The link between social anxiety and intimate loneliness is stronger for older adults than for younger adults.

OBJECTIVES: The current study focuses on the association between social anxiety (e.g. fear of social interactions or negative judgment by others) and intimate loneliness (lacking meaningful relationships, i.e. having low quantity/quality of intimate companionship) in older and younger adults. We assessed whether social anxiety, a factor which hampers intimacy, may be associated with intimate loneliness to a greater extent in older adults versus younger adults. METHOD: Measures of loneliness (Revised UCLA loneliness scale) and social anxiety (Leibowitz social anxiety scale) were obtained from 342 participants (220 younger adults, age = 19-40, and 122 older adults, age = 61-89). RESULTS: Age differences were evident for non-intimate types of loneliness but not for intimate loneliness. Further, older adults were less socially anxious. Critically, the strength of the social anxiety-intimate loneliness link was more robust among older adults. Effects remained significant after controlling for demographic and computer/social media variables. CONCLUSIONS: Older adults with high levels of social anxiety displayed greater intimate loneliness relative to younger adults. On a theoretical level, the results reveal that the pruning mechanism of investing more in closer and more rewarding relationships among older adults may be challenged under high social anxiety. The results suggest that older adults with higher intimate loneliness may benefit from interventions aimed at decreasing their social anxiety.

Targeting mitochondrial translation by inhibiting DDX3: a novel radiosensitization strategy for cancer treatment.

DDX3 is a DEAD box RNA helicase with oncogenic properties. RK-33 is developed as a small-molecule inhibitor of DDX3 and showed potent radiosensitizing activity in preclinical tumor models. This study aimed to assess DDX3 as a target in breast cancer and to elucidate how RK-33 exerts its anti-neoplastic effects. High DDX3 expression was present in 35% of breast cancer patient samples and correlated with markers of aggressiveness and shorter survival. With a quantitative proteomics approach, we identified proteins involved in the mitochondrial translation and respiratory electron transport pathways to be significantly downregulated after RK-33 or DDX3 knockdown. DDX3 localized to the mitochondria and DDX3 inhibition with RK-33 reduced mitochondrial translation. As a consequence, oxygen consumption rates and intracellular ATP concentrations decreased and reactive oxygen species (ROS) increased. RK-33 antagonized the increase in oxygen consumption and ATP production observed after exposure to ionizing radiation and reduced DNA repair. Overall, we conclude that DDX3 inhibition with RK-33 causes radiosensitization in breast cancer through inhibition of mitochondrial translation, which results in reduced oxidative phosphorylation capacity and increased ROS levels, culminating in a bioenergetic catastrophe.

Asynchronous replication and allelic exclusion in the immune system.

The development of mature B cells involves a series of molecular decisions which culminate in the expression of a single light-chain and heavy-chain antigen receptor on the cell surface. There are two alleles for each receptor locus, so the ultimate choice of one receptor type must involve a process of allelic exclusion. One way to do this is with a feedback mechanism that downregulates rearrangement after the generation of a productive receptor molecule, but recent work suggests that monoallelic epigenetic changes may also take place even before rearrangement. To better understand the basis for distinguishing between alleles, we have analysed DNA replication timing. Here we show that all of the B-cell-receptor loci (mu, kappa and lambda) and the TCRbeta locus replicate asynchronously. This pattern, which is established randomly in each cell early in development and maintained by cloning, represents an epigenetic mark for allelic exclusion, because it is almost always the early-replicating allele which is initially selected to undergo rearrangement in B cells. These results indicate that allelic exclusion in the immune system may be very similar to the process of X chromosome inactivation.

Synergy of SF1 and RAR in activation of Oct-3/4 promoter.

The Oct-3/4 transcription factor is expressed in the earliest stages of embryogenesis, and is thus likely to play an important role in regulation of initial decisions in development. For the first time, we have shown that SF1 and Oct-3/4 are co-expressed in embryonal carcinoma (EC) P19 cells, and their expression is down-regulated with very similar kinetics following retinoic acid (RA) induced differentiation of these cells, suggesting a functional relationship between the two. Previously, we have shown that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive regulators of transcription, such as RAR and RXR. In this study we have identified in the Oct-3/4 promoter two novel SF1-binding sites: SF1(a) and SF1(b). The proximal site, SF1(a), is located within the RAREoct, and the distal site, SF1(b), is located between nucleotide -193 and -209 of the Oct-3/4 promoter. Both sites contribute to activation of Oct-3/4 promoter in EC cells, with SF1(a) playing a more crucial role. SF1, and its isoforms ELP2 and ELP3 bind to both SF1 sites and activate the Oct-3/4 promoter. This activation depends on the presence of SF1 DNA-binding domain. Thus, Oct-3/4 is the first EC-specific gene reported that is regulated by SF1. Interestingly, SF1 and RAR form a novel complex on the RAREoct sequence that synergistically activate the Oct-3/4 promoter. Both RARE and SF1 cis regulatory elements, as well as the SF1 DNA-binding domain, are needed for this synergism. SF1 and Oct-3/4 transcription factors play a role in the same developmental regulatory cascade.

Allelic exclusion in B and T lymphopoiesis.

The plasticity of the immune system relies on stochastic, i.e. random, decisions as well as on controlled events. V(D)J rearrangement of antigen receptors on B and T cells are mediated through the action of compound elements containing enhancer sequences. These elements function in a developmentally stage-specific and a cell-type-specific manner to attract machineries that demethylate DNA, remodel chromatin structure, and induce V(D)J recombination on one allele preferentially.

Developmental regulation of immune system gene rearrangement.

Rearrangement of antigen receptors in the immune system is mediated through the action of complex enhancers which function in both a developmentally stage-specific and a cell-type-specific manner to demethylate DNA, open chromatin structure and tether the recombination machinery to one preferred allele at each locus.

The mutant plasmacytoma cell line S107 allows the identification of distinct pathways leading to NF-kappaB activation.

Studies on the mechanisms of inducible and constitutive activity of NF-kappaB transcription factors have been hampered by the lack of appropriate mutant cell lines. We have analyzed the defect in the murine S107 plasmacytoma cell line, which was previously found to lack both constitutive and inducible NF-kappaB activity. Our analysis shows that these cells bear a specific defect that interferes with NF-kappaB induction by many diverse stimuli, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, UV light, x-rays, and H2O2. This does not however represent a general signal transduction defect, because AP-1 transcription factors are readily induced by the same stimuli. Phosphatase inhibitors such as okadaic acid as well as calyculin A can efficiently induce NF-kappaB in S107 cells via a pathway apparently insensitive to the radical scavenger pyrrolidine dithiocarbamate. Furthermore, MEKK1 a protein kinase supposedly induced by some of the above stimuli, is also capable of activating NF-kappaB. Interestingly, both the potent physiological inducer of NF-kappaB TNFalpha as well as endoplasmic reticulum overload can induce NF-kappaB via a PDTC sensitive pathway. In all cases, DNA-binding NF-kappaB complexes are comprised predominantly of p50-RelA heterodimers, and NF-kappaB activation results in the induction of transiently transfected or resident reporter genes. In summary, these results suggest that the pathways for many NF-kappaB-inducing stimuli converge at a specific junction, and this pivotal step is mutated in the S107 cell line. Yet there are alternative routes bypassing this critical step that also lead to NF-kappaB induction. These routes utilized by tumor necrosis factor alpha and endoplasmic reticulum overload are still intact in this cell line.

DNA demethylation: turning genes on.

The regulation of eukaryotic gene expression is a complicated process involving the interaction of a large number of transacting factors with specific cis-regulatory elements. DNA methylation plays a role in this scheme by acting in cis to modulate protein-DNA interactions. Several lines of evidence indicate that methylation serves to silence transcription, mainly through indirect mechanisms involving the assembly of repressive nucleoprotein complexes. DNA demethylation is mostly an active enzymatic process, controlled by cis regulatory elements which provide binding sites for trans demethylation factors. In the immune system DNA methylation plays multiple roles, such as regulating both gene expression and gene rearrangement

Kappa chain monoallelic demethylation and the establishment of allelic exclusion.

Allelic exclusion in kappa light-chain synthesis is thought to result from a feedback mechanism by which the expression of a functional kappa light chain on the surface of the B cell leads to an intracellular signal that down-regulates the V(D)J recombinase, thus precluding rearrangement of the other allele. Whereas such a feedback mechanism clearly plays a role in the maintenance of allelic exclusion, here we provide evidence suggesting that the initial establishment of allelic exclusion involves differential availability of the two kappa alleles for rearrangement. Analysis of kappa+ B-cell populations and of individual kappa+ B cells that have rearranged only one allele demonstrates that in these cells, critical sites on the rearranged allele are unmethylated, whereas the nonrearranged allele remains methylated. This pattern is apparently generated by demethylation that is initiated at the small pre-B cell stage, on a single allele, in a process that occurs prior to rearrangement and requires the presence in cis of both the intronic and 3' kappa enhancers. Taken together with data demonstrating that undermethylation is required for rearrangement, these results indicate that demethylation may actually underly the process of allelic exclusion by directing the initial choice of a single kappa allele for rearrangement.

Emergence of obsessive--compulsive symptoms and tics during clozapine withdrawal.

Treatment with clozapine may be associated with the appearance of obsessive-compulsive (OC) symptoms in up to 10% of schizophrenic patients. We present the first report of the emergence of de novo OC symptoms during clozapine withdrawal in two schizophrenic patients, associated in one with Tourette's syndrome-like tics. Resumption of clozapine led to the complete disappearance of the OC symptoms and a substantial alleviation of the tics. It is suggested that an imbalance between the dopamine and serotonin systems may account for this complication.

Rex-1, a gene encoding a transcription factor expressed in the early embryo, is regulated via Oct-3/4 and Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an adjacent site.

The Rex-1 (Zfp-42) gene, which encodes an acidic zinc finger protein, is expressed at high levels in embryonic stem (ES) and F9 teratocarcinoma cells. Prior analysis identified an octamer motif in the Rex-1 promoter which is required for promoter activity in undifferentiated F9 cells and is involved in retinoic acid (RA)-associated reduction in expression. We show here that the Oct-3/4 transcription factor, but not Oct-1, can either activate or repress the Rex-1 promoter, depending on the cellular environment. Rex-1 repression is enhanced by E1A. The protein domain required for Oct-3/4 activation was mapped to amino acids 1 to 35, whereas the domain required for Oct-3/4 repression was mapped to amino acids 61 to 126, suggesting that the molecular mechanisms underlying transcriptional activation and repression differ. Like Oct-3/4, Oct-6 can also lower the expression of the Rex-1 promoter via the octamer site, and the amino-terminal portion of Oct-6 mediates this repression. In addition to the octamer motif, a novel positive regulatory element, located immediately 5' of the octamer motif, was identified in the Rex-1 promoter. Mutations in this element greatly reduce Rex-1 promoter activity in F9 cells. High levels of a binding protein(s), designated Rox-1, recognize this novel DNA element in F9 cells, and this binding activity is reduced following RA treatment. Taken together, these results indicate that the Rex-1 promoter is regulated by specific octamer family members in early embryonic cells and that a novel element also contributes to Rex-1 expression.

RelB is a key player for both kappa B-dependent transcription and demethylation in B cells.

NF-kappa B was originally identified as a B cell-specific nuclear factor binding to the intronic kappa-light-chain enhancer element. It is found constitutively in the nucleus of mature B and plasma cells. In other cell types including pre-B cells, NF-kappa B is sequestered in the cytoplasm but can be induced by a variety of stimuli. In contrast to essentially all other mature B cells and plasma cell lines, the S107 plasmacytoma cell line lacks both constitutive and inducible kappa B-binding activity. A molecular characterization of the defect in these S107 cells suggests that the primary defect lies in the signal transduction pathway leading to NF-kappa B induction. Ectopic expression of RelB after stable transfection of these cells restores constitutive nuclear kappa B-binding activity. Moreover, kappa B-dependent transcription is also restored. Finally we demonstrate, that in contrast to parental S107 cells, the stable RelB transfectants have also regained the ability to specifically demethylate a transfected immunoglobulin kappa-locus. These data suggest that RelB is critically involved in both B cell-specific transcription and demethylation directed by the intronic kappa-enhancer element.

A role for nuclear NF-kappaB in B-cell-specific demethylation of the Igkappa locus.

The immunoglobulin kappa gene is specifically demethylated during B-cell maturation in a process which utilizes discrete cis-acting modules such as the intronic kappa enhancer element and the matrix attachment region (MAR). While any MAR sequence is sufficient for this reaction, mutation analysis indicates that tissue specificity is mediated by kappaB binding sequences within the kappa intronic enhancer. The plasmacytoma cell line S107 lacks kappaB binding activity and fails to demethylate the kappa locus. However, B-cell specific demethylation is restored by the introduction of an active kappaB binding protein gene relB. This represents the first demonstration of a trans-acting factor involved in cell-type-specific demethylation, and suggests that the same protein-DNA recognition system used for transcription may also contribute to the earlier developmental events that bring about activation of the kappa locus.

Direct and indirect mechanisms of repression participate in suppression of T-cell-specific gene expression in T x L-cell hybrids.

Expression of tissue-specific genes can be altered upon fusion of mammalian cells of different types. To resolve the genetic basis of this phenomenon and to identify components of the regulatory circuits that are involved, we have established a series of somatic cell hybrids between mouse T cells and L cells. These hybrids have an unusual and interesting phenotype. Unlike many hybrid cells studied, in which the expression of an entire set of tissue-specific genes was coordinately extinguished, in our T x L-cell hybrids only two out of seven T-cell-restricted genes were completely extinguished, whereas the other genes were repressed to various degrees. These hybrids extinguish the production of TCR beta and Thy-1 mRNA, repress the expression of TCR alpha, GATA-3, TCF-1, and LEF-1 genes to different extents, exhibit small changes in the level of CD3-epsilon mRNA, and continue to express the fibroblast-specific fibronectin gene, and the ets-1 gene. In this study we have evaluated for the first time the molecular mechanisms that underlie the repression of TCR alpha and TCR beta chain genes in T x L-cell hybrids. We have shown that multiple repression mechanisms, both direct and indirect, contribute to TCR alpha and TCR beta suppression. Repression of the expression of these genes correlated not only with the downregulation of GATA-3, TCF-1, and LEF-1 transcription factor expression, and with a change in the chromatin structure, but more importantly, with the activation of the silencer activity. Our study provides evidence for the existence of at least two negatively regulating elements, located at the TCR alpha enhancer-containing fragment and at the silencer region, which are active in our hybrid cells. We have shown that there was no correlation between the levels of GATA-3, TCF-1, and LEF-1 expression versus the level of TCR alpha mRNA in the independent hybrids. In contrast, both the silencer activity and the ability of the TCR alpha enhancer to downregulate thymidine kinase (TK) promoter activity were found to be in an inverse correlation with the ability of the different hybrid cells to express TCR alpha mRNA.

Coordinate suppression of myeloma-specific genes and expression of fibroblast-specific genes in myeloma X fibroblast somatic cell hybrids.

In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of the differentiation-specific traits of the non-fibroblast parental cell. To explore the genetic basis of this phenomenon, we have studied a series of somatic cell hybrids between mouse myeloma and fibroblasts. All the hybrids were adherent having a fibroblast-like phenotype. Molecular analysis revealed that plasma cell specific genes like the productively rearranged Ig genes, the J chain gene and genes for the cell surface markers CD20 and PC1, were extinguished in the hybrids. In contrast, fibroblast specific genes like fibronectin, alpha 2(I) and III collagens, as well as the receptor for fibroblast growth factor (flg), were expressed. Extinction was not due to chromosomal loss or lack of the relevant genes. To learn about the mechanism(s) of this phenomenon we have looked for the presence of positive and negative transcription factors in our hybrids. Expression of the PU.1 transcription factor, a member of the Ets transcription factor family normally expressed in B cells and macrophages, was lost in the cell hybrids. Interestingly, we found that the B-cell-specific Oct-2 transcription factor was still expressed at somewhat variable levels in several of the hybrid cell lines. In contrast, expression of the recently identified octamer coactivator BOB.1/OBF.1 was extinguished in all cell hybrids. This supports a critical role of this transcriptional coactivator for B-cell-specific gene expression. In addition, the Id and HLH462 genes coding for proteins known to repress bHLH transcription factors by formation of heterodimers, were found to be expressed at increased levels in fibroblasts and in the hybrids, indicating that their increased levels might also contribute to the suppression of myeloma-specific genes. Our results show that in myeloma x fibroblast hybrids, the phenotype of the fibroblast is dominant. It is suggested that fibroblasts contain regulatory "master" genes that are responsible for activation of the fibroblast differentiation pathway and suppress differentiation programs of other cell types.

A dynamic balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and retinoic acid receptor:retinoid X receptor heterodimers regulates Oct-3/4 expression in embryonal carcinoma cells.

The Oct-3/4 transcription factor is a member of the POU family of transcription factors and, as such, probably plays a crucial role in mammalian embryogenesis and differentiation. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Similarly, Oct-3/4 is expressed in embryonal carcinoma (EC) cells and is repressed in retinoic acid (RA)-differentiated EC cells. Previously we have shown that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for negative regulators. Our present results demonstrate that in P19 and RA-treated P19 cells, the orphan receptors ARP-1/COUP-TFII and EAR-3/COUP-TFI repress Oct-3/4 promoter activity through the RAREoct site in a dose-dependent manner. While the N-terminal region of the ARP-1/COUP-TFII receptor is dispensable for this repression, the C-terminal domain harbors the silencing region. Interestingly, three different RA receptor:retinoid X receptor (RAR:RXR) heterodimers, RAR alpha:RXR alpha, RAR beta:RXR alpha, and RAR beta:RXR beta, specifically bind and activate Oct-3/4 promoter through the RAREoct site in a ligand-dependent manner. We have shown that antagonism between ARP-1/COUP-TFII or EAR-3/COUP-TFI and the RAR:RXR heterodimers and their intracellular balance modulate Oct-3/4 expression. Oct-3/4 transcriptional repression by the orphan receptors can be overcome by increasing amounts of RAR:RXR heterodimers. Conversely, activation of Oct-3/4 promoter by RAR:RXR heterodimers was completely abolished by EAR-3/COUP-TFI and by ARP-1/COUP-TFII. The orphan receptors bind the RAREoct site with a much higher affinity than the RAR:RXR heterodimers. This high binding affinity provides ARP-1/COUP-TFII and EAR-3/COUP-TFI with the ability to compete with and even displace RAR:RXR from the RAREoct site and subsequently to actively silence the Oct-3/4 promoter. We have shown that RA treatment of EC cells results in up-regulation of ARP-1/COUP-TFII and EAR-3/COUP-TFI expression. Most interestingly, in RA-treated EC cells, the kinetics of Oct-3/4 repression inversely correlates with the kinetics of ARP-1/COUP-TFII and EAR-3/COUP-TFI activation. These findings are in accordance with the suggestion that these orphan receptors participate in controlling a network of transcription factors, among which Oct-3/4 is included, which may establish the pattern of normal gene expression during development.

B cell-specific demethylation: a novel role for the intronic kappa chain enhancer sequence.

We studied the molecular mechanism of demethylation and its role in kappa chain gene regulation. Following transfection into B cell cultures, this gene undergoes regional demethylation in a process that is developmentally regulated in a lineage- and stage-specific manner. Although a germline V kappa promoter is not required for the demodification activity, a fragment containing the intronic kappa chain transcriptional enhancer and the nearby matrix attachment region is essential. In its natural location downstream to the J kappa 5 sequence, this element induces bidirectional demodification of plasmid constructs in a distance- and orientation-independent manner. When this enhancer is placed in an upstream position, however, the kappa gene remains modified and transcriptionally inactive, demonstrating that demethylation is required for kappa chain activation. These studies suggest that the kappa enhancer plays a dual role in regulating B cell differentiation by inducing demethylation and by promoting tissue-specific transcription.

Retinoic acid represses Oct-3/4 gene expression through several retinoic acid-responsive elements located in the promoter-enhancer region.

The Oct-3/4 gene product, which belongs to the POU family of transcription factors, is a good candidate for regulating initial differentiation decisions. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Retinoic acid (RA)-induced differentiation of embryonal carcinoma (EC) cells is accompanied by decreased expression of the Oct-3/4 gene. Previous findings show that sequences in the Oct-3/4 enhancer region (designated RARE1) are targets for RA-mediated repression (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Our present results demonstrate conclusively that the TATA-less Oct-3/4 promoter is also a target for RA-induced repression. We identified a novel cis element in the Oct-3/4 promoter harbors a putative Sp1 binding site and a RA-responsive element (designated RAREoct), which are juxtaposed to one another. Protein binding to the Sp1 site is independent of protein binding to the RAREoct sequence. Unlike the RARE1 situated in the Oct-3/4 enhancer which does not contain a typical RAR recognition site, the RAREoct identified in this study consists of three directly repeated motifs that exhibit extensive homology to RARE sequences located in RA-responsive genes. Moreover, the RAREoct shows different DNA-binding characteristics and DNase I footprint patterns with nuclear proteins isolated from undifferentiated versus RA-differentiated EC cells. This suggests that the RAREoct element binds different nuclear proteins in RA-treated and untreated EC cells which most probably belong to the RA receptor, retinoid X receptor, or orphan receptor families of transcription factors. Using site-directed mutagenesis, we show that the RAREoct contributes to the transcriptional activation of Oct-3/4 promoter in P19 cells and, most interestingly, mediates the RA-induced repression in RA-differentiated EC cells. Thus, the RAREoct element could be one of the points of integration of several signalling pathways influencing Oct-3/4 expression. In accordance with the suggestion that suppression of Oct-3/4 expression is a crucial step during embryogenesis, the Oct-3/4 upstream region contains multiple targets for RA-induced repression, probably to ensure accurate and prompt repression of Oct-3/4 expression. It is possible that these repressors are differentially used at specific stages of development in response to various signals.