Dynamic changes in nuclear import of a nuclear localisation signal-bearing substrate in 8-cell stage porcine embryos

2015 ◽  
Vol 27 (2) ◽  
pp. 385 ◽  
Author(s):  
Yanfang Li ◽  
Ki-Eun Park ◽  
Ryan A. Cabot
Keyword(s):  
Nuclear Import ◽  
In Vitro Assays ◽  
Cell Stage ◽  
Nuclear Trafficking ◽  
Nuclear Localisation ◽  
Cellular Events ◽  
Stage Of Development ◽  
Importin Α

Coordinated intracellular trafficking is critically important for proper timing of major cellular events during embryogenesis. Nuclear import mediated by the karyopherin α/β (importin α/β) heterodimer is perhaps the best characterised nuclear trafficking system in eukaryotic cells. Seven karyopherin α subtypes have been identified in the domestic pig, and although each karyopherin α subtype transports proteins bearing classical nuclear localisation signals (NLSs), individual karyopherin α subtypes have been shown to preferentially transport specific cargoes. The aim of the present study was to determine the mechanism by which BRN2, a transcription factor previously reported to be transported by the karyopherin α/β heterodimer, gains access to the nucleus in porcine oocytes and embryos. Using a combination of in vivo and in vitro assays, we tested the hypothesis that discrete karyopherin α subtypes transport BRN2 into the nuclei of porcine oocytes and cleavage stage embryos. Our results show that ectopically expressed BRN2 adopts a nuclear localisation in all nuclei through the 4-cell stage of development, whereas only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. This pattern is unique to BRN2 because another ectopically expressed NLS-containing protein is able to adopt a nuclear localisation in all blastomeres of 8-cell stage embryos.

scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

scholarly journals GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

2011 ◽  
Vol 286 (41) ◽  
pp. 35553-35561 ◽  
Author(s):  
Lidija Staresincic ◽  
Jane Walker ◽  
A. Barbara Dirac-Svejstrup ◽  
Richard Mitter ◽  
Jesper Q. Svejstrup
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Import ◽  
Chromatin Immunoprecipitation ◽  
Nuclear Transport ◽  
Proteomic Screen ◽  
Genome Wide ◽  
Importin Α

We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin α/β pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5′-3-O-(thio)triphosphate (GTPγS). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.

159 USE OF PHYTOHEMAGGLUTININ-L, AS AN AGGLUTINATOR AGENT, TO PRODUCE CHIMERAS OF IVF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 238
Author(s):  
I. P. Emanuelli ◽  
B. F. Agostinho ◽  
M. P. M. Mancini ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Stage Of Development ◽  
Fisher’S Exact Test

Embryonic chimeras have been used as a tool to understand embryogenesis and organogenesis, as well as to prove, in vivo, the pluripotency of the embryonic stem cells. One of the techniques used to obtain embryonic chimeras is aggregation, which can be performed with intact or half-embryos and in different stages of the development, produced by in vivo or in vitro systems and in different wells. However, its efficiency tends to reduce when advanced stages, such as morulae and blastocysts, are used. The aim of this work was to evaluate the effect of the treatment with an agglutinating agent (phytohemagglutinin-L; PHA) in the percentage of chimeras produced with IVF bovine embryos. Bovine ovaries (from abattoir) were used to obtain 270 COC that were matured in drops (90 μL) of TCM-199 bicarbonate medium, supplemented with 10% of FCS, and incubated in vitro for 22 to 24 h. The fertilization occurred in TALP-IVF medium, and the COC were maintained in the incubator for 18 h. After fertilization, the presumptive zygotes were transferred to SOF culture medium to in vitro culture. In vitro maturation, fertilization, and culture were performed under 38.5°C, 5% CO2 in air and saturated humidity. The chimerism by aggregation was tested between 2 intact (zona-free) 8- to 16-cell stage embryos in the presence (G1, n = 16) or absence of PHA (G2, n = 14) and between one half-morula and one half-blastocyst with (G3, n = 15) or without PHA (G4, n = 12). The embryos in groups G1 and G3 were treated with PHA in a concentration of 500 μLg mL-1 for 3 min. After PHA treatment, the pairs of embryos were allocated in wells, under previously described culture conditions, until expanded blastocyst stage could be observed (Day 7 of culture). At 24 h of culture, embryonic aggregation pairs were first evaluated to detect only cohesive masses of cells. The results (chimerism rate) were 62.5%, 42.9%, 40.0%, and 25.0%, respectively, for groups G1, G2, G3, and G4. There were no significant differences neither among groups (chi-square, P = 0.252) nor between G1 and G2 (P = 0.464), G3, and G4 (P = 0.683; Fisher’s exact test). Main effects as use of PHA (G1 + G3 v. G2 + G4, P = 0.284) and stage of embryos (G1 + G2 v. G3 + G4, P = 0.183; Fisher’s exact test) were not statistically significant. However, when all groups were compared, the power of the performed test (0.354) was below the desired power of 0.800 (i.e. one must be cautious in over-interpreting the lack of difference among them). In the conditions of this study, it was concluded that the treatment with PHA did not increase the rate of aggregation in the embryonic chimera production, even for half-embryos in advanced stage of development (morulae and blastocysts). Granted by FAPESP, Brazil: 06/06491-2 and 07/07705-9 (MFGN) and 07/04291-9 (MPMM).

scholarly journals The Nup358-RanGAP Complex Is Required for Efficient Importin α/β-dependent Nuclear Import

2008 ◽  
Vol 19 (5) ◽  
pp. 2300-2310 ◽  
Author(s):  
Saskia Hutten ◽  
Annette Flotho ◽  
Frauke Melchior ◽  
Ralph H. Kehlenbach
Keyword(s):  
Nuclear Import ◽  
Protein Transport ◽  
Nucleocytoplasmic Transport ◽  
Limiting Factor ◽  
Dual Function ◽  
Nuclear Pore ◽  
Transport Reaction ◽  
Importin Α

In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin α/β-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin β, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin β strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore–associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin β recycling and reformation of novel import complexes.

142 CHIMERISM BY AGGREGATION OF BISECTED IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 175
Author(s):  
E. M. Razza ◽  
I. P. Emanuelli ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Aggregation Rate ◽  
Stage Of Development

Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

scholarly journals 252A COMPARATIVE EXPRESSION ANALYSIS OF GENES IN PREIMPLANTATION DEVELOPMENTAL STAGES OF BOVINE EMBRYOS PRODUCED IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 246 ◽  
Author(s):  
D. Tesfaye ◽  
K. Wimmers ◽  
M. Gilles ◽  
S. Ponsuksili ◽  
K. Schellander
Keyword(s):  
Developmental Stage ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
The Novel ◽  
Cell Stage ◽  
Novel Transcript ◽  
Stage Of Development ◽  
Significant Difference

A comparative analysis of mRNA expression patterns between embryos produced under different in vitro and in vivo culture systems allows the isolation of genes associated with embryo quality and investigation of the effect of culture environment on the embryonic gene expression. In this study, expression analysis of four known (PSCD2, TCF7L2, NADH-subunit and PAIP1) genes and one novel transcript, derived from differential display PCR, was performed in in vitro (Ponsuksili et al., 2002, Theriogenology 57, 1611–1624) or in vivo- (Moesslacher et al., 2001 Reprod. Dom. Anim. 32, 37) produced bovine 2-, 4-, 8-, 16-cell, morula and blastocyst stage embryos using real time PCR technology. Poly(A) RNA was isolated from four separate individual embryos from each developmental stage and embryo group (in vitro or in vivo) using Dynabeads mRNA kit (Dynal, Oslo, Norway). After reverse transcription, quantitative PCR was performed with sequence specific primers in an ABI PRISM® 7000 Sequence Detection System instrument (Applied Biosystems, Foster City, CA, USA) using SYBR® Green as a double-strand DNA-specific fluorescent dye. Standard curves were generated for target and endogenous genes using serial dilutions of plasmid DNA. Final quantification was done using the relative standard curve method, and results were reported as relative expression or n-fold difference to the calibrator cDNA (i.e., the blastocyst stage) after normalization with the endogenous control (Histone2a). Data were analyzed using SAS version 8.0 (SAS Institute Inc., NC, USA) software package. Analysis of variance was performed with the main effects being the developmental stage and embryo source (in vitro or in vivo) and their interactions followed by multiple pairwise comparisons using Tukey’s test. No significant difference was observed in the relative abundance of the PSCD2 gene between the two embryo groups. However, its expression was higher (20-fold) (P&lt;0.05) at the 8-cell stage than the other developmental stages among in vitro embryos. Higher expression (P&lt;0.05) of NADH-subunit mRNA was detected in vivo than in vitro at the 2-cell stage of development. The TCF7L2 mRNA was expressed in the in vitro embryos but not in the in vivo ones. PAIP1 mRNA was higher (P&lt;0.05) in in vitro (1500-fold) than in the in vivo embryos (500-fold) at the 2-cell developmental stage compared to the calibrator. The novel transcript was also detected at higher level (P&lt;0.05) in the in vitro than in the in vivo embryos at the 2-cell stage of development. However, the PAIP1 and the novel transcript showed no significant difference in their expression between the two embryo groups beyond the 2-cell developmental stage. Both PAIP1 and the novel transcript were detected only up to 8-cell stage in both embryo groups, suggesting their maternal origin. In conclusion, the variations in the expression of studied genes between in vitro and in vivo may reflect the effect of the two culture systems on the transcriptional activity of early embryos.

In vitro embryo culture in the production of identical merino lambs by nuclear transplantation

1990 ◽  
Vol 2 (6) ◽  
pp. 619 ◽  
Author(s):  
KJ McLaughlin ◽  
L Davies ◽  
RF Seamark
Keyword(s):  
Nuclear Transplantation ◽  
Cell Stage ◽  
Fluid Medium ◽  
Stage Of Development ◽  
Simple Medium ◽  
Pregnancy Status ◽  
Oviduct Fluid ◽  
Similar Stage

This study examined the viability of embryos developed in vitro from 8- to 16-cell stage blastomeres fused with enucleated oocytes. Of 209 blastomeres recovered and subjected to manipulation and electrofusion procedures, 190 (91%) fused successfully, with 86 (45%) of those undergoing cleavage up to the 4- to 16-cell stage when cultured for 66 h in a synthetic oviduct fluid medium. The viability of the embryos was examined by transferring them to recipient ewes and determining the ewes' pregnancy status by ultrasound on Day 45. Of 86 embryos transferred, 14 developed to fetuses in 8 of the 36 recipients, including four sets of identical twins and one set of quads. In contrast, with uncultured and unmanipulated embryos, 15 fetuses developed from 19 embryos transferred at a similar stage of development. The viability of embryos derived from manipulated zygotes cultured in vitro was comparable to that previously reported for studies employing in vivo culture, indicating the potential of in vitro culture systems based on a simple medium for nuclear-transplantation embryos.

scholarly journals Sequence Elements in Both Subunits of the DNA Fragmentation Factor Are Essential for Its Nuclear Transport

2007 ◽  
Vol 282 (49) ◽  
pp. 35821-35830 ◽  
Author(s):  
Sonja Neimanis ◽  
Werner Albig ◽  
Detlef Doenecke ◽  
Joerg Kahle
Keyword(s):  
Dna Fragmentation ◽  
Nuclear Import ◽  
Dna Cleavage ◽  
Nuclear Accumulation ◽  
Proliferating Cells ◽  
Nuclear Localization Signals ◽  
Importin Α ◽  
The Individual

DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin α/β-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin α/β-heterodimer. However, importin α/β binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin α/β more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin α/β with the DFF complex are presented.

A in Vivo Assay for Standardizing Heparin Preparations

1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy
Keyword(s):  
In Vitro Assays ◽  
British Pharmacopoeia ◽  
In Vitro Method ◽  
Standard Preparation ◽  
Reliable Estimation ◽  
Assay Procedure ◽  
Accuracy And Precision ◽  
Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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