Nuclear function

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Glu-108 in Saccharomyces cerevisiae Rad51 Is Critical for DNA Damage-Induced Nuclear Function

Glu-108 in Saccharomyces cerevisiae Rad51 Is Critical for DNA Damage-Induced Nuclear Function

Generation of RAD51 mutant strain based on the molecular docking studies between yHsp90 and Rad51. Earlier studies in our lab demonstrated that yHsp90 and Rad51 can physically interact (14). Unlike other chaperones, there is no specific binding pocket present in Hsp90 through which it binds to the client proteins. Hence, in order to understand the point of contacts between yHsp90 and Rad51, we employed a bioinformatics approach. To that end, Rad51 proteins (PDB identifier [ID] 1SZP) having various combinations of monomers, dimers, and hexamers were allowed to dock with yHsp90 (PDB ID 2CG9) using the fully automated web-based program ClusPro 2.0 (18), which employs the improved fast Fourier transform (FFT)-based rigid docking program PIPER (19). Thirty models of the protein-protein complex for each type of interaction, namely, balanced, electrostatic favored, hydrophobic favored, and van der Waal’s plus electrostatic, were generated for each docking. It was found that a hydrophobic- favored interaction showed the lowest energy scores; hence, the corresponding protein complex model with the largest cluster was chosen. The surface view of the three- dimensional structure of Rad51 displays a characteristic pocket in each of the mono- mers into which the yHsp90 is found to dock. The docked complex models showed that the N-terminal residue of the Rad51 E chain, Glu 108 (1.88 Å), has the shortest bond distance with yHsp90 C-terminal residues. We conducted a multiple-sequence align- ment of Rad51 (Fig. 1A) and found that E108, which is predicted to have the strongest association with Hsp90, is evolutionarily conserved. In Rad51, the amino acid residue E108 is present in the N-terminal domain of Rad51, which lies outside its catalytic domain (Fig. 1B). To explore whether the Hsp90 and Rad51 association mediates Rad51 nuclear function under DNA-damaging conditions, one approach may be the genera- tion of a Rad51 mutant with a reduced affinity for Hsp90. However, as Rad51 is a client of Hsp90, we reasoned that any mutant of Rad51 that fails to interact with Hsp90 due to a low affinity would be unstable in the cell. Hence, we designed a strong-affinity mutant to establish our hypothesis. By in silico mutation, we created four single mutants of Rad51 where the glutamic acid at the 108th position was replaced by neutral residues (glycine, alanine, leucine, and isoleucine). Table 1 displays a compar- ison of the parameters of yHsp90 docking with the wild-type and mutant Rad51 based on ClusPro results. Our study shows that the mutant Rad51 E108L and Hsp90 docked
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Pressuromodulation at the cell membrane as the basis for small molecule hormone and peptide regulation of cellular and nuclear function

Pressuromodulation at the cell membrane as the basis for small molecule hormone and peptide regulation of cellular and nuclear function

and increases the exocytosis of pre-synthesized vesicular endogolgi peptides and small molecules as well as nuclear-to-rough endoplasmic reticulum membrane proteins to the CM, with the potential to simultaneously increase the NM-associated chromatin DNA transcription of higher molecular weight protein forms, secretory and CM-destined, mitochondrial and nuclear, including the highest molecular weight nuclear proteins, Ki67 (359 kDa) and Separase (230 kDa), with the latter leading to mitogenesis and cell division; while, in the case of growth factors or cytokines with external cationomodulation capability, CM Receptor External Cationomodulation of CM receptors ( ≥ 3 + → 1 + ) results in cationic extracellular interaction ( ≥ 3 + ) with extracellular matrix heparan sulfates ( ≥ 3 + → 1 + ) concomitant with lamellopodesis and cell migration. It can be surmised that the modulation of cellular, and nuclear, function is mostly a reactive process, governed, primarily, by small molecule hormone and peptide inter- actions at the cell membrane, with CM receptors and the CM itself. These insights taken together, provide valuable translationally applicable knowledge.
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PIP5K: Role in Plant Nuclear Function and Characterization of MORN Domain Structure.

PIP5K: Role in Plant Nuclear Function and Characterization of MORN Domain Structure.

In Arabidopsis thaliana, the MORN sequence containing protein ARC3 had three C- terminal repeated MORN repeats (Shimada et al., 2004). ARC3 is important for chloroplast division (Maple et al., 2007)..It is possible that the C-terminal MORN domain or ARC3 is important for establishing membrane binding for a scaffold of chloroplast fission proteins to bind to the ARC3 Ftz domain or that the MORN domain functions as a protein/protein interaction domain. The fact that a point mutation arc3-3, which produces a truncated protein without the C-terminal MORN domain, results in a giant chloroplast phenotype suggests that the MORN domain of ARC3 is important for chloroplast fission (Cho et al., 2012). More recently, four proteins containing only a MORN domain have been annotated in the A. thaliana genome, and one, EMB1211, has been identified as an embryonic lethal mutant and was important for chloroplast fission (Liang et al., 2010). These data and those describing the function of the MORN1 protein in T. gondii imply a conserved, ancient function of the MORN repeats in the fission of membranes and organelles.
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Immunohistochemical studies of cyclic guanosine monophosphate and nuclear function

Immunohistochemical studies of cyclic guanosine monophosphate and nuclear function

immunofluorescent technique. These aggregates correspond to the areas of condensation of DNA, demonstrable by either Felugen's or acridine orange stain. Treatment with DNAase eliminated DNA and cGMP staining. Staining for RNA, with a human anti-RNA antibody, demonstrated RNA to be distributed diffusely throughout the nucleus and not preferentially in the areas of discrete cGMP aggregates. The diffuse stain for nuclear RNA was eliminated by pretreatment with RNAase but not DNAase, but aggregates of cGMP were not affected by pretreatment with RNAase. Sites of active RNA synthesis were determined by
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Defects in nuclear structure and function promote dilated cardiomyopathy in lamin A/C–deficient mice

Defects in nuclear structure and function promote dilated cardiomyopathy in lamin A/C–deficient mice

heterochromatin; these changes are present but less severe in left atrial nuclei. Electron microscopy of Lmna –/– cardiomyocytes shows disorganization and detachment of desmin filaments from the nuclear surface with progressive disruption of the cytoskeletal desmin network. Alterations in nuclear architecture are associated with defective nuclear function evidenced by decreased SREBP1 import, reduced PPARg expression, and a lack of hypertrophic gene activation. These findings suggest a model in which the primary pathophysiological mechanism in Lmna –/– mice is defective force transmission resulting from disruption of lamin interactions with the muscle-specific desmin network and loss of cytoskeletal tension. Despite severe DCM, defects in nuclear function prevent Lmna –/– cardiomyocytes from developing compensatory hypertrophy and accelerate disease progression.
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Defective nuclear IKKα function in patients with ectodermal dysplasia with immune deficiency

Defective nuclear IKKα function in patients with ectodermal dysplasia with immune deficiency

Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-κB essential modulator (NEMO; also known as IκB kinase γ subunit [IKKγ]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-κB proteins, resulting in defective expression of NF-κB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-κB. However, cells deficient in full-length NEMO were defective in expression of NF-κB–regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-κB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKα-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-κB heterodimers RelA and cRel to the promoter. Expression of a super-active form of IKKα restored IL-12 pro- duction in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKα and offer new insights into the mechanisms underlying diminished NF-κB signaling in patients with EDI.
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Nuclear-Cytoplasmic Shuttling Is Not Required for the Epstein-Barr Virus EBNA-LP Transcriptional Coactivation Function

Nuclear-Cytoplasmic Shuttling Is Not Required for the Epstein-Barr Virus EBNA-LP Transcriptional Coactivation Function

FIG. 5. Coactivation-functional and -nonfunctional EBNA-LP mutants shuttle in a heterokaryon assay. (A) Comparison of EBNA-LP primary amino acid sequences from EBV types 1 and 2 (EBV1 and EBV2) and several nonhuman primate lymphocryptoviruses (LCV). Ch, chimpanzee; Go, gorilla; Rh, rhesus macaque; Ba, baboon. Amino acid residues encoded by the W1, W2, Y1, and Y2 exons are indicated above. The sequence is shown with a single W repeat, which is comprised of W1 and W2. The arrowheads above the sequence indicate conserved serine residues, and below the sequences the various CRs are indicated. The black and gray bars below show regions of the protein required for coactivation function and nuclear localization (NLS). At the bottom of the diagram, the various mutants tested are shown and have been described previously (21, 31). The coactivation-competent ⌬ Y1Y2 EBNA-LP contains four repeats because a smaller version with two repeats failed to localize efficiently in the nucleus. Nuclear localization, coactivation, and nuclear-cytoplasmic (N/C) shuttling statuses are indicated for each mutant. ⫹ , present; ⫺ , absent. (B) Heterokaryons between HeLa cells transfected with the indicated EBNA-LP proteins and murine 3T3 cells. Heterokaryons were stained with anti-EBNA-LP (green) in the images on the left. The images on the right show Hoechst (white) and phalloidin (red) staining to visualize the heterokaryons. WT, wild type. Human and mouse nuclei are indicated (H and M, respectively).
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A novel function for the IκB inhibitor Cactus in promoting Dorsal nuclear localization and activity in the Drosophila embryo

A novel function for the IκB inhibitor Cactus in promoting Dorsal nuclear localization and activity in the Drosophila embryo

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the I κ B inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NF κ B effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NF κ B inhibitor, ensuring a correct response to Toll signals.
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Molecular anatomy of tunicate senescence: reversible function of mitochondrial and nuclear genes associated with budding cycles

Molecular anatomy of tunicate senescence: reversible function of mitochondrial and nuclear genes associated with budding cycles

Zooids of the asexual strain of Polyandrocarpa misakiensis have a lifespan of 4-5 months; before dying, they produce many buds, enabling continuation of the strain. This study was designed to investigate the nature of gene inactivation and reactivation during this continuous process of senescence and budding. During senescence, the zooidal epidermis showed acid -galactosidase activity, lost proliferating cell nuclear antigen immunoreactivity and became ultrastructurally worn, indicating that the epidermis is a major tissue affected by the ageing process. Semi-quantitative PCR analysis showed that the genes encoding mitochondrial respiratory chains (MRCs) engaged in decreased transcriptional activity in senescent adults compared with younger adults. The results of in situ hybridization showed that the epidermis dramatically attenuates MRC expression during ageing but restores gene activity when budding commences. During budding and ageing, the nuclear gene Eed (a polycomb group component) was activated and inactivated in a pattern similar to that observed in MRCs. In buds, RNA interference (RNAi) of Eed attenuated Eed transcripts but did not affect the gene expression of pre-activated MRCs. A tunicate humoral factor, TC14-3, could induce Eed , accompanying the reactivation of MRC in adult zooids. When RNAi of Eed and Eed induction were performed simultaneously, zooidal cells and tissues failed to engage in MRC reactivation, indicating the involvement of Eed in MRC activation. Results of this study provide evidence that the mitochondrial gene activities of Polyandrocarpa can be reversed during senescence and budding, suggesting that they are regulated by nuclear polycomb group genes.
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Dynamic Nuclear Architecture and Chromatin Function (EuroDYNA)

Dynamic Nuclear Architecture and Chromatin Function (EuroDYNA)

“Thanks to EuroDYNA, nine research projects were funded that may otherwise not have been funded. Therefore, European research in the area of nuclear dynamics and architecture has been stimulated. Without EuroDYNA I would not have been able to perform the research I have carried out over the last three years. One aspect of EuroDYNA that I like a lot is the lack of bureaucratic burden compared to other research programmes. Another very important aspect is that the EUROCORES programmes are suggested by the scientists themselves (bottom-up approach). Finally, a great added value is the willingness at ESF to stimulate discussion among scientists, by organizing conferences, workshops and brainstorm meetings. As a EuroDYNA member I have benefited enormously from this valuable resource,” said Niels Galjart, Department of Cell Biology and Genetics, Erasmus University, Rotterdam and the Project Leader of “Role of multi zinc finger proteins CTCF and BORIS in the dynamic change of the nuclear architecture and chromatin function during cell cycle and differentiation”. There is no doubt that EuroDYNA has achieved some great results and many of these results stem directly from the EUROCORES Programmes’ focus on networking and collaboration. David Shore, University of Geneva and the Project Leader of “Environmental stress-induced dynamic modulation of chromatin structure, gene expression and nuclear architecture in yeast” commented, “My project recently entered into collaboration with a lab in Vienna. This wouldn’t have happened if it weren’t for EuroDYNA. The Vienna group is interested in understanding how arsenic affects cells and of course this has important global health implications. Arsenic is a pollutant in drinking water in many places in the world. Our collaboration began at a EuroDYNA meeting in Brno; we presented a gene we were working on that’s involved in growth regulation in yeast cells and also in the cellular response to stress (which is what our project is aimed at understanding) when we were approached by a researcher from the Vienna group. The Vienna researcher noticed that this gene had also come up in his studies as a regulator of the cellular response to arsenic poisoning. As a result, we got together and did some more work which has now led to a manuscript ready for submission”.
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Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev export function

Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev export function

moter used [27]. The report that NF90ctv arrests HIV-1 replication in human osteosarcoma-derived cells [22], supports the argument that ectopic expression of NF90ctv results in overall stimulation of IFN response genes. We reason that the attenuation of HIV-1 replication in NF90ctv expressing cells may in part be due to the affinity of NF90ctv protein to the structured RNA molecules essential in viral life cycle. That is, in addition to general- ized induction of the innate antiviral response pathway in NF90ctv-expressing cells [22], the binding properties of NF90ctv to structured RNAs are likely to interfere with specific steps of viral life cycle, particularly those that rely on precise protein:RNA interactions. Thus, the overall antiviral response of NF90ctv may result from the stimu- lated innate immunity of the host following virus infec- tion as well as from the ability of NF90ctv to interfere with specific steps in the viral life cycle. Here we examined the protein:protein and protein-dsRNA interaction properties of NF90ctv that inhibits HIV-1 replication by blocking Rev function. The results suggest that NF90ctv negatively affects the export of HIV-1 transcripts regulated by Rev. The NF90ctv-mediated inhibition of Rev involved three specific regions of NF90ctv protein.
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An Unconventional Nuclear Localization Motif Is Crucial for Function of the Drosophila Wnt/Wingless Antagonist Naked Cuticle

An Unconventional Nuclear Localization Motif Is Crucial for Function of the Drosophila Wnt/Wingless Antagonist Naked Cuticle

Wnt/ b -catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, we first must understand how Wnt signals are normally controlled. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/b-catenin signaling that is crucial for segmentation in the model genetic organism, the fruit fly Drosophila melanogaster. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not un- derstood. Here we show that nkd mutants exhibit elevated levels of the b-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of- function assays. While Dsh-binding regions of Nkd contribute to its activity, we identify a conserved 30- amino-acid motif, separable from Dsh-binding regions, that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. Our studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal.
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Mapping of Sequences in Pseudorabies Virus pUL34 That Are Required for Formation and Function of the Nuclear Egress Complex

Mapping of Sequences in Pseudorabies Virus pUL34 That Are Required for Formation and Function of the Nuclear Egress Complex

The nuclear egress complex (NEC) is required for efficient translocation of newly synthesized herpesvirus nucleocapsids from the nucleus to the cytosol. It consists of the type II membrane protein pUL34 which interacts with pUL31 at the inner nuclear membrane (INM). To map regions within pUL34 required for nuclear membrane targeting and pUL31 interaction, we con- structed deletion/substitution mutations. Previously, we showed that 50 C-terminal amino acids (aa) of pseudorabies virus (PrV) pUL34, including the transmembrane domain, could be functionally replaced by cellular lamina-associated polypeptide 2 ␤ (Lap2 ␤ ) sequences. In contrast, replacement of the C-terminal 100 aa abrogated complementation but not pUL31 interac- tion. To further delineate essential sequences within this region, C-terminal pUL34 truncations of 60, 70, 80, 85, and 90 aa fused to Lap2␤ sequences were generated. While truncations up to 85 aa were functional, deletion of the C-terminal 90 aa abrogated function, which indicates that the important region is located between aa 171 and 176. Amino acids 173 to 175 represent RQR, a motif suggested to mediate INM targeting. Mutagenesis to RQG revealed that the mutant protein exhibited pronounced Golgi localization, but a fraction still reached the INM. Deletion mutations in the N-terminal domain of pUL34 demonstrated that absence of the first 4 aa was tolerated, while removal of 9 or more residues resulted in a nonfunctional protein. In addition, mu- tation of three conserved cysteines did not abrogate pUL34 function, whereas alteration of a conserved glutamine/tyrosine se- quence yielded a nonfunctional protein.
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Arsenic trioxide inhibits nuclear receptor function via SEK1/JNK mediated 
RXRα phosphorylation

Arsenic trioxide inhibits nuclear receptor function via SEK1/JNK mediated RXRα phosphorylation

ble that the high level of phosphorylation within the DE domain masks a single induced phosphorylated amino acid, but we have excluded 2 serine sites suggested by previous reports. Adam-Sti- tah et al. reported that UV activation, as well as overexpression of JNK, leads to phosphorylation of murine RXRα in the AB region (30). They defined 3 residues in the AB region and 1 residue in the E region as targets for this inducible phosphorylation. How- ever, they found that mutation of these amino acids did not alter the ability of 9-cis RA to induce transactivation of either a DR1 reporter or the murine RARβ2 reporter constructs. When we mutated the corresponding serines in the human RXRα (S56A, S70A, and S260), we also saw no impact on the ability of RA to induce transactivation nor on the inhibition of nuclear receptor function by As 2 O 3 . This is further supported by a recent report
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Hepatic function in a family with a nonsense mutation (R154X) in the hepatocyte nuclear factor 4alpha/MODY1 gene

Hepatic function in a family with a nonsense mutation (R154X) in the hepatocyte nuclear factor 4alpha/MODY1 gene

of the young (MODY), a monogenic form of diabetes mellitus characterized by autosomal dominant inheritance and early onset, usually before 25 yr of age (1, 2). The subtype of MODY resulting from mutations in the HNF-4 a gene is termed MODY1. HNF-4 a is a member of the nuclear receptor superfamily, a family of transcription factors that plays an im- portant role in cellular regulation linking extracellular signals and transcriptional responses (3, 4). HNF-4 a was identified during the course of characterizing the transcription factors that were responsible for the tissue-specific regulation of gene expression in adult liver (3). It is not restricted to liver, how- ever, and is also found in other tissues, including kidney, intes- tine, and pancreatic b -cells (2–4).
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Identification of Conserved Amino Acids in pUL34 Which Are Critical for Function of the Pseudorabies Virus Nuclear Egress Complex

Identification of Conserved Amino Acids in pUL34 Which Are Critical for Function of the Pseudorabies Virus Nuclear Egress Complex

The dileucine motif L166/167 was speculated to be part of an arginine-based ER sorting motif (RQR) (20). Therefore, we mu- tated either the more highly conserved L167 or both leucine resi- dues to alanine. Although a point mutation of RQR to RQG re- sulted in pUL34 accumulation in the Golgi apparatus (20), localization of pUL34 –L167A or -L166/167A was indistinguish- able from that of native pUL34, indicating that these residues are not required for nuclear rim localization or ER retention. More- over, speckles could be observed after cotransfection with pUL31, correlating with the location of both residues outside the pUL31 interaction domain. Nevertheless, mutation of both leucine resi- dues had a dramatic effect on NEC function, and RK13-UL34 – L166/L167A cells completely failed to complement the UL34 de- letion mutant, while the single mutation L167 had no effect. Therefore, this motif is apparently not involved in the proper translocation of pUL34 to the INM, as was previously hypothe- sized (20), but, rather, plays an important role in another step, e.g., in capsid recruitment or interaction with other factors in- volved in nuclear egress. Whether the L166A substitution is suffi- cient for this effect needs to be tested. Interestingly, both leucine residues are also present in MCMV and HCMV pUL34 homologs (10, 19), and a linker insertion at the corresponding site in MCMV UL50 also resulted in a nonfunctional protein. Thus, the location of the dileucine motif seems to separate the essential N-terminal FIG 7 Complementation of direct cell-to-cell transmission. Direct viral cell-to-cell spread was analyzed by infecting the different pUL34-expressing RK13 cell lines with PrV-Ka or PrV- ⌬ UL34 under plaque assay conditions. After 2 days, samples were fixed and the diameters of 30 plaques of each cell line and virus were measured. Plaque diameters of PrV-Ka on a given cell line were set equal to 100%, and the diameters of the plaques induced by PrV- ⌬ UL34 were calculated accordingly. Values represent the means of three independent experiments. The corresponding standard deviations are indicated, and statistically significant differences of the infection with PrV- ⌬ UL34 compared to wild-type PrV-Ka infection of each transgenic cell line are indicated: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.
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Determination of the nuclear level densities and radiative strength function for 43 nuclei in the mass interval 28≤A≤200

Determination of the nuclear level densities and radiative strength function for 43 nuclei in the mass interval 28≤A≤200

Unfortunately, an existence of the sources of uncertainties of the sought ρ and Γ functions is a fundamental problem, and it is inevitable for any nuclear model used for experimental data analysis and for predictions of the spectra and cross sections. There are also fluctuations of the intensities of gamma-transitions in different nuclei, which has a contribution to the systematical error. Nevertheless, the practical model showed one possibility to describe the data of the two-step experiments with the accuracy that exceeds the statistical one.

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Characterization of the HIV 1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

Characterization of the HIV 1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

Identification of proteins associated with HIV-1 RNA As we described above, we used the MS2 tagging for the purpose of HIV-1 RNA affinity purification. Next, to identify nuclear factors associated with viral RNA, we proceeded as follows: U2OS HIVexo and U2OS HIVin- tro stable cell lines together with wild type U2OS were transfected with vectors expressing Tat-CFP and flag- MS2nls proteins. Since we were interested in the identi- fication of factors involved in nuclear HIV-1 RNA meta- bolism, we subjected the cells to biochemical fractionation for the extraction of the nucleoplasmic fraction (NF) (Figure 2A). Indeed, the procedure resulted in clean preparation of NF as controlled by immunoblotting with nuclear (tubulin) and cytoplasmic (RecQ) markers as shown in Figure 2B. The nuclear fraction was further subjected to flag-immunoprecipita- tion. IPs were extensively washed in the presence of nonspecific competitors as described in Materials and Methods, and the specificity of pulldown was assessed by immunoblotting as shown in Figure 2C. Lastly, IPs were subjected to mass spectrometry analysis as described in details in Materials and Methods. We were interested in proteins that associated with both HIVexo and HIVintro RNAs because they represent hits obtained from two totally independent procedures. The combined results of two immunoprecipitations led to the identification of 32 proteins that were specific for the stable cell lines carrying the virus (Table 1). Indeed, most of the identified proteins have been characterized in RNA binding and/or regulation. Proteins such as BAT1, FUS and hnRNPs have been already found in large-scale proteomic analysis of the human spliceosome [46,47]. BAT2 and CAPRIN1 were shown to associate with pre-mRNA, although their role in pre-mRNA pro- cessing is yet to be demonstrated [48,49]. Interestingly, many of the identified proteins have been already shown to be involved in various steps of HIV-1 RNA metabo- lism. DBPA and RPL3 were shown to interact with the TAR while ILF3 interacts with both - the TAR and the RRE [50-52]. DDX3X, SFPQ and Upf1 were shown to regulate Rev-dependent unspliced and partially spliced viral transcripts while PTB was shown to regulate Rev- independent, multiply spliced HIV-1 RNA [10,23,53,54]. MOV10 belongs to a family of Upf1-like RNA helicases, and it has been shown to inhibit viral replication at mul- tiple stages although its activity on viral RNA is yet to
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The Sf1 related nuclear hormone receptor Hr39 regulates
Drosophila female reproductive tract development and function

The Sf1 related nuclear hormone receptor Hr39 regulates Drosophila female reproductive tract development and function

Some molecular pathways of sex determination evolve rapidly whereas others are relatively conserved (reviewed by Marin and Baker, 1998). The roles played by steroid hormones in vertebrates and invertebrates appear to be among the most divergent. Multiple steroid hormones serve non-autonomously as master regulators of male or female sexual development in mammals, whereas the Drosophila molting hormone ecdysone acts sex-specifically only during adult oogenesis (Buszczak et al., 1999; Carney and Bender, 2000; Hackney et al., 2007; Li et al., 2000). Steroid-producing (‘steroidogenic’) tissues arise early in mammalian embryos under the control of the nuclear receptor steroidogenic factor 1 (SF1) (reviewed by Val et al., 2003) and function throughout life. The closely related protein LRH1 (NR5A2) also plays an important role, especially in ovarian function (reviewed by Fayard et al., 2004). The Drosophila genome encodes two proteins that are closely related to SF1 and LRH1, FTZ-F1 and HR39, which bind to similar target sequences (Ohno et al., 1994), but neither has been implicated in sex determination (reviewed by King-Jones and Thummel, 2005). Steroid production in the ovary, the only known site of adult steroidogenesis, is not known to depend on either gene.
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DETERMINATION OF THE ORDER OF GENE FUNCTION IN THE YEAST NUCLEAR DIVISION PATHWAY USING cs AND ts MUTANTS

DETERMINATION OF THE ORDER OF GENE FUNCTION IN THE YEAST NUCLEAR DIVISION PATHWAY USING cs AND ts MUTANTS

The positions of the CDC9 and CDC44 execution points are ambiguous with respect to the branch required to make CDC45 and CD.C.26 independent; otherwise the positions of the f[r]

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