cytoplasmic genomes has been limited, and their role with respect to grapevine germplasm diversity has not been elucidated y...

  12. Ubiquitin-dependent distribution of the transcriptional coactivator p300 in cytoplasmic inclusion bodies.

    PubMed

    Chen, Jihong; Halappanavar, Sabina; Th' ng, John P H; Li, Qiao

    2007-01-01

    The protein level of transcriptional coactivator p300, an essential nuclear protein, is critical to a broad array of cellular activities including embryonic development, cell differentiation and proliferation. We have previously established that histone deacetylase inhibitor such as valproic acid induces p300 degradation through the 26S proteasome pathway. Here, we report the roles of cellular trafficking and spatial redistribution in valproic acid-induced p300 turnover. Our study demonstrates that p300 is redistributed to the cytoplasm prior to valproic acid-induced turnover. Inhibition of proteasome-dependent protein degradation, does not prevent nucleo-cytoplasmic shuttling of p300, rather sequesters the cytoplasmic p300 to a distinct perinuclear region. In addition, the formation of p300 aggregates in the perinuclear region depends on functional microtubule networks and correlates with p300 ubiquitination. Our work establishes, for the first time, that p300 is also a substrate of the cytoplasmic ubiquitin-proteasome system and provides insight on how cellular trafficking and spatial redistribution regulate the availability and activity of transcriptional coactivator p300.

  13. Diffusive Promotion by Velocity Gradient of Cytoplasmic Streaming (CPS) in Nitella Internodal Cells.

    PubMed

    Kikuchi, Kenji; Mochizuki, Osamu

    2015-01-01

    Cytoplasmic streaming (CPS) is well known to assist the movement of nutrients, organelles and genetic material by transporting all of the cytoplasmic contents of a cell. CPS is generated by motility organelles that are driven by motor proteins near a membrane surface, where the CPS has been found to have a flat velocity profile in the flow field according to the sliding theory. There is a consistent mixing of contents inside the cell by CPS if the velocity gradient profile is flattened, which is not assisted by advection diffusion but is only supported by Brownian diffusion. Although the precise flow structure of the cytoplasm has an important role for cellular metabolism, the hydrodynamic mechanism of its convection has not been clarified. We conducted an experiment to visualise the flow of cytoplasm in Nitella cells by injecting tracer fluorescent nanoparticles and using a flow visualisation system in order to understand how the flow profile affects their metabolic system. We determined that the velocity field in the cytosol has an obvious velocity gradient, not a flattened gradient, which suggests that the gradient assists cytosolic mixing by Taylor-Aris dispersion more than by Brownian diffusion.

  14. Cytoplasmic RNA viruses as potential vehicles for the delivery of therapeutic small RNAs

    PubMed Central

    2013-01-01

    Viral vectors have become the best option for the delivery of therapeutic genes in conventional and RNA interference-based gene therapies. The current viral vectors for the delivery of small regulatory RNAs are based on DNA viruses and retroviruses/lentiviruses. Cytoplasmic RNA viruses have been excluded as viral vectors for RNAi therapy because of the nuclear localization of the microprocessor complex and the potential degradation of the viral RNA genome during the excision of any virus-encoded pre-microRNAs. However, in the last few years, the presence of several species of small RNAs (e.g., virus-derived small interfering RNAs, virus-derived short RNAs, and unusually small RNAs) in animals and cell cultures that are infected with cytoplasmic RNA viruses has suggested the existence of a non-canonical mechanism of microRNA biogenesis. Several studies have been conducted on the tick-borne encephalitis virus and on the Sindbis virus in which microRNA precursors were artificially incorporated and demonstrated the production of mature microRNAs. The ability of these viruses to recruit Drosha to the cytoplasm during infection resulted in the efficient processing of virus-encoded microRNA without the viral genome entering the nucleus. In this review, we discuss the relevance of these findings with an emphasis on the potential use of cytoplasmic RNA viruses as vehicles for the efficient delivery of therapeutic small RNAs. PMID:23759022

  15. Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.

    PubMed

    Gatla, Himavanth R; Singha, Bipradeb; Persaud, Valerie; Vancurova, Ivana

    2014-01-01

    Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.

  16. Characterization of aconitate hydratase from mitochondria and cytoplasm of ascites tumor cells.

    PubMed

    Hernanz, A; de la Fuente, M

    1988-07-01

    This paper describes the characterization of aconitate hydratase (EC 4.2.1.3) in cytoplasmic and mitochondrial extracts from Ehrlich ascites tumor cells carried by BALB/C mice. The results show a similar distribution of aconitate hydratase in both extracts, with specific activities much lower than those found in pig and mouse tissues. Mitochondrial aconitate hydratase shows a substrate inhibition by citrate with a Km similar to that found in cytoplasm (Km = 1.0 mM and 0.9 mM, respectively). Oxalacetate produces a mixed type of inhibition in both cytoplasmic and mitochondrial aconitate hydratases with different inhibition constants (Ki = 0.3 mM and 1.0 mM, respectively). Moreover, the specific activities of aconitate hydratase in both cytoplasm and mitochondria decrease when the tumor progresses in the peritoneum of BALB/C mice, as well as the percentage of aconitate hydratase activity in the presence of oxalacetate as the inhibitor. These results indicate that the activity and kinetics of aconitate hydratase are markedly altered by neoplastic transformation as occurs in Ehrlich ascites tumor cells. Since aconitate hydratase is not a key enzyme, these unexpected data are of interest in the study of cancer biochemistry. PMID:3179020

  17. [Rna transport from the nucleus to the cytoplasm in Acetabularia mediterranea].

    PubMed

    Pressman, E K; Razumova, V P; Sandakhchiev, L S; Levin, I M

    1975-01-01

    The biosynthesis of nuclear RNA, its transport from the nucleus to the cytoplasm and distribution in the cytoplasm were studied in Acetabularia mediterranea under different light conditions. It was shown that the nuclear RNA incorportate 3H-uracil more rapidly in the darkness and the transport of labeled RNA from the nucleus slowed down after the transfer of plants in the cold medium in the darkness. To study the distribution of nuclear RNA in the cytoplasm, the 3H-uracil labeled nuclei were transplanted in the rhizoids of unlabeled plants, the dikaryons obtained were kept for different time in the light and in the darkness and the content of 3H-RNA was determined in different stem regions. It was shown that the transport of 3H-RNA in the cytoplasm is slowed down in the darkness and it is distributed by the basal-apical gradient. RNA is rapidly accumulated in the apical stem zone in the light and redistributed afterwards in the basal stem zones. The problem of relationship between the polarity and nuclear RNA distribution in Acetabularia is discussed.

  18. Studies on independent synthesis of cytoplasmic ribonucleic acids in Acetabularia mediterranea.

    PubMed

    NAORA, H; BRACHET, J

    1960-07-01

    1. The RNA content of anucleate and nucleate fragments of Acetabularia has been measured. It was found that there is a net synthesis of RNA in nucleate fragments. On the other hand, the RNA content of anucleate fragments did not change significantly after enucleation. 2. Anucleate fragments, however, can readily incorporate (14)C-labeled adenine, orotic acid, and carbon dioxide into their cytoplasmic RNA. 3. The results of experiments on (14)CO(2) incorporation into the RNA of anucleate and nucleate fragments suggest that there is a mechanism for de novo synthesis of RNA in anucleate cytoplasm. 4. In Acetabularia, 81 per cent of the cytoplasmic RNA is bound to a large granule fraction, consisting mainly of chloroplasts. Even after removal of the nucleus, RNA is synthesized in this "chloroplast" fraction. The chloroplasts are thus a major site of RNA synthesis in the cytoplasm of these algae. Synthesis of "chloroplastic" RNA, in anucleate fragments, possibly occurs at the expense of the RNA present in other fractions (microsomes and supernatant). 5. 8-Azaguanine stimulates regeneration and cap formation in anucleate fragments and does not inhibit RNA synthesis in these fragments.

  19. Effect of cytoplasmic diversity on post anthesis heat tolerance in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nuclear genomes of ten alloplasmic lines were substituted by backcrossing four or five times using ‘Karl 92’, ‘Ventnor’, ‘U1275’ and ‘Jagger’ as recurrent parents to study the cytoplasmic effects on heat tolerance. During the final backcross, reciprocal crosses were made to develop NILs (Near Is...

  20. Cytoplasmic membrane changes during adaptation of the fresh water cyanobacterium Synechococcus 6311 to salinity

    NASA Technical Reports Server (NTRS)

    Lefort-Tran, M.; Pouphile, M.; Spath, S.; Packer, L.

    1988-01-01

    In this investigation, changes were characterized in cell structure and cytoplasmic membrane organization that occur when the freshwater cyanobacterium Synechococcus 6311 is transferred from 'low salt' (0.03 molar NaCl) to 'high salt' (0.5 molar NaCl) media (i.e. sea water concentration). Cells were examined at several time points after the imposition of the salt stress and compared to control cells, in thin sections and freeze fracture electron microscopy, and by flow cytometry. One minute after exposure to high salt, i.e. 'salt shock', virtually all intracellular granules disappeared, the density of the cytoplasm decreased, and the appearance of DNA material was changed. Glycogen and other granules, however, reappeared by 4 hours after salt exposure. The organization of the cytoplasmic membrane undergoes major reorganization following salt shock. Freeze-fracture electron microscopy showed that small intramembrane particles (diameter 7.5 and 8.5 nanometers) are reduced in number by two- to fivefold, whereas large particles, (diameters 14.5 and 17.5 nanometers) increase two- to fourfold in frequency, compared to control cells grown in low salt medium. The changes in particle size distribution suggest synthesis of new membrane proteins, in agreement with the known increases in respiration, cytochrome oxidase, and sodium proton exchange activity of the cytoplasmic membrane.

  1. Human Cytomegalovirus UL97 Kinase and Nonkinase Functions Mediate Viral Cytoplasmic Secondary Envelopment ▿

    PubMed Central

    Goldberg, Miri D.; Honigman, Alik; Weinstein, Jacob; Chou, Sunwen; Taraboulos, Albert; Rouvinski, Alexander; Shinder, Vera; Wolf, Dana G.

    2011-01-01

    Previous studies have revealed critical roles for the human cytomegalovirus (HCMV) UL97 kinase in viral nuclear maturation events. We have shown recently that UL97 affects the morphology of the viral cytoplasmic assembly compartment (AC) (M. Azzeh, A. Honigman, A. Taraboulos, A. Rouvinski, and D. G. Wolf, Virology 354:69-79, 2006). Here, we employed a comprehensive ultrastructural analysis to dissect the impact of UL97 on cytoplasmic steps of HCMV assembly. Using UL97 deletion (ΔUL97) and kinase-null (K355M) mutants, as well as the UL97 kinase inhibitor NGIC-I, we demonstrated that the loss of UL97 kinase activity resulted in a unique combination of cytoplasmic features: (i) the formation of pp65-rich aberrant cytoplasmic tegument aggregates, (ii) distorted intracytoplasmic membranes, which replaced the normal architecture of the AC, and (iv) a paucity of cytoplasmic tegumented capsids and dense bodies (DBs). We further showed that these abnormal assembly intermediates did not result from impaired nuclear capsid maturation and egress per se by using 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl) benzimidizole (BDCRB) to induce the artificial inhibition of nuclear maturation and the nucleocytoplasmic translocation of capsids. The specific abrogation of UL97 kinase activity under low-multiplicity-of-infection conditions resulted in the improved release of extracellular virus compared to that of ΔUL97, despite similar rates of viral DNA accumulation and similar effects on nuclear capsid maturation and egress. The only ultrastructural correlate of the growth difference was a higher number of cytoplasmic DBs, tegumented capsids, and clustered viral particles observed upon the specific abrogation of UL97 kinase activity compared to that of ΔUL97. These combined findings reveal a novel role for UL97 in HCMV cytoplasmic secondary envelopment steps, with a further distinction of kinase-mediated function in the formation of the virus-induced AC and a nonkinase function

  2. Tumor cell characterization and classification based on cellular specific membrane capacitance and cytoplasm conductivity.

    PubMed

    Zhao, Y; Zhao, X T; Chen, D Y; Luo, Y N; Jiang, M; Wei, C; Long, R; Yue, W T; Wang, J B; Chen, J

    2014-07-15

    This paper reports a microfluidic system that enables the characterization of tumor cell electrical properties where cells were aspirated through a constriction channel (cross-section area smaller than that of biological cells) with cellular impedance profiles measured and translated to specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm). Two batches of H1299 cells were quantified by the microfluidic platform with different constriction channel cross-section areas, recording no differences with statistical significance (p<0.001) in both Cspecific membrane (1.63±0.52 vs. 1.65±0.43 μF/cm(2)) and σcytoplasm (0.90±0.19 vs. 0.92±0.15S/m), and thus confirming the reliability of the microfluidic platform. For paired high- and low-metastatic carcinoma strains 95D (ncell=537) and 95C cells (ncell=486), significant differences in both Cspecific membrane (2.00±0.43 vs. 1.62±0.39 μF/cm(2)) and σcytoplasm (0.88±0.46 vs. 1.25±0.35S/m) were observed. Statistically significant difference only in Cspecific membrane (2.00±0.43 vs. 1.58±0.30 μF/cm(2)) was observed for 95D cells (ncell=537) and 95D CCNY-KD cells with single oncogene CCNY down regulation (ncell=479, CCNY is a membrane-associated protein). In addition, statistically significant difference only in σcytoplasm (0.73±0.17 vs. 1.01±0.17S/m) was observed for A549 cells (ncell=487) and A549 CypA-KD cells with single oncogene CypA down regulation (ncell=597, CypA is a cytosolic protein). These results validated the developed microfluidic platform for Cspecific membrane and σcytoplasm quantification and confirmed the feasibility of using Cspecific membrane and σcytoplasm for tumor cell classification. PMID:24594591

  3. Cytoplasmic effects on DNA methylation between male sterile lines and the maintainer in wheat (Triticum aestivum L.).

    PubMed

    Ba, Qingsong; Zhang, Gaisheng; Niu, Na; Ma, Shoucai; Wang, Junwei

    2014-10-01

    Male sterile cytoplasm plays an important role in hybrid wheat, and three-line system including male sterile (A line), its maintainer (B line) and restoring (R line) has played a major role in wheat hybrid production. It is well known that DNA methylation plays an important role in gene expression regulation during biological development in wheat. However, no reports are available on DNA methylation affected by different male sterile cytoplasms in hybrid wheat. We employed a methylation-sensitive amplified polymorphism technique to characterize nuclear DNA methylation in three male sterile cytoplasms. A and B lines share the same nucleus, but have different cytoplasms which is male sterile for the A and fertile for the B. The results revealed a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A lines and B line was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. K-type cytoplasm affected the methylation to a much greater degree than T-type and S-type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated sites, and polymorphism between A lines and B line for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the K-type is much greater than for the T- and S-types because the former is between Aegilops genus and Triticum genus and the latter is within Triticum genus between Triticum spelta and Triticum timopheevii species. Thus, this difference in genetic distance may be responsible for the variation in methylation that we observed.

  4. Multimerization potential of the cytoplasmic domain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41.

    PubMed

    Lee, S F; Wang, C T; Liang, J Y; Hong, S L; Huang, C C; Chen, S S

    2000-05-26

    We previously demonstrated that an envelope mutant of human immunodeficiency virus type 1 lacking the entire cytoplasmic domain interferes in trans with the production of infectious virus by inclusion of the mutant envelope into the wild-type envelope complex. We also showed that the envelope incorporation into virions is not affected when the wild-type envelope is coexpressed with the mutant envelope. These results suggest that an oligomeric structure of the cytoplasmic domain is functionally required for viral infectivity. To understand whether the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 has the potential to self-assemble as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3' to the Escherichia coli malE gene, which encodes a monomeric maltose-binding protein. The expressed fusion protein was examined by chemical cross-linking, sucrose gradient centrifugation, and gel filtration. The results showed that the cytoplasmic domain of gp41 assembles into a high-ordered structural complex. The intersubunit interaction of the cytoplasmic domain was also confirmed by a mammalian two-hybrid system that detects protein-protein interactions in eucaryotic cells. A cytoplasmic domain fragment expressed in eucaryotic cells was pulled down by glutathione-Sepharose 4B beads via its association with another cytoplasmic domain fragment fused to the C terminus of the glutathione S-transferase moiety. We also found that sequences encompassing the lentiviral lytic peptide-1 and lentiviral lytic peptide-2, which are located within residues 828-856 and 770-795, respectively, play a critical role in cytoplasmic domain self-assembly. Taken together, the results from the present study indicate that the cytoplasmic domain of gp41 by itself is sufficient to assemble into a multimeric structure. This finding supports the hypothesis that a multimeric form of the gp41 cytoplasmic domain plays a

  5. Protein kinases are associated with multiple, distinct cytoplasmic granules in quiescent yeast cells.

    PubMed

    Shah, Khyati H; Nostramo, Regina; Zhang, Bo; Varia, Sapna N; Klett, Bethany M; Herman, Paul K

    2014-12-01

    The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival.

  6. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    PubMed

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  7. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    PubMed

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  8. Critical Role of the Fusion Protein Cytoplasmic Tail Sequence in Parainfluenza Virus Assembly

    PubMed Central

    Stone, Raychel; Takimoto, Toru

    2013-01-01

    Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F (rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites. PMID:23593451

  9. Protein Kinases Are Associated with Multiple, Distinct Cytoplasmic Granules in Quiescent Yeast Cells

    PubMed Central

    Shah, Khyati H.; Nostramo, Regina; Zhang, Bo; Varia, Sapna N.; Klett, Bethany M.; Herman, Paul K.

    2014-01-01

    The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival. PMID:25342717

  10. Visualization of the cytoplasmic surface of Torpedo postsynaptic membranes by freeze-etch and immunoelectron microscopy

    PubMed Central

    1987-01-01

    The synapse-specific Mr 43,000 protein (43K protein) and the acetylcholine receptor were visualized by freeze-etch immunoelectron microscopy in preparations of purified Torpedo postsynaptic membranes. Vesicles were immobilized on glass and then sheared open by sonication to expose the cytoplasmic surface. Membranes were labeled with monoclonal antibodies to the 43K protein or the acetylcholine receptor. The cytoplasmic surface was devoid of filamentous structure, and the 43K protein and the cytoplasmic projection of the acetylcholine receptor were associated with prominent surface particles. Acetylcholine receptor and 43K protein, in membrane surfaces in direct contact with glass coated with polyornithine, segregated into dense particle aggregates separated by smooth membrane patches, whereas those in contact with glass coated with Alcian Blue underwent little or no detectable rearrangement. After treatment of vesicles at alkaline pH to remove the 43K protein, the cytoplasmic surfaces were still covered by a dense array of particles that were more uniform in shape and appeared slightly shorter than those seen on unextracted membranes, but similar in height to the extracellular projection. Monoclonal antibodies to the acetylcholine receptor labeled these particles, while antibodies to 43K protein did not. We conclude that the 43K protein is in direct association with the receptor and that complexes of the receptor and 43K protein can undergo surface-induced lateral redistribution. In addition, the cytoplasmic projection of the acetylcholine receptor is sufficiently large to be readily detected by freeze-etch electron microscopy and is similar in height to the extracellular projection. PMID:3312239

  11. Role of "active" potassium transport in the regulation of cytoplasmic pH by nonanimal cells.

    PubMed Central

    Blatt, M R; Slayman, C L

    1987-01-01

    High-affinity potassium uptake in Neurospora occurs by symport with protons [Km (apparent) = 15 microM at pH 5.8], for which a large inward gradient (approximately 400 mV) is generated by the H+-extruding ATPase of the plasma membrane. Operating in parallel, the two transport systems yield a net 1:1 exchange of K+ for cytoplasmic H+. Since this exchange could play a role in cytoplasmic pH (pHi) regulation, the coordinated functioning of the K+-H+ symport and H+ pump has been examined during acid stress. Cytoplasmic acid loads were imposed by injection and by exposure to extracellular permeant weak acid. Multibarrelled microelectrodes were used to monitor membrane potential (Vm), pHi, and the current-voltage (I-V) characteristics of the cells. The behaviors of the H+ pump and K+-H+ symport were resolved, respectively, by fitting whole membrane I-V curves to an explicit kinetic model of the Neurospora membrane and by subtracting I-V curves obtained in the absence from those obtained in the presence of 5-200 microM K+ outside. Proton pumping accelerates nearly in proportion with the cytoplasmic H+ concentration, but pHi recovery from imposed acid loads is dependent on micromolar K+ outside. Potassium import via the symport leads to a measurable alkalinization of the cytoplasm in accordance with stoichiometric (1:1) K+/H+ exchange. Potassium transport is accelerated at low pHi, but in a manner consistent with its inherent voltage sensitivity and changes in Vm resulting from an increased rate of H+ extrusion by the pump. The primary response to acid stress thus rests with the H+ pump, but K+ transport introduces an essential kinetic "valve" that can regulate net H+ export. Images PMID:3472234

  12. Nucleus and cytoplasm segmentation in microscopic images using K-means clustering and region growing

    PubMed Central

    Sarrafzadeh, Omid; Dehnavi, Alireza Mehri

    2015-01-01

    Background: Segmentation of leukocytes acts as the foundation for all automated image-based hematological disease recognition systems. Most of the time, hematologists are interested in evaluation of white blood cells only. Digital image processing techniques can help them in their analysis and diagnosis. Materials and Methods: The main objective of this paper is to detect leukocytes from a blood smear microscopic image and segment them into their two dominant elements, nucleus and cytoplasm. The segmentation is conducted using two stages of applying K-means clustering. First, the nuclei are segmented using K-means clustering. Then, a proposed method based on region growing is applied to separate the connected nuclei. Next, the nuclei are subtracted from the original image. Finally, the cytoplasm is segmented using the second stage of K-means clustering. Results: The results indicate that the proposed method is able to extract the nucleus and cytoplasm regions accurately and works well even though there is no significant contrast between the components in the image. Conclusions: In this paper, a method based on K-means clustering and region growing is proposed in order to detect leukocytes from a blood smear microscopic image and segment its components, the nucleus and the cytoplasm. As region growing step of the algorithm relies on the information of edges, it will not able to separate the connected nuclei more accurately in poor edges and it requires at least a weak edge to exist between the nuclei. The nucleus and cytoplasm segments of a leukocyte can be used for feature extraction and classification which leads to automated leukemia detection. PMID:26605213

  13. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development

    PubMed Central

    Peremyslov, Valera V.; Cole, Rex A.; Fowler, John E.; Dolja, Valerian V.

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  14. Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells

    PubMed Central

    Baldassarre, Gustavo; Belletti, Barbara; Bruni, Paola; Boccia, Angelo; Trapasso, Francesco; Pentimalli, Francesca; Barone, Maria Vittoria; Chiappetta, Gennaro; Vento, Maria Teresa; Spiezia, Stefania; Fusco, Alfredo; Viglietto, Giuseppe

    1999-01-01

    The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27–cyclin D3–Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A–E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells. PMID:10510327

  15. Nonsense-mediated mRNA decay factor Upf2 exists in both the nucleoplasm and the cytoplasm.

    PubMed

    Tatsuno, Takanori; Nakamura, Yuka; Ma, Shaofu; Tomosugi, Naohisa; Ishigaki, Yasuhito

    2016-07-01

    Upf2 protein predominantly localizes to the cytoplasmic fraction, and binds to the exon junction complex (EJC) on spliced mRNA. The present study aimed to determine the cellular site where the interaction between Upf2 and EJC occurs. First, the cell lysate was fractionated into the cytoplasm and nucleoplasm, and western blotting to detect levels of Upf2 protein was performed. Upf2 was clearly detected in the cytoplasm and in the nucleoplasm. Secondly, immunostaining was performed, and the majority of Upf2 was detected in the cytoplasmic perinuclear region; a small quantity of Upf2 was detected in the intranuclear region. RNase treatment of the cells reduced the Upf2 immunostained signal. The immunopurified fractions containing nuclear and cytoplasmic Upf2 also contained one of the EJC core factors, RBM8A. These results implied the existence of Upf2 in the nucleoplasm and the cytoplasm, and it appeared to be involved in the construction of the mRNA complex. In order to verify the construction of Upf2‑binding EJC in the nucleoplasm, an in situ proximity ligation assay was performed with anti‑Upf2 and anti‑RBM8A antibodies. These results demonstrated that their interaction occurred not only in the cytoplasmic region, but also in the intranuclear region. Taken together, these results suggested that Upf2 combines with EJC in both the cytoplasmic and the intranuclear fractions, and that it is involved in mRNA metabolism in human cells. PMID:27221324

  16. Nonsense-mediated mRNA decay factor Upf2 exists in both the nucleoplasm and the cytoplasm

    PubMed Central

    TATSUNO, TAKANORI; NAKAMURA, YUKA; MA, SHAOFU; TOMOSUGI, NAOHISA; ISHIGAKI, YASUHITO

    2016-01-01

    Upf2 protein predominantly localizes to the cytoplasmic fraction, and binds to the exon junction complex (EJC) on spliced mRNA. The present study aimed to determine the cellular site where the interaction between Upf2 and EJC occurs. First, the cell lysate was fractionated into the cytoplasm and nucleoplasm, and western blotting to detect levels of Upf2 protein was performed. Upf2 was clearly detected in the cytoplasm and in the nucleoplasm. Secondly, immunostaining was performed, and the majority of Upf2 was detected in the cytoplasmic perinuclear region; a small quantity of Upf2 was detected in the intranuclear region. RNase treatment of the cells reduced the Upf2 immunostained signal. The immune-purified fractions containing nuclear and cytoplasmic Upf2 also contained one of the EJC core factors, RBM8A. These results implied the existence of Upf2 in the nucleoplasm and the cytoplasm, and it appeared to be involved in the construction of the mRNA complex. In order to verify the construction of Upf2-binding EJC in the nucleoplasm, an in situ proximity ligation assay was performed with anti-Upf2 and anti-RBM8A antibodies. These results demonstrated that their interaction occurred not only in the cytoplasmic region, but also in the intranuclear region. Taken together, these results suggested that Upf2 combines with EJC in both the cytoplasmic and the intranuclear fractions, and that it is involved in mRNA metabolism in human cells. PMID:27221324

  17. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

    PubMed Central

    Ansseau, Eugénie; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q.; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  18. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.

    PubMed

    Ansseau, Eugénie; Eidahl, Jocelyn O; Lancelot, Céline; Tassin, Alexandra; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  19. Manumycin A inhibits triple-negative breast cancer growth through LC3-mediated cytoplasmic vacuolation death.

    PubMed

    Singha, P K; Pandeswara, S; Venkatachalam, M A; Saikumar, P

    2013-01-17

    Therapy resistance can be attributed to acquisition of anti-apoptotic mechanisms by the cancer cells. Therefore, developing approaches that trigger non-apoptotic cell death in cancer cells to compensate for apoptosis resistance will help to treat cancer effectively. Triple-negative breast cancers (TNBC) are among the most aggressive and therapy resistant to breast tumors. Here we report that manumycin A (Man A), an inhibitor of farnesyl protein transferase, reduces cancer cell viability through induction of non-apoptotic, non-autophagic cytoplasmic vacuolation death in TNBC cells. Man A persistently induced cytoplasmic vacuolation and cell death through the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins along with endoplasmic reticulum (ER) stress markers, Bip and CHOP, and accumulation of ubiquitinated proteins. As inhibitors of apoptosis and autophagy failed to block cytoplasmic vacuolation and its associated protein expression or cell death, it appears that these processes are not involved in the death induced by Man A. Ability of thiol antioxidant, NAC in blocking Man A-induced vacuolation, death and its related protein expression suggests that sulfhydryl homeostasis may be the target of Man A. Surprisingly, normal human mammary epithelial cells failed to undergo cytoplasmic vacuolation and cell death, and grew normally in presence of Man A. In conjunction with its in vitro effects, Man A also reduced tumor burden in vivo in xenograft models that showed extensive cytoplasmic vacuoles and condensed nuclei with remarkable increase in the vacuolation-associated protein expression together with increase of p21, p27, PTEN and decrease of pAkt. Interestingly, Man A-mediated upregulation of p21, p27 and PTEN and downregulation of pAkt and tumor growth suppression were also mimicked by LC3 knockdown in MDA-MB-231 cells. Overall, these results suggest novel therapeutic actions by Man A through the induction of non-apoptotic and non

  20. Characterization of mpl cytoplasmic domain sequences required for myeloproliferative leukemia virus pathogenicity.

    PubMed Central

    Bénit, L; Courtois, G; Charon, M; Varlet, P; Dusanter-Fourt, I; Gisselbrecht, S

    1994-01-01

    v-mpl is a truncated form of a receptor-like chain which belongs to the cytokine receptor superfamily. This sequence has been transduced in the myeloproliferative leukemia virus as an env-mpl fusion gene responsible for an acute myeloproliferative disorder in mice. We constructed a series of viral mutants in the mpl sequence. Analysis of their oncogenic potential in vivo indicated that a critical 69-amino-acid-long cytoplasmic domain of v-Mpl is required for myoproliferative leukemia virus pathogenicity. We also developed an in vitro assay and showed that expression of the env-mpl gene confers growth factor independence to murine as well as to human hematopoietic growth factor-dependent cell lines. These findings strongly suggest that v-Mpl delivers a constitutive proliferative signal through a limited region of its cytoplasmic domain. Images PMID:8035524

  1. Ooplasm donation in humans: the need to investigate the transmission of mitochondrial DNA following cytoplasmic transfer.

    PubMed

    St John, Justin C

    2002-08-01

    The use of cytoplasmic transfer as an assisted reproductive technique has generated much attention. This arises as donor mitochondria are introduced into the cytoplasm of the recipient oocyte. The consequences are the possible transmission of two mitochondrial (mt)DNA populations to the offspring. This pattern of inheritance is in contrast to the strictly maternal manner in which mtDNA is transmitted following natural fertilization and ICSI. This paper discusses the advantages of using such a technique to enhance embryonic development from poor quality oocytes with respect to the low copy number of mtDNA found in some oocytes following superovulation protocols. However, it also cautions against using such a technique before a clearer understanding of the patterns of inheritance and transmission of mtDNA has been established and suggests that animal models be utilised to do so. PMID:12151420

  2. Estrogen action and cytoplasmic signaling cascades. Part I: membrane-associated signaling complexes

    PubMed Central

    Segars, James H.; Driggers, Paul H.

    2014-01-01

    Remarkable progress in recent years has suggested that estrogen action in vivo is complex and often involves activation of cytoplasmic signaling cascades in addition to genomic actions mediated directly through estrogen receptors α and β. Rather than a linear response mediated solely through estrogen-responsive DNA elements, in vivo estrogen might simultaneously activate distinct signaling cascades that function as networks to coordinate tissue responses to estrogen. This complex signaling system provides for exquisite control and plasticity of response to estrogen at the tissue level, and undoubtedly contributes to the remarkable tissue-specific responses to estrogens. In part I of this series, we summarize cytoplasmic signaling modules involving estrogen or estrogen receptors, with particular focus on recently described membrane-associated signaling complexes. PMID:12217492

  3. Direct exposure of chromosomes to nonactivated ovum cytoplasm is effective for bovine somatic cell nucleus reprogramming.

    PubMed

    Tani, T; Kato, Y; Tsunoda, Y

    2001-01-01

    We examined the in vitro developmental potential of nonactivated and activated enucleated ova receiving cumulus cells at various stages of the cell cycle. Eleven to 29% of activated ova receiving donor cells stopped developing at the 8-cell stage but 21% to 50% of nonactivated ova receiving donor cells at either the G(0), G(1), G(2), or M phase, or cycling cells developed into blastocysts. One normal calf was born after transferring five blastocysts that had developed from ova receiving donor cells at the M phase. The present study demonstrated that direct exposure of donor chromosomes to nonactivated ovum cytoplasm is effective for somatic cell nucleus reprogramming, and activated ovum cytoplasm does not reprogram the nucleus.

  4. Anthracycline drug targeting: cytoplasmic versus nuclear--a fork in the road.

    PubMed

    Lothstein, L; Israel, M; Sweatman, T W

    2001-06-01

    The anthracycline antibiotics doxorubicin (Adriamycin; DOX) and daunorubicin (DNR) continue to be essential components of first-line chemotherapy in the treatment of a variety of solid and hematopoietic tumors. The overall efficacies of DOX and DNR are, however, impeded by serious dose-limiting toxicities, including cardiotoxicity, and the selection of multiple mechanisms of cellular drug resistance. These limitations have necessitated the development of newer anthracyclines whose structural and functional modifications circumvent these impediments. In this review, we will present recent strategies in anthracycline design and assess their potential therapeutic merits. Current anthracycline design has diverged to target either cytoplasmic or nuclear sites. Nuclear targets have been broadened to include not only topoisomerase II (topo II) inhibition through ternary complex stabilization and catalytic inhibition, but also topoisomerase I (topo I) inhibition and transcriptional inhibition. In contrast, cytoplasmic targeting focuses on anthracycline binding to protein kinase C (PKC) regulatory domain with consequent modulation of activity.

  5. A deep learning based framework for accurate segmentation of cervical cytoplasm and nuclei.

    PubMed

    Song, Youyi; Zhang, Ling; Chen, Siping; Ni, Dong; Li, Baopu; Zhou, Yongjing; Lei, Baiying; Wang, Tianfu

    2014-01-01

    In this paper, a superpixel and convolution neural network (CNN) based segmentation method is proposed for cervical cancer cell segmentation. Since the background and cytoplasm contrast is not relatively obvious, cytoplasm segmentation is first performed. Deep learning based on CNN is explored for region of interest detection. A coarse-to-fine nucleus segmentation for cervical cancer cell segmentation and further refinement is also developed. Experimental results show that an accuracy of 94.50% is achieved for nucleus region detection and a precision of 0.9143±0.0202 and a recall of 0.8726±0.0008 are achieved for nucleus cell segmentation. Furthermore, our comparative analysis also shows that the proposed method outperforms the related methods.

  6. Interaction with Ppil3 leads to the cytoplasmic localization of Apoptin in tumor cells

    SciTech Connect

    Huo Dehua; Yi Lina; Yang Jine

    2008-07-18

    Apoptin, a small protein encoded by chicken anemia virus (CAV), induces cell death specifically in cancer cells. In normal cells, Apoptin remains in the cytoplasm; whereas in cancerous cells, it migrates into the nucleus and kills the cell. Cellular localization appears to be crucial. Through a yeast two-hybrid screen, we identified human Peptidyl-prolyl isomerase-like 3 (Ppil3) as one of the Apoptin-associated proteins. Ppil3 could bind Apoptin directly, and held Apoptin in cytoplasm even in tumor cells. We then demonstrated that the nuclearcytoplasmic distribution of Apoptin is related to the expression level of intrinsic Ppil3. Moreover, extrinsic modifying of Ppil3 levels also resulted in nuclearcytoplasmic shuffling of Apoptin. The Apoptin P109A mutant, located between the putative nuclear localization and export signals, could significantly impair the function of Ppil3. Our results suggest a new direction for the localization mechanism study of Apoptin in cells.

  7. Intracellular Trafficking of Plasmids for Gene Therapy: Mechanisms of Cytoplasmic Movement and Nuclear Import

    PubMed Central

    Dean, David A.

    2015-01-01

    Under physiologically relevant conditions, the levels of non-viral gene transfer are low at best. The reason for this is that many barriers exist for the efficient transfer of genes to cells, even before any gene expression can occur. While many transfection strategies focus on DNA condensation and overcoming the plasma membrane, events associated with the intracellular trafficking of the DNA complexes have not been as extensively studied. Once internalized, plasmids must travel potentially long distances through the cytoplasm to reach their next barrier, the nuclear envelope. This review summarizes the current progress on the cytoplasmic trafficking and nuclear transport of plasmids used for gene therapy applications. Both of these processes utilize specific and defined mechanisms to facilitate movement of DNA complexes through the cell. The continued elucidation and exploitation of these mechanisms will lead to improved strategies for transfection and successful gene therapy. PMID:17168698

  8. Evidence for nuclear-cytoplasmic incompatibility between Allium fistulosum and A. cepa.

    PubMed

    Ulloa-G, M; Corgan, J N; Dunford, M

    1995-04-01

    An F2 population (Allium fistulosum x A. cepa) of 20plants, 10 BC1,[(A. fistulosum x A. cepa) x A. cepa], and 50 BC2 plants, [(A. fistulosum x A. cepa) x A. cepa] x A. cepa were studied cytogenetically and characterized for four isozyme alleles plus various morphological characteristics. All of the progenies were in A. fistulosum (the bunching onion) cytoplasm. In the F2 population we observed non-random chromosomal and allelic segregation, suppression of bulb onion allelic expression, and abnormalities in mitosis and meiosis. Most BC2 plants resembled A. cepa (the bulbing onion) morphologically, but anthers, filaments, pistils, and petals were abnormal. Only 3 plants, and these were most nearly like the F1 hybrid morphologically, produced any seeds.The data and observations support the hypothesis of nuclear-cytoplasmic incompatibility interactions between the bunching and bulb onion species. PMID:24174037

  9. Actin based processes that could determine the cytoplasmic architecture of plant cells.

    PubMed

    van der Honing, Hannie S; Emons, Anne Mie C; Ketelaar, Tijs

    2007-05-01

    Actin polymerisation can generate forces that are necessary for cell movement, such as the propulsion of a class of bacteria, including Listeria, and the protrusion of migrating animal cells. Force generation by the actin cytoskeleton in plant cells has not been studied. One process in plant cells that is likely to depend on actin-based force generation is the organisation of the cytoplasm. We compare the function of actin binding proteins of three well-studied mammalian models that depend on actin-based force generation with the function of their homologues in plants. We predict the possible role of these proteins, and thus the role of actin-based force generation, in the production of cytoplasmic organisation in plant cells.

  10. The human immunodeficiency virus type 1 Rev protein shuttles between the cytoplasm and nuclear compartments.

    PubMed Central

    Kalland, K H; Szilvay, A M; Brokstad, K A; Saetrevik, W; Haukenes, G

    1994-01-01

    A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (HIV). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation. Images PMID:7935458

  11. A new cytoplasmic male sterile genotype in the sugar beet Beta vulgaris L.: a molecular analysis.

    PubMed

    Mann, V; McIntosh, L; Theurer, C; Hirschberg, J

    1989-08-01

    Mitochondrial DNA (mtDNA) from fertile (N) and possibly new cytoplasmic male sterile (CMS) genotypes was studied in the sugar beet Beta vulgaris L. It was found by restriction endonuclease analysis that BMC-CMS, a cytoplasm that was derived from the wild beet Beta maritima, contained a unique type of mtDNA which is distinguishable from both the N and S-CMS, the only other CMS genotype that is currently availabe in B. vulgaris L. The organization of three genes: coxI, coxII and cob, was analyzed by hybridization with heterologous probes from maize. These genes have a similar structure in N and BMC-CMS that is different from S-CMS. It is concluded that BMC-CMS is a novel CMS genotype in the sugar beet.

  12. Cytoplasmic constriction and vesiculation after axotomy in the squid giant axon.

    PubMed

    Gallant, P E; Hammar, K; Reese, T S

    1995-12-01

    The squid giant axon responded to a transection injury by producing a gradient of cytoplasmic and vesicular changes at the cut end. At the immediate opening of the cut axon the cytoplasm was fragmented and dispersed and the vesicles in this region were in rapid Brownian movement. Approximately 0.1 mm further in, at the site of maximal axonal constriction, the axoplasm was condensed into a compact, constricted mass containing many large vesicles. The axoplasm was normal a few millimetres beyond this constricted, vesiculated end. It appears that transection triggered the transformation of normal axoplasm into a tightly constricted, highly vesiculated structure. This modified axoplasm at the cut end may slow the spread of damage and degeneration by preventing the bulk outflow of axoplasm, by slowing down the loss of intracellular molecules and by slowing down the influx of destructive extracellular ions (like calcium and chloride).

  13. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton.

    PubMed

    Lyi, Sangbom Michael; Tan, Min Jie Alvin; Parrish, Colin R

    2014-05-01

    Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.

  14. THE AAA3 DOMAIN OF CYTOPLASMIC DYNEIN ACTS AS A SWITCH TO FACILITATE MICROTUBULE RELEASE

    PubMed Central

    Dewitt, Mark A.; Cypranowska, Caroline A.; Cleary, Frank B.; Belyy, Vladislav; Yildiz, Ahmet

    2014-01-01

    Cytoplasmic dynein is an AAA+ motor responsible for intracellular cargo transport and force generation along microtubules (MTs). Unlike kinesin and myosin, dynein contains multiple ATPase subunits, with AAA1 serving as the primary catalytic site. ATPase activity at AAA3 is also essential for robust motility, but its role in dynein’s mechanochemical cycle remains unclear. Here, we introduced transient pauses in Saccharomyces cerevisiae dynein motility by using a slowly hydrolyzing ATP analog. Analysis of pausing behavior revealed that AAA3 hydrolyzes nucleotide an order of magnitude slower than AAA1 and the two sites do not coordinate. ATPase mutations to AAA3 abolish the ability of dynein to modulate MT release. Nucleotide hydrolysis at AAA3 lifts this “MT gate” to fast motility. These results suggest that AAA3 acts as a switch that repurposes cytoplasmic dynein for fast cargo transport and MT anchoring tasks in cells. PMID:25486306

  15. Cytoplasmic translocation of HuR contributes to angiotensin II induced cardiac fibrosis.

    PubMed

    Bai, Danna; Ge, Lan; Gao, Yan; Lu, Xiaozhao; Wang, Haichang; Yang, Guodong

    2015-08-01

    Cardiac fibrosis is one of the key structural changes of the hypertrophied left ventricle in hypertensive heart disease. Increased angiotensin II was found to be important in the hypertension related fibrosis, while the underlying mechanism is unknown. In this study, we found that angiotensin II dose-dependently increased the expression of Col1a1, Col3a1 and α-smooth muscle actin, which were blocked by ROS (reactive oxygen species) scavenger N-acetyl cysteine (NAC). Mechanistically, angiotensin II induced robust ROS generation, which in turn induced cytoplasmic translocation of RNA binding protein HuR. Cytoplasmic translocated HuR increased TGFβ pathway activity and subsequent collagen synthesis. In contrast, knockdown of HuR nearly blocked angiotensin II induced TGFβ activation and collagen synthesis. Taken together, we here identified that angiotensin II promotes collagen synthesis in cardiac fibroblast through ROS-HuR-TGFβ pathway.

  16. A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells

    PubMed Central

    Castanotto, Daniela; Lin, Min; Kowolik, Claudia; Wang, LiAnn; Ren, Xiao-Qin; Soifer, Harris S.; Koch, Troels; Hansen, Bo Rode; Oerum, Henrik; Armstrong, Brian; Wang, Zhigang; Bauer, Paul; Rossi, John; Stein, C.A.

    2015-01-01

    Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing. PMID:26433227

  17. Cytoplasmic streaming emerges naturally from hydrodynamic self-organisation of a microfilament suspension

    NASA Astrophysics Data System (ADS)

    Woodhouse, Francis; Goldstein, Raymond

    2013-03-01

    Cytoplasmic streaming is the ubiquitous phenomenon of deliberate, active circulation of the entire liquid contents of a plant or animal cell by the walking of motor proteins on polymer filament tracks. Its manifestation in the plant kingdom is particularly striking, where many cells exhibit highly organised patterns of flow. How these regimented flow templates develop is biologically unclear, but there is growing experimental evidence to support hydrodynamically-mediated self-organisation of the underlying microfilament tracks. Using the spirally-streaming giant internodal cells of the characean algae Chara and Nitella as our prototype, we model the developing sub-cortical streaming cytoplasm as a continuum microfilament suspension subject to hydrodynamic and geometric forcing. We show that our model successfully reproduces emergent streaming behaviour by evolving from a totally disordered initial state into a steady characean ``conveyor belt'' configuration as a consequence of the cell geometry, and discuss applicability to other classes of steadily streaming plant cells.

  18. Correlations between nuclear morphology and bundles of cytoplasmic fibrils in 50 cases of acute myeloid leukaemia.

    PubMed Central

    Pearson, E C

    1986-01-01

    An electron microscopic examination was carried out of peripheral blood or bone marrow samples, or both, from 50 patients entered into the Medical Research Council 9th Acute Myeloid Leukaemia Trial. The results showed a striking correlation between the presence of conspicuous bundles of fibrils within the cytoplasm of the leukaemic cells and the degree of convolution or lobulation of the nuclei. In none of the samples were predominantly convoluted or lobed nuclei observed in the absence of prominent fibrillar bundles and in only two cases were nuclei of a more regular outline seen in association with many conspicuous bundles of cytoplasmic fibrils. No correlation was found between the apparent degree of maturity of the nuclei, as assessed by the degree of chromatin condensation, and the absence or abundance of fibrillar bundles. Images PMID:3456357

  19. Attractant Signaling by an Aspartate Chemoreceptor Dimer with a Single Cytoplasmic Domain

    NASA Astrophysics Data System (ADS)

    Gardina, Paul J.; Manson, Michael D.

    1996-10-01

    Signal transduction across cell membranes often involves interactions among identical receptor subunits, but the contribution of individual subunits is not well understood. The chemoreceptors of enteric bacteria mediate attractant responses by interrupting a phosphotransfer circuit initiated at receptor complexes with the protein kinase CheA. The aspartate receptor (Tar) is a homodimer, and oligomerized cytoplasmic domains stimulate CheA activity much more than monomers do in vitro. Intragenic complementation was used to show in Escherichia coli that heterodimers containing one full-length and one truncated Tar subunit mediated responses to aspartate in the presence of full-length Tar homodimers that could not bind aspartate. Thus, a Tar dimer containing only one cytoplasmic domain can initiate an attractant (inhibitory) signal, although it may not be able to stimulate kinase activity of CheA.

  20. Kluyveromyces lactis killer system: analysis of cytoplasmic promoters of the linear plasmids.

    PubMed Central

    Schickel, J; Helmig, C; Meinhardt, F

    1996-01-01

    All of the 14 genes encoded by the cytoplasmic linear killer plasmids of Kluyveromyces lactis are preceded by upstream conserved sequences (UCSs), cis-acting elements involved in plasmid gene transcription. Using the bacterial glucose-dehydrogenase gene as a reporter, expression driven by seven cytoplasmic promoters was determined. The level of expression ranged from 0.5 to 6 nkat. The highest activity was displayed by UCS 6 of pGKL2 whereas the lowest level was obtained with UCS2 of pGKL2, all other values were in between. Sequences located 5' upstream the UCSs do not influence expression. As exemplified for UCS5 and UCS10, deletion led to an almost complete loss of expression. PMID:8657569

  1. Enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers.

    PubMed

    Parodi, Alessandro; Corbo, Claudia; Cevenini, Armando; Molinaro, Roberto; Palomba, Roberto; Pandolfi, Laura; Agostini, Marco; Salvatore, Francesco; Tasciotti, Ennio

    2015-07-01

    Nanocarriers are designed to specifically accumulate in diseased tissues. In this context, targeting of intracellular compartments was shown to enhance the efficacy of many drugs and to offer new and more effective therapeutic approaches. This is especially true for therapies based on biologicals that must be encapsulated to favor cell internalization, and to avoid intracellular endosomal sequestration and degradation of the payload. In this review, we discuss specific surface modifications designed to achieve cell cytoplasm delivery and to improve targeting of major organelles; we also discuss the therapeutic applications of these approaches. Last, we describe some integrated strategies designed to sequentially overcome the biological barriers that separate the site of administration from the cell cytoplasm, which is the drug's site of action.

  2. The transmission of nuclear pore complexes to daughter cells requires a cytoplasmic pool of Nsp1.

    PubMed

    Colombi, Paolo; Webster, Brant M; Fröhlich, Florian; Lusk, C Patrick

    2013-10-28

    Nuclear pore complexes (NPCs) are essential protein assemblies that span the nuclear envelope and establish nuclear-cytoplasmic compartmentalization. We have investigated mechanisms that control NPC number in mother and daughter cells during the asymmetric division of budding yeast. By simultaneously tracking existing NPCs and newly synthesized NPC protomers (nups) through anaphase, we uncovered a pool of the central channel nup Nsp1 that is actively targeted to the bud in association with endoplasmic reticulum. Bud targeting required an intact actin cytoskeleton and the class V myosin, Myo2. Selective inhibition of cytoplasmic Nsp1 or inactivation of Myo2 reduced the inheritance of NPCs in daughter cells, leading to a daughter-specific loss of viability. Our data are consistent with a model in which Nsp1 releases a barrier that otherwise prevents NPC passage through the bud neck. It further supports the finding that NPC inheritance, not de novo NPC assembly, is primarily responsible for controlling NPC number in daughter cells.

  3. Cytoplasmic motion induced by cytoskeleton stretching and its effect on cell mechanics.

    PubMed

    Zhang, T

    2011-09-01

    Cytoplasmic motion assumed as a steady state laminar flow induced by cytoskeleton stretching in a cell is determined and its effect on the mechanical behavior of the cell under externally applied forces is demonstrated. Non-Newtonian fluid is assumed for the multiphase cytoplasmic fluid and the analytical velocity field around the macromolecular chain is obtained by solving the reduced nonlinear momentum equation using homotopy technique. The entropy generation by the fluid internal friction is calculated and incorporated into the entropic elasticity based 8-chain constitutive relations. Numerical examples showed strengthening behavior of cells in response to externally applied mechanical stimuli. The spatial distribution of the stresses within a cell under externally applied fluid flow forces were also studied. PMID:21977515

  4. Cytoplasmic translocation of HuR contributes to angiotensin II induced cardiac fibrosis.

    PubMed

    Bai, Danna; Ge, Lan; Gao, Yan; Lu, Xiaozhao; Wang, Haichang; Yang, Guodong

    2015-08-01

    Cardiac fibrosis is one of the key structural changes of the hypertrophied left ventricle in hypertensive heart disease. Increased angiotensin II was found to be important in the hypertension related fibrosis, while the underlying mechanism is unknown. In this study, we found that angiotensin II dose-dependently increased the expression of Col1a1, Col3a1 and α-smooth muscle actin, which were blocked by ROS (reactive oxygen species) scavenger N-acetyl cysteine (NAC). Mechanistically, angiotensin II induced robust ROS generation, which in turn induced cytoplasmic translocation of RNA binding protein HuR. Cytoplasmic translocated HuR increased TGFβ pathway activity and subsequent collagen synthesis. In contrast, knockdown of HuR nearly blocked angiotensin II induced TGFβ activation and collagen synthesis. Taken together, we here identified that angiotensin II promotes collagen synthesis in cardiac fibroblast through ROS-HuR-TGFβ pathway. PMID:26093296

  5. Flow-induced channel formation in the cytoplasm of motile cells

    NASA Astrophysics Data System (ADS)

    Guy, Robert D.; Nakagaki, Toshiyuki; Wright, Grady B.

    2011-07-01

    A model is presented to explain the development of flow channels within the cytoplasm of the plasmodium of the giant amoeba Physarum polycephalum. The formation of channels is related to the development of a self-organizing tubular network in large cells. Experiments indicate that the flow of cytoplasm is involved in the development and organization of these networks, and the mathematical model proposed here is motivated by recent experiments involving the observation of development of flow channel in small cells. A model of pressure-driven flow through a polymer network is presented in which the rate of flow increases the rate of depolymerization. Numerical solutions and asymptotic analysis of the model in one spatial dimension show that under very general assumptions this model predicts the formation of channels in response to flow.

  6. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

    SciTech Connect

    Lyi, Sangbom Michael; Tan, Min Jie Alvin Parrish, Colin R.

    2014-05-15

    Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.

  7. Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

    SciTech Connect

    Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

    1988-03-01

    Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added /sup 14/CO/sub 2/ was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.

  8. Evidence for nuclear-cytoplasmic incompatibility between Allium fistulosum and A. cepa.

    PubMed

    Ulloa-G, M; Corgan, J N; Dunford, M

    1995-04-01

    An F2 population (Allium fistulosum x A. cepa) of 20plants, 10 BC1,[(A. fistulosum x A. cepa) x A. cepa], and 50 BC2 plants, [(A. fistulosum x A. cepa) x A. cepa] x A. cepa were studied cytogenetically and characterized for four isozyme alleles plus various morphological characteristics. All of the progenies were in A. fistulosum (the bunching onion) cytoplasm. In the F2 population we observed non-random chromosomal and allelic segregation, suppression of bulb onion allelic expression, and abnormalities in mitosis and meiosis. Most BC2 plants resembled A. cepa (the bulbing onion) morphologically, but anthers, filaments, pistils, and petals were abnormal. Only 3 plants, and these were most nearly like the F1 hybrid morphologically, produced any seeds.The data and observations support the hypothesis of nuclear-cytoplasmic incompatibility interactions between the bunching and bulb onion species.

  9. Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA.

    PubMed

    Woerner, Andreas C; Frottin, Frédéric; Hornburg, Daniel; Feng, Li R; Meissner, Felix; Patra, Maria; Tatzelt, Jörg; Mann, Matthias; Winklhofer, Konstanze F; Hartl, F Ulrich; Hipp, Mark S

    2016-01-01

    Amyloid-like protein aggregation is associated with neurodegeneration and other pathologies. The nature of the toxic aggregate species and their mechanism of action remain elusive. Here, we analyzed the compartment specificity of aggregate toxicity using artificial β-sheet proteins, as well as fragments of mutant huntingtin and TAR DNA binding protein-43 (TDP-43). Aggregation in the cytoplasm interfered with nucleocytoplasmic protein and RNA transport. In contrast, the same proteins did not inhibit transport when forming inclusions in the nucleus at or around the nucleolus. Protein aggregation in the cytoplasm, but not the nucleus, caused the sequestration and mislocalization of proteins containing disordered and low-complexity sequences, including multiple factors of the nuclear import and export machinery. Thus, impairment of nucleocytoplasmic transport may contribute to the cellular pathology of various aggregate deposition diseases. PMID:26634439

  10. Evidence for a species of nuclear actin distinct from cytoplasmic and muscles actins.

    PubMed

    Bremer, J W; Busch, H; Yeoman, L C

    1981-03-31

    Nuclear actin (protein BJ) has been isolated from the chromatin of Novikoff hepatoma ascites cells and purified to homogeneity by selective extraction, Sepharose CL-6B chromatography, and preparative polyacrylamide gel electrophoresis. A comparison of nuclear and cytoplasmic actins from Novikoff hepatoma cells and rabbit muscle actin was made by amino acid analysis, isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional peptide mapping procedures. By these criteria, all of the proteins compared are actins, but each is chemically distinct. It was concluded, therefore, that nuclear actin is similar to, but not identical with, cytoplasmic actin isolated from Novikoff hepatoma cells. A striking similarity in peptide charge and migration as shown by peptide map analysis was observed for nuclear and rabbit skeletal muscle actins. This may indicate that nuclear actin has the capacity for contractile function. In addition, the actins synthesized in Novikoff hepatoma cells may results from more than two structural genes.

  11. Cytoplasmic Ig-Domain Proteins: Cytoskeletal Regulators with a Role in Human Disease

    PubMed Central

    Otey, Carol A.; Dixon, Richard; Stack, Christianna; Goicoechea, Silvia M.

    2009-01-01

    Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain proteins are more than structural elements of the cell, and may have evolved to fill different needs in different cellular compartments. PMID:19466753

  12. The CMS-associated 16 kDa protein encoded by orfH522 in the PET1 cytoplasm is also present in other male-sterile cytoplasms of sunflower.

    PubMed

    Horn, R; Hustedt, J E; Horstmeyer, A; Hahnen, J; Zetsche, K; Friedt, W

    1996-02-01

    In sunflower plants carrying the PET1 cytoplasm male sterility (CMS) is associated with a new open reading frame (orfH522) in the 3'-flanking region of the atpA gene and an additional 16 kDa protein. Twenty-seven male-sterile cytoplasms of different origin were studied for the expression of the 16 kDa protein. In addition to the PET1 cytoplasm nine other male-sterile cytoplasms express the CMS-associated protein. These CMS sources originate from different interspecific crosses, from spontaneously occurring male-sterile plants in wild sunflower and from induced mutagenesis. Polyclonal antisera were raised against fusion proteins which contain 421 bp of the 3'-coding region of orfH522 to verify by immunological methods the identity of the other CMS cytoplasms. The anti-ORFH522 antiserum showed a positive reaction in the immunoblot with all CMS cytoplasms which expressing the 16 kDa protein. Investigations of the mitochondrial DNA demonstrated that all ten CMS cytoplasms which express the 16 kDa protein have the same organization at the atpA locus. OrfH522 as probes gave the same transcript pattern for the investigated CMS cytoplasms, just as for PET1. The MAX1 cytoplasm has an orfH522-related sequence but does not synthesize the 16 kDa protein. Using the sodium carbonate treatment the 16 kDa protein proved to be membrane-bound. Computer analyses predict that the hydrophobic N-terminal region of ORFH522 may form a transmembrane helix functioning as membrane anchor. PMID:8605303

  13. Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix

    PubMed Central

    1987-01-01

    We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy- requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p- [dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages. PMID:3693397

  14. Cytoplasmic male sterility contributes to hybrid incompatibility between subspecies of Arabidopsis lyrata.

    PubMed

    Aalto, Esa A; Koelewijn, Hans-Peter; Savolainen, Outi

    2013-10-03

    In crosses between evolutionarily diverged populations, genomic incompatibilities may result in sterile hybrids, indicating evolution of reproductive isolation. In several plant families, crosses within a population can also lead to male sterile progeny because of conflict between the maternally and biparentally inherited genomes. We examined hybrid fertility between subspecies of the perennial outcrossing self-incompatible Lyrate rockcress (Arabidopsis lyrata) in large reciprocal F2 progenies and three generations of backcrosses. In one of the reciprocal F2 progenies, almost one-fourth of the plants were male-sterile. Correspondingly, almost one-half of the plants in one of the four reciprocal backcross progenies expressed male sterility. In an additional four independent F2 and backcross families, three segregated male sterility. The observed asymmetrical hybrid incompatibility is attributable to male sterility factors in one cytoplasm, for which the other population lacks effective fertility restorers. Genotyping of 96 molecular markers and quantitative trait locus mapping revealed that only 60% of the plants having the male sterile cytoplasm and lacking the corresponding restorers were phenotypically male-sterile. Genotyping data showed that there is only one restorer locus, which mapped to a 600-kb interval at the top of chromosome 2 in a region containing a cluster of pentatricopeptide repeat genes. Male fertility showed no trade-off with seed production. We discuss the role of cytoplasm and genomic conflict in incipient speciation and conclude that cytoplasmic male sterility-lowering hybrid fitness is a transient effect with limited potential to form permanent reproductive barriers between diverged populations of hermaphrodite self-incompatible species.

  15. Intratumoral injection of taxol in vivo suppresses A549 tumor showing cytoplasmic vacuolization.

    PubMed

    Wang, Chaoyang; Chen, Tongsheng

    2012-04-01

    Based on our recent in vitro studies, this report was designed to explore the mechanism by which high concentration of taxol (70 µM) induced paraptosis-like cell death in human lung carcinoma (A549) cells, and to evaluate the therapeutic efficacy of taxol using A549 tumor-bearing mice in vivo. Exposure of cells to taxol induced time-dependent cytotoxicity and cytoplasmic vacuolization without the involvement of Bax, Bak, Mcl-1, Bcl-XL, and caspase-3. Although taxol treatment induced activating transcription factor 6 (ATF6) cleavage indicative of endoplasmic reticulum (ER) stress, silencing ATF6 by shATF6 did not prevent taxol-induced both cytotoxcity and cytoplasmic vacuolization, suggesting that taxol-induced cytoplasmic vacuolization and cell death were not due to ER stress. Moreover, taxol-treated cells did not show DNA fragmentation and loss of mitochondrial membrane potential, the typical characteristics of apoptosis. In addition, taxol-induced cytoplasmic vacuolization did not show the cellular lysis, the characteristics of oncosis, and positive of β-galactosidase, the characteristic of senescence, indicating that taxol induced paraptosis-like cell death is neither oncosis nor senescence. Moreover, our in vivo data showed that intratumoral injection of taxol (50 mg/kg) in A549 tumor xenograft mice on day 1 and day 19 potently suppressed tumor growth showing significant ER vacuolization without toxicity. In conclusion, high concentration of taxol exhibits a significant anticancer activity by inducing paraptosis-like cell death in vitro and in vivo, without significant toxicity, suggesting a promising therapeutic strategy for apoptosis-resistance cancer by inducing ER vacuolization.

  16. Surface bound or cytoplasmic immunoglobulins: interpretation of the immunofluorescence observed in cytocentrifuge slides of human lymphocytes.

    PubMed Central

    Schuit, H R; Hijmans, W; Jansen, J

    1984-01-01

    Results of immunofluorescence observations in the study of normal and malignant blood lymphocytes are described. Data which support the proposition that most membrane bound immunoglobulin molecules are stable enough to remain intact during cytocentrifuge slide preparation are presented. Therefore not all positive cells in a fixed cytocentrifuge slide should be considered as containing cytoplasmic immunoglobulins. A correct interpretation is essential because of its bearing on our concepts of B lymphocyte differentiation. Images Fig. 3 PMID:6378456

  17. An automated image processing routine for segmentation of cell cytoplasms in high-resolution autofluorescence images

    NASA Astrophysics Data System (ADS)

    Walsh, Alex J.; Skala, Melissa C.

    2014-02-01

    The heterogeneity of genotypes and phenotypes within cancers is correlated with disease progression and drug-resistant cellular sub-populations. Therefore, robust techniques capable of probing majority and minority cell populations are important both for cancer diagnostics and therapy monitoring. Herein, we present a modified CellProfiler routine to isolate cytoplasmic fluorescence signal on a single cell level from high resolution auto-fluorescence microscopic images.

  18. The Architecture of the Cytoplasmic Region of Type III Secretion Systems

    PubMed Central

    Makino, Fumiaki; Shen, Dakang; Kajimura, Naoko; Kawamoto, Akihiro; Pissaridou, Panayiota; Oswin, Henry; Pain, Maria; Murillo, Isabel; Namba, Keiichi; Blocker, Ariel J.

    2016-01-01

    Type III secretion systems (T3SSs) are essential devices in the virulence of many Gram-negative bacterial pathogens. They mediate injection of protein effectors of virulence from bacteria into eukaryotic host cells to manipulate them during infection. T3SSs involved in virulence (vT3SSs) are evolutionarily related to bacterial flagellar protein export apparatuses (fT3SSs), which are essential for flagellar assembly and cell motility. The structure of the external and transmembrane parts of both fT3SS and vT3SS is increasingly well-defined. However, the arrangement of their cytoplasmic and inner membrane export apparatuses is much less clear. Here we compare the architecture of the cytoplasmic regions of the vT3SSs of Shigella flexneri and the vT3SS and fT3SS of Salmonella enterica serovar Typhimurium at ~5 and ~4 nm resolution using electron cryotomography and subtomogram averaging. We show that the cytoplasmic regions of vT3SSs display conserved six-fold symmetric features including pods, linkers and an ATPase complex, while fT3SSs probably only display six-fold symmetry in their ATPase region. We also identify other morphological differences between vT3SSs and fT3SSs, such as relative disposition of their inner membrane-attached export platform, C-ring/pods and ATPase complex. Finally, using classification, we find that both types of apparatuses can loose elements of their cytoplasmic region, which may therefore be dynamic. PMID:27686865

  19. The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

    PubMed Central

    Latifi, Ali Mohammad; Khajeh, Khosro; Farnoosh, Gholamreza; Hassanpour, Kazem; Khodi, Samaneh

    2015-01-01

    Background: Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process. Objectives: In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b). Materials and Methods: The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd. Results: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band. Conclusions: The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step. PMID:26870308

  20. Casein kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in the cytoplasm.

    PubMed

    Golden, D; Cantley, L G

    2015-09-01

    Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during developmental epithelial-mesenchymal transition (EMT) and has been associated with EMT in malignant epithelia. Our laboratory has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2 (CK2) phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and phosphorylates this site in vitro. Knockdown or inhibition of the CK2α/α' kinase subunit in epithelial cells causes de novo accumulation of Foxc2 in the nucleus. Mutation of serine 124 to leucine promotes constitutive nuclear localization of Foxc2 and expression of mesenchymal genes, whereas an S124D phosphomimetic leads to constitutive cytoplasmic localization and epithelial maintenance. In malignant breast cancer cells, the CK2β regulatory subunit is downregulated and FOXC2 is found in the nucleus, correlating with an increase in α-smooth muscle actin (SMA) expression. Restoration of CK2β expression in these cells results in cytoplasmic localization of Foxc2, decreased α-SMA expression and reduced cell migration and invasion. In contrast, knockdown of CK2β in normal breast epithelial cells leads to FOXC2 nuclear localization, decreased E-cadherin expression, increased α-SMA and vimentin expression, and enhanced cell migration and invasion. Based on these findings, we propose that Foxc2 is functionally maintained in the cytoplasm of normal epithelial cells by CK2α/α'-mediated phosphorylation at serine 124, which is dependent on proper targeting of the holoenzyme via the CK2β regulatory subunit.

  1. Cytoplasmic sensing by the inner membrane histidine kinase EnvZ

    PubMed Central

    Foo, Yong Hwee; Gao, Yunfeng; Zhang, Hongfang; Kenney, Linda J.

    2016-01-01

    Two-component regulatory systems drive signal transduction in bacteria. The simplest of these employs a membrane sensor kinase and a cytoplasmic response regulator. Environmental sensing is typically coupled to gene regulation. The histidine kinase EnvZ and its cognate response regulator OmpR regulate expression of outer membrane proteins (porins) in response to osmotic stress. We used hydrogen:deuterium exchange mass spectrometry to identify conformational changes in the cytoplasmic domain of EnvZ (EnvZc) that were associated with osmosensing. The osmosensor localized to a seventeen amino acid region of the four-helix bundle of the cytoplasmic domain and flanked the His243 autophosphorylation site. High osmolality increased autophosphorylation of His243, suggesting that these two events were linked. The transmembrane domains were not required for osmosensing, but mutants in the transmembrane domains altered EnvZ activity. A photoactivatable fusion protein composed of EnvZc fused to the fluorophore mEos2 (EnvZc-mEos2) was as capable as EnvZc in supporting OmpR-dependent ompF and ompC transcription. Over-expression of EnvZc reduced activity, indicating that the EnvZ/OmpR system is not robust. Our results support a model in which osmolytes stabilize helix one in the four-helix bundle of EnvZ by increased hydrogen bonding of the peptide backbone, increasing autophosphorylation and downstream signaling. The likelihood that additional histidine kinases use similar cytoplasmic sensing mechanisms is discussed. PMID:25937465

  2. Human autoantibodies to diacyl-phosphatidylethanolamine recognize a specific set of discrete cytoplasmic domains

    PubMed Central

    Laurino, C C F C; Fritzler, M J; Mortara, R A; Silva, N P; Almeida, I C; Andrade, L E C

    2006-01-01

    The aim of this study was to characterize a novel human autoantibody–autoantigen system represented as cytoplasmic discrete speckles (CDS) in indirect immunofluorescence (IIF). A distinct CDS IIF pattern represented by 3–20 discrete speckles dispersed throughout the cytoplasm was identified among other cytoplasmic speckled IIF patterns. The cytoplasmic domains labelled by human anti-CDS-1 antibodies did not co-localize with endosome/lysosome markers EEA1 and LAMP-2, but showed partial co-localization with glycine–tryptophan bodies (GWB). CDS-1 sera did not react with several cellular extracts in immunoblotting and did not immunoprecipitate recombinant GW182 or EEA1 proteins. The typical CDS-1 IIF labelling pattern was abolished after delipidation of HEp-2 cells. Moreover, CDS-1 sera reacted strongly with a lipid component co-migrating with phosphatidylethanolamine (PE) in high performance thin-layer chromatography (HPTLC)-immunostaining of HEp-2 cell total lipid extracts. The CDS-1 major molecular targets were established by electrospray ionization–mass spectrometry (ESI-MS), HPTLC-immunostaining and chemiluminescent enzyme-linked immunosorbent assay as diacyl-PE species, containing preferentially a cis-C18 : 1 fatty acid chain at C-2 of the glycerol moiety, namely 1,2-cis-C18 : 1-PE and 1-C16 : 0-2-cis-C18 : 1-PE. The clinical association of CDS-1 sera included a variety of systemic and organ-specific autoimmune diseases but they were also observed in patients with no evidence of autoimmune disease. PMID:16487257

  3. CALMODULIN ANTAGONISTS EFFECT ON Ca2+ LEVEL IN THE MITOCHONDRIA AND CYTOPLASM OF MYOMETRIUM CELLS.

    PubMed

    Shlykov, S G; Babich, L G; Yevtushenko, M E; Karakhim, S O; Kosterin, S O

    2015-01-01

    It is known that Ca(2+)-dependent regulation of this cation exchange in mitochondria is carried out with participation of calmodulin. We had shown in a previous work using two experimental models: isolated mitochondria and intact myometrium cells, that calmodulin antagonists reduce the level of mitochondrial membrane polarization. The aim of this work was to investigate the influence of calmodulin antagonists on the level of ionized Ca in mitochondria and cytoplasm of uterine smooth muscle cells using spectrofluorometry and confocal microscopy. It was shown that myometrium mitochondria, in the presence of ATP and MgCl2 in the incubation medium, accumulate Ca ions in the matrix. Incubation of mitochondria in the presence of CCCP inhibited cation accumulation, but did not cease it. Calmodulin antagonist such as trifluoperazine (100 μm) considerably increased the level of ionized Ca in the mitochondrial matrix. Preliminary incubation of mitochondria with 100 μM Ca2+, before adding trifluoperazine to the incubation medium, partly prevented influence of the latter on the cation level in the matrix. Incubation of myometrium cells (primary culture) with another calmodulin antagonist calmidazolium (10 μM was accompanied by depolarization of mitochondrial membrane and an increase in the concentration of ionized Ca in cytoplasm. Thus, using two models, namely, isolated mitochondria and intact myometrium cells, it has been shown that calmodulin antagonists cause depolarization of mitochondrial membranes and an increase of the ionized Ca concentration in both the mitochondrial matrix and the cell cytoplasm.

  4. Characterization and localization of the cytoplasmic dynein heavy chain in Aspergillus nidulans.

    PubMed

    Xiang, X; Roghi, C; Morris, N R

    1995-10-10

    Migration of nuclei throughout the mycelium is essential for the growth and differentiation of filamentous fungi. In Aspergillus nidulans, the nudA gene, which is involved in nuclear migration, encodes a cytoplasmic dynein heavy chain. In this paper we use antibodies to characterize the Aspergillus cytoplasmic dynein heavy chain (ACDHC) and to show that the ACDHC is concentrated at the growing tip of the fungal mycelium. We demonstrate that four temperature-sensitive mutations in the nudA gene result in a striking decrease in ACDHC protein. Cytoplasmic dynein has been implicated in nuclear division in animal cells. Because the temperature-sensitive nudA mutants are able to grow slowly with occasional nuclei found in the mycelium and are able to undergo nuclear division, we have created a deletion/disruption nudA mutation and a tightly downregulated nudA mutation. These mutants exhibit a phenotype very similar to that of the temperature-sensitive nudA mutants with respect to growth, nuclear distribution, and nuclear division. This suggests that there are redundant backup motor proteins for both nuclear migration and nuclear division.

  5. Endocytosis by the asialoglycoprotein receptor is independent of cytoplasmic serine residues.

    PubMed Central

    Geffen, I; Fuhrer, C; Spiess, M

    1991-01-01

    The human asialoglycoprotein (ASGP) receptor, like most other plasma membrane receptors, has previously been shown to be phosphorylated at serine residues within the cytoplasmic domain. Phorbol esters, which activate protein kinase C, cause hyperphosphorylation and down-regulation of the ASGP receptor in HepG2 cells. To test the importance of serine residues for receptor traffic and function, we have mutated all the cytoplasmic serines of the two receptor subunits H1 (at positions 16 and 37) and H2 (at positions 12, 13, and 55) to alanines or glycines. Stable transfected fibroblast cell lines expressing either mutant H1 alone or both mutant subunits together were created and compared to cell lines expressing the respective wild-type proteins. Mutant and wild-type subunits were found to have very similar distributions between the cell surface and intracellular compartments. Constitutive internalization of H1 alone and ligand uptake and degradation by cells expressing both receptor subunits were not affected by the mutations. Cytoplasmic serines and serine phosphorylation are thus not essential for receptor function and intracellular traffic. Analysis of individual serine mutations identified serine-12 of subunit H2 as the major site of phosphorylation in the ASGP receptor. Images PMID:1924301

  6. A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice.

    PubMed

    Luo, Dangping; Xu, Hong; Liu, Zhenlan; Guo, Jingxin; Li, Heying; Chen, Letian; Fang, Ce; Zhang, Qunyu; Bai, Mei; Yao, Nan; Wu, Hong; Wu, Hao; Ji, Chonghui; Zheng, Huiqi; Chen, Yuanling; Ye, Shan; Li, Xiaoyu; Zhao, Xiucai; Li, Riqing; Liu, Yao-Guang

    2013-05-01

    Plant cytoplasmic male sterility (CMS) results from incompatibilities between the organellar and nuclear genomes and prevents self pollination, enabling hybrid crop breeding to increase yields. The Wild Abortive CMS (CMS-WA) has been exploited in the majority of 'three-line' hybrid rice production since the 1970s, but the molecular basis of this trait remains unknown. Here we report that a new mitochondrial gene, WA352, which originated recently in wild rice, confers CMS-WA because the protein it encodes interacts with the nuclear-encoded mitochondrial protein COX11. In CMS-WA lines, WA352 accumulates preferentially in the anther tapetum, thereby inhibiting COX11 function in peroxide metabolism and triggering premature tapetal programmed cell death and consequent pollen abortion. WA352-induced sterility can be suppressed by two restorer-of-fertility (Rf) genes, suggesting the existence of different mechanisms to counteract deleterious cytoplasmic factors. Thus, CMS-related cytoplasmic-nuclear incompatibility is driven by a detrimental interaction between a newly evolved mitochondrial gene and a conserved, essential nuclear gene.

  7. Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

    PubMed Central

    Slabodnick, Mark; Prevo, Bram; Gross, Peter; Sheung, Janet; Marshall, Wallace

    2013-01-01

    Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a "double decker" microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm. PMID:24378633

  8. Visualizing cytoplasmic flow during single-cell wound healing in Stentor coeruleus.

    PubMed

    Slabodnick, Mark; Prevo, Bram; Gross, Peter; Sheung, Janet; Marshall, Wallace

    2013-01-01

    Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a "double decker" microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm. PMID:24378633

  9. Cooperative regulation of cytoplasmic streaming and ca fluxes by pfr and photosynthesis in vallisneria mesophyll cells.

    PubMed

    Takagi, S; Yamamoto, K T; Furuya, M; Nagai, R

    1990-12-01

    In mesophyll cells of Vallisneria gigantea Graebner, Ca(2+) regulates the induction and cessation of cytoplasmic streaming. Streaming is induced when the level of calcium in the cytoplasm is lowered through light-accelerated release of Ca(2+) from the cells (S Takagi, R Nagai [1988] Plant Physiol 88: 228-232). We have now initiated an investigation on the nature of the photoreceptor(s) that are involved in the regulation of Ca(2+) movements across the cell membrane and of streaming. Streaming is induced only when phytochrome exists in the phytochrome-far redabsorbing form (Pfr)-and photosynthesis is allowed to take place for at least 4 minutes. The former effect is typically photoreversible by red and far-red light, and phytochrome is spectro-photometrically detectable in the crude extract from the leaves. The latter effect is assessed in terms of the wavelength dependency and the effects of diuron and atrazine, two inhibitors of photosynthesis. A similar requirement for Pfr and photosynthesis is found to be associated with the acceleration of Ca(2+) efflux in the protoplasts. The results suggest that phytochrome and photosynthetic pigment(s) cooperatively regulate cytoplasmic streaming via modulation of the Ca(2+) transport in the cell membrane. PMID:16667905

  10. Cytoplasmic hnRNPK interacts with GSK3β and is essential for the osteoclast differentiation

    PubMed Central

    Fan, Xiaoqin; Xiong, Haiting; Wei, Jinmei; Gao, Xuejuan; Feng, Yuan; Liu, Xiaohui; Zhang, Gong; He, Qing-Yu; Xu, Jiake; Liu, Langxia

    2015-01-01

    Osteoclast differentiation is a complex and finely regulated physiological process that involves a variety of signaling pathways and factors. Recent studies suggested that the Ser9 phosphorylation of Glycogen synthase kinase-3β (GSK3β) is required for the osteoclast differentiation. However, the precise underlying mechanism remains unclear. We have previously identified the heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a putative GSK3β interactor. In the present study, we demonstrate that, during the RANKL-induced osteoclast differentiation, the PI3K/Akt-mediated Ser9 phosphorylation of GSK3β provokes the nuclear-cytoplasmic translocation of hnRNPK in an ERK-dependent manner, enhancing the cytoplasmic co-localization and interaction of GSK3β and hnRNPK. We show that hnRNPK is essential for the osteoclast differentiation, and is involved in several reported functions of GSK3β, including the activation of NF-κB, the expression of NFATc1, and the acetylation of tubulin, all known to be critical for osteoclast differentiation and functions. We find that hnRNPK is localized in the actin belt, and is important for the mature osteoclast formation. Taken together, we demonstrate here the critical role of hnRNPK in osteoclast differentiation, and depict a model in which the cytoplasmic hnRNPK interacts with GSK3β and regulates its function. PMID:26638989

  11. Autophagy regulates cytoplasmic remodeling during cell reprogramming in a zebrafish model of muscle regeneration

    PubMed Central

    Saera-Vila, Alfonso; Kish, Phillip E.; Louie, Ke'ale W.; Grzegorski, Steven J.; Klionsky, Daniel J.; Kahana, Alon

    2016-01-01

    ABSTRACT Cell identity involves both selective gene activity and specialization of cytoplasmic architecture and protein machinery. Similarly, reprogramming differentiated cells requires both genetic program alterations and remodeling of the cellular architecture. While changes in genetic and epigenetic programs have been well documented in dedifferentiating cells, the pathways responsible for remodeling the cellular architecture and eliminating specialized protein complexes are not as well understood. Here, we utilize a zebrafish model of adult muscle regeneration to study cytoplasmic remodeling during cell dedifferentiation. We describe activation of autophagy early in the regenerative response to muscle injury, while blocking autophagy using chloroquine or Atg5 and Becn1 knockdown reduced the rate of regeneration with accumulation of sarcomeric and nuclear debris. We further identify Casp3/caspase 3 as a candidate mediator of cellular reprogramming and Fgf signaling as an important activator of autophagy in dedifferentiating myocytes. We conclude that autophagy plays a critical role in cell reprogramming by regulating cytoplasmic remodeling, facilitating the transition to a less differentiated cell identity. PMID:27467399

  12. The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers.

    PubMed

    Nikulina, Karina; Patel-King, Ramila S; Takebe, Sachiko; Pfister, K Kevin; King, Stephen M

    2004-04-01

    The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis. Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella.

  13. Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus.

    PubMed

    Horie, Ryo; Yoneda, Misako; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

    2016-10-01

    Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21-24 and 110-139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60-75 and 72-75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm. PMID:27501340

  14. Phage shock proteins B and C prevent lethal cytoplasmic membrane permeability in Yersinia enterocolitica.

    PubMed

    Horstman, N Kaye; Darwin, Andrew J

    2012-08-01

    The bacterial phage shock protein (Psp) stress response system is activated by events affecting the cytoplasmic membrane. In response, Psp protein levels increase, including PspA, which has been implicated as the master effector of stress tolerance. Yersinia enterocolitica and related bacteria with a defective Psp system are highly sensitive to the mislocalization of pore-forming secretin proteins. However, why secretins are toxic to psp null strains, whereas some other Psp inducers are not, has not been explained. Furthermore, previous work has led to the confounding and disputable suggestion that PspA is not involved in mitigating secretin toxicity. Here we have established a correlation between the amount of secretin toxicity in a psp null strain and the extent of cytoplasmic membrane permeability to large molecules. This leads to a morphological change resembling cells undergoing plasmolysis. Furthermore, using novel strains with dis-regulated Psp proteins has allowed us to obtain unequivocal evidence that PspA is not required for secretin-stress tolerance. Together, our data suggest that the mechanism by which secretin multimers kill psp null cells is by causing a profound defect in the cytoplasmic membrane permeability barrier. This allows lethal molecular exchange with the environment, which the PspB and PspC proteins can prevent. PMID:22646656

  15. Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

    SciTech Connect

    Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji Saeki, Yasushi

    2013-07-05

    Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

  16. Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

    PubMed Central

    Alameda, Josefa P.; Gaspar, Miriam; Ramírez, Ángel; Navarro, Manuel; Page, Angustias; Suárez-Cabrera, Cristian; Fernández, M. Guadalupe; Mérida, Jose R.; Paramio, Jesús M.; García-Fernández, Rosa A.; Fernández-Aceñero, M. Jesús; Casanova, M. Llanos

    2016-01-01

    Nonmelanoma skin cancers (NMSC) are the most common human malignancies. IKKα is an essential protein for skin development and is also involved in the genesis and progression of NMSC, through mechanisms not fully understood. While different studies show that IKKα protects against skin cancer, others indicate that it promotes NMSC. To resolve this controversy we have generated two models of transgenic mice expressing the IKKα protein in the nucleus (N-IKKα mice) or the cytoplasm (C-IKKα mice) of keratinocytes. Chemical skin carcinogenesis experiments show that tumors developed by both types of transgenic mice exhibit histological and molecular characteristics that make them more prone to progression and invasion than those developed by Control mice. However, the mechanisms through which IKKα promotes skin tumors are different depending on its subcellular localization; while IKKα of cytoplasmic localization increases EGFR, MMP-9 and VEGF-A activities in tumors, nuclear IKKα causes tumor progression through regulation of c-Myc, Maspin and Integrin-α6 expression. Additionally, we have found that N-IKKα skin tumors mimic the characteristics associated to aggressive human skin tumors with high risk to metastasize. Our results show that IKKα has different non-overlapping roles in the nucleus or cytoplasm of keratinocytes, and provide new targets for intervention in human NMSC progression. PMID:27121058

  17. The clinical presentation and therapy of diseases related to anti-neutrophil cytoplasmic antibodies (ANCA).

    PubMed

    Weiner, Maria; Segelmark, Mårten

    2016-10-01

    Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies that react with proteins predominantly expressed in cytoplasmic granules of polymorphonuclear neutrophil granulocytes (PMNs). ANCA was initially detected using indirect immunofluorescence, allowing for different patterns such as p-ANCA (perinuclear) and c-ANCA (cytoplasmic) to be distinguished. Today it is common to detect the antibodies by immunochemical assays such as ELISA using purified proteins as antigens. The strongest association with ANCA is found in the pauci-immune small vessel vasculitides granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). There is compelling evidence that ANCA contributes to the pathogenesis in these conditions. ANCA also occurs in 30%-40% of patients with eosinophilic granulomatosis with polyangiitis (EGPA) and anti-GBM disease, but is uncommon in other forms of vasculitis. ANCA with different specificities have been described with varying frequencies in diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, inflammatory bowel disease, endocarditis, chronic infections and hematopoietic malignancies. ANCA can also develop as an adverse event during pharmacological treatment. These entities are treated quite differently, with therapies ranging from immunosuppressive agents over antibiotics to simply removing the causative drug. A positive ANCA test thus requires a careful diagnostic work-up. PMID:27481040

  18. Genetic studies on cytoplasmic male sterility in maize. Progress report, April 15, 1990--April 14, 1992

    SciTech Connect

    Laughnan, J.R.

    1992-05-01

    Our research concerns the basic mechanisms of cytoplasmic male sterility (CMS) and fertility restoration in maize. The molecular determination of CMS is in the DNA of the mitochondria (mtDNA) but specific nuclear restorer-of-fertility (Rf) genes can overrule the male-sterile effect of the cytoplasm. Our approach to the study of the Rf genes is threefold. We are attempting to tag the cms-S Rf genes and the cms-T Rf2 gene with controlling elements (CEs). Since we have identified a number of spontaneous Rf genes for cms-S and have demonstrated that they are themselves transposable, we are also searching for cases in which an Rf gene is inserted into a wild-type gene. The other aspect of our research involves the nuclear control over the organization of the mitochondrial genome. We found that the changes in mtDNA organization upon cytoplasmic reversion to fertility were characteristic of the nuclear background in which the reversion event occurred. We have investigated whether these differences are a reflection of differences in the organization of the mtDNA genome before reversion.

  19. Diffusion, Crowding & Protein Stability in a Dynamic Molecular Model of the Bacterial Cytoplasm

    PubMed Central

    McGuffee, Sean R.; Elcock, Adrian H.

    2010-01-01

    A longstanding question in molecular biology is the extent to which the behavior of macromolecules observed in vitro accurately reflects their behavior in vivo. A number of sophisticated experimental techniques now allow the behavior of individual types of macromolecule to be studied directly in vivo; none, however, allow a wide range of molecule types to be observed simultaneously. In order to tackle this issue we have adopted a computational perspective, and, having selected the model prokaryote Escherichia coli as a test system, have assembled an atomically detailed model of its cytoplasmic environment that includes 50 of the most abundant types of macromolecules at experimentally measured concentrations. Brownian dynamics (BD) simulations of the cytoplasm model have been calibrated to reproduce the translational diffusion coefficients of Green Fluorescent Protein (GFP) observed in vivo, and “snapshots” of the simulation trajectories have been used to compute the cytoplasm's effects on the thermodynamics of protein folding, association and aggregation events. The simulation model successfully describes the relative thermodynamic stabilities of proteins measured in E. coli, and shows that effects additional to the commonly cited “crowding” effect must be included in attempts to understand macromolecular behavior in vivo. PMID:20221255

  20. Regulation of Mouse Oocyte Microtubule and Organelle Dynamics by PADI6 and the Cytoplasmic Lattices

    PubMed Central

    Kan, Rui; Yurttas, Piraye; Kim, Boram; Jin, Mei; Wo, Luccie; Lee, Bora; Gosden, Roger; Coonrod, Scott A.

    2010-01-01

    Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement. PMID:21147087

  1. Chronic Psychosocial Stress and Negative Feedback Inhibition: Enhanced Hippocampal Glucocorticoid Signaling despite Lower Cytoplasmic GR Expression

    PubMed Central

    Füchsl, Andrea M.; Reber, Stefan O.

    2016-01-01

    Chronic subordinate colony housing (CSC), a pre-clinically validated mouse model for chronic psychosocial stress, results in increased basal and acute stress-induced plasma adrenocorticotropic hormone (ACTH) levels. We assessed CSC effects on hippocampal glucocorticoid (GC) receptor (GR), mineralocorticoid receptor (MR), and FK506 binding protein (FKBP51) expression, acute heterotypic stressor-induced GR translocation, as well as GC effects on gene expression and cell viability in isolated hippocampal cells. CSC mice showed decreased GR mRNA and cytoplasmic protein levels compared with single-housed control (SHC) mice. Basal and acute stress-induced nuclear GR protein expression were comparable between CSC and SHC mice, as were MR and FKBP51 mRNA and/or cytoplasmic protein levels. In vitro the effect of corticosterone (CORT) on hippocampal cell viability and gene transcription was more pronounced in CSC versus SHC mice. In summary, CSC mice show an, if at all, increased hippocampal GC signaling capacity despite lower cytoplasmic GR protein expression, making negative feedback deficits in the hippocampus unlikely to contribute to the increased ACTH drive following CSC. PMID:27057751

  2. Human Tissue Plasminogen Activator Expression in Escherichia coli using Cytoplasmic and Periplasmic Cumulative Power.

    PubMed

    Majidzadeh-A, Keivan; Mahboudi, Fereidoun; Hemayatkar, Mahdi; Davami, Fatemeh; Barkhordary, Farzaneh; Adeli, Ahmad; Soleimani, Mohammad; Davoudi, Noushin; Khalaj, Vahid

    2010-07-01

    Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm.

  3. Human Tissue Plasminogen Activator Expression in Escherichia coli using Cytoplasmic and Periplasmic Cumulative Power

    PubMed Central

    Majidzadeh-A, Keivan; Mahboudi, Fereidoun; Hemayatkar, Mahdi; Davami, Fatemeh; Barkhordary, Farzaneh; Adeli, Ahmad; Soleimani, Mohammad; Davoudi, Noushin; Khalaj, Vahid

    2010-01-01

    Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5′ site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm. PMID:23408156

  4. In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum

    PubMed Central

    Bureau, Thomas E.; Brown, R. Malcolm

    1987-01-01

    The cytoplasmic and outer membranes of Acetobacter xylinum (ATCC 53582) were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme (EC 3.2.1.17) and trypsin (EC 3.4.21.4) were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxyoctulosonic acid was used to identify the outer membranes. Cellulose synthetase (UDP-glucose:1,4-β-D-glucan 4-β-D-glucosyltransferase; EC 2.4.1.12) activity was assayed as the conversion of radioactivity from UDP-[14C]glucose into an alkali-insoluble β-1,4-D-[14C]glucan. This activity was predominantly found in the cytoplasmic membrane. The cellulose nature of the product was demonstrated by (i) enzymatic hydrolysis followed by TLC, (ii) methylation analysis followed by TLC, and (iii) GC/MS. Further, the weight-average and number-average degree of polymerization of the in vitro product, determined by high-performance gel permeation chromatography, were 4820 and 5270, respectively. In addition, x-ray diffraction analysis indicated that the in vitro product is cellulose II, which is in contrast to the in vivo product—namely, cellulose I. Images PMID:16593877

  5. Spectral domain phase microscopy: a new tool for measuring cellular dynamics and cytoplasmic flow

    NASA Astrophysics Data System (ADS)

    McDowell, Emily J.; Choma, Michael A.; Ellerbee, Audrey K.; Izatt, Joseph A.

    2005-03-01

    Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved interferometric phase information via coherence gating. Here a phase sensitive technique called spectral domain phase microscopy (SDPM) is presented. SDPM is a functional extension of spectral domain optical coherence tomography that allows for the detection of cellular motions and dynamics with nanometer-scale sensitivity. This sensitivity is made possible by the inherent phase stability of spectral domain OCT combined with common-path interferometry. The theory that underlies this technique is presented, the sensitivity of the technique is demonstrated by the measurement of the thermal expansion coefficient of borosilicate glass, and the response of an Amoeba proteus to puncture of its cell membrane is measured. We also exploit the phase stability of SDPM to perform Doppler flow imaging of cytoplasmic streaming in A. proteus. We show reversal of cytoplasmic flow in response to stimuli, and we show that the cytoplasmic flow is laminar (i.e. parabolic) in nature. We are currently investigating the use of SDPM in a variety of different cell types.

  6. Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus.

    PubMed

    Horie, Ryo; Yoneda, Misako; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

    2016-10-01

    Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21-24 and 110-139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60-75 and 72-75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm.

  7. The roles of vitamin A for cytoplasmic maturation of bovine oocytes.

    PubMed

    Ikeda, Shuntaro; Kitagawa, Masayuki; Imai, Hiroshi; Yamada, Masayasu

    2005-02-01

    Vitamin A is one of the micronutrients which have been implicated in cattle reproduction. In cattle, ingested vitamin A, mainly as beta-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells. PMID:15750294

  8. Disassembly of simian virus 40 during passage through the endoplasmic reticulum and in the cytoplasm.

    PubMed

    Kuksin, Dmitry; Norkin, Leonard C

    2012-02-01

    The nonenveloped polyomavirus simian virus 40 (SV40) is taken up into cells by a caveola-mediated endocytic process that delivers the virus to the endoplasmic reticulum (ER). Within the ER lumen, the capsid undergoes partial disassembly, which exposes its internal capsid proteins VP2 and VP3 to immunostaining with antibodies. We demonstrate here that the SV40 genome does not become accessible to detection while the virus is in the ER. Instead, the genome becomes accessible two distinct detection procedures, one using anti-bromodeoxyuridine antibodies and the other using a 5-ethynyl-2-deoxyuridine-based chemical reaction, only after the emergence of partially disassembled SV40 particles in the cytoplasm. These cytoplasmic particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. Thus, SV40 particles undergo discrete disassembly steps during entry that are separated temporally and topologically. First, a partial disassembly of the particles occurs in the ER, which exposes internal capsid proteins VP2 and VP3. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection.

  9. Disassembly of Simian Virus 40 during Passage through the Endoplasmic Reticulum and in the Cytoplasm

    PubMed Central

    Kuksin, Dmitry

    2012-01-01

    The nonenveloped polyomavirus simian virus 40 (SV40) is taken up into cells by a caveola-mediated endocytic process that delivers the virus to the endoplasmic reticulum (ER). Within the ER lumen, the capsid undergoes partial disassembly, which exposes its internal capsid proteins VP2 and VP3 to immunostaining with antibodies. We demonstrate here that the SV40 genome does not become accessible to detection while the virus is in the ER. Instead, the genome becomes accessible two distinct detection procedures, one using anti-bromodeoxyuridine antibodies and the other using a 5-ethynyl-2-deoxyuridine-based chemical reaction, only after the emergence of partially disassembled SV40 particles in the cytoplasm. These cytoplasmic particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. Thus, SV40 particles undergo discrete disassembly steps during entry that are separated temporally and topologically. First, a partial disassembly of the particles occurs in the ER, which exposes internal capsid proteins VP2 and VP3. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection. PMID:22090139

  10. BRCA2 mediates centrosome cohesion via an interaction with cytoplasmic dynein

    PubMed Central

    Malik, Sadiya; Saito, Hiroko; Takaoka, Miho; Miki, Yoshio; Nakanishi, Akira

    2016-01-01

    BRCA2 is responsible for familial breast and ovarian cancer and has been linked to DNA repair and centrosome duplication. Here we analyzed the mechanism by which the centrosomal localization signal (CLS) of BRCA2 interacts with cytoplasmic dynein 1 to localize BRCA2 to the centrosome. In vitro pull-down assays demonstrated that BRCA2 directly binds to the cytoplasmic dynein 1 light intermediate chain 2. A dominant-negative HA-CLS-DsRed fusion protein, the depletion of dynein by siRNA, and the inactivation of dynein by EHNA, inhibited the localization of BRCA2 at centrosomes and caused the separation of centrosome pairs during the S-phase. The double depletion of BRCA2 and C-Nap1 caused a larger dispersion of centrosome distances than the silencing of C-Nap1. These results suggest that cytoplasmic dynein 1 binds to BRCA2 through the latter's CLS and BRCA2 mediates the cohesion between centrosomes during the S phase, potentially serving as a cell-cycle checkpoint. PMID:27433848

  11. Structural and Thermodynamic Characterization of a Cytoplasmic Dynein Light Chain-Intermediate Chain Complex

    SciTech Connect

    Williams,J.; Roulhac, P.; Roy, A.; Vallee, R.; Fitzgerald, M.; Hendrickson, W.

    2007-01-01

    Cytoplasmic dynein is a microtubule-based motor protein complex that plays important roles in a wide range of fundamental cellular processes, including vesicular transport, mitosis, and cell migration. A single major form of cytoplasmic dynein associates with membranous organelles, mitotic kinetochores, the mitotic and migratory cell cortex, centrosomes, and mRNA complexes. The ability of cytoplasmic dynein to recognize such diverse forms of cargo is thought to be associated with its several accessory subunits, which reside at the base of the molecule. The dynein light chains (LCs) LC8 and TcTex1 form a subcomplex with dynein intermediate chains, and they also interact with numerous protein and ribonucleoprotein partners. This observation has led to the hypothesis that these subunits serve to tether cargo to the dynein motor. Here, we present the structure and a thermodynamic analysis of a complex of LC8 and TcTex1 associated with their intermediate chain scaffold. The intermediate chains effectively block the major putative cargo binding sites within the light chains. These data suggest that, in the dynein complex, the LCs do not bind cargo, in apparent disagreement with a role for LCs in dynein cargo binding interactions.

  12. Plant vegetative and animal cytoplasmic actins share functional competence for spatial development with protists.

    PubMed

    Kandasamy, Muthugapatti K; McKinney, Elizabeth C; Roy, Eileen; Meagher, Richard B

    2012-05-01

    Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin's competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.

  13. The cytoplasmic extension of the integrin β6 subunit regulates epithelial-to-mesenchymal transition.

    PubMed

    Lee, Carlin; Lee, Casey; Lee, Stacey; Siu, Amanda; Ramos, Daniel M

    2014-02-01

    Prognosis for oral cancer patients has not improved in over 60 years due to invasion and recurrence. To understand the invasive behavior of this tumor, we evaluated the role of the αvβ6 integrin. Invasive oral SCC cells express the αvβ6 integrin, which contains an 11-amino-acid extension on its β-subunit unique to the integrin family. We determined that this β6 cytoplasmic extension regulates the composition of the intermediate filament network and the organization of signaling structures called focal contacts. The auto-phosphorylation of FAK, which is localized to focal contacts, was also regulated by the β6-cytoplasmic tail, as were the transcription factors Notch and STAT3. Lastly, we also determined that activation of MAPK required the full-length β6 integrin. Together these results indicate that the signaling critical to epithelial-to-mesenchymal transition (EMT) is regulated by the β6 integrin cytoplasmic domain. PMID:24510996

  14. The clinical presentation and therapy of diseases related to anti-neutrophil cytoplasmic antibodies (ANCA).

    PubMed

    Weiner, Maria; Segelmark, Mårten

    2016-10-01

    Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies that react with proteins predominantly expressed in cytoplasmic granules of polymorphonuclear neutrophil granulocytes (PMNs). ANCA was initially detected using indirect immunofluorescence, allowing for different patterns such as p-ANCA (perinuclear) and c-ANCA (cytoplasmic) to be distinguished. Today it is common to detect the antibodies by immunochemical assays such as ELISA using purified proteins as antigens. The strongest association with ANCA is found in the pauci-immune small vessel vasculitides granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). There is compelling evidence that ANCA contributes to the pathogenesis in these conditions. ANCA also occurs in 30%-40% of patients with eosinophilic granulomatosis with polyangiitis (EGPA) and anti-GBM disease, but is uncommon in other forms of vasculitis. ANCA with different specificities have been described with varying frequencies in diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, inflammatory bowel disease, endocarditis, chronic infections and hematopoietic malignancies. ANCA can also develop as an adverse event during pharmacological treatment. These entities are treated quite differently, with therapies ranging from immunosuppressive agents over antibiotics to simply removing the causative drug. A positive ANCA test thus requires a careful diagnostic work-up.

  15. A novel role for DNA methyltransferase 1 in regulating oocyte cytoplasmic maturation in pigs.

    PubMed

    Huan, Yanjun; Xie, Bingteng; Liu, Shichao; Kong, Qingran; Liu, Zhonghua

    2015-01-01

    Maternal factors are required for oocyte maturation and embryo development. To better understand the role of DNA methyltransferase 1 (Dnmt1) in oocyte maturation and embryo development, small interfering RNA (siRNA) was conducted in porcine oocytes. In this study, our results showed that Dnmt1 localized in oocyte cytoplasm and its expression displayed no obvious change during oocyte maturation. When siRNAs targeting Dnmt1 were injected into germinal vesicle (GV) stage oocytes, Dnmt1 transcripts significantly decreased in matured oocytes (P<0.05). After Dnmt1 knockdown in GV stage oocytes, the significant reduction of glutathione content, mitochondrial DNA copy number, glucose-6-phosphate dehydrogenase activity and expression profiles of maternal factors and the severely disrupted distribution of cortical granules were observed in MII stage oocytes (P<0.05), leading to the impaired oocyte cytoplasm. Further study displayed that Dnmt1 knockdown in GV stage oocytes significantly reduced the development of early embryos generated through parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (P<0.05). In conclusion, Dnmt1 was indispensable for oocyte cytoplasmic maturation, providing a novel role for Dnmt1 in the regulation of oocyte maturation. PMID:26009894

  16. Cytoplasmic PML promotes TGF-β-associated epithelial–mesenchymal transition and invasion in prostate cancer

    PubMed Central

    Buczek, M E; Miles, A K; Green, W; Johnson, C; Boocock, D J; Pockley, A G; Rees, R C; Hulman, G; van Schalkwyk, G; Parkinson, R; Hulman, J; Powe, D G; Regad, T

    2016-01-01

    Epithelial–mesenchymal transition (EMT) is a key event that is involved in the invasion and dissemination of cancer cells. Although typically considered as having tumour-suppressive properties, transforming growth factor (TGF)-β signalling is altered during cancer and has been associated with the invasion of cancer cells and metastasis. In this study, we report a previously unknown role for the cytoplasmic promyelocytic leukaemia (cPML) tumour suppressor in TGF-β signalling-induced regulation of prostate cancer-associated EMT and invasion. We demonstrate that cPML promotes a mesenchymal phenotype and increases the invasiveness of prostate cancer cells. This event is associated with activation of TGF-β canonical signalling pathway through the induction of Sma and Mad related family 2 and 3 (SMAD2 and SMAD3) phosphorylation. Furthermore, the cytoplasmic localization of promyelocytic leukaemia (PML) is mediated by its nuclear export in a chromosomal maintenance 1 (CRM1)-dependent manner. This was clinically tested in prostate cancer tissue and shown that cytoplasmic PML and CRM1 co-expression correlates with reduced disease-specific survival. In summary, we provide evidence of dysfunctional TGF-β signalling occurring at an early stage in prostate cancer. We show that this disease pathway is mediated by cPML and CRM1 and results in a more aggressive cancer cell phenotype. We propose that the targeting of this pathway could be therapeutically exploited for clinical benefit. PMID:26549027

  17. On the nature of hyaline zones in the cytoplasm of Amoeba proteus.

    PubMed

    Korohoda, W; Stockem, W

    1975-07-01

    Investigations with the Nomarski DIC (differential interferece contrast) microscope and the electron microscope on the nature of hyaline zones in the cytoplasm of Amoeba proteus revealed that these regions represent pure ground cytoplasm. Differences between specimens 1) treated with 2% ethanol, 2) released from high hydrostatic pressure or 3) preincubated at 35 degrees C for 30 minutes could not be observed. Only dying cells undergoing lysis contained a watery solution within the zones of hyaline appearance. The existence of a so-called cell surface complex composed of the plasma membrane and an electron dense filamentous layer of groundplasm was demonstrated by the electron microscopical analysis of narcotized and pre-heated amoebae. This complex corresponds morphologically to the cell surface complexes in tissue cells. Hence it seems possible that the cell surface complex of amoebae is also responsible for changes of the cell shape and movements of the cell membrane. Observations with the DIC microscope also revealed the existence of two types of hyaline caps in A. proteus: in pseudopodia extending during normal locomotion the hyaline cap consists of pure ground cytoplasm, whereas in specimens showing fountain-like streaming the cap is built up by a large vacuole containing a watery fluid. PMID:1196142

  18. Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

    SciTech Connect

    Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki . E-mail: mikeda.emb@tmd.ac.jp

    2006-02-03

    The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16{sup INK4a}, a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.

  19. Proteins contribute insignificantly to the intrinsic buffering capacity of yeast cytoplasm

    SciTech Connect

    Poznanski, Jaroslaw; Szczesny, Pawel; Ruszczynska, Katarzyna; Zielenkiewicz, Piotr; Paczek, Leszek

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We predicted buffering capacity of yeast proteome from protein abundance data. Black-Right-Pointing-Pointer We measured total buffering capacity of yeast cytoplasm. Black-Right-Pointing-Pointer We showed that proteins contribute insignificantly to buffering capacity. -- Abstract: Intracellular pH is maintained by a combination of the passive buffering of cytoplasmic dissociable compounds and several active systems. Over the years, a large portion of and possibly most of the cell's intrinsic (i.e., passive non-bicarbonate) buffering effect was attributed to proteins, both in higher organisms and in yeast. This attribution was not surprising, given that the concentration of proteins with multiple protonable/deprotonable groups in the cell exceeds the concentration of free protons by a few orders of magnitude. Using data from both high-throughput experiments and in vitro laboratory experiments, we tested this concept. We assessed the buffering capacity of the yeast proteome using protein abundance data and compared it to our own titration of yeast cytoplasm. We showed that the protein contribution is less than 1% of the total intracellular buffering capacity. As confirmed with NMR measurements, inorganic phosphates play a crucial role in the process. These findings also shed a new light on the role of proteomes in maintaining intracellular pH. The contribution of proteins to the intrinsic buffering capacity is negligible, and proteins might act only as a recipient of signals for changes in pH.

  20. Mechanisms for independent cytoplasmic inheritance of mitochondria and plastids in angiosperms.

    PubMed

    Nagata, Noriko

    2010-03-01

    The inheritance of mitochondria and plastids in angiosperms has been categorized into three modes:maternal, biparental and paternal. Many mechanisms have been proposed for maternal inheritance, including: (1) physical exclusion of the organelle itself during pollenmitosis I (PMI); (2) elimination of the organelle by formation of enucleated cytoplasmic bodies (ECB); (3) autophagic degradation of organelles during male gametophyte development; (4) digestion of the organelle after fertilization; and (5)--the most likely possibility--digestion of organellar DNA in generative cells just after PMI. In detailed cytological observations, the presence or absence of mitochondrial and plastid DNA in generative cells corresponds to biparental/paternal inheritance or maternal inheritance of the respective organelle examined genetically. These improved cytological observations demonstrate that the replication or digestion of organellar DNA in young generative cells just after PMI is a critical point determining the mode of cytoplasmic inheritance. This review describes the independent control mechanisms in mitochondria and plastids that lead to differences in cytoplasmic inheritance in angiosperms.

  1. A new source of cytoplasmic male sterility found in wild beet and its relationship to other CMS types.

    PubMed

    Kawanishi, Yuki; Shinada, Hiroshi; Matsunaga, Muneyuki; Masaki, Yusuke; Mikami, Tetsuo; Kubo, Tomohiko

    2010-04-01

    We found a number of male-sterile plants in a wild beet (Beta vulgaris L. subsp. maritima) accession line, FR4-31. The inheritance study of the male sterility indicated the trait to be of the cytoplasmic type. The mitochondrial genome of FR4-31 proved to lack the male-sterility-associated genes preSatp6 and orf129, which are characteristic of the Owen CMS and I-12CMS(3) cytoplasms of beets, respectively. Instead, the truncated cox2 gene involved in G CMS originating from wild beets was present in the FR4-31 mitochondrial genome. In Southern hybridization using four mitochondrial gene probes, the FR4-31 cytoplasm showed patterns similar to those typical of the G cytoplasm. It is thus likely that the FR4-31 cytoplasm has a different CMS mechanism from both Owen CMS and I-12CMS(3), and that the FR4-31 and G cytoplasms resemble each other closely. A restriction map of the FR4-31 mitochondrial DNA was generated and aligned with those published for the Owen and normal fertile cytoplasms. The FR4-31 mitochondrial genome was revealed to differ extensively in arrangement from the Owen and normal genomes, and the male-sterile Owen and FR4-31 genomes seem to be derived independently from an ancestral genome.

  2. Particle-rich cytoplasmic structure (PaCS): identification, natural history, role in cell biology and pathology.

    PubMed

    Solcia, Enrico; Sommi, Patrizia; Necchi, Vittorio; Vitali, Agostina; Manca, Rachele; Ricci, Vittorio

    2014-09-22

    Cytoplasmic structures showing a selective concentration of both polyubiquitinated proteins and proteasome have been described in various epithelial, hematopoietic, mesenchymal and neural cells in vitro or in fetal tissues, as well as in chronically-infected, mutated preneoplastic and neoplastic tissues. These cytoplasmic structures differ from other ubiquitin-reactive cytoplasmic bodies, like sequestosomes, aggresome-like-induced structures in dendritic cells (DALIS)/non-dendritic cells (ALIS) and aggresomes in showing distinctive ultrastructural organization (particle-rich cytoplasmic structure or PaCS), a cytochemical pattern and a functional profile. Their formation can be induced in vitro in dendritic or natural killer cells by trophic factors and interleukin treatment. They originate in close connection with ribosomes, while, as a result of their growth, the cytoskeleton and other surrounding organelles are usually dislocated outside their core. Interestingly, these particulate cytoplasmic structures are often found to fill cytoplasmic blebs forming proteasome- and polyubiquitinated protein-discharging vesicles, called ectosomes, which are found to detach from the cell and freely float in the extracellular space. To clearly point out the importance of the polyubiquitinated proteins and proteasome containing cytoplasmic structures, their role in cell biology and pathology has been carefully analyzed.

  3. Expression of aquaporin1, a water channel protein, in cytoplasm is negatively correlated with prognosis of breast cancer patients

    PubMed Central

    Shao, Ying; Liu, Xiaoli; Yang, Limin; Huang, Yong; Fu, Li; Gu, Feng; Ma, Yongjie

    2016-01-01

    Aquaporin1 (AQP1) belongs to a highly conserved family of aquaporin proteins which facilitate water flux across cell membranes. Although emerging evidences indicated the cytoplasm was important for AQP1 localization, the function of AQP1 corresponding to its cytoplasmic distribution has rarely been explored until present. In our clinical study, we reported for the first time that AQP1 was localized dominantly in the cytoplasm of cancer cells of invasive breast cancer patients and cytoplasmic AQP1 was an independent prognostic factor. High expression of AQP1 indicated a shorter survival, especially in luminal subtype. Moreover, in line with our findings in clinic, cytoplasmic expression of AQP1 was further validated in both primary cultured breast cancer cells and AQP1 over-expressing cell lines, in which the functional importance of cytoplasmic AQP1 was confirmed in vitro. In conclusion, our study provided the first evidence that cytoplasmic expression of AQP1 promoted breast cancer progression and it could be a potential prognostic biomarker for breast cancer. PMID:26812884

  4. A new source of cytoplasmic male sterility found in wild beet and its relationship to other CMS types.

    PubMed

    Kawanishi, Yuki; Shinada, Hiroshi; Matsunaga, Muneyuki; Masaki, Yusuke; Mikami, Tetsuo; Kubo, Tomohiko

    2010-04-01

    We found a number of male-sterile plants in a wild beet (Beta vulgaris L. subsp. maritima) accession line, FR4-31. The inheritance study of the male sterility indicated the trait to be of the cytoplasmic type. The mitochondrial genome of FR4-31 proved to lack the male-sterility-associated genes preSatp6 and orf129, which are characteristic of the Owen CMS and I-12CMS(3) cytoplasms of beets, respectively. Instead, the truncated cox2 gene involved in G CMS originating from wild beets was present in the FR4-31 mitochondrial genome. In Southern hybridization using four mitochondrial gene probes, the FR4-31 cytoplasm showed patterns similar to those typical of the G cytoplasm. It is thus likely that the FR4-31 cytoplasm has a different CMS mechanism from both Owen CMS and I-12CMS(3), and that the FR4-31 and G cytoplasms resemble each other closely. A restriction map of the FR4-31 mitochondrial DNA was generated and aligned with those published for the Owen and normal fertile cytoplasms. The FR4-31 mitochondrial genome was revealed to differ extensively in arrangement from the Owen and normal genomes, and the male-sterile Owen and FR4-31 genomes seem to be derived independently from an ancestral genome. PMID:20616856

  5. A cytoplasmic activator of DNA replication is involved in signal transduction in antigen-specific T cell lines.

    PubMed

    Wong, R L; Clark, R B; Gutowski, J K; Katz, M E; Fresa, K L; Cohen, S

    1990-05-01

    Cytoplasmic extracts prepared from T cell lines undergoing antigen-specific, interleukin-2 (IL-2)-dependent proliferation were tested for their ability to induce DNA synthesis in isolated, quiescent nuclei. A tetanus toxoid (TET)-specific T cell line, established from peripheral blood of a normal human volunteer, was stimulated in the presence of relevant antigen and 1 unit/ml IL-2. Cytoplasmic extracts prepared from these cells were capable of inducing DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated nuclei correlated positively with the degree of proliferation induced in these cells. In contrast, incubation of this T cell line in the absence of antigen failed to induce proliferation and cytoplasmic extracts prepared from these cells induced little to no DNA synthesis in isolated, quiescent nuclei. The factor present in the cytoplasm of T cells stimulated with relevant antigen in the presence of IL-2 is similar, if not identical, to a factor which we have previously demonstrated in cytoplasmic extracts prepared from transformed lymphoblastoid cell lines and from mitogenically stimulated normal human peripheral blood mononuclear cells. This factor, which we have called activator of DNA replication (ADR) is a heat-labile protein, and is inactivated by treatment with protease inhibitors, including aprotinin. The ability of cytoplasmic extracts from T cells undergoing antigen-specific, IL-2-dependent proliferation to induce DNA synthesis in isolated, quiescent nuclei was markedly inhibited in the presence of aprotinin, providing strong evidence that a cytoplasmic activator of DNA replication, ADR, is involved in the signal transduction process for antigen-specific, IL-2-dependent T cell proliferation. ADR may represent a common intracellular mediator of DNA synthesis in activated and transformed lymphocytes

  6. Cell nuclei and cytoplasm joint segmentation using the sliding band filter.

    PubMed

    Quelhas, Pedro; Marcuzzo, Monica; Mendonça, Ana Maria; Campilho, Aurélio

    2010-08-01

    Microscopy cell image analysis is a fundamental tool for biological research. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. It is still common practice to perform analysis tasks by visual inspection of individual cells which is time consuming, exhausting and prone to induce subjective bias. This makes automatic cell image analysis essential for large scale, objective studies of cell cultures. Traditionally the task of automatic cell analysis is approached through the use of image segmentation methods for extraction of cells' locations and shapes. Image segmentation, although fundamental, is neither an easy task in computer vision nor is it robust to image quality changes. This makes image segmentation for cell detection semi-automated requiring frequent tuning of parameters. We introduce a new approach for cell detection and shape estimation in multivariate images based on the sliding band filter (SBF). This filter's design makes it adequate to detect overall convex shapes and as such it performs well for cell detection. Furthermore, the parameters involved are intuitive as they are directly related to the expected cell size. Using the SBF filter we detect cells' nucleus and cytoplasm location and shapes. Based on the assumption that each cell has the same approximate shape center in both nuclei and cytoplasm fluorescence channels, we guide cytoplasm shape estimation by the nuclear detections improving performance and reducing errors. Then we validate cell detection by gathering evidence from nuclei and cytoplasm channels. Additionally, we include overlap correction and shape regularization steps which further improve the estimated cell shapes. The approach is evaluated using two datasets with different types of data: a 20 images benchmark set of simulated cell culture images, containing 1000 simulated cells; a 16 images Drosophila melanogaster Kc167 dataset containing 1255 cells, stained for DNA and

  7. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains.

    PubMed

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxPhi domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1(NL4.3) compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

  8. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

    SciTech Connect

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the Yxx{phi} domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1{sub NL4.3} compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and

  9. Regulation of adherence and virulence by the Entamoeba histolytica lectin cytoplasmic domain, which contains a beta2 integrin motif.

    PubMed

    Vines, R R; Ramakrishnan, G; Rogers, J B; Lockhart, L A; Mann, B J; Petri, W A

    1998-08-01

    Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the beta2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin's role in virulence.

  10. Heterozygous alleles restore male fertility to cytoplasmic male-sterile radish (Raphanus sativus L.): a case of overdominance.

    PubMed

    Wang, Zhi Wei; De Wang, Chuan; Wang, Chuan; Gao, Lei; Mei, Shi Yong; Zhou, Yuan; Xiang, Chang Ping; Wang, Ting

    2013-04-01

    The practice of hybridization has greatly contributed to the increase in crop productivity. A major component that exploits heterosis in crops is the cytoplasmic male sterility (CMS)/nucleus-controlled fertility restoration (Rf) system. Through positional cloning, it is shown that heterozygous alleles (RsRf3-1/RsRf3-2) encoding pentatricopeptide repeat (PPR) proteins are responsible for restoring fertility to cytoplasmic male-sterile radish (Raphanus sativus L.). Furthermore, it was found that heterozygous alleles (RsRf3-1/RsRf3-2) show higher expression and RNA polymerase II occupancy in the CMS cytoplasmic background compared with their homozygous alleles (RsRf3-1/RsRf3-1 or RsRf3-2/RsRf3-2). These data provide new insights into the molecular mechanism of fertility restoration to cytoplasmic male-sterile plants and illustrate a case of overdominance.

  11. Structure of the cytoplasmic domain of Yersinia pestis YscD, an essential component of the type III secretion system

    PubMed Central

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2012-01-01

    The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1–121), a transmembrane linker (122–142) and a large periplasmic domain (143–419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are required for binding phosphothreonine and is therefore unlikely to function as a true FHA domain. PMID:22349221

  12. Structure of the cytoplasmic domain of Yersinia pestis YscD, an essential component of the type III secretion system

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2012-09-17

    The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1-121), a transmembrane linker (122-142) and a large periplasmic domain (143-419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are required for binding phosphothreonine and is therefore unlikely to function as a true FHA domain.

  13. Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes

    NASA Technical Reports Server (NTRS)

    Johannes, E.; Collings, D. A.; Rink, J. C.; Allen, N. S.; Brown, C. S. (Principal Investigator)

    2001-01-01

    In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism.

  14. Ion flux interaction with cytoplasmic streaming in branchlets of Chara australis.

    PubMed

    Babourina, Olga; Voltchanskii, Konstantin; Newman, Ian

    2004-12-01

    Both parts of the actin-myosin complex involved in cytoplasmic streaming could be regulated by mineral ions. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell wall and plasma membrane. The transport of K(+) and Ca(2+) along pH bands in Chara branchlet internodal cells was characterized by using the MIFE system for non-invasive microelectrode measurement of ion fluxes. Branchlets formed acidic and alkaline bands with the pH ranging from 5 to 8. Different pH patterns were observed for different sides of the branchlets. Sides with cyclosis streaming acropetally generally showed greater variation in the profiles of pH and H(+) fluxes. Although a high correlation was not found between pH bands and Ca(2+) or K(+) fluxes, there was a positive correlation between Ca(2+) and K(+) fluxes themselves for both sides of the branchlets. Application of cytochalasin D, an inhibitor of cyclosis, had no immediate effect on pH and ion fluxes, however, the time of cyclosis cessation corresponded with a dramatic change in Ca(2+) and K(+) fluxes; pH profiles and H(+) fluxes were affected within 2 h. The evidence suggests that, in Chara branchlets, pH band formation and Gd(3+)-insensitive Ca(2+) transport systems are linked to the cyclosis machinery: (i) the pH band amplitude for the acropetally streaming side was larger than that for the basipetally streaming side; (ii) cessation of cytoplasmic streaming after cytochalasin D application resulted in changed pH banding profiles and H(+), Ca(2+) and K(+) fluxes; and (iii) the application of GdCl(3) or incubation in GdCl(3) solutions did not lead to the cessation of cytoplasmic streaming, although external Ca(2+) fluxes changed.

  15. Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes.

    PubMed

    Johannes, E; Collings, D A; Rink, J C; Allen, N S

    2001-09-01

    In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism. PMID:11553740

  16. Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes.

    PubMed

    Johannes, E; Collings, D A; Rink, J C; Allen, N S

    2001-09-01

    In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism.

  17. A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.

    PubMed

    Li, Linwei; Mirza, Shamaruh; Richardson, Spencer J; Gallant, Esther M; Thekkedam, Chris; Pace, Suzy M; Zorzato, Francesco; Liu, Dan; Beard, Nicole A; Dulhunty, Angela F

    2015-03-01

    Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1-S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156. PMID:25609705

  18. The mRNA of human cytoplasmic arginyl-tRNA synthetase recruits prokaryotic ribosomes independently.

    PubMed

    Yang, Fang; Ji, Quan-Quan; Ruan, Liang-Liang; Ye, Qing; Wang, En-Duo

    2014-07-25

    There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (NhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the NhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the “AGGA” core of the Shine-Dalgarno sequence and the “A-rich” sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells.

  19. Recombinant Expression and Characterization of the Cytoplasmic Rice β-Glucosidase Os1BGlu4

    PubMed Central

    Rouyi, Chen; Baiya, Supaporn; Lee, Sang-Kyu; Mahong, Bancha; Jeon, Jong-Seong; Ketudat-Cairns, James R.; Ketudat-Cairns, Mariena

    2014-01-01

    The Os1BGlu4 β-glucosidase is the only glycoside hydrolase family 1 member in rice that is predicted to be localized in the cytoplasm. To characterize the biochemical function of rice Os1BGlu4, the Os1bglu4 cDNA was cloned and used to express a thioredoxin fusion protein in Escherichia coli. After removal of the tag, the purified recombinant Os1BGlu4 (rOs1BGlu4) exhibited an optimum pH of 6.5, which is consistent with Os1BGlu4's cytoplasmic localization. Fluorescence microscopy of maize protoplasts and tobacco leaf cells expressing green fluorescent protein-tagged Os1BGlu4 confirmed the cytoplasmic localization. Purified rOs1BGlu4 can hydrolyze p-nitrophenyl (pNP)-β-d-glucoside (pNPGlc) efficiently (kcat/Km  =  17.9 mM−1·s−1), and hydrolyzes pNP-β-d-fucopyranoside with about 50% the efficiency of the pNPGlc. Among natural substrates tested, rOs1BGlu4 efficiently hydrolyzed β-(1,3)-linked oligosaccharides of degree of polymerization (DP) 2–3, and β-(1,4)-linked oligosaccharide of DP 3–4, and hydrolysis of salicin, esculin and p-coumaryl alcohol was also detected. Analysis of the hydrolysis of pNP-β-cellobioside showed that the initial hydrolysis was between the two glucose molecules, and suggested rOs1BGlu4 transglucosylates this substrate. At 10 mM pNPGlc concentration, rOs1BGlu4 can transfer the glucosyl group of pNPGlc to ethanol and pNPGlc. This transglycosylation activity suggests the potential use of Os1BGlu4 for pNP-oligosaccharide and alkyl glycosides synthesis. PMID:24802508

  20. Cytoplasmic filaments of Amoeba proteus. I. The role of filaments in consistency changes and movement.

    PubMed

    Pollard, T D; Ito, S

    1970-08-01

    The role of filaments in consistency changes and movement in a motile cytoplasmic extract of Amoeba proteus was investigated by correlating light and electron microscopic observations with viscosity measurements. The extract is prepared by the method of Thompson and Wolpert (1963). At 0 degrees C, this extract is nonmotile and similar in structure to ameba cytoplasm, consisting of groundplasm, vesicles, mitochondria, and a few 160 A filaments. The extract undergoes striking ATP-stimulated streaming when warmed to 22 degrees C. Two phases of movement are distinguished. During the first phase, the apparent viscosity usually increases and numerous 50-70 A filaments appear in samples of the extract prepared for electron microscopy, suggesting that the increase in viscosity in caused, at least in part, by the formation of these thin filaments. During this initial phase of ATP-stimulated movement, these thin filaments are not detectable by phase-contrast or polarization microscopy, but later, in the second phase of movement, 70 A filaments aggregate to form birefringent microscopic fibrils. A preparation of pure groundplasm with no 160 A filaments or membranous organelles exhibits little or no ATP-stimulated movement, but 50-70 A filaments form and aggregate into birefringent fibrils. This observation and the structural relationship of the 70 A and the 160 A filaments in the motile extract suggest that both types of filaments may be required for movement. These two types of filaments, 50-70 A and 160 A, are also present in the cytoplasm of intact amebas. Fixed cells could not be used to study the distribution of these filaments during natural ameboid movement because of difficulties in preserving the normal structure of the ameba during preparation for electron microscopy.