Promoter- and RNA polymerase II-dependent hsp-16 gene association with nuclear pores in Caenorhabditis elegans.

Some inducible yeast genes relocate to nuclear pores upon activation, but the general relevance of this phenomenon has remained largely unexplored. Here we show that the bidirectional hsp-16.2/41 promoter interacts with the nuclear pore complex upon activation by heat shock in the nematode Caenorhabditis elegans. Direct pore association was confirmed by both super-resolution microscopy and chromatin immunoprecipitation. The hsp-16.2 promoter was sufficient to mediate perinuclear positioning under basal level conditions of expression, both in integrated transgenes carrying from 1 to 74 copies of the promoter and in a single-copy genomic insertion. Perinuclear localization of the uninduced gene depended on promoter elements essential for induction and required the heat-shock transcription factor HSF-1, RNA polymerase II, and ENY-2, a factor that binds both SAGA and the THO/TREX mRNA export complex. After induction, colocalization with nuclear pores increased significantly at the promoter and along the coding sequence, dependent on the same promoter-associated factors, including active RNA polymerase II, and correlated with nascent transcripts.

Immunohistochemical localization of RANK, RANKL and OPG in healthy and arthritic canine elbow joints

OBJECTIVE: To determine if the receptor activator of nuclear factor-kappaB-receptor activator of nuclear factor-kappaB ligand-osteoprotegerin (RANK-RANKL-OPG) system is active in bone remodeling in dogs and, if so, whether differences in expression of these mediators occur in healthy and arthritic joints. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Fragmented processus coronoidei (n=20) were surgically removed from dogs with elbow arthritis and 5 corresponding healthy samples from dogs euthanatized for reasons other than elbow joint disease. METHODS: Bright-field immunohistochemistry and high-resolution fluorescence microscopy were used to investigate the distribution of RANK, RANKL, and OPG in healthy and arthritic joints. RESULTS: All 3 molecules were identified by immunostaining of canine bone tissue. In elbow dysplasia, the number of RANK-positive osteoclasts was increased. In their vicinity, cells expressing RANKL, a mediator of osteoclast activation, were abundant whereas the number of osteoblasts having the potential to limit osteoclastogenesis and bone resorption via OPG was few. CONCLUSIONS: The RANK-RANKL-OPG system is active in bone remodeling in dogs. In elbow dysplasia, a surplus of molecules promoting osteoclastogenesis was evident and is indicative of an imbalance between the mediators regulating bone resorption and bone formation. Both OPG and neutralizing antibodies against RANKL have the potential to counterbalance bone resorption. CLINICAL RELEVANCE: Therapeutic use of neutralizing antibodies against RANKL to inhibit osteoclast activation warrants further investigation.

Intracellular localization of the BCL-2 family member BOK and functional implications

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.

Microscopic analysis of chromatin localization and dynamics in C. elegans

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system.

The sequence of addition of IL-3 and IgE-dependent/IgE-independent stimuli regulates RANKL expression in human basophils

Background: Receptor Activator of Nuclear Factor kappaB Ligand (RANKL), a member of the TNF superfamily, contributes to the imbalance of bone resorption and immunoregulation in rheumatoid arthritis. In mice, collagen induced arthritis was exacerbated by IL-3 and anti-IgER antibodies, two mediators activating basophils that are known as effector cells of allergy. Interestingly, our unpublished microarray data revealed that IL-3 induces RANKL mRNA in human basophils. Here we further investigate under which conditions human basophils express surface and/or soluble RANKL. Methods: One part of purified human basophils was co-stimulated with IL-3 and either IgE-dependent or IgE-independent stimuli. The other part of purified basophils was first primed with IL-3 and subsequently triggered with IgE-dependent or IgE-independent stimuli. Expression of surface and soluble RANKL were detected by flow cytometry, ELISA and real-time PCR. Results: By flow cytometry we show that IL-3 induces de novo expression of surface RANKL on human basophils in a time and dose dependent manner. Co-stimulation of basophils with IL-3 and an IgE-dependent stimulus reduces IL-3-induced expression of surface RANKL in a dose dependent manner while IgE-independent stimuli have no effect. In contrast, both IgE-dependent and IgE-independent stimuli enhance expression of surface and soluble RANKL in basophils that were first primed with IL-3 and then triggered. Real-time PCR analysis shows that surface hRANKL1 and soluble hRANKL3 are induced by IL-3 and reduced by co-stimulation with IL-3 and an IgE-dependent stimulus and thus confirms our flow cytometry data. Conclusion: RANKL expression in human basophils is not only dependent on IL-3 and IgE-dependent/IgE-independent stimuli but also on the sequence of their addition to cell culture. Based on our data, we suggest that basophils might have previously unidentified functions in bone resorption or immunoregulation via RANKL.

Microfabric memory of vein quartz for strain localization in detachment faults: A case study on the Simplon fault zone

This manuscript deals with the adaptation of quartz-microfabrics to changing physical deformation conditions, and discusses their preservation potential during subsequent retrograde deformation. Using microstructural analysis, a sequence of recrystallization processes in quartz, ranging from Grain-Boundary Migration Recrystallization (GBM) over Subgrain-Rotation Recrystallization (SGR) to Bulging Nucleation (BLG) is detected for the Simplon fault zone (SFZ) from the low strain rim towards the internal high strain part of the large-scale shear zone. Based on: (i) the retrograde cooling path; (ii) estimates of deformation temperatures; and (iii) spatial variation of dynamic recrystallization processes and different microstructural characteristics, continuous strain localization with decreasing temperature is inferred. In contrast to the recrystallization microstructures, crystallographic preferred orientations (CPO) have a longer memory. CPO patterns indicative of prism and rhomb glide systems in mylonitic quartz veins, overprinted at low temperatures (�400 �C), suggest inheritance of a high-temperature deformation. In this way, microstructural, textural and geochemical analyses provide information for several million years of the deformation history. The reasons for such incomplete resetting of the rock texture is that strain localization is caused by change in effective viscosity contrasts related to temporal large- and small-scale temperature changes during the evolution of such a long-lived shear zone. The spatially resolved, quantitative investigation of quartz microfabrics and associated recrystallization processes therefore provide great potential for an improved understanding of the geodynamics of large-scale shear zones.

Investigation of IL-23 (p19, p40) and IL-23R identifies nuclear expression of IL-23 p19 as a favorable prognostic factor in colorectal cancer: a retrospective multicenter study of 675 patients.

IL-23 is a heterodimeric cytokine involved in inflammatory diseases; its role in cancer progression is controversial. Here we analyse the expression of IL-23 subunits (p40 and p19) and IL-23R in colorectal cancer with regard to disease progression, clinical-pathological and molecular aspects. Immunohistochemistry for IL-23p19, IL-23p40, IL-23R and CD8 was performed on a multi-punch tissue microarray of 195 colorectal cancers (cohort 1), matched normal tissue, adenoma and lymph node metastases. Results were compared with clinical-pathological features and CD8+ T-cell counts, then validated on two patient cohorts (cohort 2: n=341, cohort 3: n=139). Cytoplasmic/membranous expression of IL-23 (p19 and p40 subunits) and IL-23R, respectively were over-expressed in carcinomas versus adenomas and normal tissues (p<0.0001) but were reduced in lymph node metastases (p<0.0001). Nuclear IL-23p19 expression was observed in 23.1% and was associated with early TNM stage (p=0.0186), absence of venous (p=0.0124) and lymphatic invasion (p=0.01493), favorable survival (p=0.014) and absence of distant metastasis (p=0.0146; specificity: 100%). This unexpected cellular localization was confirmed by cell fractionation. The beneficial effect of nuclear IL-23p19 was restricted to tumours with CD8+ high counts. Results were validated on Cohorts 2/3. This multicenter study underlines the possible CD8(+)--dependency and beneficial effect of nuclear IL-23p19 on overall patient survival.

Evolutionary conservation of the U7 small nuclear ribonucleoprotein in Drosophila melanogaster.

The U7 snRNP involved in histone RNA 3' end processing is related to but biochemically distinct from spliceosomal snRNPs. In vertebrates, the Sm core structure assembling around the noncanonical Sm-binding sequence of U7 snRNA contains only five of the seven standard Sm proteins. The missing Sm D1 and D2 subunits are replaced by U7-specific Sm-like proteins Lsm10 and Lsm11, at least the latter of which is important for histone RNA processing. So far, it was unknown if this special U7 snRNP composition is conserved in invertebrates. Here we describe several putative invertebrate Lsm10 and Lsm11 orthologs that display low but clear sequence similarity to their vertebrate counterparts. Immunoprecipitation studies in Drosophila S2 cells indicate that the Drosophila Lsm10 and Lsm11 orthologs (dLsm10 and dLsm11) associate with each other and with Sm B, but not with Sm D1 and D2. Moreover, dLsm11 associates with the recently characterized Drosophila U7 snRNA and, indirectly, with histone H3 pre-mRNA. Furthermore, dLsm10 and dLsm11 can assemble into U7 snRNPs in mammalian cells. These experiments demonstrate a strong evolutionary conservation of the unique U7 snRNP composition, despite a high degree of primary sequence divergence of its constituents. Therefore, Drosophila appears to be a suitable system for further genetic studies of the cell biology of U7 snRNPs.

The Ste20-like kinase misshapen functions together with Bicaudal-D and dynein in driving nuclear migration in the developing drosophila eye.

Nuclear translocation, driven by the motility apparatus consisting of the cytoplasmic dynein motor and microtubules, is essential for cell migration during embryonic development. Bicaudal-D (Bic-D), an evolutionarily conserved dynein-interacting protein, is required for developmental control of nuclear migration in Drosophila. Nothing is known about the signaling events that coordinate the function of Bic-D and dynein during development. Here, we show that Misshapen (Msn), the fly homolog of the vertebrate Nck-interacting kinase is a component of a novel signaling pathway that regulates photoreceptor (R-cell) nuclear migration in the developing Drosophila compound eye. Msn, like Bic-D, is required for the apical migration of differentiating R-cell precursor nuclei. msn displays strong genetic interaction with Bic-D. Biochemical studies demonstrate that Msn increases the phosphorylation of Bic-D, which appears to be necessary for the apical accumulation of both Bic-D and dynein in developing R-cell precursor cells. We propose that Msn functions together with Bic-D to regulate the apical localization of dynein in generating directed nuclear migration within differentiating R-cell precursor cells.

Localization of Hidden Insulinomas with 68Ga-DOTA-Exendin-4 PET/CT: A Pilot Study.

UNLABELLED (111)In-DOTA-exendin-4 SPECT/CT has been shown to be highly efficient in the detection of insulinomas. We aimed at determining whether novel PET/CT imaging with [Nle(14),Lys(40)(Ahx-DOTA-(68)Ga)NH2]exendin-4 ((68)Ga-DOTA-exendin-4) is feasible and sensitive in detecting benign insulinomas. METHODS (68)Ga-DOTA-exendin-4 PET/CT and (111)In-DOTA-exendin-4 SPECT/CT were performed in a randomized cross-over order on 5 patients with endogenous hyperinsulinemic hypoglycemia. The gold standard for comparison was the histologic diagnosis after surgery. RESULTS In 4 patients histologic diagnosis confirmed a benign insulinoma, whereas one patient refused surgery despite a positive (68)Ga-DOTA-exendin-4 PET/CT scan. In 4 of 5 patients, previously performed conventional imaging (CT or MR imaging) was not able to localize the insulinoma. (68)Ga-DOTA-exendin-4 PET/CT correctly identified the insulinoma in 4 of 4 patients, whereas (111)In-DOTA-exendin-4 SPECT/CT correctly identified the insulinoma in only 2 of 4 patients. CONCLUSION These preliminary data suggest that the use of (68)Ga-DOTA-exendin-4 PET/CT in detecting hidden insulinomas is feasible.

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs.

In several forms of beta-thalassemia, mutations in the second intron of the beta-globin gene create aberrant 5' splice sites and activate a common cryptic 3' splice site upstream. As a result, the thalassemic beta-globin pre-mRNAs are spliced almost exclusively via the aberrant splice sites leading to a deficiency of correctly spliced beta-globin mRNA and, consequently, beta-globin. We have designed a series of vectors that express modified U7 snRNAs containing sequences antisense to either the aberrant 5' or 3' splice sites in the IVS2-705 thalassemic pre-mRNA. Transient expression of modified U7 snRNAs in a HeLa cell line stably expressing the IVS2-705 beta-globin gene restored up to 65% of correct splicing in a sequence-specific and dose-dependent manner. Cell lines that stably coexpressed IVS2-705 pre-mRNA and appropriately modified U7 snRNA exhibited up to 55% of permanent restoration of correct splicing and expression of full-length beta-globin protein. This novel approach provides a potential alternative to gene replacement therapies.

Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA

Three U7 RNA-related sequences were isolated from mouse genomic DNA libraries. Only one of the sequences completely matches the published mouse U7 RNA sequence, whereas the other two apparently represent pseudogenes. The matching sequence represents a functional gene, as it is expressed after microinjection into Xenopus laevis oocytes. Sequence variations of the conserved cis-acting 5' and 3' elements of U RNA genes may partly explain the low abundance of U7 RNA.

Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes.

In Xenopus oocytes in vitro transcribed mouse U7 RNA is assembled into small nuclear ribonucleoproteins (snRNPs) that are functional in histone RNA 3' processing. If the special Sm binding site of U7 (AAUUUGUCUAG, U7 Sm WT) is converted into the canonical Sm sequence derived from the major snRNAs (AAUUUUUGGAG, U7 Sm OPT) the RNA assembles into a particle which accumulates more efficiently in the nucleus, but which is non-functional. U7 RNA with a heavily mutated Sm binding site (AACGCGUCAUG, U7 Sm MUT) is deficient in nuclear accumulation and function. By UV cross-linking U7 Sm WT RNA can be linked to three proteins, i.e. the common snRNP proteins G and B/B' and an apparently U7-specific protein of 40 kDa. As a result of altering the Sm binding site, U7 Sm OPT RNA cannot be cross-linked to the 40 kDa protein and no cross-links are obtained with U7 Sm MUT RNA. The fact that the Sm site also interacts with at least one U7-specific protein is so far unique to U7 RNA and may provide an explanation for the atypical sequence of this site. All described RNA-protein interactions, including that with the 40 kDa protein, already occur in the cytoplasm. An additional cytoplasmic photoadduct obtained with U7 Sm WT and U7 Sm OPT, but not U7 Sm MUT, RNAs is indicative of a protein of 60-80 kDa. The m7G cap structure of U7 Sm WT and U7 Sm OPT RNA becomes hypermethylated. However, the 3mG cap enhances, but is not required for, nuclear accumulation. Finally, U7 Sm WT RNA is functional in histone RNA processing even when bearing an ApppG cap.

Structures of four human pseudogenes for U7 small nuclear RNA.

Four U7 RNA-related sequences were isolated from a human genomic DNA library. None of the sequences completely match the published human U7 RNA sequence and all of them contain features typical of reverse-transcribed pseudogenes.