Leucine Carboxyl Methyltransferase 1 (LCMT1)-dependent Methylation Regulates the Association of Protein Phosphatase 2A and Tau Protein with Plasma Membrane Microdomains in Neuroblastoma Cells

Down-regulation of protein phosphatase 2A (PP2A) methylation occurs in Alzheimer disease (AD). However, the regulation of PP2A methylation remains poorly understood. We have reported that altered leucine carboxyl methyltransferase (LCMT1)-dependent PP2A methylation is associated with down-regulation of PP2A holoenzymes containing the Bα subunit (PP2A/Bα) and subsequent accumulation of phosphorylated Tau in N2a cells, in vivo and in AD. Here, we show that pools of LCMT1, methylated PP2A, and PP2A/Bα are co-enriched in cholesterol-rich plasma membrane microdomains/rafts purified from N2a cells. In contrast, demethylated PP2A is preferentially distributed in non-rafts wherein small amounts of the PP2A methylesterase PME-1 are exclusively present. A methylation-incompetent PP2A mutant is excluded from rafts. Enhanced methylation of PP2A promotes the association of PP2A and Tau with the plasma membrane. Altered PP2A methylation following expression of a catalytically inactive LCMT1 mutant, knockdown of LCMT1, or alterations in one-carbon metabolism all result in a loss of plasma membrane-associated PP2A and Tau in N2a cells. This correlates with accumulation of soluble phosphorylated Tau, a hallmark of AD and other tauopathies. Thus, our findings reveal a distinct compartmentalization of PP2A and PP2A regulatory enzymes in plasma membrane microdomains and identify a novel methylation-dependent mechanism involved in modulating the targeting of PP2A, and its substrate Tau, to the plasma membrane. We propose that alterations in the membrane localization of PP2A and Tau following down-regulation of LCMT1 may lead to PP2A and Tau dysfunction in AD.     

Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes

Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.     

Low immune response to hepatitis B vaccine among children in Dakar, Senegal

HBV vaccine was introduced into the Expanded Programme on Immunization (EPI) in Senegal and Cameroon in 2005. We conducted a cross-sectional study in both countries to assess the HBV immune protection among children. All consecutive children under 4 years old, hospitalized for any reason between May 2009 and May 2010, with an immunisation card and a complete HBV vaccination, were tested for anti-HBs and anti-HBc. A total of 242 anti-HBc-negative children (128 in Cameroon and 114 in Senegal) were considered in the analysis. The prevalence of children with anti-HBs ≥ 10 IU/L was higher in Cameroon with 92% (95% CI: 87%-97%) compared to Senegal with 58% (95% CI: 49%-67%), (p<0.001). The response to vaccination in Senegal was lower in 2006-2007 (43%) than in 2008-2009 (65%), (p = 0.028). Our results, although not based on a representative sample of Senegalese or Cameroonian child populations, reveal a significant problem in vaccine response in Senegal. This response problem extends well beyond hepatitis B: the same children who have not developed an immune response to the HBV vaccine are also at risk for diphtheria, tetanus, pertussis (DTwP) and Haemophilus influenzae type b (Hib). Field biological monitoring should be carried out regularly in resource-poor countries to check quality of the vaccine administered.     

In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b⁺ F4/80⁺ MHC-II⁺ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS⁺ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.     

Biochemistry and comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent.

The bacterial acyltransferases of the SxxK superfamily vary enormously in sequence and function, with conservation of particular amino acid groups and all-alpha and alpha/beta folds. They occur as independent entities (free-standing polypeptides) and as modules linked to other polypeptides (protein fusions). They can be classified into three groups. The group I SxxK D,D-acyltransferases are ubiquitous in the bacterial world. They invariably bear the motifs SxxK, SxN(D), and KT(S)G. Anchored in the plasma membrane with the bulk of the polypeptide chain exposed on the outer face of it, they are implicated in the synthesis of wall peptidoglycans of the most frequently encountered (4-->3) type. They are inactivated by penicillin and other beta-lactam antibiotics acting as suicide carbonyl donors in the form of penicillin-binding proteins (PBPs). They are components of a morphogenetic apparatus which, as a whole, controls multiple parameters such as shape and size and allows the bacterial cells to enlarge and duplicate their particular pattern. Class A PBP fusions comprise a glycosyltransferase module fused to an SxxK acyltransferase of class A. Class B PBP fusions comprise a linker, i.e., protein recognition, module fused to an SxxK acyltransferase of class B. They ensure the remodeling of the (4-->3) peptidoglycans in a cell cycle-dependent manner. The free-standing PBPs hydrolyze D,D peptide bonds. The group II SxxK acyltransferases frequently have a partially modified bar code, but the SxxK motif is invariant. They react with penicillin in various ways and illustrate the great plasticity of the catalytic centers. The secreted free-standing PBPs, the serine beta-lactamases, and the penicillin sensors of several penicillin sensory transducers help the D,D-acyltransferases of group I escape penicillin action. The group III SxxK acyltransferases are indistinguishable from the PBP fusion proteins of group I in motifs and membrane topology, but they resist penicillin. They are referred to as Pen(r) protein fusions. Plausible hypotheses are put forward on the roles that the Pen(r) protein fusions, acting as L,D-acyltransferases, may play in the (3-->3) peptidoglycan-synthesizing molecular machines. Shifting the wall peptidoglycan from the (4-->3) type to the (3-->3) type could help Mycobacterium tuberculosis and Mycobacterium leprae survive by making them penicillin resistant.     

Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells

BACKGROUND: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations.Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha. MATERIALS AND METHODS: The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN. RESULTS: Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa. CONCLUSION: In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.