A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammalian cell lines.

The tetracycline-dependent expression system has gained increasing popularity for the expression of any gene of interest. Careful choice of the expression vector has been suggested to exploit the full potential of this system. A novel tetracycline-sensitive expression vector based on a modified mouse mammary tumor virus promoter achieved considerably improved regulatory properties in a series of cell lines tested under transient and stable conditions. Therefore, the applicability of the tetracycline-dependent expression system can be largely enhanced by careful adaptation of the expression vector to the host cell line.     

Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland and the brain.

The proliferation rate of a cell population reflects a balance between cell division, cell cycle arrest, differentiation and apoptosis. The regulation of these processes is central to development and tissue homeostasis, whereas dysregulation may lead to overt pathological outcomes, notably cancer and neurodegenerative disorders. We report here the cloning of a novel zinc finger protein which regulates apoptosis and cell cycle arrest and was accordingly named Zac1. In vitro Zac1 inhibited proliferation of tumor cells, as evidenced by measuring colony formation, growth rate and cloning in soft agar. In vivo Zac1 abrogated tumor formation in nude mice. The antiproliferative activity of Zac1 was due to induction of extensive apoptosis and of G1 arrest, which proceeded independently of retinoblastoma protein and of regulation of p21(WAF1/Cip1), p27Kip1, p57Kip2 and p16INK4a expression. Zac1-mediated apoptosis was unrelated to cell cycle phase and G1 arrest was independent of apoptosis, indicating separate control of apoptosis and cell cycle arrest. Zac1 is thus the first gene besides p53 which concurrently induces apoptosis and cell cycle arrest.     

Alkaline fixation drastically improves the signal of in situ hybridization

In situ hybridization (ISH) is widely used to detect DNA and RNA sequences within the cell and tissue sections. The important step in performing this technique is tissue fixation. We investigated the influence of the pH of the fixative on the outcome of ISH. Our studies indicate that alkaline formaldehyde dramatically increases the ISH signal with RNA probes. The increase in signal was observed for detection of low as well as for high abundance messages. The sensitivity of the method was increased 5- to 6-fold.     

Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8

Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.     

Reconstitution of cyc- S49 membranes by in vitro translated Gs alpha. Membrane anchorage and functional implications

After ADP-ribosylation by cholera toxin which promotes dissociation of the subunits, the alpha-subunit of Gs (Gs alpha) remained strongly associated with plasma membranes of wild-type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH greater than or equal to 11.5. In vitro translated Gs alpha could interact with plasma membranes from the cyc- mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTP gamma S did not alter anchorage of Gs alpha. The characteristics of the association of in vitro translated Gs alpha with cyc- membranes after GTP gamma S treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP-ribosylated form in wild-type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc- membranes by in vitro translated alpha-subunit of Gs.     

Cultural Sensitive Care Provision in a Public Child and Adolescent Mental Health Centre: A Case Study from the Toulouse University Hospital Intercultural Consultation

Child and adolescent mental health services in Europe are confronted with children with increasingly diverse socio-cultural backgrounds. Clinicians encounter cultural environments of hyperdiversity in terms of languages and countries of origin, growing diversity within groups, and accelerated change with regards to social and administrational situations (Hannah, in: DelVecchio Good et al. (eds) Shattering culture: American medicine responds to cultural diversity, Russel Sage Foundation, New York, 2011). Children and families who live in these complex constellations face multiple vulnerabilizing factors related to overlapping or intersecting social identities (Crenshaw in Univ Chic Leg Forum 140:139–167, 1989). Mobilizing existing resources in terms of social and family support, and encouraging creative strategies of interculturation in therapeutic work (Denoux, in: Blomart and Krewer (eds) Perspectives de l’interculturel, L’Harmattan, Paris, 1994) may be helpful in order to enhance resilience. Drawing from experiences in the context of French transcultural and intercultural psychiatry, and inspired by the Mc Gill Cultural Consultation in Child Psychiatry, we developed an innovative model, the Intercultural Consultation Service (ICS). This consultation proposes short term interventions to children and families with complex migration experiences. It has been implemented into a local public health care structure in Toulouse, the Medical and Psychological Centre la Grave. The innovation includes the creation of a specific setting for short term therapeutic interventions and team training via shared case discussions. Our objectives are (a) to improve outcomes of mental health care for the children through a better understanding of the child’s family context (exploration of family dynamics and their relatedness to complex migration histories), (b) to enhance intercultural competencies in professionals via shared case discussions, and, (c) to improve the therapeutic relationship between children and professionals on the basis of the work with the family and the dialogue with the team. In our paper, we present the rationale and functioning of the ICS and illustrate our work with a case study. The presentation of the case uses the Mc Gill B-version of the Cultural Formulation, combined with a relational and process oriented reflection on the intercultural dynamics that unfold during the encounter with a family.