Allelic repertoire of the humanMHC class IMICA gene

 The hallmark of the classical major histocompatibility complex (MHC) class I molecules is their astonishing level of polymorphism, a characteristic not shared by the nonclassical MHC class I genes. A distinct family of MHC class I genes has been recently identified within the human MHC class I region. The MICA (MHC class I chain-related A) gene in this family is a highly divergent member of the MHC class I family and has a unique pattern of tissue expression. We have sequenced exons encoding the extracellular α1, α2, and α3 domains of the MICA gene from twenty HLA homozygous typing cell lines and four unrelated individuals. We report the identification of eleven new alleles defined by a total of twenty-two amino acid substitutions. Thus, the total number of MICA alleles is sixteen. Interestingly, a tentative superimposition of MICA variable residues on the HLA-A2 structure reveals a unique pattern of distribution, concentrated primarily on the outer edge of the MICA putative antigen binding cleft, apparently bordering an invariant ligand binding site. Received: 13 May 1996 / Revised: 29 May 1996     

Enantioselective high-performance liquid chromatography determination of methadone enantiomers and its major metabolite in human biological fluids using a new derivatized cyclodextrin-bonded phase

The simultaneous determination of methadone (Mtd) enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in human urine and serum by enantioselective HPLC using a new Cyclobond I-2000 RSP column is described. After alkaline extraction from urine or serum with estazolam as an internal standard, Mtd enantiomers and its metabolite (EDDP) are separated on the previous column with reversed-mobile phase and detected at 210 nm. Peak resolutions are about 2.0 for Mtd enantiomers. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 0.5 and 4.5%. Most drugs of abuse are shown not to interfere with this technique. The method has been applied to study levels of each Mtd enantiomer and of its racemic metabolite in urine and serum of patients under maintenance treatment for opiate dependence. In urine, R-(−)-Mtd levels are always higher (about 2±0.5-fold_ than those of S-(+)-Mtd and in most cases, metabolite concentrations are greater than those of global Mtd enantiomers. However, the R-(−) enantiomer levels of residual drug in serum of some patients were lower than those of its antipode. This method is suitable for pharmacokinetic and toxicological studies of Mtd enantiomers and its major metabolite in biological fluids.     

Cmv4, a new locus linked to the NK cell gene complex, controls innate resistance to cytomegalovirus in wild-derived mice

CMV can cause life-threatening disease in immunodeficient hosts. Experimental infection in mice has revealed that the genetically determined natural resistance to murine CMV (MCMV) may be mediated either by direct recognition between the NK receptor Ly49H and the pathogen-encoded glycoprotein m157 or by epistatic interaction between Ly49P and the host MHC H-2D(k). Using stocks of wild-derived inbred mice as a source of genetic diversity, we found that PWK/Pas (PWK) mice were naturally resistant to MCMV. Depletion of NK cells subverted the resistance. Analysis of backcrosses to susceptible BALB/c mice revealed that the phenotype was controlled by a major dominant locus effect linked to the NK gene complex. Haplotype analysis of 41 polymorphic markers in the Ly49h region suggested that PWK mice may share a common ancestral origin with C57BL/6 mice; in the latter, MCMV resistance is dependent on Ly49H-m157 interactions. Nevertheless, PWK mice retained viral resistance against m157-defective mutant MCMV. These results demonstrate the presence of yet another NK cell-dependent viral resistance mechanism, named Cmv4, which most likely encodes for a new NK activating receptor. Identification of Cmv4 will expand our understanding of the specificity of the innate recognition of infection by NK cells.     

NK cell receptor/H2-Dk-dependent host resistance to viral infection is quantitatively modulated by H2q inhibitory signals

The cytomegalovirus resistance locus Cmv3 has been linked to an epistatic interaction between two loci: a Natural Killer (NK) cell receptor gene and the major histocompatibility complex class I (MHC-I) locus. To demonstrate the interaction between Cmv3 and H2(k), we generated double congenic mice between MA/My and BALB.K mice and an F(2) cross between FVB/N (H-2(q)) and BALB.K (H2(k)) mice, two strains susceptible to mouse cytomegalovirus (MCMV). Only mice expressing H2(k) in conjunction with Cmv3(MA/My) or Cmv3(FVB) were resistant to MCMV infection. Subsequently, an F(3) cross was carried out between transgenic FVB/H2-D(k) and MHC-I deficient mice in which only the progeny expressing Cmv3(FVB) and a single H2-D(k) class-I molecule completely controlled MCMV viral loads. This phenotype was shown to be NK cell-dependent and associated with subsequent NK cell proliferation. Finally, we demonstrated that a number of H2(q) alleles influence the expression level of H2(q) molecules, but not intrinsic functional properties of NK cells; viral loads, however, were quantitatively proportional to the number of H2(q) alleles. Our results support a model in which H-2(q) molecules convey Ly49-dependent inhibitory signals that interfere with the action of H2-D(k) on NK cell activation against MCMV infection. Thus, the integration of activating and inhibitory signals emanating from various MHC-I/NK cell receptor interactions regulates NK cell-mediated control of viral load.     

Pyruvate shuttle in muscle cells: high-affinity pyruvate transport sites insensitive to trans-lactate efflux

The specificity of the transport mechanisms for pyruvate and lactate and their sensitivity to inhibitors were studied in L6 skeletal muscle cells. Trans- and cis-lactate effects on pyruvate transport kinetic parameters were examined. Pyruvate and lactate were transported by a multisite carrier system, i.e., by two families of sites, one with low affinity and high capacity (type I sites) and the other with high affinity and low capacity (type II). The multisite character of transport kinetics was not modified by either hydroxycinnamic acid (CIN) or p-chloromercuribenzylsulfonic acid (PCMBS), which exert different types of inhibition. The transport efficiency (TE) ratios of maximal velocity to the trans-activation dissociation constant (Kt) showed that lactate and pyruvate were preferentially transported by types I and II sites, respectively. The cis-lactate effect was observed with high Ki values for both sites. The trans-lactate effect on pyruvate transport occurred only on type I sites and exhibited an asymmetric interaction pattern (Kt of inward lactate > Kt of outward lactate). The inability of lactate to trans-stimulate type II sites suggests that intracellular lactate cannot recruit these sites. The high-affinity type II sites act as a specific pyruvate shuttle and constitute an essential relay for the intracellular lactate shuttle.     

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

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Genome-Wide Mouse Mutagenesis Reveals CD45-Mediated T Cell Function as Critical in Protective Immunity to HSV-1

Herpes simplex encephalitis (HSE) is a lethal neurological disease resulting from infection with Herpes Simplex Virus 1 (HSV-1). Loss-of-function mutations in the UNC93B1, TLR3, TRIF, TRAF3, and TBK1 genes have been associated with a human genetic predisposition to HSE, demonstrating the UNC93B-TLR3-type I IFN pathway as critical in protective immunity to HSV-1. However, the TLR3, UNC93B1, and TRIF mutations exhibit incomplete penetrance and represent only a minority of HSE cases, perhaps reflecting the effects of additional host genetic factors. In order to identify new host genes, proteins and signaling pathways involved in HSV-1 and HSE susceptibility, we have implemented the first genome-wide mutagenesis screen in an in vivo HSV-1 infectious model. One pedigree (named P43) segregated a susceptible trait with a fully penetrant phenotype. Genetic mapping and whole exome sequencing led to the identification of the causative nonsense mutation L3X in the Receptor-type tyrosine-protein phosphatase C gene (Ptprc^L3X), which encodes for the tyrosine phosphatase CD45. Expression of MCP1, IL-6, MMP3, MMP8, and the ICP4 viral gene were significantly increased in the brain stems of infected Ptprc^L3X mice accounting for hyper-inflammation and pathological damages caused by viral replication. Ptprc^L3X mutation drastically affects the early stages of thymocytes development but also the final stage of B cell maturation. Transfer of total splenocytes from heterozygous littermates into Ptprc ^L3X mice resulted in a complete HSV-1 protective effect. Furthermore, T cells were the only cell population to fully restore resistance to HSV-1 in the mutants, an effect that required both the CD4⁺ and CD8⁺ T cells and could be attributed to function of CD4⁺ T helper 1 (Th1) cells in CD8⁺ T cell recruitment to the site of infection. Altogether, these results revealed the CD45-mediated T cell function as potentially critical for infection and viral spread to the brain, and also for subsequent HSE development.