Isoprenylation of the G protein gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C

We have previously shown that isoprenylation and/or additional pest-translational processing of the G protein gamma(1) subunit carboxyl terminus is required for beta(1) gamma(1) subunit stimulation of phospholipase C-beta(2) (PLC beta(2)) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171-178]. To examine whether isoprenylation of the gamma(1) subunit alone is sufficient for beta(1) gamma(1)-mediated PLC beta(2) stimulation or whether any of the two subsequent modifications, proteolytic removal of the carboxyl-terminal tripeptide and/or carboxylmethylation, is required for this effect, nonisoprenylated recombinant beta(1) gamma(1) dimers were produced in baculovirus-infected insect cells, purified to near homogeneity, and then isoprenylated in vitro using purified recombinant protein farnesyltransferase. Analysis of the beta(1) gamma(1) dimer after in vitro farnesylation by reversed phase high-performance liquid chromatography followed by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry confirmed that the gamma(1) subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated beta(1) gamma(1) dimers with a recombinant PLC beta(2) isozyme revealed that farnesylation rendered recombinant nonisoprenylated beta(1) gamma(1) dimers capable of stimulating PLC beta(2) and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated beta(1) gamma(1) dimers than for fully modified native beta(1) gamma(1) purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.

Restorative justice conferencing for reducing recidivism in young offenders (aged 7 to 21)

Background
Restorative justice is “a process whereby parties with a stake in a specific offence resolve collectively how to deal with the aftermath of the offence and its implications for the future” (Marshall 2003). Despite the increasing use of restorative justice programmes as an alternative to court proceedings, no systematic review has been undertaken of the available evidence on the effectiveness of these programmes with young offenders. Recidivism in young offenders is a particularly worrying problem, as recent surveys have indicated
the frequency of re-offences for young offenders has ranged from 40.2% in 2000 to 37.8% in 2007 (Ministry of Justice 2009)

Objectives
To evaluate the effects of restorative justice conferencing programmes for reducing recidivism in young offenders.

Search methods
We searched the following databases up to May 2012: CENTRAL, 2012 Issue 5, MEDLINE (1978 to current), Bibliography of Nordic Criminology (1999 to current), Index to Theses (1716 to current), PsycINFO (1887 to current), Social Sciences Citation Index (1970 to current), Sociological Abstracts (1952 to current), Social Care Online (1985 to current), Restorative Justice Online (1975 to current), Scopus (1823 to current), Science Direct (1823 to current), LILACS (1982 to current), ERIC (1966 to current), Restorative Justice Online (4May 2012),WorldCat (9May 2012), ClinicalTrials.gov (19May 2012) and ICTRP (19May 2012). ASSIA,National Criminal Justice Reference Service and Social Services Abstracts were searched up to May 2011. Relevant bibliographies, conference programmes and journals were also searched.

Selection criteria
Randomised controlled trials (RCTs) or quasi-RCTs of restorative justice conferencing versus management as usual, in young offenders.

Data collection and analysis
Two authors independently assessed the risk of bias of included trials and extracted the data. Where necessary, original investigators were contacted to obtain missing information.

Main results
Four trials including a total of 1447 young offenders were included in the review. Results failed to find a significant effect for restorative justice conferencing over normal court procedures for any of the main analyses, including number re-arrested (odds ratio (OR) 1.00, 95% confidence interval (CI) 0.59 to 1.71; P = 0.99), monthly rate of reoffending (standardised mean difference (SMD) -0.06, 95% CI -0.28 to 0.16; P = 0.61), young person’s remorse following conference (OR 1.73, 95% CI 0.97 to 3.10; P = 0.06), young person’s recognition of wrongdoing following conference (OR 1.97, 95% CI 0.81 to 4.80; P = 0.14), young person’s self-perception following conference (OR 0.95, 95% CI 0.55 to 1.63; P = 0.85), young person’s satisfaction following conference (OR 0.42, 95% CI 0.04 to 4.07; P = 0.45) and victim’s satisfaction following conference (OR 4.05, 95%CI 0.56 to 29.04; P = 0.16). A small number of sensitivity analyses did indicate significant effects, although all are to be interpreted with caution.

Authors’ conclusions
There is currently a lack of high quality evidence regarding the effectiveness of restorative justice conferencing for young offenders. Caution is urged in interpreting the results of this review considering the small number of included studies, subsequent low power and high risk of bias. The effects may potentially be more evident for victims than offenders. The need for further research in this area is highlighted.

SLA Brokering and Bandwidth Reservation Negotiation Schemes for QoS-aware Internet

We present a novel Service Level Agreement (SLA)-driven service provisioning architecture, which enables dynamic and flexible bandwidth reservation schemes on a per-user or per-application basis. Various session level SLA negotiation schemes involving bandwidth allocation, service start time and service duration parameters are introduced and analyzed. The results show that these negotiation schemes can be utilized for the benefit of both end users and network providers in achieving the highest individual SLA optimization in terms of key Quality of Service (QoS) metrics and price. The inherent characteristics of software agents such as autonomy, adaptability and social abilities offer many advantages in this dynamic, complex, and distributed network environment especially when performing Service Level Agreements (SLA) definition negotiations and brokering tasks. This article also presents a service broker prototype based on Fujitsu's Phoenix Open Agent Mediator (OAM) agent technology, which was used to demonstrate a range of SLA brokering scenarios.

The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130–230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130–230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.

HIF-2alpha Associated Familial Erythrocytosis Supports the PHD2-HIF2-alpha-VHL Axis as the Major Regulator of Erythropoietin Production

Transcriptionally erythropoietin (Epo) synthesis is tightly regulated by the hypoxia inducible factor (HIF), which is composed of one alpha and one beta subunit that are constitutively expressed. The beta subunit is non-variable, but three different alpha subunits give rise to three isoforms of HIF. The alpha subunit is proteasomally regulated in the presence of oxygen by hydroxylation of the proline in the LXXLAP motif of the oxygen dependent degradation (ODD) domain of HIFalpha, catalysed by members of the prolyl hydroxylase domain (PHD) family of enzymes. This allows the von Hippel Lindau (VHL) protein to associate with the alpha subunit, which is subsequently tagged with ubiquitin and degraded by the proteasome. Any defect in the oxygen sensing pathway that allows the alpha subunit to escape proteasomal regulation leads to elevated expression of HIF target genes.

Recently mutations in both VHL and PHD2 have been identified in a cohort of patients with erythrocytosis, but no mutations were found in the ODD domain of HIF1alpha. Instead, investigation of the homologous region in HIF-2alpha revealed four different mutations, Pro534Leu, Met535Val, Gly537Arg and Gly537Trp in seven individuals/families. Affected individuals presented at a young age with elevated serum Epo. Several individuals have a clinical history of thrombosis, but no evidence of a von Hippel Lindau-like syndrome.

To define how the four mutations relate to the erythrocytosis phenotype functional assays were performed in vitro. Binding of PHD2 to the four HIF-2alpha mutants was impaired to varying degrees, with both the Gly537 mutants showing the greatest reduction. The association of VHL with the hydroxylated Met535Val mutant peptide was similar to wild type HIF- 2alpha, but was decreased in the other three HIF-2alpha mutants. Expression of three HIF- 2alpha target genes, adrenomedullin, NDRG1 and VEGF, was significantly up-regulated in cells stably transfected with the mutants under normoxia compared to wild type HIF-2alpha. Mutations in the ODD domain of HIF-2alpha disrupt proteasomal regulation by reducing the association with PHD2 and hence hydroxylation. Furthermore the binding of VHL is also impaired, even when HIF-2alpha is hydroxylated. Examination of the three-dimensional structure of hydroxylated HIF-1alpha bound to VHL confirms that amino acids close to site of hydroxylation (Pro-531 in isoform 2) are important for this association. These observations, together with recent studies utilising murine models of erythrocytosis, support the PHD2-HIF-2alpha-VHL axis as the major regulator of erythropoietin.