Intranuclear Localization Research Articles

Cyclophilins contribute to Stat3 signaling and survival of multiple myeloma cells

Signal transducer and activator of transcription 3 (Stat3) is the major mediator of interleukin-6 (IL-6) family cytokines. In addition, Stat3 is known to be involved in the pathophysiology of many malignancies. Here, we show that the cis-trans peptidyl-prolyl isomerase cyclophilin (Cyp) B specifically interacts with Stat3, whereas the highly related CypA does not. CypB knockdown inhibited the IL-6-induced transactivation potential but not the tyrosine phosphorylation of Stat3. Binding of CypB to Stat3 target promoters and alteration of the intranuclear localization of Stat3 on CypB depletion suggested a nuclear function of Stat3/CypB interaction. By contrast, CypA knockdown inhibited Stat3 IL-6-induced tyrosine phosphorylation and nuclear translocation. The Cyp inhibitor cyclosporine A (CsA) caused similar effects. However, Stat1 activation in response to IL-6 or interferon-gamma was not affected by Cyp silencing or CsA treatment. As a result, Cyp knockdown shifted IL-6 signaling to a Stat1-dominated pathway. Furthermore, Cyp depletion or treatment with CsA induced apoptosis in IL-6-dependent multiple myeloma cells, whereas an IL-6-independent line was not affected. Thus, Cyps support the anti-apoptotic action of Stat3. Taken together, CypA and CypB both play pivotal roles, yet at different signaling levels, for Stat3 activation and function. These data also suggest a novel mechanism of CsA action.

Properties of virion transactivator proteins encoded by primate cytomegaloviruses

BackgroundHuman cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in situations where the immune system is immature or compromised. HCMV immediate early (IE) gene expression is stimulated by the virion phosphoprotein pp71, encoded by open reading frame (ORF) UL82, and this transactivation activity is important for the efficient initiation of viral replication. It is currently recognized that pp71 acts to overcome cellular intrinsic defences that otherwise block viral IE gene expression, and that interactions of pp71 with the cell proteins Daxx and ATRX are important for this function. A further property of pp71 is the ability to enable prolonged gene expression from quiescent herpes simplex virus type 1 (HSV-1) genomes. Non-human primate cytomegaloviruses encode homologs of pp71, but there is currently no published information that addresses their effects on gene expression and modes of action.ResultsThe UL82 homolog encoded by simian cytomegalovirus (SCMV), strain Colburn, was identified and cloned. This ORF, named S82, was cloned into an HSV-1 vector, as were those from baboon, rhesus monkey and chimpanzee cytomegaloviruses. The use of an HSV-1 vector enabled expression of the UL82 homologs in a range of cell types, and permitted investigation of their abilities to direct prolonged gene expression from quiescent genomes. The results show that all UL82 homologs activate gene expression, and that neither host cell type nor promoter target sequence has major effects on these activities. Surprisingly, the UL82 proteins specified by non-human primate cytomegaloviruses, unlike pp71, did not direct long term expression from quiescent HSV-1 genomes. In addition, significant differences were observed in the intranuclear localization of the UL82 homologs, and in their effects on Daxx. Strikingly, S82 mediated the release of Daxx from nuclear domain 10 substructures much more rapidly than pp71 or the other proteins tested. All UL82 homologs stimulated the early release of ATRX from nuclear domain 10.ConclusionAll of the UL82 homolog proteins analysed activated gene expression, but surprising differences in other aspects of their properties were revealed. The results provide new information on early events in infection with cytomegaloviruses.

Inhibition of poly(ADP-ribose) polymerase activity affects its subcellular localization and DNA strand break rejoining.

Poly(ADP-ribose) polymerase (PARP) plays a crucial role in DNA repair. Modulation of its activity by stimulation or inhibition is considered as a potentially important strategy in clinical practice, especially to sensitize tumor cells to chemo- and radiotherapy through inhibition of DNA repair. Here we studied the effect of the three PARP inhibitors, 5-iodo-6-amino-benzopyrone (INH(2)BP), 1,5-isoquinolinediol (1,5-dihydroxyisoquinolinediol (1,5-IQD) and 8-hydroxy-2-methylquinazolin-4-[3H]one (NU1025), and for two of them the efficiency in slowing the rejoining of DNA strand breaks induced by H(2)O(2) was compared. Inhibition of PARP changed its intranuclear localization markedly; cells exposed to the inhibitor NU1025 showed a significant tendency to accumulate PARP in large foci, whereas in untreated cells its distribution was more uniform. The speed and efficiency of rejoining of H(2)O(2)-induced DNA strand breaks were lower in cells incubated with a PARP inhibitor, and the kinetics of rejoining were modulated in a different manner by each inhibitor. At a concentration of 100 microM the efficiency of the inhibitors could be ranked in the order NU1025 > IQD > INH(2)BP. The two first compounds were able to decrease the overall PARP activity below the level detected in control cells, while INH(2)BP showed up to 40% PARP activity after exposure to H(2)O(2).

Endogenous avidin biotin activity (EABA) in thyroid pathology: immunohistochemical study

BackgroundImmunohistochemical methods based on the high affinity of avidin and biotin (e.g. ABC, LSAB) are characterized by high sensitivity and are widely used for detection of immunologic reaction. However, a non-specific reaction, observed in frozen tissues and in paraffin-embedded material, increasing after heat induced epitope retrieval (HIER), and caused either by endogenous biotin or any another chemical compound with high affinity for avidin, may lead to diagnostic mistakes. The aim of our investigation is to study presence of endogenous avidin biotin activity (EABA) in thyrocytes originating from various thyroid pathological lesions (neoplastic and non-neoplastic).Materials and methodsThe immunohistochemical study was performed on paraffin-embedded specimens of surgically resected thyroid tissue from 97 patients with thyroid diseases: 65 patients with papillary carcinoma (PTC), 11 patients with nodular goiter in whom features of benign papillary hyperplasia were found, 9 with lymphocytic thyroiditis (LT), 8 with follicular adenoma, and 4 patients with follicular carcinoma. In PTC immunohistochemical study was performed both in primary tumors and in lymph node metastases. After HIER, incubation with streptavidin from LSAB+ (DakoCytomation) kit was done.ResultsStrong cytoplasmic EABA was observed in 56 of 65 (87.5%) PTC and in oxyphilic cells in 8 of 9 cases of LT. Significant correlation between EABA in primary PTC tumor and EABA in lymph node metastases was stated. Normal surrounding thyroid tissues showed absence or weak EABA. Aberrant intranuclear localization of biotin was noted in morules of cribriform-morular variant of PTC. No statistically significant correlation between patient's age, sex, metastases presence and EABA was observed.ConclusionAmong thyroid lesions, false positive reactions are highly probable in papillary thyroid carcinoma and in lymphocytic thyroiditis if immunohistochemical detection is used on systems containing (strept)avidin. The most probable reason is the high endogenous biotin content.

5-Lipoxygenase: mechanisms of regulation

5-lipoxygenase (5-LO) catalyzes two steps in biosynthesis of leukotrienes (LTs), a group of lipid mediators of inflammation derived from arachidonic acid (AA). LT antagonists are used in treatment of asthma; more recently a potential role also in atherosclerosis has raised considerable interest. Furthermore, possible effects of 5-LO metabolites in relation to tumorigenesis have emerged. Thus, an understanding of the biochemistry of this lipoxygenase has potential implications for treatment of various diseases.

Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity

Human cytomegalovirus encodes a number of phosphorylation-regulated proteins, including the autophosphorylating protein kinase pUL97 and the nuclear mRNA export factor pUL69. Recently, it was reported that the kinase inhibitor roscovitine induces an intranuclear aggregation of pUL69 in infected fibroblasts. Here, we demonstrate that pUL97-specific kinase inhibitors induce a similar pUL69 aggregation. Furthermore, a direct pUL69-pUL97 interaction was demonstrated by coimmunoprecipitation analyses. Deletion mapping identified the domains required for interaction in both proteins (1-140/478-532 in pUL69 and 231-336 in pUL97). Further analysis of the immunoprecipitates by in vitro kinase assays demonstrated the phosphorylation of pUL69 by pUL97. However, catalytically inactive mutants of pUL97 and interaction-negative fragments of pUL69 were phosphorylation-negative. Moreover, an analysis of the pUL69-mediated nuclear RNA export indicated a correlation of the export efficiency with the presence of active pUL97 kinase. These data suggest a specific pUL69-pUL97 interaction and pUL97-mediated phosphorylation which influences the regulatory activities of pUL69.

Comparison between arginine conjugated PAMAM dendrimers with structural diversity for gene delivery systems

Mono- and di-arginine conjugated PAMAM dendrimers (G = 3 or 4) were synthesized to examine the structure-activity relationships of arginine conjugation for gene delivery systems. Number of conjugated arginines was examined by 1H NMR (PAMAM3-R: 31, PAMAM4-R: 60, PAMAM3-R2: 59, PAMAM4-R2: 116). They could retard pDNA at a charge ratio of 2 and form polyplexes with sizes less than 250 nm from a charge ratio of 4. Di-arginine conjugated dendrimers showed higher Zeta-potential values and polyplex stabilities than mono-arginine conjugates. PAMAM3-R and PAMAM4-R showed low cytotoxicities even at high concentration but PAMAM3-R2 and PAMAM4-R2 exhibited significant cytotoxicities at high concentration. PAMAM3-R2 displayed greater transfection efficiency than PAMAM3-R, although the transfection efficiency of PAMAM4-R2 was not higher than that of PAMAM4-R in all condition. PAMAM3-R2 and PAMAM4-R2 polyplexes were observed to show the good intra-nuclear localization in comparison with PAMAM3-R and PAMAM4-R. It is concluded that di-arginine conjugation to PAMAM dendrimers can improve polyplex stability, intra-nuclear localization, and transfection efficiency but also induce charge density- and generation-dependent cytotoxicity. Therefore, a novel strategy for highly densed arginine conjugation maintaining low cytotoxicity will be needed for the development of efficient gene delivery carriers.

Novel 7-mer peptide mediates nuclear targeting in MCF7 breast cancer cells.

Abstract Abstract #6023 Nonviral gene therapy strives to eliminate cancer by correcting the molecular cause of the disease. Corrective DNA sequences, in the form of plasmids (pDNA), must localize to the cell nucleus to obtain transgene expression. This low probability event contributes to inefficient transgene expression following nonviral gene delivery. Previous studies using a bacteriophage (phage) display approach modified for the discovery of intracellular ligands yielded a panel of 7-mer peptides associated with the nuclei of MCF7 human mammary epithelial cells. The phage clone with the highest frequency of nuclear recovery displayed the QPSPSPT peptide. This study confirms the nuclear targeting capability of QPSPSPT and suggests the potential for QPSPSPT as a novel nonviral gene delivery ligand to modulate transgene expression in human breast cancer cells.
 The capacity for nuclear localization mediated by QPSPSPT was confirmed by image analysis of in vitro cell cultures exposed to fluorescently-labeled M13 phage displaying the heptapeptide. Control, wild-type M13 phage and phage displaying the QPSPSPT heptapeptide were labeled using an AlexaFluor 488 through conjugation to the pVIII coat proteins. Fluorescence microscopy revealed that 12.2% of MCF7 cell nuclei were associated with fluorescently labeled phage displaying the QPSPSPT peptide, but that wild-type M13 phage failed to localize to the cell nucleus.
 Quantitative analysis of confocal fluorescence micrographs enabled determination of intracellular and intranuclear phage localization. The number of phage per MCF7 nucleus was 3.3-fold greater for samples displaying the QPSPSPT peptide relative to the wild type M13 population. The increase in nuclear localization was mediated by both an increase in the amount of QPSPSPT-phage per cell (1.7-fold) and the intracellular translocation of QPSPSPT-phage to the nucleus (1.9-fold) as compared to the wild-type M13 phage. The equivalent spherical diameter of an M13 phage is approximately 40 nm, suggesting that the QPSPSPT heptapeptide has the capacity to mediate the cellular entry and nuclear localization of payloads consistent with the size of pDNA or formulated nonviral gene delivery vehicles.
 A commercial pDNA (pDualGC, Stratagene) was modified to enable the expression of fusion products consisting of a green fluorescent protein (GPF) bearing an N-terminus triple repeat of either the QPSPSPT or the positive control YSPTSPS. Transfection of these new constructs was performed independently in cultured MCF7 cells using Lipofectamine (Invitrogen). Six hours after transfection, the GFP-QPSPSPTx3 and GFP-YSPTSPSx3 fusion proteins appeared predominately in the cell nucleus in comparison with the cytosolic distribution of wild-type GFP. QPSPSPT has the capacity to preferentially localize proteins and phage to the cell nucleus, suggesting a potential role for this novel heptapeptide as a nonviral gene delivery adjuvant in breast cancer therapy.
 This work was funded by the BCRP-CDMRP under awards BC023276 and BC044010. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6023.

Granulosa cells express three inositol 1,4,5-trisphosphate receptor isoforms: cytoplasmic and nuclear Ca2+ mobilization

BackgroundGranulosa cells play an important endocrine role in folliculogenesis. They mobilize Ca2+ from intracellular stores by a coordinated action between 1,4,5 inositol trisphosphate and ryanodine receptors (IP3R and RyR). The aim of this study was to explore the isoforms of IP3Rs expressed in mouse C57BL/6 NHsd granulosa cells, characterizing their intranuclear localization and the relation with other Ca2+-handling proteins.MethodsOvarian tissue and granulosa cells were analyzed by multiphotonic and confocal microscopy to determine the intracellular presence of IP3R types 1, 2 and 3, RyR, thapsigargin-sensitive Ca2+-ATPase, and endomembranes. Cellular fractionation and Western blot assays were also used to further confirm the nuclear occurrence of the three IP3R isoforms. Free nuclear and cytosolic Ca2+ concentrations were measured using Fluo-4 AM by confocal microscopy.ResultsBy using antibodies and specific fluorophores, was shown that granulosa cells endomembranes contain three isoforms of IP3R, the RyR, and the thapsigargin-sensitive Ca2+-ATPase (SERCA). Interestingly, all these proteins were also detected in the nuclear envelope and in well-defined intranuclear structures. Microsomal membranes depicted characteristic bands of the 3 types of IP3R, but also variants of lower molecular weight. Analysis of nuclear membranes and nucleoplasmic fraction confirmed the nuclear localization of the IP3R types 1, 2 and 3. We demonstrated ATP-induced Ca2+ transients in the nuclear and cytoplasmic compartments. Remarkably, the inhibitory effect on ATP-induced Ca2+ mobilization of brefeldin A was more accentuated in the cytoplasm than in the nucleus.ConclusionThese findings provide evidence that granulosa cells, including nuclei, express the Ca2+-handling proteins that allow Ca2+ mobilization. All three IP3R were also detected in ovarian slices, including the nuclei of granulosa cells, suggesting that these cells use the three IP3R in situ to achieve their physiological responses.

Causal Role of Apoptosis-Inducing Factor for Neuronal Cell Death Following Traumatic Brain Injury

Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact; and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether apoptosis-inducing factor (AIF), a pro-apoptotic mitochondrial molecule and the key factor in the caspase-independent, cell death signaling pathway, plays a causal role in neuronal death following TBI. Using an in vitro model of neuronal stretch injury, we demonstrated that AIF translocated from mitochondria to the nucleus of neurons displaying axonal disruption, chromatin condensation, and nuclear pyknosis in a caspase-independent manner, whereas astrocytes remained unaffected. Similar findings were observed following experimental TBI in mice, where AIF translocation to the nucleus coincided with delayed neuronal cell death in both cortical and hippocampal neurons. Down-regulation of AIF in vitro by siRNA significantly reduced stretch-induced neuronal cell death by 67%, a finding corroborated in vivo using AIF-deficient harlequin mutant mice, where secondary contusion expansion was significantly reduced by 44%. Hence, our current findings demonstrate that caspase-independent, AIF-mediated signaling pathways significantly contribute to post-traumatic neuronal cell death and may therefore represent novel therapeutic targets for the treatment of TBI.

Matrix Metalloproteinase-13 is Activated and is found in the Nucleus of Neural Cells after Cerebral Ischemia

Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of ischemic stroke. In this study, we investigated the time course of gelatinolytic activation in a rat model of permanent ischemia. We observed an activation of MMPs as early as 30 mins after the ischemic insult, mainly in the nuclei of brain cells. Besides, we explored MMP-13 expression in brain samples of the animal model and stroke deceased patients. We observed an upregulation of active MMP-13 in rat brains (P<0.05) after 90 mins of cerebral ischemia. Human infarct/periinfarct samples also showed higher levels of active MMP-13 (P<0.05) compared with contralateral ones. Interestingly, we found that MMP-13 colocalized with 46-diamidino-2-phenyl indole signal by immunohistochemistry in both humans and rats, suggesting an intranuclear localization for MMP-13. Immunohistochemistry also revealed that MMP-13 was mainly produced by neurons, in both species, but also by oligodendrocytes in rats, and by astrocytes in humans. Finally we subjected a rat primary neuronal culture to oxygen and glucose deprivation (OGD) and we reproduced the nuclear translocation of MMP-13 in vitro. Nuclear extracts from cells confirmed upregulation of active MMP-13 after OGD (P<0.05). These results suggest that MMP-13 activation and its nuclear translocation is an early consequence of an ischemic stimulus.

Toxoplasmosis in Bennett’s wallabies ( Macropus rufogriseus) in Spain

Toxoplasmosis is one of the more common parasitic zoonoses world-wide. In this study, an epizootic of toxoplasmosis among captive Bennett’s wallabies ( Macropus rufogriseus) from different locations is reported. By means of light microscopy, Toxoplasma gondii-like tachyzoites were observed associated to interstitial pneumonia, non-suppurative myocarditis, cholangiohepatitis and severe gastroenteritis. The protozoa stained positively with a T. gondii antibody and ultrastructurally were similar to T. gondii. Strikingly, tachyzoites appeared sometimes in an intranuclear location within granulocyte-like cells. Feral cats or reactivation of a latent infection are discussed as the possible sources of infection. As far as we know, this is the first confirmed report of toxoplasmosis in Bennett’s wallabies in Spain and Europe, and may constitute a risk of infection for humans since new alimentary habits are being imposed in our countries.

Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization

Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.

Thematic Review Series: Sphingolipids. Nuclear sphingolipids: metabolism and signaling

Sphingolipids are most prominently expressed in the plasma membrane, but recent studies have pointed to important signaling and regulatory roles in the nucleus. The most abundant nuclear sphingolipid is sphingomyelin (SM), which occurs in the nuclear envelope (NE) as well as intranuclear sites. The major metabolic product of SM is ceramide, which is generated by nuclear sphingomyelinase and triggers apoptosis and other metabolic changes. Ceramide is further hydrolyzed to free fatty acid and sphingosine, the latter undergoing conversion to sphingosine phosphate by action of a specific nuclear kinase. Gangliosides are another type of sphingolipid found in the nucleus, members of the a-series of gangliotetraose gangliosides (GM1, GD1a) occurring in the NE and endonuclear compartments. GM1 in the inner membrane of the NE is tightly associated with a Na(+)/Ca(2+) exchanger whose activity it potentiates, thereby contributing to regulation of Ca(2+) homeostasis in the nucleus. This was shown to exert a cytoprotective role as absence or inactivation of this nuclear complex rendered cells vulnerable to apoptosis. This was demonstrated in the greatly enhanced kainite-induced seizure activity in knockout mice lacking gangliotetraose gangliosides. The pathology included apoptotic destruction of neurons in the CA3 region of the hippocampus. Ca(2+) homeostasis was restored in these animals with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger. Some evidence suggests the presence of uncharged glycosphingolipids in the nucleus.

Degradation of a Polyadenylated rRNA Maturation By-Product Involves One of the Three RRP6-Like Proteins in Arabidopsis thaliana

Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6Delta strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5' part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.

Interindividual differences and alterations in the topology of chromosomes in human sperm nuclei of fertile donors and carriers of reciprocal translocations

Recently it has been shown that the nucleus of the human spermatozoon appears to possess a specific architecture. The current prevailing view is that spatial organization of the male genome contains information critical for the spermatozoon's function as well as for early embryonic development. The purpose of this study was to determine whether there are alterations in intranuclear localization of centromeres in spermatozoa of chromosomes associated with particular reciprocal chromosome translocations (RCT). We analyzed the longitudinal and spatial localization of centromeres of selected chromosomes in sperm nuclei of four control males with normal karyotypes as well as in six carriers of reciprocal chromosome translocations: t(1;7), t(7;2), t(7;13), t(7;9), t(9;14), and t(4;13). Our study revealed that chromosomes with translocations may have shifted their intranuclear localization and that these translocations may influence the localization of other chromosomes in sperm nuclei. The chromocenter in sperm nuclei of translocation carriers was widened toward the apical side in comparison with chromocenter sites visible in control males. Our study also revealed interindividual differences in the localization of the Y chromosome centromere in the chromocenter area of sperm from fertile individuals.

Human galectin-2: nuclear presence in vitro and its modulation by quiescence/stress factors.

Galectins have the particular capacity to interact with distinct proteins, in addition to the typical reactivity of lectins with glycans. Therefore, they can be functionally active when residing at places other than the membrane or extracellular matrix. In fact, nuclear presence of galectins-1 and -3 is solidly documented but it is an open question whether these two cases are exceptional within this lectin family. Thus, galectin-2, which shares 43% sequence identity on the protein level with galectin-1, warrants study in this respect. Based on initial immunohistochemical evidence we herein address the issue as to whether this galectin can join the category of nuclear lectins. To do so we studied different types of cell in vitro using an antibody preparation free of cross-reactivity against other tested galectins. The immunocytochemical experiments revealed that galectin-2 was present in nuclei of murine 3T3 fibroblasts and also genetically engineered human colon carcinoma cells with stable ectopic expression. Transport of galectin-2 to the nucleus could be enhanced by physical (UV light), chemical (mitomycin C, serum withdrawal) or cell biological (coculture with stromal cells) treatment modalities. As a means of further characterizing the staining profile cytochemically, a series of markers with well-defined site of residency within the nuclear compartment was tested in parallel. Importantly, no colocalization with galectins-1 and -3 and the splicing factor SC35 was detectable, the former cases also serving as inherent specificity control. In contrast, a similarity was uncovered in the case of the promyelocytic leukemia (PML) protein as marker of PML nuclear bodies. In aggregate, nuclear localization is documented for galectin-2. This attribute should thus not be considered as an exceptional finding confined to galectins-1 and -3. That even closely related family members, here galectins-1 and -2, exhibit distinct intranuclear localization patterns gives ensuing research a clear direction.

Identification of a New Human Adenovirus Protein Encoded by a Novel Late l -Strand Transcription Unit

A short open reading frame named the "U exon," located on the adenovirus (Ad) l-strand (for leftward transcription) between the early E3 region and the fiber gene, is conserved in mastadenoviruses. We have observed that Ad5 mutants with large deletions in E3 that infringe on the U exon display a mild growth defect, as well as an aberrant Ad E2 DNA-binding protein (DBP) intranuclear localization pattern and an apparent failure to organize replication centers during late infection. Mutants in which the U exon DNA is reconstructed have a reversed phenotype. Chow et al. (L. T. Chow et al., J. Mol. Biol. 134:265-303, 1979) described mRNAs initiating in the region of the U exon and spliced to downstream sequences in the late DBP mRNA leader and the DBP-coding region. We have cloned this mRNA (as cDNA) from Ad5 late mRNA; the predicted protein is 217 amino acids, initiating in the U exon and continuing in frame in the DBP leader and in the DBP-coding region but in a different reading frame from DBP. Polyclonal and monoclonal antibodies generated against the predicted U exon protein (UXP) showed that UXP is approximately 24K in size by immunoblot and is a late protein. At 18 to 24 h postinfection, UXP is strongly associated with nucleoli and is found throughout the nucleus; later, UXP is associated with the periphery of replication centers, suggesting a function relevant to Ad DNA replication or RNA transcription. UXP is expressed by all four species C Ads. When expressed in transient transfections, UXP complements the aberrant DBP localization pattern of UXP-negative Ad5 mutants. Our data indicate that UXP is a previously unrecognized protein derived from a novel late l-strand transcription unit.

Functional Sequestration of Transcription Factor Activity by Repetitive DNA

Higher eukaryote genomes contain repetitive DNAs, often concentrated in transcriptionally inactive heterochromatin. Although repetitive DNAs are not typically considered as regulatory elements that directly affect transcription, they can contain binding sites for some transcription factors. Here, we demonstrate that binding of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) to the mouse major alpha-satellite repetitive DNA sequesters C/EBPalpha in the transcriptionally inert pericentromeric heterochromatin. We find that this sequestration reduces the transcriptional capacity of C/EBPalpha. Functional sequestration of C/EBPalpha was demonstrated by experimentally reducing C/EBPalpha binding to the major alpha-satellite DNA, which elevated the concentration of C/EBPalpha in the non-heterochromatic subcompartment of the cell nucleus. The reduction in C/EBPalpha binding to alpha-satellite DNA was induced by the co-expression of the transcription factor Pit-1, which removes C/EBPalpha from the heterochromatic compartment, and by the introduction of an altered-specificity mutation into C/EBPalpha that reduces binding to alpha-satellite DNA but permits normal binding to sites in some gene promoters. In both cases the loss of alpha-satellite DNA binding coincided with an elevation in the binding of C/EBPalpha to a promoter and an increased transcriptional output from that promoter. Thus, the binding of C/EBPalpha to this highly repetitive DNA reduced the amount of C/EBPalpha available for binding to and regulation of this promoter. The functional sequestration of some transcription factors through binding to repetitive DNAs may represent an underappreciated mechanism controlling transcription output.

Intranuclear localization and isoform-dependent translocation of 14-3-3 proteins in human brain with infarction

Immunolocalization of 14-3-3 proteins in human brains with infarction was investigated using isoform-specific antibodies. Neurons around acute or subacute ischemic foci exhibited an enhanced immunoreactivity for 14-3-3 proteins either in the cytoplasm (especially for its sigma isoform) or in the nucleus (especially for its beta isoform), and sometimes in both. 14-3-3-like immunoreactivity was evaluated in each neuron, which enabled us to identify into three patterns: intense cytoplasmic staining with or without nuclear staining; a predominant nuclear staining with weak cytoplasmic staining; and an exclusive nuclear staining without cytoplamic staining. Quantification of 1500 neurons in relation to the severity of ischemia estimated by the relative distance from ischemic foci clarified that nuclear immunoreactivity for 14-3-3 proteins was more frequent in neurons near the ischemic core. Although the cytoplasm of astrocytes was similarly positive for the sigma and the epsilon isoform, their nuclei were only immunopositive for the gamma isoform. In the cerebral white matter with ischemia, axonal swelling and some nuclei of oligodendrocytes were positive for the zeta isoform. Isoform-specific translocation of 14-3-3 proteins into nuclei is a cellular reaction to ischemic stress that may be related to survival of neurons and their protection against cell death.