A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN

Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrat

Remission in schizophrenia: validity, frequency, predictors, and patients' perspective 5 years later

In March 2005, the Remission in Schizophrenia Working Group (RSWG) proposed a consensus definition of symptomatic remission in schizophrenia and developed specific operational criteria for its assessment. They pointed out, however, that the validity and the relationship to other outcome dimensions required further examination. This article reviews studies on the validity, frequency, and predictors of symptomatic remission in schizophrenia and studies on patients' perspectives. These studies have demonstrated that the RSWG remission criteria appear achievable and sustainable for a significant proportion of patients, and are related to a better overall symptomatic status and functional outcome and, to a less clear extent, to a better quality of life and cognitive performance. However, achieving symptomatic remission is not automatically concurrent with an adequate status in other outcome dimensions. The results of the present review suggest that the RSWG remission criteria are valid and useful. As such, they should be consistently applied in clinical trials. However, the lack of consensus definitions of functional remission and adequate quality of life hampers research on their predictive validity on these outcome dimensions. Future research should therefore search for criteria of these dimensions and test whether the RSWG remission criteria consistently predict a “good” outcome with respect to functioning and quality of life

A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN

Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrate

Service users’ perceptions of relevant and helpful components of an integrated care concept (ACCESS) for psychosis

IntroductionPsychotic disorders have a significant impact on patients’ lives and their families, and long-term treatment with individually tailored multimodal combinations of therapies is often required. Integrated care (IC) concepts such as the “Hamburg Model (ACCESS)” with a focus on psychotic disorders, includes different (therapeutic) components with pharmaco- and psychotherapy, family involvement, home treatment and the option of using a 24/7 crisis hotline. All components are offered by a therapeutically-oriented assertive community treatment (TACT) team in a need-adapted manner. So far, however, little is known about which specific components are regarded as especially relevant and helpful by the users of IC.MethodsPatients currently participating in IC completed a questionnaire as part of the continuous quality assurance study (ACCESS II) in which they were asked to rate the different components of treatment according to their relevance and helpfulness, considering the individual’s unique experiences with IC and needs in mental health care. Furthermore, they were asked to make suggestions regarding additional helpful components of treatment.ResultsFifty patients participated in this survey (23% of the patients currently participating in the IC concept). For participants, the most helpful and important factors were having the same therapist in the long-term and the 24/7 crisis telephone. Additional components suggested by patients included more addiction-specific therapies and increased focus on vocational rehabilitation and integration.ConclusionFrom the perspective of the users of IC, long-term care from a trusted therapist with whom there is a therapeutic relationship and the possibility to reach someone they already know from the TACT team 24/7 serves as the best basis for effective care, fostering trust, understanding, and open communication. In contrast, home treatment remains a relevant aspect of evidence-based care for people with severe mental illness, but perhaps surprisingly, is not viewed as the most important issue

Domain orientation in the RNA helicase YxiN and the role of conformational changes for RNA unwinding

The RNA helicase YxiN from Bacillus subtilis is a member of the family of DEAD box proteins. YxiN is able to unwind RNA double strands in an ATP-dependent manner. The ability to catalyse RNA rearrangement is in vivo presumably necessary for the bacterial ribosome biogenesis. YxiN comprises a two-domain helicase core region and a C-terminal RNA binding domain. While crystal structures of the C-terminal core domain and the RNA binding domain separately have been determined before, the structure of full-length YxiN is not known. In the current project the orientation of these three domains to each other was determined employing fluorescence resonance energy transfer (FRET) experiments at the single-molecule level. Therefore the approximate architecture of the full-length enzyme in solution can now be described. The two core domains exhibit a conformation similar to the crystal structure of the DEAD box protein MjDeaD. The RNA binding domain is adjacent to the C-terminal core domain. Presumably the central -sheet of the RNA binding domain faces towards a patch of the core domain that is formed by loops. During catalysis YxiN undergoes a conformational change. The conformation of the core domains mentioned above is adopted in the absence of substrates and in the presence of RNA, ADP, ATP or ADPNP. In the presence of both RNA and ATP (or ADPNP) the core domains approach each other constituting a closed conformation. During the catalytic cycle this conformational change takes place initially after binding of RNA and ATP. The conformational change is necessary for RNA unwinding. But it is not sufficient since the YxiN_K52Q mutant adopts the closed conformation upon binding of RNA and ATP (or ADPNP) but is unwinding deficient. Transitions between the open and the closed conformation could only rarely been detected in the FRET experiments on a confocal microscope due to a limited observation time. To be able to monitor the conformation of YxiN on longer timescale the protein was engineered for FRET experiments on a total internal reflection microscope. A protocol was developed that comprises fluorophore double labelling of YxiN and the attachment of a biotin at the protein’s C-terminus. The biotinylation procedure is based on the reaction type of expressed protein ligation. The labelled and biotinylated YxiN construct could be specifically immobilized on a streptavidin coated surface for total internal reflection microscopy. Subsequently, YxiN could be monitored for up to a few seconds. Expressed protein ligation was furthermore employed to develop a specific fluorophore double labelling strategy for FRET experiments. Employing this strategy a YxiN construct could be generated that carries one certain fluorophore exclusively at one position in the protein. A different fluorophore can attach to the same position or to one further site within the protein. The procedure was therefore termed semi-site-specific double labelling. In comparison with statistic labelling procedures the semi-site-specific double labelling allows for decreasing the sample heterogeneity in FRET experiments. Taken together, this study revealed the conformation of the three-domain RNA helicase YxiN, its conformational change during catalysis which is essential for the activity of the helicase and the study established protein preparation techniques that provide the basis for further studies on the helicase mechanism

Immunohistochemical Findings on the Influence of Immunosuppressive Therapy after Penetrating Keratoplasty in the Rat

EINLEITUNG 6 1\. Hornhauttransplantation 6 2\. Die Pathogenese der Abstossungsreaktion 8 3\. Makropathologie und Histologie der Transplantatabstossung 16 4\. Prävention und Behandlung der Abstossung in der Klinik 17 5\. Tiermodelle ? Kaninchen, Ratte, Maus 18 6\. Experimentelle Therapieformen 20 7\. Fragestellung 28 MATERIAL UND METHODEN 29 ERGEBNISSE 38 1\. Aus der Studie ausgeschlossene Tiere 38 2\. Normale (nicht operierte) Rattenaugen 38 3\. Hornhauttransplantierte Augen 38 DISKUSSION 54 ZUSAMMENFASSUNG 71 ANHANG 72 LITERATURVERZEICHNIS 75In dieser Arbeit ist mit immunhistochemischen Methoden untersucht worden, welche Zellen in welchem Verhältnis zueinander und in welcher zeitlichen Abfolge die Hornhaut nach Keratoplastik infiltrieren, nachdem auf verschiedene Weise immunsuppressiv bzw. tolerogen therapiert wurde. Spender- und Empfängerstamm des verwendeten Rattenmodells sind hinsichtlich ihrer Histokompatibilitätsantigene vollständig inkompatibel, und es kommt bei unbehandelten Tieren immer zur Abstoßung des Transplantates. Die Blockade des CD4-Moleküls der T-Helferzellen durch den monoklonalen Anti- CD4-Antikörper RIB5/2 führt zu einem Rückgang der Zahl aller das Transplantat infiltrierenden Zellpopulationen. Dem liegt höchstwahrscheinlich ein Überwiegen immunsuppressiv wirkender Th2-Zytokine zugrunde, die nach Antigenerkennung bei blockiertem CD4-Molekül von T-Zellen vermehrt sezerniert werden. Diese histologische entspricht der klinischen Beobachtung, daß ein Großteil der so behandelten Tiere ihre Transplantate nicht abstoßen. Die Behandlung mit Leflunomid bzw. Cyclosporin A blieb in diesem Modell ohne Effekt. Die Arbeit gibt einen Hinweis darauf, daß eine Störung der T-Helferzellen an zentraler Stelle in die Abstoßungsreaktion an der Hornhaut eingreift und unterstützt die These, daß CD4+ Zellen, wenn auch nicht als Effektorzellen im Sinne von die Gewebezerstörung letztlich ausführende Zellen, doch durch ihre steuernde Funktion eine zentrale Position innerhalb der Abstoßungsreaktion einnehmen. Anti-CD4-Antikörper stellen somit eine erfolgversprechende Therapiealternative zur Verhinderung der Transplantatabstoßung nach Keratoplastik dar.Utilising immunohistochemistry it has been investigated which cells of the immune system in which proportion to each other and in which timely sequence are infiltrating the graft after penetrating keratoplasty in the rat eye. After transplantation the rats had been treated with different immunosuppressing or tolerogenic agents. The rat strains used as donor and recipient are different in their MHC antigens so that transplantation in untreated animals always leads to allograft rejection. Blocking the CD4 antigen expressed on T-helper cells by treating the animals with the monoclonal anti-CD4 antibody RIB 5/2 leads to much less infiltration of the graft by all cell populations investigated. This is probably due to the increased secretion of Th2-cytokines by T-helper cells on antigen recognition after blocking the CD4-molecule. Th2-cytokines are immunosuppressing in the context of allograft rejection. This is corresponding to the clinical observation that most of the animals treated with the anti-CD4 antibody are not rejecting their graft. Treatment with Leflunomide or with Cyclosporine remained without effect in this setting. These results support the view that CD4+ T-cells although not destroying the infiltrated tissue themselves are of central importance in allograft rejection because of their initialising role and that blocking normal T-cell function may interrupt allograft reaction in the cornea. Treatment with anti-CD4 antibodies seems to be a promising alternative in preventing allograft rejection after penetrating keratoplasty

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains

RNA helicases mediate structural rearrangements of RNA or RNA–protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis–Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication