Sequence analysis and expression of the attachment and fusion proteins of canine distemper virus wild-type strain A75/17.

The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

Etude de validation d'une version francophone du Q-Sort d'attachement de Waters et Deane = A validation study of the french adaptation of Waters and Deane's attachment Q-sort

Ce texte relate l'étude de validation d'une adaptation francophone du Q-Sort d'attachement de Waters et Deane (cette adaptation est présentée dans ce même numéro). Le Q-Sort rempli par les parents ne paraît pas tenir ses promesses de méthode alternative à la Situation étrange pour évaluer la qualité de la relation d'attachement. Cependant, lorsqu'il est rempli par un observateur extérieur, il semble mieux refléter la catégorisation des comportements dans la Situation étrange. Au travers du Q-Sort, les parents semblent décrire davantage le tempérament de l'enfant que la qualité de la relation d'attachement.

Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity.

Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor alpha. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARgamma but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARgamma in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.

Trauma and disorganized attachment in refugee children : integrating theories and exploring treatment options

Refugee families incur many different types of stressors in the course of the phases prior to flight, those of flight, and resettlement. Multiple and varied negative life events and traumas, such as those experienced by refugee families, may give rise to negative changes in attachment between children and their parents. However, such negative changes in attachment may be countered through the use of culturally appropriate counselling theories and their respective interventions. The integration of attachment theory with family systems, trauma systems, and cognitive behavioural theories and the use of cognitive behavioural caregiver support, filial therapy training, and play therapy interventions are discussed as a treamtent framework for promoting more positive and secure attachments between refugee children and their caregivers.

Cilengitide modulates attachment and viability of human glioma cells, but not sensitivity to irradiation or temozolomide in vitro.

Cilengitide is a cyclic peptide antagonist of integrins alphavbeta3 and alphavbeta5 that is currently being evaluated as a novel therapeutic agent for recurrent and newly diagnosed glioblastoma. Its mode of action is thought to be mainly antiangiogenic but may include direct effects on tumor cells, notably on attachment, migration, invasion, and viability. In this study we found that, at clinically relevant concentrations, cilengitide (1-100 microM) induces detachment in some but not all glioma cell lines, while the effect on cell viability is modest. Detachment induced by cilengitide could not be predicted by the level of expression of the cilengitide target molecules, alphavbeta3 and alphavbeta5, at the cell surface. Glioma cell death induced by cilengitide was associated with the generation of caspase activity, but caspase activity was not required for cell death since ectopic expression of cytokine response modifier (crm)-A or coexposure to the broad-spectrum caspase inhibitor zVAD-fmk was not protective. Moreover, forced expression of the antiapoptotic protein marker Bcl-X(L) or altering the p53 status did not modulate cilengitide-induced cell death. No consistent effects of cilengitide on glioma cell migration or invasiveness were observed in vitro. Preliminary clinical results indicate a preferential benefit from cilengitide added to temozolomide-based radiochemotherapy in patients with O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation. Accordingly, we also examined whether the MGMT status determines glioma cell responses to cilengitide alone or in combination with temozolomide. Neither ectopic expression of MGMT in MGMT-negative cells nor silencing the MGMT gene in MGMT-positive cells altered glioma cell responses to cilengitide alone or to cilengitide in combination with temozolomide. These data suggest that the beneficial clinical effects derived from cilengitide in vivo may arise from altered perfusion, which promotes temozolomide delivery to glioma cells.

Development of stable cell lines for production or regulated expression using matrix attachment regions.

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.

Another way of thinking about ADHD: the predictive role of early attachment deprivation in adolescents' level of symptoms.

PURPOSE: Attention deficit and hyperactivity disorder (ADHD) is one of the most frequent disorders in childhood and adolescence. Both neurocognitive and environmental factors have been related to ADHD. The current study contributes to the documentation of the predictive relation between early attachment deprivation and ADHD. METHOD: Data were collected from 641 adopted adolescents (53.2 % girls) aged 11-16 years in five countries, using the DSM oriented scale for ADHD of the Child Behavior Checklist (CBCL) (Achenbach and Rescorla, Manual for the ASEBA school-age forms and profiles. University of Vermont, Research Center for Children, Youth and Families, Burlington, 2001). The influence of attachment deprivation on ADHD symptoms was initially tested taking into consideration several key variables that have been reported as influencing ADHD at the adoptee level (age, gender, length of time in the adoptive family, parents' educational level and marital status), and at the level of the country of origin and country of adoption (poverty, quality of health services and values). The analyses were computed using the multilevel modeling technique. RESULTS: The results showed that an increase in the level of ADHD symptoms was predicted by the duration of exposure to early attachment deprivation, estimated from the age of adoption, after controlling for the influence of adoptee and country variables. The effect of the age of adoption was also demonstrated to be specific to the level of ADHD symptoms in comparison to both the externalizing and internalizing behavior scales of the CBCL. CONCLUSION: Deprivation of stable and sensitive care in infancy may have long-lasting consequences for children's development.

The Effect of Sexual Abuse on Children's Attachment Representations in Chile.

Child sexual abuse is associated with problems in children's emotional development, particularly increased insecurity of attachment. However, few studies have examined its effect on the organization of attachment representations in preschoolers, and the findings of those that have been conducted have not been entirely consistent. Therefore, this study aims to analyze the effect of child sexual abuse on attachment representation quality in a sample of children 3 to 7 years old in Chile. The results indicate child sexual abuse does affect children's attachment representation quality. The attachment narratives of child sexual abuse victims scored significantly higher than nonvictims on the hyperactivity and disorganization dimensions of attachment. These results are discussed in terms of attachment theory, clinical findings on child sexual abuse, and clinical implications.

Molecular characterization of a human matrix attachment region epigenetic regulator.

Matrix attachment regions (MAR) generally act as epigenetic regulatory sequences that increase gene expression, and they were proposed to partition chromosomes into loop-forming domains. However, their molecular mode of action remains poorly understood. Here, we assessed the possible contribution of the AT-rich core and adjacent transcription factor binding motifs to the transcription augmenting and anti-silencing effects of human MAR 1-68. Either flanking sequences together with the AT-rich core were required to obtain the full MAR effects. Shortened MAR derivatives retaining full MAR activity were constructed from combinations of the AT-rich sequence and multimerized transcription factor binding motifs, implying that both transcription factors and the AT-rich microsatellite sequence are required to mediate the MAR effect. Genomic analysis indicated that MAR AT-rich cores may be depleted of histones and enriched in RNA polymerase II, providing a molecular interpretation of their chromatin domain insulator and transcriptional augmentation activities.

Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells.

Gene transfer in eukaryotic cells and organisms suffers from epigenetic effects that result in low or unstable transgene expression and high clonal variability. Use of epigenetic regulators such as matrix attachment regions (MARs) is a promising approach to alleviate such unwanted effects. Dissection of a known MAR allowed the identification of sequence motifs that mediate elevated transgene expression. Bioinformatics analysis implied that these motifs adopt a curved DNA structure that positions nucleosomes and binds specific transcription factors. From these observations, we computed putative MARs from the human genome. Cloning of several predicted MARs indicated that they are much more potent than the previously known element, boosting the expression of recombinant proteins from cultured cells as well as mediating high and sustained expression in mice. Thus we computationally identified potent epigenetic regulators, opening new strategies toward high and stable transgene expression for research, therapeutic production or gene-based therapies.