Hyperpolarizing gases via dynamic nuclear polarization and sublimation.

A high throughput method was designed to produce hyperpolarized gases by combining low-temperature dynamic nuclear polarization with a sublimation procedure. It is illustrated by applications to 129Xe nuclear magnetic resonance in xenon gas, leading to a signal enhancement of 3 to 4 orders of magnitude compared to the room-temperature thermal equilibrium signal at 7.05 T.

FHY1 mediates nuclear import of the light-activated phytochrome A photoreceptor.

The phytochrome (phy) family of photoreceptors is of crucial importance throughout the life cycle of higher plants. Light-induced nuclear import is required for most phytochrome responses. Nuclear accumulation of phyA is dependent on two related proteins called FHY1 (Far-red elongated HYpocotyl 1) and FHL (FHY1 Like), with FHY1 playing the predominant function. The transcription of FHY1 and FHL are controlled by FHY3 (Far-red elongated HYpocotyl 3) and FAR1 (FAr-red impaired Response 1), a related pair of transcription factors, which thus indirectly control phyA nuclear accumulation. FHY1 and FHL preferentially interact with the light-activated form of phyA, but the mechanism by which they enable photoreceptor accumulation in the nucleus remains unsolved. Sequence comparison of numerous FHY1-related proteins indicates that only the NLS located at the N-terminus and the phyA-interaction domain located at the C-terminus are conserved. We demonstrate that these two parts of FHY1 are sufficient for FHY1 function. phyA nuclear accumulation is inhibited in the presence of high levels of FHY1 variants unable to enter the nucleus. Furthermore, nuclear accumulation of phyA becomes light- and FHY1-independent when an NLS sequence is fused to phyA, strongly suggesting that FHY1 mediates nuclear import of light-activated phyA. In accordance with this idea, FHY1 and FHY3 become functionally dispensable in seedlings expressing a constitutively nuclear version of phyA. Our data suggest that the mechanism uncovered in Arabidopsis is conserved in higher plants. Moreover, this mechanism allows us to propose a model explaining why phyA needs a specific nuclear import pathway.

The effect of in vitro heating on the distribution of nuclear matrix polypeptides in HeLa cells.

The in situ nuclear matrix was obtained from HeLa cells. After permeabilization with nonionic detergent, the resulting structures were incubated for 1 h at 37 degrees C to determine whether or not such an incubation might result in the redistribution of nuclear polypeptides which resisted extraction with buffers of high-ionic strength (1.6 M NaCl or 0.25 M (NH4)2SO4 as well as DNase I digestion. Using indirect immunofluorescence experiments and monoclonal antibodies we show that heating to 37 degrees C changes the distribution of a 160 kDa protein previously shown to be a component of the inner matrix network. On the other hand, a 125 kDa polypeptide was not affected at all by the incubation. Our results clearly indicate that the inclusion of a 37 degrees C incubation (for example during digestion with DNase I) in the protocol to obtain the in situ nuclear matrix can result in the formation of in vitro artifacts.

Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand.

In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.

Expression of prox1, lymphatic endothelial nuclear transcription factor, in Kaposiform hemangioendothelioma and tufted angioma.

Kaposiform hemangioendothelioma (KHE) and tufted angioma (TA) are rare tumors mainly occurring in early childhood. Our recent results showed that ectopic overexpression of human Prox1 gene, a lymphatic endothelial nuclear transcription factor, promoted an aggressive behavior in 2 murine models of KHE. This dramatic Prox1-induced phenotype prompted us to investigate immunohistochemical staining pattern of Prox1, podoplanin (D2-40), LYVE-1, and Prox1/CD34 as well as double immunofluorescent staining pattern of LYVE-1/CD31 in KHE and TA, compared with other pediatric vascular tumors. For this purpose, we examined 75 vascular lesions: KHE (n=18), TA (n=13), infantile hemangioma (n=13), pyogenic granuloma (n=18), and granulation tissue (n=13). Overall, KHE and TA shared an identical endothelial immunophenotype: the neoplastic spindle cells were Prox1, podoplanin, LYVE-1, CD31, and CD34, whereas endothelial cells within glomeruloid foci were Prox1, podoplanin, LYVE-1, CD31, and CD34. The lesional cells of all infantile hemangiomas and pyogenic granulomas were negative for Prox1 in the presence of positive internal control. These findings provide immunophenotypic evidence to support a preexisting notion that KHE and TA are closely related, if not identical. Overall, our results show, for the first time, that Prox1 is an immunohistochemical biomarker helpful in confirming the diagnosis of KHE/TA and in distinguishing it from infantile hemangioma and pyogenic granuloma.

Oxidants positively or negatively regulate nuclear factor kappaB in a context-dependent manner.

Redox-based mechanisms play critical roles in the regulation of multiple cellular functions. NF-kappaB, a master regulator of inflammation, is an inducible transcription factor generally considered to be redox-sensitive, but the modes of interactions between oxidant stress and NF-kappaB are incompletely defined. Here, we show that oxidants can either amplify or suppress NF-kappaB activation in vitro by interfering both with positive and negative signals in the NF-kappaB pathway. NF-kappaB activation was evaluated in lung A549 epithelial cells stimulated with tumor necrosis factor alpha (TNFalpha), either alone or in combination with various oxidant species, including hydrogen peroxide or peroxynitrite. Exposure to oxidants after TNFalpha stimulation produced a robust and long lasting hyperactivation of NF-kappaB by preventing resynthesis of the NF-kappaB inhibitor IkappaB, thereby abrogating the major negative feedback loop of NF-kappaB. This effect was related to continuous activation of inhibitor of kappaB kinase (IKK), due to persistent IKK phosphorylation consecutive to oxidant-mediated inactivation of protein phosphatase 2A. In contrast, exposure to oxidants before TNFalpha stimulation impaired IKK phosphorylation and activation, leading to complete prevention of NF-kappaB activation. Comparable effects were obtained when interleukin-1beta was used instead of TNFalpha as the NF-kappaB activator. This study demonstrates that the influence of oxidants on NF-kappaB is entirely context-dependent, and that the final outcome (activation versus inhibition) depends on a balanced inhibition of protein phosphatase 2A and IKK by oxidant species. Our findings provide a new conceptual framework to understand the role of oxidant stress during inflammatory processes.

Mitotic functions for SNAP45, a subunit of the small nuclear RNA-activating protein complex SNAPc.

The small nuclear RNA-activating protein complex SNAP(c) is required for transcription of small nuclear RNA genes and binds to a proximal sequence element in their promoters. SNAP(c) contains five types of subunits stably associated with each other. Here we show that one of these polypeptides, SNAP45, also known as PTF delta, localizes to centrosomes during parts of mitosis, as well as to the spindle midzone during anaphase and the mid-body during telophase. Consistent with localization to these mitotic structures, both down- and up-regulation of SNAP45 lead to a G(2)/M arrest with cells displaying abnormal mitotic structures. In contrast, down-regulation of SNAP190, another SNAP(c) subunit, leads to an accumulation of cells with a G(0)/G(1) DNA content. These results are consistent with the proposal that SNAP45 plays two roles in the cell, one as a subunit of the transcription factor SNAP(c) and another as a factor required for proper mitotic progression.

IB1, a JIP-1-related nuclear protein present in insulin-secreting cells.

JIP-1 is a cytoplasmic inhibitor of the c-Jun amino-terminal kinase activated pathway recently cloned from a mouse brain cDNA library. We report herein the expression cloning of a rat cDNA encoding a JIP-1-related nuclear protein from a pancreatic beta-cell cDNA library that we named IB1 for Islet-Brain 1. IB1 was isolated by its ability to bind to GTII, a cis-regulatory element of the GLUT2 promoter. The IB1 cDNA encodes a 714-amino acid protein, which differs from JIP-1 by the insertion of 47 amino acids in the carboxyl-terminal part of the protein. The remaining 667 amino acids are 97% identical to JIP-1. The 47-amino acid insertion contains a truncated phosphotyrosine interaction domain and a putative helix-loop-helix motif. Recombinant IB1 (amino acids 1-714 and 280-714) was shown to bind in vitro to GTII. Functionally IB1 transactivated the GLUT2 gene. IB1 was localized within the cytoplasm and the nucleus of insulin-secreting cells or COS-7 cells transfected with an expression vector encoding IB1. Using a heterologous GAL4 system, we localized an activation domain of IB1 within the first 280 amino acids of the protein. These data demonstrate that IB1 is a DNA-binding protein related to JIP-1, which is highly expressed in pancreatic beta-cells where it functions as a transactivator of the GLUT2 gene.

Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I.

Efficient initiation by the DNA polymerase of adenovirus type 2 requires nuclear factor I (NFI), a cellular sequence-specific transcription factor. Three functions of NFI--dimerization, DNA binding, and activation of DNA replication--are colocalized within the N-terminal portion of the protein. To define more precisely the role of NFI in viral DNA replication, a series of site-directed mutations within the N-terminal domain have been generated, thus allowing the separation of all three functions contained within this region. Impairment of the dimerization function prevents sequence-specific DNA binding and in turn abolishes the NFI-mediated activation of DNA replication. NFI DNA-binding activity, although necessary, is not sufficient to activate the initiation of adenovirus replication. A distinct class of NFI mutations that abolish the recruitment of the viral DNA polymerase to the origin also prevent the activation of replication. Thus, a direct interaction of NFI with the viral DNA polymerase complex is required to form a stable and active preinitiation complex on the origin and is responsible for the activation of replication by NFI.

Retention half times in the skeleton of plutonium and 90Sr from above-ground nuclear tests: a retrospective study of the Swiss population.

Plutonium and (90)Sr are considered to be among the most radiotoxic nuclides produced by the nuclear fission process. In spite of numerous studies on mammals and humans there is still no general agreement on the retention half time of both radionuclides in the skeleton in the general population. Here we determined plutonium and (90)Sr in human vertebrae in individuals deceased between 1960 and 2004 in Switzerland. Plutonium was measured by sensitive SF-ICP-MS techniques and (90)Sr by radiometric methods. We compared our results to the ones obtained for other environmental compartments to reveal the retention half time of NBT fallout (239)Pu and (90)Sr in trabecular bones of the Swiss population. Results show that plutonium has a retention half time of 40+/-14 years. In contrast (90)Sr has a shorter retention half time of 13.5+/-1.0 years. Moreover (90)Sr retention half time in vertebrae is shown to be linked to the retention half time in food and other environmental compartments. These findings demonstrate that the renewal of the vertebrae through calcium homeostatic control is faster for (90)Sr excretion than for plutonium excretion. The precise determination of the retention half time of plutonium in the skeleton will improve the biokinetic model of plutonium metabolism in humans.

Nuclear scaffold proteins are differently sensitive to stabilizing treatment by heat or Cu++.

The distribution of three nuclear scaffold proteins (of which one is a component of a particular class of nuclear bodies) has been studied in intact K562 human erythroleukemia cells, isolated nuclei, and nuclear scaffolds. Nuclear scaffolds were obtained by extraction with the ionic detergent lithium diidosalicylate (LIS), using nuclei prepared in the absence of divalent cations (metal-depleted nuclei) and stabilized either by a brief heat exposure (20 min at 37C or 42C) or by Cu++ ions at 0C. Proteins were visualized by in situ immunocytochemistry and confocal microscopy. Only a 160-kD nuclear scaffold protein was unaffected by all the stabilization procedures performed on isolated nuclei. However, LIS extraction and scaffold preparation procedures markedly modified the distribution of the polypeptide seen in intact cells, unless stabilization had been performed by Cu++. In isolated nuclei, only Cu++ treatment preserved the original distribution of the two other antigens (M(r), 125 and 126 kD), whereas in heat-stabilized nuclei we detected dramatic changes. In nuclear scaffolds reacted with antibodies to 125 and 126-kD proteins, the fluorescent pattern was always disarranged regardless of the stabilization procedure. These results, obtained with nuclei prepared in the absence of Mg+2 ions, indicate that heat treatment per se can induce changes in the distribution of nuclear proteins, at variance with previous suggestions. Nevertheless, each of the proteins we have studied behaves in a different way, possibly because of its specific association with the nuclear scaffold.

CD10 is inversely associated with nuclear factor-kappa B and predicts biochemical recurrence after radical prostatectomy.

INTRODUCTION: The cell surface endopeptidase CD10 (neutral endopeptidase) and nuclear factor-κB (NF-κB) have been independently associated with prostate cancer (PC) progression. We investigated the correlations between these two factors and their prognostic relevance in terms of biochemical (prostate-specific antigen, PSA) relapse after radical prostatectomy (RP) for localized PC. PATIENTS AND METHODS: The immunohistochemical expression of CD10 and NF-κB in samples from 70 patients who underwent RP for localized PC was correlated with the preoperative PSA level, Gleason score, pathological stage and time to PSA failure. RESULTS: CD10 expression was inversely associated with NF-κB expression (p < 0.001), stage (p = 0.03) and grade (p = 0.003), whereas NF-κB was directly related with stage (p = 0.006) and grade (p = 0.002). The median time to PSA failure was 56 months. CD10 and NF-κB were directly (p < 0.001) and inversely (p < 0.001) correlated with biochemical recurrence-free survival, respectively. CD10 expression (p = 0.022) and stage (p = 0.018) were independently associated with time to biochemical recurrence. CONCLUSION: Low CD10 expression is an adverse prognostic factor for biochemical relapse after RP in localized PC, which is also associated with high NF-κB expression. Decreased CD10 expression which would lead to increased neuropeptide signaling and NF-κB activity may be present in a subset of early PCs.

Nuclear and cytoplasmic triiodothyronine-binding sites in primary sensory neurons and Schwann cells: radioautographic study during development.

The effects of the thyroid hormones on target cells are mediated through nuclear T3 receptors. In the peripheral nervous system, nuclear T3 receptors were previously detected with the monoclonal antibody 2B3 mAb in all the primary sensory neurons throughout neuronal life and in peripheral glia at the perinatal period only (Eur. J. Neurosci. 5, 319, 1993). To determine whether these nuclear T3 receptors correspond to functional ones able to bind T3, cryostat sections and in vitro cell cultures of dorsal root ganglion (DRG) or sciatic nerve were incubated with 0.1 nM [125I]-labeled T3, either alone to visualize the total T3-binding sites or added with a 10(3) fold excess of unlabeled T3 to estimate the part due to the non-specific T3-binding. After glutaraldehyde fixation, radioautography showed that the specific T3-binding sites were largely prevalent. The T3-binding capacity of peripheral glia in DRG and sciatic nerve was restricted to the perinatal period in vivo and to Schwann cells cultured in vitro. In all the primary sensory neurons, specific T3-binding sites were disclosed in foetal as well as adult rats. The detection of the T3-binding sites in the nucleus indicated that the nuclear T3 receptors are functional. Moreover the concomitant presence of both T3-binding sites and T3 receptors alpha isoforms in the perikaryon of DRG neurons infers that: 1) [125I]-labeled T3 can be retained on the T3-binding 'E' domain of nascent alpha 1 isoform molecules newly-synthesized on the perikaryal ribosomes; 2) the alpha isoforms translocated to the nucleus are modified by posttranslational changes and finally recognized by 2B3 mAb as nuclear T3 receptor. In conclusion, the radioautographic visualization of the T3-binding sites in peripheral neurons and glia confirms that the nuclear T3 receptors are functional and contributes to clarify the discordant intracellular localization provided by the immunocytochemical detection of nuclear T3 receptors and T3 receptor alpha isoforms.

Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex.

The in vitro adenovirus (Ad) DNA replication system provides an assay to study the interaction of viral and host replication proteins with the DNA template in the formation of the preinitiation complex. This initiation system requires in addition to the origin DNA sequences 1) Ad DNA polymerase (Pol), 2) Ad preterminal protein (pTP), the covalent acceptor for protein-primed DNA replication, and 3) nuclear factor I (NFI), a host cell protein identical to the CCAAT box-binding transcription factor. The interactions of these proteins were studied by coimmunoprecipitation and Ad origin DNA binding assays. The Ad Pol can bind to origin sequences only in the presence of another protein which can be either pTP or NFI. While NFI alone can bind to its origin recognition sequence, pTP does not specifically recognize DNA unless Ad Pol is present. Thus, protein-protein interactions are necessary for the targetting of either Ad Pol or pTP to the preinitiation complex. DNA footprinting demonstrated that the Ad DNA site recognized by the pTP.Pol complex was within the first 18 bases at the end of the template which constitutes the minimal origin of replication. Mutagenesis studies have defined the Ad Pol interaction site on NFI between amino acids 68-150, which overlaps the DNA binding and replication activation domain of this factor. A putative zinc finger on the Ad Pol has been mutated to a product that fails to bind the Ad origin sequences but still interacts with pTP. These results indicate that both protein-protein and protein-DNA interactions mediate specific recognition of the replication origin by Ad DNA polymerase.