Essential role of Ca2+/calmodulin in Early Endosome Antigen-1 localization

Ca2+ is an essential requirement in membrane fusion, acting through binding proteins such as calmodulin (CaM). Ca2+/CaM is required for early endosome fusion in vitro, however, the molecular basis for this requirement is unknown. An additional requirement for endosome fusion is the protein Early Endosome Antigen 1 (EEA1), and its recruitment to the endosome depends on phosphatidylinositol 3-phosphate [PI(3)P] and the Rab5 GTPase. Herein, we demonstrate that inhibition of Ca2+/CaM, by using either chemical inhibitors or specific antibodies directed to CaM, results in a profound inhibition of EEA1 binding to endosomal membranes both in live cells and in vitro. The concentration of Ca2+/CaM inhibitors required for a full dissociation of EEA1 from endosomal membranes had no effect on the activity of phosphatidylinositol 3-kinases or on endogenous levels of PI(3)P. However, the interaction of EEA1 with liposomes containing PI(3)P was decreased by Ca2+/CaM inhibitors. Thus, Ca2+/CaM seems to be required for the stable interaction of EEA1 with endosomal PI(3)P, perhaps by directly or indirectly stabilizing the quaternary organization of the C-terminal FYVE domain of EEA1. This requirement is likely to underlie at least in part the essential role of Ca2+/CaM in endosome fusion.     

Mimicry and autoantibody-mediated neuronal cell signaling in Sydenham chorea

Streptococcus pyogenes-induced acute rheumatic fever (ARF) is one of the best examples of postinfectious autoimmunity due to molecular mimicry between host and pathogen. Sydenham chorea is the major neurological manifestation of ARF but its pathogenesis has remained elusive, with no candidate autoantigen or mechanism of pathogenesis described. Chorea monoclonal antibodies showed specificity for mammalian lysoganglioside and N-acetyl-beta-D-glucosamine (GlcNAc), the dominant epitope of the group A streptococcal (GAS) carbohydrate. Chorea antibodies targeted the surface of human neuronal cells, with specific induction of calcium/calmodulin-dependent protein (CaM) kinase II activity by monoclonal antibody 24.3.1 and sera from active chorea. Convalescent sera and sera from other streptococcal diseases in the absence of chorea did not activate the kinase. The new evidence implicates antibody-mediated neuronal cell signaling in the immunopathogenesis of Sydenham chorea and will lead to a better understanding of other antibody-mediated neurological disorders.     

HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation

The viral infectivity factor (Vif) encoded by HIV-1 neutralizes a potent antiviral pathway that occurs in human T lymphocytes and several leukemic T-cell lines termed nonpermissive, but not in other cells termed permissive. In the absence of Vif, this antiviral pathway efficiently inactivates HIV-1. It was recently reported that APOBEC3G (also known as CEM-15), a cytidine deaminase nucleic acid-editing enzyme, confers this antiviral phenotype on permissive cells. Here we describe evidence that Vif binds APOBEC3G and induces its rapid degradation, thus eliminating it from cells and preventing its incorporation into HIV-1 virions. Studies of Vif mutants imply that it contains two domains, one that binds APOBEC3G and another with a conserved SLQ(Y/F)LA motif that mediates APOBEC3G degradation by a proteasome-dependent pathway. These results provide promising approaches for drug discovery.     

Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo

Interleukin (IL)-2 is currently used to enhance T-cell immunity but can have both positive and negative effects on T cells. To determine whether these opposing results are due to IL-2 acting differently on T cells depending on their stage of differentiation, we examined the effects of IL-2 therapy during the expansion, contraction and memory phases of the T-cell response in lymphocytic choriomeningitis virus (LCMV)-infected mice. IL-2 treatment during the expansion phase was detrimental to the survival of rapidly dividing effector T cells. In contrast, IL-2 therapy was highly beneficial during the death phase, resulting in increased proliferation and survival of virus-specific T cells. IL-2 treatment also increased proliferation of resting memory T cells in mice that controlled the infection. Virus-specific T cells in chronically infected mice also responded to IL-2 resulting in decreased viral burden. Thus, timing of IL-2 administration and differentiation status of the T cell are critical parameters in designing IL-2 therapies.     

The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases?

The COP9 signalosome (CSN) is a highly conserved protein complex implicated in diverse biological functions that involve ubiquitin-mediated proteolysis. Paradoxically, conserved enzymatic activities associated with CSN inhibit cullin ubiquitin ligase activity in vitro, whereas mutational analysis suggests that CSN promotes cullin-dependent proteolysis in vivo. This apparent paradox can be resolved in a model that proposes CSN-mediated cullin inhibition is a prerequisite for the proper assembly and maintenance of active cullin ubiquitin ligase complexes.     

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Background

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.

Results

We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.

Conclusion

Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.     

The use and abuse of commercial kits used to detect autoantibodies

The detection of autoantibodies in human sera is an important approach to the diagnosis and management of patients with autoimmune conditions. To meet market demands, manufacturers have developed a wide variety of easy to use and cost-effective diagnostic kits that are designed to detect a variety of human serum autoantibodies. A number of studies over the past two decades have suggested that there are limitations and concerns in the use and clinical application of test results derived from commercial kits. It is important to appreciate that there is a complex chain of users and circumstances that contributes to variations in the apparent reliability of commercial kits. The goal of this review is to identify the principal links in this chain, to identify the factors that weaken the chain and to propose a plan of resolution. It is suggested that a higher level of commitment and partnership between all of the participants is required to achieve the goal of improving the quality of patient care through the use of autoantibody testing and analysis.     

Effects of cadmium on the reproductive axis of Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubquitous element and a significant inorganic pollutant that has previously been found to bioaccumulate in reproductive organs of fish and disrupt important endocrine processes, especially those involved in synthesis, release and metabolism of hormones. Clearly, there is potential for reproductive effects in fish populations exposed to Cd, however, few studies have investigated the non-lethal consequences of Cd in fish. To this extent, adult male and female Japanese medaka were exposed to 0-10 ppb Cd for 7 weeks. Reproductive endpoints were monitored during weeks 6 and 7 of exposure and compared to physiological responses along the hypothalamus-pituitary-gonadal (HPG) axis, including plasma vitellogenin (VTG), hepatic estrogen receptor (ER), plasma steroids, gonadal-somatic indices (GSI), and gonadal steroid release. There were no observed effects on VTG and ER by long-term Cd exposure. However, gonadal steroid release was significantly decreased in males and females at all exposure concentrations and female plasma estradiol levels were significantly altered at concentrations higher than 5 ppb Cd. Overall, responses along the HPG axis were more sensitive to Cd exposure than the reproductive and developmental endpoints, which were not affected in this study, indicating that higher level impairment in fish might be relatively protected.