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Nucleophosmin (NPM1) is a key nucleolar protein released from the nucleolus in response to stress stimuli. NPM1 functions as a stress regulator with nucleic acid and protein chaperone activities, rapidly shuttling between the nucleus and cytoplasm. NPM1 is ubiquitously expressed in tissues and can be found in the nucleolus, nucleoplasm, cytoplasm, and extracellular environment. It plays a central role in various biological processes such as ribosome biogenesis, cell cycle regulation, cell proliferation, DNA damage repair, and apoptosis. In addition, it is highly expressed in cancer cells and solid tumors, and its mutation is a major cause of acute myeloid leukemia (AML). This review focuses on NPM1's structural features, functional diversity, subcellular distribution, and role in stress modulation. [ABSTRACT FROM AUTHOR]

Nucleoplasmin (NPM) histone chaperones regulate distinct processes in the nucleus and nucleolus. While intrinsically disordered regions (IDRs) are hallmarks of NPMs, it is not clear whether all NPM functions require these unstructured features. We assessed the importance of IDRs in a yeast NPM‐like protein and found that regulation of rDNA copy number and genetic interactions with the nucleolar RNA surveillance machinery require the highly conserved FKBP prolyl isomerase domain, but not the NPM domain or IDRs. By contrast, transcriptional repression in the nucleus requires IDRs. Furthermore, multiple lysines in polyacidic serine/lysine motifs of IDRs are required for both lysine polyphosphorylation and NPM‐mediated transcriptional repression. These results demonstrate that this NPM‐like protein relies on IDRs only for some of its chromatin‐related functions. [ABSTRACT FROM AUTHOR]

The nucleoplasm and nucleolus are the two main territories of the nucleus. While specific functions are associated with each of these territories (such as mRNA synthesis in the nucleoplasm and ribosomal rRNA synthesis in the nucleolus), some proteins are known to be located in both. Here, we investigated the molecular function of REBELOTE (RBL), an Arabidopsis thaliana protein previously characterized as a regulator of floral meristem termination. We show that RBL displays a dual localization, in the nucleolus and nucleoplasm. Moreover, we used direct and global approaches to demonstrate that RBL interacts with nucleic acid‐binding proteins. It binds to the NOC proteins SWA2, AtNOC2 and AtNOC3 in both the nucleolus and nucleoplasm, and also to OBE1 and VFP3/ENAP1. Taking into account the identities of these RBL interactors, we hypothesize that RBL acts both in ribosomal biogenesis and in the regulation of gene expression. [ABSTRACT FROM AUTHOR]

Fertilization requires decondensation of promatine-condensed sperm chromatin, a dynamic process serving as an attractive system for the study of chromatin reprogramming. Nucleoplasmin is a key factor in regulating nucleosome assembly as a chaperone during fertilization process. However, knowledge on nucleoplasmin in chromatin formation remains elusive. Herein, magnetic tweezers (MT) and a chromatin assembly system were used to study the nucleoplasmin-mediated DNA decondensation/condensation at the single-molecular level in vitro . We found that protamine induces DNA condensation in a stepwise manner. Once DNA was condensed, nucleoplasmin, polyglutamic acid, and RNA could remove protamine from the DNA at different rates. The affinity binding of the different polyanions with protamine suggests chaperone-mediated chromatin decondensation activity occurs through protein–protein interactions. After decondensation, both RNA and polyglutamic acid prevented the transfer of histones onto the naked DNA. In contrast, nucleoplasmin is able to assist the histone transfer process, even though it carries the same negative charge as RNA and polyglutamic acid. These observations imply that the chaperone effects of nucleoplasmin during the decondensation/condensation process may be driven by specific spatial configuration of its acidic pentamer structure, rather than by electrostatic interaction. Our findings offer a novel molecular understanding of nucleoplasmin in sperm chromatin decondensation and subsequent developmental chromatin reprogramming at individual molecular level. [ABSTRACT FROM AUTHOR]

Summary The question of whether retained histones in the sperm genome localize to gene-coding regions or gene deserts has been debated for years. Previous contradictory observations are likely caused by the non-uniform sensitivity of sperm chromatin to micrococcal nuclease (MNase) digestion. Sperm chromatin has a highly condensed but heterogeneous structure and is composed of 90%∼99% protamines and 1%∼10% histones. In this study, we utilized nucleoplasmin (NPM) to improve the solubility of sperm chromatin by removing protamines in vitro . NPM treatment efficiently solubilized histones while maintaining quality and quantity. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses using NPM-treated sperm demonstrated the predominant localization of H4 to distal intergenic regions, whereas modified histones exhibited a modification-dependent preferential enrichment in specific genomic elements, such as H3K4me3 at CpG-rich promoters and H3K9me3 in satellite repeats, respectively, implying the existence of machinery protecting modified histones from eviction. [ABSTRACT FROM AUTHOR]

Nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowed us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites. [ABSTRACT FROM AUTHOR]

Mammals is a powerful tool to study pathophysiologic process of cardiovascular disease, but for a long time it is just limited to large animals, like pigs, rabbits, and big rats. The mice with reproduction fast cycle, lower costing, and close to human genes, can be showed about some phenomenon quickly, also more easy to form model of atherosclerosis. Thus, a mouse model of vascular injury is vitally important for researching the pathophysiological mechanisms to restenosis after percutaneous transluminal coronary angioplasty (PTCA) as well as translational approaches. In this Experiment, we established a unique murine model of femoral artery injury through a home-made wire. The mouse model of vascular injury may be used to explore the molecular mechanism of post-angioplasty restenosis with lowest costing. [ABSTRACT FROM AUTHOR]

Nucleoplasmin (Npm) is the most abundant protein in the nucleus of oocytes which can promote in vitro nucleosome formation. In this study, to improve the efficiency of SCNT in Banna Mini-pig Inbred Line (BMI), we obtained the recombinant nucleoplasmin (Npm) by using a prokaryotic expression system and compared the difference on the developmental effects between exogenous Npm and its structural analog, polyglutamic acid (PGA). We chose the pMD18-T vector for Npm expression and transformed E. coli BL21 (DE3) cells to construct a prokaryotic expression system. The results showed that the recombinant Npm protein of 32 kDa could be obtained. Recombinant Npm or PGA was injected into porcine oocytes at different concentrations. The results showed that the blastocyst rates in injection 560 ng/μL Npm and 1000 ng/μL PGA groups were higher than those of other groups. Moreover, the blastocyst rate was highest in injection 560 ng/μL Npm group and was lowest in injection 100 ng/μL PGA group. These data suggested that addition of recombinant Npm obtained by prokaryotic expression or PGA could improve the development of SCNT embryos in the BMI. [ABSTRACT FROM AUTHOR]

Maternal effect genes (MEGs) are expressed in oocytes and embryos and play an important role in activation of the embryonic genome. An abnormality in the expression of these genes may lead to arrest of embryonic cleavage or to altered transcription of factors responsible for further embryonic development. In vitro –produced porcine embryos have a lower developmental potential than embryos produced in vivo . We hypothesized that in vitro embryo culture conditions have an effect on the expression of MEGs at various developmental stages, which may affect their developmental potential. Here, using real-time polymerase chain reaction, we examined mRNA profiles of the MEGs, zygote arrest 1 ( ZAR-1 ), nucleoplasmin 2 ( NPM2 ), and developmentally associated pluripotency protein 3 ( DPPA3 ), in porcine oocytes and embryos produced in vitro and in vivo . Further, we evaluated the effect of the combined addition of EGF, interleukin 1β, and leukemia inhibitory factor to the porcine in vitro embryo production system on mRNA profiles of selected MEGs. Finally, we studied localization of the MEG protein products in in vitro –obtained oocytes and embryos using confocal microscopy. We found that the ZAR-1 mRNA profile differed throughout in vitro and in vivo embryo development. In the embryos produced in vitro , the decrease in ZAR-1 mRNA levels was observed at the 2-cell stage, whereas in in vivo embryos, ZAR-1 mRNA levels declined significantly starting at the 4-cell stage (P < 0.05). In vitro culture conditions affected transiently also DPPA3 mRNA levels at the 4-cell stage (P < 0.05). There was no difference in the NPM2 mRNA profile during in vitro and in vivo embryo development. The ZAR-1 and DPPA3 proteins were localized in the cytoplasm of the oocytes and embryos, whereas the NPM2 protein was found both in the cytoplasm and in the nucleus. All proteins were expressed until blastocyst stage. The addition of EGF and cytokines to the culture medium decreased DPPA3 mRNA levels in 8-cell embryos (P < 0.05). This study indicated that IVC conditions affect ZAR-1 mRNA levels before the 4-cell stage, which may disturb the activation of the embryonic genome in pigs. The expression of the proteins after the 4-cell to 8-cell transition indicates that these factors play a role beyond activation of the embryonic genome. Supplementation of the culture media with EGF and cytokines affects DPPA3 mRNA levels after maternal to embryonic transition. [ABSTRACT FROM AUTHOR]

Ribosomes and protein synthesis have been reported to be altered in the cerebral cortex at advanced stages of Alzheimer's disease (AD). Modifications in the hippocampus with disease progression have not been assessed. Sixty-seven cases including middle-aged (MA) and AD stages I-VI were analyzed. Nucleolar chaperones nucleolin, nucleophosmin and nucleoplasmin 3, and upstream binding transcription factor RNA polymerase I gene ( UBTF) mRNAs are abnormally regulated and their protein levels reduced in AD. Histone modifications dimethylated histone H3K9 (H3K9me2) and acetylated histone H3K12 (H3K12ac) are decreased in CA1. Nuclear tau declines in CA1 and dentate gyrus (DG), and practically disappears in neurons with neurofibrillary tangles. Subunit 28 ribosomal RNA (28S rRNA) expression is altered in CA1 and DG in AD. Several genes encoding ribosomal proteins are abnormally regulated and protein levels of translation initiation factors eIF2α, eIF3η and eIF5, and elongation factor eEF2, are altered in the CA1 region in AD. These findings show alterations in the protein synthesis machinery in AD involving the nucleolus, nucleus and ribosomes in the hippocampus in AD some of them starting at first stages (I-II) preceding neuron loss. These changes may lie behind reduced numbers of dendritic branches and reduced synapses of CA1 and DG neurons which cause hippocampal atrophy. [ABSTRACT FROM AUTHOR]

Molecular chaperones, also known as heat-shock proteins, refold misfolded proteins and help other proteins reach their native conformation. Thanks to these abilities, some chaperones, such as the Hsp90 protein or the chaperonin GroEL, can buffer the deleterious phenotypic effects of mutations that alter protein structure and function. Hsp70 chaperones use a chaperoning mechanism different from that of Hsp90 and GroEL, and it is not known whether they can also buffer mutations. Here, we show that they can. To this end, we performed a mutation accumulation experiment in Escherichia coli, followed by whole-genome resequencing. Overexpression of the Hsp70 chaperone DnaK helps cells cope with mutational load and completely avoid the extinctions we observe in lineages evolving without chaperone overproduction. Additionally, our sequence data show that DnaK overexpression increases mutational robustness, the tolerance of its clients to nonsynonymous nucleotide substitutions. We also show that this elevated mutational buffering translates into differences in evolutionary rates on intermediate and long evolutionary time scales. Specifically, we studied the evolutionary rates of DnaK clients using the genomes of E. coli, Salmonella enterica, and 83 other gamma-proteo-bacteria. We find that clients that interact strongly with DnaK evolve faster than weakly interacting clients. Our results imply that all three major chaperone classes can buffer mutations and affect protein evolution. They illustrate how an individual protein like a chaperone can have a disproportionate effect on the evolution of a proteome. [ABSTRACT FROM AUTHOR]

1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK- dnaJ- grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool. [ABSTRACT FROM AUTHOR]

Alpha solenoid proteins play a key role in regulating the classical nuclear import pathway, recognizing a target protein and transporting it into the nucleus. Importin-α (Impα) is the solenoid responsible for cargo protein recognition, and it has been extensively studied by X-ray crystallography to understand the binding specificity. To comprehend the main motions of Impα and to extend the information about the critical interactions during carrier-cargo recognition, we surveyed different conformational states based on molecular dynamics (MD) and normal mode (NM) analyses. Our model of study was a crystallographic structure of Impα complexed with the classical nuclear localization sequence (cNLS) from nucleoplasmin (Npl), which was submitted to multiple 100 ns of MD simulations. Representative conformations were selected for calculating the 87 lowest frequencies NMs of vibration, and a displacement approach was applied along each NM. Based on geometric criteria, using the radius of curvature and inter-repeat angles as the reference metrics, the main motions of Impα were described. Moreover, we determined the salt bridges, hydrogen bonds and hydrophobic interactions in the Impα-NplNLS interface. Our results show the bending and twisting motions participating in the recognition of nuclear proteins, allowing the accommodation and adjustment of a classical bipartite NLS sequence. The essential contacts for the nuclear import were also described and were mostly in agreement with previous studies, suggesting that the residues in the cNLS linker region establish important contacts with Impα adjusting the cNLS backbone. The MD simulations combined with NM analysis can be applied to the Impα-NLS system to help understand interactions between Impα and cNLSs and the analysis of non-classic NLSs. [ABSTRACT FROM AUTHOR]

ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology withknownISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previouslyshownto play a role inEScontrol. In addition,weidentifytwo novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically foundonchromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are alsofound at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear toform a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution. [ABSTRACT FROM AUTHOR]

The aim of the present study, was to investigate the involvement of the endoplasmic reticulum (ER) in post-traumatic stress disorder (PTSD) by detecting changes of ER chaperone protein 78 and ER-resident caspase 12 in the basolateral amygdala after exposure to single prolonged stress (SPS). The established rat model of PTSD was generated by exposure of the animals to SPS. The expression of glucose-regulated protein 78 (GRP78) was examined by immunofluorescence, western blot and reverse transcription-polymerase chain reaction (RT-PCR), and the expression of caspase 12 was examined by western blot and RT-PCR. The morphological changes of the ER were detected by transmission electron microscopy. The results showed that GRP78 expression significantly increased when compared to that in the control group 1 day after SPS exposure (P<0.05). The expression of caspase 12 was also significantly upregulated after SPS exposure and peaked at 7 days following SPS (P<0.05). Morphological evaluation showed that a tumescent ER, ER vacuolization and degranulation of the ER were present following SPS. In conclusion, the findings of the present study suggested that SPS induced GRP78 and caspase 12 upregulation and morphological changes of the ER in the amygdala, which may play important roles in the pathogenesis of PTSD rats. [ABSTRACT FROM AUTHOR]

Thyroid cancer is one of the fastest growing cancer types worldwide. Using the cytokinesis-block micronucleus (CBMN) and comet assays, we performed a case-control study of 23 untreated papillary thyroid cancer (PTC) patients and 23 healthy volunteers. PTC patients showed higher basal DNA damage in peripheral blood lymphocytes. The CBMN assay indicated that the numbers of micronuclei, nuclear buds, and nucleoplasmic bridges among the cases were 2.67-, 2.79-, and 7.72-fold higher, respectively, than among the controls ( p < 0.05). Comet assay tail lengths and tail intensities were 1.20- and 1.94-fold higher, respectively ( p < 0.05). In additional, 14 thyroid tissues from PTC patients were probed for Raf-B and Ret expression; all samples were positive for at least one of these proteins. [ABSTRACT FROM AUTHOR]

TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo pro-teolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by Tl,R7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA. [ABSTRACT FROM AUTHOR]

Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals. [ABSTRACT FROM AUTHOR]

The nucleoplasmin family of histone chaperones is a key player in governing the dynamic architecture of chromatin, thereby regulating various DNA-templated processes. The crystal structure of the N-terminal domain of Arabidopsis thaliana FKBP43 (AtFKBP43), an FK506-binding immunophilin protein, revealed a characteristic nucleoplasmin fold, thus confirming it to be a member of the FKBP nucleoplasmin class. Small-Angle X-ray Scattering (SAXS) analyses confirmed its pentameric nature in solution, and additional studies confirmed the nucleoplasmin fold to be highly stable. Unlike its homolog AtFKBP53, the AtFKBP43 nucleoplasmin core domain could not interact with histones and required the acidic arms, C-terminal to the core, for histone association. However, SAXS generated low-resolution envelope structure, ITC, and AUC results revealed that an AtFKBP43 pentamer with C-terminal extensions interacts with H2A/H2B dimer and H3/H4 tetramer in an equimolar ratio, like AtFKBP53. Put together, AtFKBP43 belongs to a hitherto unreported subclass of FKBP nucleoplasmins that requires the C-terminal acidic stretches emanating from the core domain for histone interaction. • The crystal structure of AtFKBP43 NTD revealed a characteristic nucleoplasmin fold with a pentameric organization. • The N-terminal nucleoplasmin core domain of AtFKBP43 could not interact with histone oligomers. • AtFKBP43 requires the acidic arms, C-terminal to the nucleoplasmin core, for histone association. • AtFKBP43 pentamer with C-terminal extensions interacts with H2A/H2B dimer and H3/H4 tetramer in a 1:1 ratio. [ABSTRACT FROM AUTHOR]

Sperm chromatin compaction is physiologically essential for sperm to acquire the fertility. However, this unique structure composed of protamines makes us unable to solubilize the chromatin due to its resistance to sonication and enzymes usually used for chromatin fragmentation in somatic cells. Even when intense enzymatic treatment is applied, it appears to solubilize only certain portions of sperm chromatin presumably because of the heterogeneous properties. To overcome this issue, we previously developed a method to treat the sperm with recombinant nucleoplasmin, a protamine remover in fertilized embryos, followed by sonication. The nucleoplasmin treatment dramatically increased the efficiency of sperm chromatin solubilization, while a relatively large amount of recombinant nucleoplasmin was required. Here, we describe an improvement of nucleoplasmin method with a less amount of recombinant protein and a shorter reaction time., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lamins are nuclear intermediate filament (IF) proteins. They assemble to fibrous structures that are positioned between the inner nuclear membrane and the peripheral chromatin. A small fraction of lamins is also present in the nucleoplasm. Lamins are required to maintain the nuclear structure and, together with their associated proteins, are involved in most nuclear activities. Mutations in lamins cause >14 distinct diseases, called laminopathies, that include heart, muscle, fat and early aging diseases. However, it is not clear how lamins are organized in vivo and how the disease mutations affect lamin organization and functions. Here, we will review structural aspects of lamin assembly, discuss differences between peripheral and nucleoplasmic lamins and describe the protein complexes that lamins form. [ABSTRACT FROM AUTHOR]

Glucose-regulated protein 94 (GRP94) has been implicated in the promotion of tumor proliferation and metastasis. Previous studies have found that GRP94 is involved in the malignant growth of gastric carcinoma cells through estrogen receptor-α36 (ER-α36)-mediated estrogen signaling, but the underlying mechanism remains unclear. In the present study, we examined the expression levels of GRP94 and ER-α36 in tumor specimens from gastric cancer patients by immunohistochemistry, and found that both GRP94 and ER-α36 were highly expressed in the cyto plasms of gastric carcinoma cells. Furthermore, treatment with 17β-estradiol at a concentration of 10-12 M for 24 h increased the expression levels of GRP94 and ER-α36, and the phosphorylation levels of Akt at the Ser473 site (Ser473-Akt). In established SGC7901 gastric cancer cells with knockdown of ER-α36 expression, the levels of GRP94 and Ser473-Akt expression were significantly reduced. Thus, the Akt signaling pathway is a potentially important signaling pathway in ER-α36-GRP94-mediated gastric carcinogenesis. [ABSTRACT FROM AUTHOR]

Background--A hallmark of heart failure is impaired cytoplasmic Ca2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca2+ handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. Methods and Results--Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca2+ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca2+ transporters. These changes were associated with altered nucleoplasmic Ca2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca2+ levels with diminished and prolonged nuclear Ca2+ transients and slowed intranuclear Ca2+ diffusion. Altered nucleoplasmic Ca2+ levels were translated to higher activation of nuclear Ca2+ calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca2+ changes that are typical of heart failure. Conclusions--During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca2+ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling. [ABSTRACT FROM AUTHOR]

Direct reprogramming of a differentiated somatic cell into a developmentally more plastic cell would offer an alternative to applications in regenerative medicine that currently depend on either embryonic stem cells (ESCs), adult stem cells, or induced pluripotent stem cells (iPSCs). Here we report the potential of select Xenopus laevis egg extract fractions, in combination with exogenous fibroblast growth factor-2 (FGF2), to affect life span, morphology, gene expression, protein translation, and cellular localization of OCT4 and NANOG transcription factors, and the developmental potential of human dermal fibroblasts in vitro. A gradual change in morphology is accompanied by translation of embryonic transcription factors and their nuclear localization and a life span exceeding 60 population doublings. Cells acquire the ability to follow adipogenic, neuronal, and osteogenic differentiation under appropriate induction conditions in vitro. Analysis of active extract fractions reveals that Xenopus egg protein and RNAs as well as exogenously supplemented FGF2 are required and sufficient for induction and maintenance of this phenotypic change. Factors so far identified in the active fractions include FGF2 itself, transforming growth factor-β, maskin, and nucleoplasmin. Identification of critical factors needed for reprogramming may allow for nonviral, chemically defined derivation of human-induced multipotent cells that can be maintained by exogenous FGF2. [ABSTRACT FROM AUTHOR]

Abstract: To model the regulation of gene expression in eukaryotes by transcriptional activators and repressors, we introduce delays in conjugation with the mass action law. Delays are associated with the time gap between the mRNA transcription in the nucleoplasm and the protein synthesis in the cytoplasm. After re-parameterisation of the m-repressilator model with the Hill cooperative parameter n, for n=1, the m-repressilator is deducible from the mass action law and, in the limit , it is a Boolean type model. With this embedding and with delays, if m is odd and , we show that there is always a choice of parameters for which the m-repressilator model has sustained oscillations (limit cycles), implying that the 1-repressilator is the simplest genetic mechanism leading to sustained oscillations in eukaryotes. If m is even and , there is always a choice of parameters for which the m-repressilator model has bistability. [ABSTRACT FROM AUTHOR]

Few studies have shown that the yields of ionising-radiation-induced nucleoplasmic bridges (NPBs) in human cells are dose dependent. However, a dose–response curve between the NPB frequency and the absorbed dose of ionising radiation has not yet been established. This study aimed to investigate NPB frequencies in human peripheral blood lymphocytes induced by cobalt-60 (60Co) γ-rays and to establish a dose–response curve. Human peripheral blood samples were collected from three healthy males, and some of these samples were irradiated with 0–6 Gy 60Co γ-rays. A cytokinesis-block micronucleus cytome assay was then carried out to analyse NPBs and micronuclei (MN) in binucleated cells. The remaining blood samples were irradiated with 0, 2 and 5 Gy of γ-rays, and unstable chromosome aberrations (dicentric chromosome, ring chromosome and acentric chromosome fragment) were analysed. The correlation between NPBs and dicentric plus ring chromosome (dic+r) induced by the same γ-ray dose was also analysed. Results showed that the NPB yields among the three subjects at each dose level were not significantly different. NPBs in binucleated cells at all γ-ray doses conformed to Poisson distribution. The dose–response curve of the γ-ray-induced NPB frequencies followed the linear-quadratic model y = (1.39×10−3)x2 + (4.94×10−3)x. A positive correlation was observed between the frequencies of NPB and dic+r, as well as between the frequencies of MN and acentric fragments. Therefore, NPB is an important biomarker of early chromosome damage event induced by ionising radiation. [ABSTRACT FROM AUTHOR]

Lamins are intermediate filament proteins of type V constituting a nuclear lamina or filamentous meshwork which lines the nucleoplasmic side of the inner nuclear membrane. This protein mesh provides a supporting scaffold for the nuclear envelope and tethers interphase chromosome to the nuclear periphery. Mutations of mainly A-type lamins are found to be causative for at least 11 human diseases collectively termed as laminopathies majority of which are characterised by aberrant nuclei with altered structural rigidity, deformability and poor mechanotransduction behaviour. But the investigation of viscoelastic behavior of lamin A continues to elude the field. In order to address this problem, we hereby present the very first report on viscoelastic properties of wild type human lamin A and some of its mutants linked with Dilated cardiomyopathy (DCM) using quantitative rheological measurements. We observed a dramatic strain-softening effect on lamin A network as an outcome of the strain amplitude sweep measurements which could arise from the large compliance of the quasi-cross-links in the network or that of the lamin A rods. In addition, the drastic stiffening of the differential elastic moduli on superposition of rotational and oscillatory shear stress reflect the increase in the stiffness of the laterally associated lamin A rods. These findings present a preliminary insight into distinct biomechanical properties of wild type lamin A protein and its mutants which in turn revealed interesting differences. [ABSTRACT FROM AUTHOR]

Background Information A Polycomb (PcG) body is an orphan nuclear subcompartment characterised by accumulations of Polycomb repressive complex 1 (PRC1) proteins. However, seemingly contradictory reports have appeared that describe the PcG bodies either as protein-based bodies in the interchromatin compartment or chromatin domains. In this respect, molecular crowding is an important factor for the assembly and stability of nuclear subcompartments. In order to settle this contradiction, crowding experiments, that represent a convenient model distinguishing between interchromatin and chromatin compartments, were carried out. Results In sucrose-hypertonically induced crowding, we observed in U-2 OS cells that PcG bodies disappeared, but persisted as nuclear domains characterised by accumulations of DNA. This phenomenon was also observed in cells hypertonically treated with sorbitol and NaCl. Importantly, the observed changes were quickly reversible after re-incubation of cells in normal medium. We found that the PcG foci disappearance and the dissociation of PRC1 proteins (BMI1 and RING1a proteins) from chromatin were associated with their hyper-phosphorylation. In addition, under hyper- and hypotonic conditions, the behaviour of the PcG bodies differed from that of the typical nucleoplasmic body. Conclusion PRC1 proteins accumulations do not represent a genuine nuclear subcompartment. The PcG body is a chromosomal domain, rather than a nucleoplasmic body. [ABSTRACT FROM AUTHOR]

Interphase prenucleolar bodies are globular bodies which accumulate in large numbers in the nucleoplasm of cultivated cells after hypotonic treatment and subsequent return to isotonic conditions; detailed studies of the role of these structures in the recovery of the nucleolus have not yet been performed. The limited mobility of interphase pronucleoli within the nucleus has been demonstrated. Exchange of the major nucleolar protein B23 between prenucleolar bodies and the surrounding nucleoplasm, rather than stable binding of this protein to the prenucleolar bodies, has been demonstrated using fluorescence recovery after photobleaching method. Gradual accumulation of B23 in the recovering nucleolus with concomitant disappearance of prenucleolar bodies has been demonstrated. [ABSTRACT FROM AUTHOR]

Abstract: Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca2+ signaling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei. Furthermore, in both cardiac myocytes and aortic endothelial cells, endocytosed ET:ETB complexes translocated to lysosomes and not the nuclear envelope. Although ETA and ETB can form heterodimers, the presence or absence of ETA did not alter ETB trafficking. Treatment of isolated nuclei with peptide: N-glycosidase F did not alter the electrophoretic mobility of ETB. The absence of N-glycosylation further indicates that these receptors did not originate at the cell surface. Intracellular photolysis of a caged ET-1 analog ([Trp-ODMNB21]ET-1) evoked an increase in nucleoplasmic Ca2+ ([Ca2+]n) that was attenuated by inositol 1,4,5-trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and prevented by pre-treatment with ryanodine. A caged cell-permeable analog of the ETB-selective antagonist IRL-2500 blocked the ability of intracellular cET-1 to increase [Ca2+]n whereas extracellular application of ETA and ETB receptor antagonists did not. These data suggest that 1) the endothelin receptor in the cardiac nuclear membranes is ETB, 2) ETB traffics directly to the nuclear membrane after biosynthesis, 3) exogenous endothelins are not ligands for ETB on nuclear membranes, and 4) ETB associated with the nuclear membranes regulates nuclear Ca2+ signaling. [Copyright &y& Elsevier]

Nucleophosmin (NPM), a ubiquitously and abundantly expressed protein, occurs in the nucleolus, shuttling between the nucleoplasm and cytoplasm. The NPM gene is mutated in almost 30% of human acute myeloid leukemia cells. NPM interacts with p53 and p19Arf, directs localization of p19Arf in the nucleolus and protects the latter from degradation. Hepatocyte odd protein shuttling (HOPS) is also a ubiquitously expressed protein that moves between the nucleus and cytoplasm. Within the nucleus of resting cells, HOPS overexpression causes cell cycle arrest in G0/G1. HOPS knockdown causes centrosome hyperamplification leading to multinucleated cells and the formation of micronuclei. We demonstrate a direct interaction of HOPS with NPM and p19Arf, resulting in a functionally active trimeric complex. NPM appeared to regulate HOPS half-life, which, in turn, stabilized p19Arf and controlled its localization in the nucleolus. These findings suggest that HOPS acts as a functional bridge in the interaction between NPM and p19Arf, providing new mechanistic insight into how NPM and p19Arf will oppose tumor cell proliferation. [ABSTRACT FROM AUTHOR]

Micronuclei have been used extensively in studies as an easily evaluated indicator of DNA damage but little is known about their association with other types of damage such as nucleoplasmic bridges and nuclear buds. Here, radiation-induced clastogenic events were evaluated via the cytokinesis-block micronucleus assay in two normal human lymphoblastoid cell lines exposed to neutrons or γ-radiation. DNA damage induced by the chemical agents mitomycin C and phleomycin was also evaluated in two normal and two mitochondrial mutant human lymphoblastoid cell lines. In addition to micronuclei, nucleoplasmic bridges and nuclear buds were enumerated by recording the coincident presence of these end points within individual cells, and the associations among these three end points were evaluated for all treatment conditions. The common odds ratios for micronuclei and nucleoplasmic bridges were found to be significantly larger than unity, indicating that the presence of one or more micronuclei in a cell imposes a significant risk of having one or more nucleoplasmic bridges in that same cell, and vice versa. The strength of this association did not change significantly with radiation dose or concentration of the chemical clastogens. Common odds ratios for association between micronuclei and buds, and between bridges and buds were also found to be significantly higher than unity. However, associations between micronuclei and buds could not be calculated for some treatments due to heterogeneity in the odds ratios and hence may depend on chemical clastogen concentration or radiation dose. This study provides evidence of how paired analyses among genetic end points in the cytokinesis-block micronucleus assay can provide information concerning abnormalities of cell division and possibly about structural chromosomal rearrangements induced by clastogens. [ABSTRACT FROM PUBLISHER]

Duplicated ribosomal protein (Rp) gene families often encode highly similar or identical proteins with redundant or unique roles. Eukaryotic-specific paralogues RpL22e and RpL22e-like-PA are structurally divergent within the N terminus and differentially expressed, suggesting tissue-specific functions. We previously identified RpL22e-like-PA as a testis Rp. Strikingly, RpL22e is detected in immunoblots at its expected molecular mass (m) of 33 kD and at increasing m of ~43- 55 kD, suggesting RpL22e post-translational modification (PTM). Numerous PTMs, including N-terminal SUMOylation, are predicted computationally. Based on S2 cell co-immunoprecipitations, bacterial-based SUMOylation assays and in vivo germline-specific RNAi depletion of SUMO, we conclude that RpL22e is a SUMO substrate. Testis-specific PTMs are evident, including a phosphorylated version of SUMOylated RpL22e identified by in vitro phosphatase experiments. In ribosomal profiles from S2 cells, only unconjugated RpL22e co-sediments with active ribosomes, supporting an extratranslational role for SUMOylated RpL22e. Ectopic expression of an RpL22e N-terminal deletion (lacking SUMO motifs) shows that truncated RpL22e co-sediments with polysomes, implying that RpL22e SUMOylation is dispensable for ribosome biogenesis and function. In mitotic germ cells, both paralogues localize within the cytoplasm and nucleolus. However, within meiotic cells, phase contrast microscopy and co-immunohistochemical analysis with nucleolar markers nucleostemin1 and fibrillarin reveals diffuse nucleoplasmic, but not nucleolar RpL22e localization that transitions to a punctate pattern as meiotic cells mature, suggesting an RpL22e role outside of translation. Germline-specific knockdown of SUMO shows that RpL22e nucleoplasmic distribution is sensitive to SUMO levels, as immunostaining becomes more dispersed. Overall, these data suggest distinct male germline roles for RpL22e and RpL22e-like-PA. [ABSTRACT FROM AUTHOR]

The passage of mRNA molecules from the site of synthesis, through the nucleoplasm and the nuclear pore, en route to the cytoplasm, might appear straightforward. Nonetheless, several decades of detailed examination of this pathway, from high resolution electron microscopy in fixed specimens, through the development of immuno-detection techniques and fluorescence toolkits, to the current era of live-cell imaging, show this to be an eventful journey. In addition to mRNAs, several species of noncoding RNAs travel and function in the nucleus, some being retained within throughout their lifetime. This review will highlight the nucleoplasmic paths taken by mRNAs and noncoding RNAs in eukaryotic cells with special focus on live-cell data and in concurrence with the biophysical nature of the nucleus. [ABSTRACT FROM AUTHOR]

Both the sequence of a promoter and the position of a gene in 3D nuclear space play crucial roles in gene regulation, but few studies address their inter-relationship. Using human and viral promoters on mini-chromosomes and RNA fluorescence in situ hybridization coupled to 'high-precision' localization, we show that promoters binding the same transcription factors and responding to the same signaling pathways tend to be co-transcribed in the same transcription factories. We go on to suggest how such spatial co-association might drive co-regulation of genes under the control of similar cis-elements. [ABSTRACT FROM AUTHOR]

Summary: Centromere clustering during interphase is a phenomenon known to occur in many different organisms and cell types, yet neither the factors involved nor their physiological relevance is well understood. Using Drosophila tissue culture cells and flies, we identified a network of proteins, including the nucleoplasmin-like protein (NLP), the insulator protein CTCF, and the nucleolus protein Modulo, to be essential for the positioning of centromeres. Artificial targeting further demonstrated that NLP and CTCF are sufficient for clustering, while Modulo serves as the anchor to the nucleolus. Centromere clustering was found to depend on centric chromatin rather than specific DNA sequences. Moreover, unclustering of centromeres results in the spatial destabilization of pericentric heterochromatin organization, leading to partial defects in the silencing of repetitive elements, defects during chromosome segregation, and genome instability. [ABSTRACT FROM AUTHOR]

A-type lamins are components of the nuclear lamina, a filamentous network of the nuclear envelope in metazoans that supports nuclear architecture. In addition, lamin A/C can also be found in the interior of the nucleus. This nucleoplasmic lamin pool is soluble in physiological buffer, depends on the presence of the lamin-binding protein, lamina-associated polypeptide 2a (LAP2α) and regulates cell cycle progression in tissue progenitor cells. ?K32 mutations in A-type lamins cause severe congenital muscle disease in humans and a muscle maturation defect in Lmna?K32/?K32 knock-in mice. Mutant ?K32 lamin A/C protein levels were reduced and all mutant lamin A/C was soluble and mislocalized to the nucleoplasm. To test the role of LAP2α in nucleoplasmic ?K32 lamin A/C regulation and functions, we deleted LAP2α in Lmna?K32/?K32 knock-in mice. In double mutant mice the Lmna?K32/?K32-linked muscle defect was unaffected. LAP2α interacted with mutant lamin A/C, but unlike wild-type lamin A/C, the intranuclear localization of ?K32 lamin A/C was not affected by loss of LAP2α. In contrast, loss of LAP2α in Lmna?K32/?K32 mice impaired the regulation of tissue progenitor cells as in lamin A/C wild-type animals. These data indicate that a LAP2α-independent assembly defect of ?K32 lamin A/C is the predominant cause of the mouse pathology, whereas the LAP2α-linked functions of nucleoplasmic lamin A/C in the regulation of tissue progenitor cells are not affected in Lmna?K32/?K32 mice. [ABSTRACT FROM AUTHOR]

Purpose: To study the possible effects of various diagnostic strategies and the relative contribution of various structures in order to determine the optimal diagnostic strategy in treating patients with noncompressive pain syndromes. Study design: Prospective, nonrandomized cohort study of 83 consecutive patients with noncompressive pain syndromes resistant to repeated courses of conservative treatment. The follow-up period was 18 months. Results: Nucleoplasty was effective in cases of discogenic pain; the consequences related to false positive results of the discography were significant. The most specific criterion was 80% pain relief after facet joint blocks, whereas 50% pain relief and any subjective pain relief were not associated with a significant increase in the success rate. A considerable rate of false negative results was associated with 80% pain relief, whereas 50% pain relief after facet joint blocks showed the optimal ratio of sensitivity and specificity. Facet joint pain was detected in 50.6% of cases (95% confidence interval 44.1%-66.3%), discogenic pain in 16.9% cases (95% confidence interval 9.5%-26.7%), and sacroiliac joint pain in 7.2% cases (95% confidence interval 2.7%-15%). It was impossible to differentiate the main source of pain in 25.3% of cases. Conclusion: It is rational to adjust the diagnostic algorithm to the probability of detecting a particular pain source and, in doing so, reduce the number of invasive diagnostic measures to evaluate a pain source. False positive results of diagnostic measures can negatively affect the overall efficacy of a particular technology; therefore, all reasons for the failure should be studied in order to reach an unbiased conclusion. In choosing diagnostic criteria, not only should the success rate of a particular technology be taken into consideration but also the rate of false negative results. Acceptable diagnostic criteria should be based on a rational balance of sensitivity and specificity. [ABSTRACT FROM AUTHOR]

Summary: Many organisms, including plants, use the circadian clock to measure the duration of day and night. Daily rhythms in the plant circadian system are generated by multiple interlocked transcriptional/translational loops and also by spatial regulations such as nuclear translocation. GIGANTEA (GI), one of the key clock components in Arabidopsis, makes distinctive nuclear bodies like other nuclear-localized circadian regulators. However, little is known about the dynamics or roles of GI subnuclear localization. Here, we characterize GI subnuclear compartmentalization and identify unexpected dynamic changes under diurnal conditions. We further identify EARLY FLOWERING 4 (ELF4) as a regulator of GI nuclear distribution through a physical interaction. ELF4 sequesters GI from the nucleoplasm, where GI binds the promoter of CONSTANS (CO), to discrete nuclear bodies. We suggest that the subnuclear compartmentalization of GI by ELF4 contributes to the regulation of photoperiodic flowering. [ABSTRACT FROM AUTHOR]

Abstract: We recently characterized a nuclear import pathway for β-dystroglycan; however, its nuclear role remains unknown. In this study, we demonstrate for the first time, the interaction of β-dystroglycan with distinct proteins from different nuclear compartments, including the nuclear envelope (NE) (emerin and lamins A/C and B1), splicing speckles (SC35), Cajal bodies (p80-coilin), and nucleoli (Nopp140). Electron microscopy analysis revealed that β-dystroglycan localized in the inner nuclear membrane, nucleoplasm, and nucleoli. Interestingly, downregulation of β-dystroglycan resulted in both mislocalization and decreased expression of emerin and lamin B1, but not lamin A/C, as well in disorganization of nucleoli, Cajal bodies, and splicing speckles with the concomitant decrease in the levels of Nopp140, and p80-coilin, but not SC35. Quantitative reverse transcription PCR and cycloheximide-mediated protein arrest assays revealed that β-dystroglycan deficiency did not change mRNA expression of NE proteins emerin and lamin B1 bud did alter their stability, accelerating protein turnover. Furthermore, knockdown of β-dystroglycan disrupted NE-mediated processes including nuclear morphology and centrosome-nucleus linkage, which provides evidence that β-dystroglycan association with NE proteins is biologically relevant. Unexpectedly, β-dystroglycan-depleted cells exhibited multiple centrosomes, a characteristic of cancerous cells. Overall, these findings imply that β-dystroglycan is a nuclear scaffolding protein involved in nuclear organization and NE structure and function, and that might be a contributor to the biogenesis of nuclear envelopathies. [Copyright &y& Elsevier]

UV damage activates cellular stress signaling pathways, causes DNA helix distortions and inhibits transcription by RNA polymerases I and II. In particular, the nucleolus, which is the site of RNA polymerase I transcription and ribosome biogenesis, disintegrates following UV damage. The disintegration is characterized by reorganization of the subnucleolar structures and change of localization of many nucleolar proteins. Here we have queried the basis of localization change of nucleophosmin (NPM), a nucleolar granular component protein, which is increasingly detected in the nucleoplasm following UV radiation. Using photobleaching experiments of NPM-fluorescent fusion protein in live human cells we show that NPM mobility increases after UV damage. However, we show that the increase in NPM nucleoplasmic abundance after UV is independent of UV-activated cellular stress and DNA damage signaling pathways. Unexpectedly, we find that proteasome activity affects NPM redistribution. NPM nucleolar expression was maintained when the UV-treated cells were exposed to proteasome inhibitors or when the expression of proteasome subunits was inhibited using RNAi. However, there was no evidence of increased NPM turnover in the UV damaged cells, or that ubiquitin or ubiquitin recycling affected NPM localization. These findings suggest that proteasome activity couples to nucleolar protein localizations in UV damage stress. [ABSTRACT FROM AUTHOR]

Abstract: Nucleolin is a multifunctional protein that carries several post-translational modifications. We characterized nucleolin acetylation and developed antibodies specific to nucleolin K88 acetylation. Using this antibody we show that nucleolin is acetylated in vivo and is not localized in the nucleoli, but instead is distributed throughout the nucleoplasm. Immunofluorescence studies indicate that acetylated nucleolin is co-localized with the splicing factor SC35 and partially with Y12. Acetylated nucleolin is expressed in all tested proliferating cell types. Our findings show that acetylation defines a new pool of nucleolin which support a role for nucleolin in the regulation of mRNA maturation and transcription by RNA polymerase II. Structured summary of protein interactions: SC35 physically interacts with Nucleolin by anti bait coimmunoprecipitation (View interaction) Nucleolin and SC35 colocalize by fluorescence microscopy (View interaction) [Copyright &y& Elsevier]

The nucleoplasmin family of histone chaperones is a key player in governing the dynamic architecture of chromatin, thereby regulating various DNA-templated processes. The crystal structure of the N-terminal domain of Arabidopsis thaliana FKBP43 (AtFKBP43), an FK506-binding immunophilin protein, revealed a characteristic nucleoplasmin fold, thus confirming it to be a member of the FKBP nucleoplasmin class. Small-Angle X-ray Scattering (SAXS) analyses confirmed its pentameric nature in solution, and additional studies confirmed the nucleoplasmin fold to be highly stable. Unlike its homolog AtFKBP53, the AtFKBP43 nucleoplasmin core domain could not interact with histones and required the acidic arms, C-terminal to the core, for histone association. However, SAXS generated low-resolution envelope structure, ITC, and AUC results revealed that an AtFKBP43 pentamer with C-terminal extensions interacts with H2A/H2B dimer and H3/H4 tetramer in an equimolar ratio, like AtFKBP53. Put together, AtFKBP43 belongs to a hitherto unreported subclass of FKBP nucleoplasmins that requires the C-terminal acidic stretches emanating from the core domain for histone interaction., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Increasing evidence shows that the spatial organization of transcription is an important epigenetic factor in eukaryotic gene regulation. The malaria parasite Plasmodium falciparum shows a remarkably complex pattern of gene expression during the erythrocytic cycle, paradoxically contrasting with the relatively low number of putative transcription factors encoded by its genome. The spatial organization of nuclear subcompartments has been correlated with the regulation of virulence genes. Here, we investigate the nuclear architecture of transcription during the asexual cycle of malaria parasites. As in mammals, transcription is organized into discrete nucleoplasmic sites in P. falciparum, but in a strikingly lower number of foci. An automated analysis of 3D images shows that the number and intensity of transcription sites vary significantly between rings and trophozoites, although the nuclear volume remains constant. Transcription sites are spatially reorganized during the asexual cycle, with a higher proportion of foci located in the outermost nuclear region in rings, whereas in trophozoites, foci are evenly distributed throughout the nucleoplasm. As in higher eukaryotes, transcription sites are predominantly found in areas of low chromatin density. Immunofluorescence analysis shows that transcription sites form an exclusive nuclear compartment, different from the compartments defined by the silenced or active chromatin markers. In conclusion, these data suggest that transcription is spatially contained in discrete foci that are developmentally regulated during the asexual cycle of malaria parasites and located in areas of low chromatin density. [ABSTRACT FROM AUTHOR]

Increasing evidence shows that the spatial organization of transcription is an important epigenetic factor in eukaryotic gene regulation. The malaria parasite Plasmodium falciparum shows a remarkably complex pattern of gene expression during the erythrocytic cycle, paradoxically contrasting with the relatively low number of putative transcription factors encoded by its genome. The spatial organization of nuclear subcompartments has been correlated with the regulation of virulence genes. Here, we investigate the nuclear architecture of transcription during the asexual cycle of malaria parasites. As in mammals, transcription is organized into discrete nucleoplasmic sites in P. falciparum, but in a strikingly lower number of foci. An automated analysis of 3D images shows that the number and intensity of transcription sites vary significantly between rings and trophozoites, although the nuclear volume remains constant. Transcription sites are spatially reorganized during the asexual cycle, with a higher proportion of foci located in the outermost nuclear region in rings, whereas in trophozoites, foci are evenly distributed throughout the nucleoplasm. As in higher eukaryotes, transcription sites are predominantly found in areas of low chromatin density. Immunofluorescence analysis shows that transcription sites form an exclusive nuclear compartment, different from the compartments defined by the silenced or active chromatin markers. In conclusion, these data suggest that transcription is spatially contained in discrete foci that are developmentally regulated during the asexual cycle of malaria parasites and located in areas of low chromatin density. [ABSTRACT FROM AUTHOR]

Abstract: Human ovarian cancer cells COC1 and COC1/DDP (cisplatin-resistant subline) were exposed to 6kV/cm nanosecond electric pulses (nsEP) with a pulse length of 8, 16 or 24ns. The potential in a subcellular unit was calculated using a multilayer dielectric spherical model, and area under the voltage–time curves (AUC) integrated with a lower limit of 0.2V. Cell viability was determined, and double-stand and total DNA breaks detected with the neutral and alkaline comet assays. nsEP evoked a higher voltage and AUC in nucleoplasm, and the levels in COC1 cells was just above those in COC1/DDP cells. Comets only appeared in the alkaline assay demonstrating single-stand DNA break. Fewer DNA break (16.51% vs. 35.13% at 24ns, p =0.0150) and more survival (22.42% vs. 13.19% at 24ns, p =0.0015) occurred in COC1/DDP cells despite an equal electric energy and almost equal cell sizes. 24-ns EP led to higher rates of cell-death and comet. The comet rate correlated with cell-death fraction in either cell line (r =0.5701, p =0.0135; r =0.5110, p =0.0302). There was no a correlation between the tail length, tail moment or Olive tail moment and cell-death rate. The data showed that response of chemosensitive cells differed from that of chemoresistant cells and DNA damage contributed to percent of cell death. [Copyright &y& Elsevier]

Background: PCV ORF2 capsid protein was predicted to contribute to the control of replication via an interaction between the Cap and Rep proteins in the nucleoplasm. We previously showed that the nuclear localization signal (NLS) on the capsid protein plays an accessory role in the replication of PCV in vitro. To further evaluate the in vivo characteristics of NLS-chimeric PCV DNA clones, BALB/C mice were inoculated intranasally and intraperitoneally with the DNA clones. Results: As expected, no gross lesions were detected during the study of the inoculated animals. The chimeric PCV12-, PCV1-NLS2- and PCV2-NLS1-inoculated animals had significantly fewer and less severe histopathological lesions in lymphoid tissues than the PCV2-inoculated animals (P < 0.05). PCV12 induced a specific antibody response against PCV2 ORF2 comparable to that induced by wild-type PCV2 but demonstrated a shorter period of viremia and much lower level of virus loads in sera than those in PCV2-inoculated mice. Remarkably, the PCV2-NLS1 and PCV1-NLS2 chimeras replicated in inoculated mice and induced specific antibody responses but failed to produce viral antigens in the lymph nodes or a detectable viremia. Conclusions: The chimeric PCV2-NLS1 and PCV1-NLS2 demonstrated a lower replication level as compared with wild type of PCV2 or PCV1 in vivo, suggesting that ORF2 NLSs played an accessory role in PCV replication. The chimeric PCV12 is a good candidate for vaccination against PCV2 infection. [ABSTRACT FROM AUTHOR]

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2-7 onto chromatin during late mitosis of the cell cycle. MCM2-7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G1 phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2-7 to facilitate the assembly of MCM2-7 onto chromatin at replication origins in late mitosis and G1 phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G1 phase cells. Thus, human And-1 facilitates loading of the MCM2-7 helicase onto chromatin during the assembly of pre-RC. [ABSTRACT FROM AUTHOR]

Nullbasic, a mutant of the HIV-1 Tat protein, has anti-HIV-1 activity through mechanisms that include inhibition of Rev function and redistribution of the HIV-1 Rev protein from the nucleolus to the nucleoplasm and cytoplasm. Here we investigate the mechanism of this effect for the first time, establishing that redistribution of Rev by Nullbasic is not due to direct interaction between the two proteins. Rather, Nullbasic affects subcellular localization of cellular proteins that regulate Rev trafficking. In particular, Nullbasic induced redistribution of exportin 1 (CRM1), nucleophosmin (B23) and nucleolin (C23) from the nucleolus to the nucleus when Rev was coexpressed, but never in its absence. Inhibition of the Rev:CRM1 interaction by leptomycin B or a non-interacting RevM10 mutant completely blocked redistribution of Rev by Nullbasic. Finally, Nullbasic did not inhibit importin b- or transportin 1-mediated nuclear import, suggesting that cytoplasmic accumulation of Rev was due to increased export by CRM1. Overall, our data support the conclusion that CRM1- dependent subcellular redistribution of Rev from the nucleolus by Nullbasic is not through general perturbation of either nuclear import or export. Rather, Nullbasic appears to interact with and disrupt specific components of a Rev trafficking complex required for its nucleocytoplasmic shuttling and, in particular, its nucleolar accumulation. [ABSTRACT FROM AUTHOR]