IAP-IAP complexes required for apoptosis resistance of C. trachomatis-infected cells

Host cells infected with obligate intracellular bacteria Chlamydia trachomatis are profoundly resistant to diverse apoptotic stimuli. The molecular mechanisms underlying the block in apoptotic signaling of infected cells is not well understood. Here we investigated the molecular mechanism by which apoptosis induced via the tumor necrosis factor (TNF) receptor is prevented in infected epithelial cells. Infection with C. trachomatis leads to the up-regulation of cellular inhibitor of apoptosis (cIAP)-2, and interfering with cIAP-2 up-regulation sensitized infected cells for TNF-induced apoptosis. Interestingly, besides cIAP-2, cIAP-1 and X-linked IAP, although not differentially regulated by infection, are required to maintain apoptosis resistance in infected cells. We detected that IAPs are constitutively organized in heteromeric complexes and small interfering RNA-mediated silencing of one of these IAPs affects the stability of another IAP. In particular, the stability of cIAP-2 is modulated by the presence of X-linked IAP and their interaction is stabilized in infected cells. Our observations suggest that IAPs are functional and stable as heteromers, a thus far undiscovered mechanism of IAP regulation and its role in modulation of apoptosis.     

RanBP2 modulates Cox11 and hexokinase I activities and haploinsufficiency of RanBP2 causes deficits in glucose metabolism

The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2^−/− are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2^+/− mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies.     

Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis

Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMKs). The expression patterns of the E. histolytica TMKs in individual trophozoites and the roles of the TMKs for sensing and responding to extracellular cues were incompletely characterised. Here we provide evidence that single cells express multiple TMKs and that TMK39 and TMK54 likely serve non-redundant cellular functions. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Anti-peptide antibodies were raised against unique regions in the extracellular domains of TMK39, TMK54 and PaTMK, and TMK expression was analysed at the protein level. Flow cytometric assays revealed that populations of trophozoites homogeneously expressed TMK39, TMK54 and PaTMK, while confocal microscopy identified different patterns of cell surface expression for TMK39 and TMK54. The functions of TMK39 and TMK54 were probed by the inducible expression of dominant-negative mutants. While TMK39 co-localised with ingested beads and expression of truncated TMK39 interfered with trophozoite phagocytosis of apoptotic lymphocytes, expression of a truncated TMK54 inhibited growth of amoebae and altered the surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilised for non-redundant functions by the parasite.     

Variable ECG signs of ischemia during controlled occlusion of the left and right coronary artery in humans

Infarct size (IS) increases with vascular occlusion time, area at risk for infarction, lack of collateral supply, absence of preconditioning, and myocardial demand for O2 supply. ECG S-T segment elevation is used as a measure of severity of ischemia and a surrogate for IS. This study in 50 patients with coronary artery disease undergoing a first 120-s balloon occlusion of a stenosis sought to determine whether S-T segment elevation, corrected for the above-mentioned variables, in the left coronary artery (LCA group, n = 36) is different from that in the right coronary artery (RCA group, n = 14) territory. After consideration of all known determinants of IS, particularly mass at risk and collateral supply, the LCA territory is more sensitive than the RCA region to a 2-min period of myocardial ischemia.     

Genetic divergence of Connecticut Melanoplus femurrubrum populations

We surveyed Melanoplus femurrubrum populations within the state of Connecticut for genetic diversity at multiple genetic markers, including three mitochondrial [cytochrome oxidase subunit 1 (COI), reduced form of nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2), and AT rich] and one nuclear [internal transcribed spacers of the ribosomal DNA cluster (ITS1)] gene regions. All markers were variable, and the AT-rich gene showed the highest sequence divergence. Analysis of molecular variance (AMOVA), fixation index (Fst) analysis, and phylogeographic patterns showed little divergence between northern and southern regions. Estimates of genetic diversity (pi) showed higher mitochondrial diversity in the northern region but nearly equal diversity for the ITS1 gene. This study shows for the first time in Melanoplus genetic variation for the ND2, AT rich, and ITS genes within a small geographic area. Our methods and results should be useful for other researchers interested in conducting population-level studies on closely related species.     

Anchoring of a large set of markers onto a BAC library for the development of a draft physical map of the grapevine genome

Five hundred and six EST-derived markers, 313 SSR markers and 26 BAC end-derived or SCAR markers were anchored by PCR on a subset of a Cabernet Sauvignon BAC library representing six genome equivalents pooled in three dimensions. In parallel, the 12,351 EST clusters of the grapevine UniGene set (build #11) from NCBI were used to design 12,125 primers pairs and perform electronic PCR on 67,543 nonredundant BAC-end sequences. This in silico experiment yielded 1,140 positive results concerning 638 different markers, among which 602 had not been already anchored by PCR. The data obtained will provide an easier access to the regulatory sequences surrounding important genes (represented by ESTs). In total, 1,731 islands of BAC clones (set of overlapping BAC clones containing at least one common marker) were obtained and 226 of them contained at least one genetically mapped anchor. These assigned islands are very useful because they will link the genetic map and the future fingerprint-based physical map and because they allowed us to indirectly place 93 ESTs on the genetic map. The islands containing two or more mapped SSR markers were also used to assess the quality of the integrated genetic map of the grapevine genome.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Didier Lamoureux and Anne Bernole contributed equally to this work.     

Solution structure of an oncogenic mutant of Cdc42Hs

Cdc42Hs(F28L) is a single-point mutant of Cdc42Hs, a member of the Ras superfamily of GTP-binding proteins, that facilitates cellular transformation brought about by an increased rate of cycling between GTP and GDP [Lin, R., et al. (1997) Curr. Biol. 7, 794-797]. Dynamics studies of Cdc42Hs(F28L)-GDP have shown increased flexibility for several residues at the nucleotide-binding site [Adams, P. D., et al. (2004) Biochemistry 43, 9968-9977]. The solution structure of Cdc42Hs-GDP (wild type) has previously been determined by NMR spectroscopy [Feltham, J. L., et al. (1997) Biochemistry 36, 8755-8766]. Here, we describe the solution structure of Cdc42Hs(F28L)-GDP, which provides insight into the structural basis for the change in affinity for GDP. Heteronuclear NMR experiments were performed to assign resonances in the protein, and distance, hydrogen bonding, residual dipolar coupling, and dihedral angle constraints were used to calculate a set of low-energy structures using distance geometry and simulated annealing refinement protocols. The overall structure of Cdc42Hs(F28L)-GDP is very similar to that of wild-type Cdc42Hs, consisting of a centrally located six-stranded beta-sheet structure surrounding the C-terminal alpha-helix [Feltham, J. L., et al. (1997) Biochemistry 36, 8755-8766]. In addition, the same three regions in wild-type Cdc42Hs that show structural disorder (Switch I, Switch II, and the Insert region) are disordered in F28L as well. Although the structure of Cdc42Hs(F28L)-GDP is very similar to that of the wild type, interactions with the nucleotide and hydrogen bonding within the nucleotide binding site are altered, and the region surrounding L28 is substantially more disordered.     

Discovering functional gene expression patterns in the metabolic network of Escherichia coli with wavelets transforms

Background  

Microarray technology produces gene expression data on a genomic scale for an endless variety of organisms and conditions. However, this vast amount of information needs to be extracted in a reasonable way and funneled into manageable and functionally meaningful patterns. Genes may be reasonably combined using knowledge about their interaction behaviour. On a proteomic level, biochemical research has elucidated an increasingly complete image of the metabolic architecture, especially for less complex organisms like the well studied bacterium Escherichia coli.     

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.