Genetic control of anti-Sm autoantibody production in NOD congenic mice narrowed to the Idd9.3 region

Anti-Smith (anti-Sm) autoantibodies are directed to proteins in the small-nuclear ribonucleoprotein (snRNP) family and are considered specific for systemic lupus erythematosus (SLE) in both humans and mice. We previously established that NOD.c3c4 mice, carrying B6 and B10 congenic segments from chromosomes 3 to 4 on an nonobese diabetic (NOD) background, and NOD.Idd9R28 mice, carrying a B10 segment on c4 alone, developed significant penetrance of anti-Sm antibody production. Here we determine autoantibody incidence in additional NOD.Idd9 congenic strains and use a congenic mapping approach to narrow the interval necessary for enhanced autoantibody production to a ∼5.6-Mb region containing insulin-dependent diabetes (Idd)9.3. The Idd9.3 interval contains the candidate molecule cluster of differentiation (CD)137, which is a member of the tumor necrosis factor (TNF) receptor superfamily, functions as an inducible costimulator of T cells, and controls T–B interactions. The NOD and B10 CD137 alleles have sequence polymorphisms and different functional effects on T cells; the NOD CD137 allele mediates weaker T cell proliferative responses and decreased interleukin (IL)-2 production after CD137-mediated costimulation. Our work establishes CD137 as a candidate gene for control of autoantibody production in NOD.Idd9.3 congenic mice.     

DNA Methylation Profile Distinguishes Clear Cell Sarcoma of the Kidney from Other Pediatric Renal Tumors

A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms’ tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing’s sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms’ tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.     

An efficient method for the preparation of preferentially heterodimerized recombinant S100A8/A9 coexpressed in Escherichia coli

It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems.     

PGC-1α-mediated changes in phospholipid profiles of exercise-trained skeletal muscle

Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α. We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle.     

LARGE2 facilitates the maturation of alpha-dystroglycan more effectively than LARGE

The LARGE gene is thought to encode a putative glycosyltransferase because of its typical topology. However, no enzyme activity has been demonstrated yet, although the gene apparently supports the functional maturation of alpha-dystroglycan by glycosylation when it is transfected into cells. A novel homologous gene to LARGE was identified and named LARGE2. LARGE2 recombinant was co-expressed with alpha-dystroglycan in human embryonic kidney 293T cells to determine its activity to support the maturation of alpha-dystroglycan. The alpha-dystroglycan co-transfected with LARGE2 was more highly glycosylated than that co-transfected with LARGE. Pull-down experiments demonstrated binding activity of LARGE2 as well as LARGE toward alpha-dystroglycan. LARGE2 was found to support the maturation of alpha-dystroglycan more effectively than LARGE. Both of them are ubiquitously expressed in many tissues, except the brain where LARGE2 was not expressed at all. This compensatory function can explain the residual functionally glycosylated alpha-dystroglycan in a patient with MDC1D whose LARGE genes are congenitally null.     

In vitro anti-mutagenic effect of magnolol against direct and indirect mutagens

Magnolol, a component of the bark of Magnolia obovata, has been reported to possess various biological activities, such as anti-carcinogenicity, anti-promotion activity and anti-oxidative activity. These findings suggest potential for this compound in cancer chemoprevention. Interestingly, there have been no reports to date on the potential anti-mutagenic activity of magnolol, involving inhibition of initiation processes of the primary stage of carcinogenicity. In this study, anti-mutagenic activity of magnolol against mutagenicity induced by direct mutagens [1-nitropyrene (1-NP), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)] and indirect mutagens [2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), benzo(a)pyrene (B(a)P), 2-aminoanthracene (2-AA) and 7,12-dimethylbenz[a]anthracene (DMBA)] were investigated using the bacterial mutagenicity test (Ames test). Results show that magnolol strongly inhibits mutagenicity induced by indirect mutagens, but does not affect direct mutagens. To elucidate the mechanism of this effect against indirect mutagens, effect of magnolol on CYP1A1- and CYP1A2-related enzyme activities of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) were investigated. Magnolol strongly and competitively suppressed these enzyme activities, suggesting it inhibited mutation induced by indirect mutagens through suppression of CYP1A1 and CYP1A2 activity.     

Structural basis of proteolytic activation of L-phenylalanine oxidase from Pseudomonas sp. P-501

The mature form of l-phenylalanine oxidase (PAOpt) from Pseudomonas sp. P-501 was generated and activated by the proteolytic cleavage of a noncatalytic proenzyme (proPAO). The crystal structures of proPAO, PAOpt, and the PAOpt-o-amino benzoate (AB) complex were determined at 1.7, 1.25, and 1.35A resolutions, respectively. The structure of proPAO suggests that the prosequence peptide of proPAO occupies the funnel (pathway) of the substrate amino acid from the outside of the protein to the interior flavin ring, whereas the funnel is closed with the hydrophobic residues at its vestibule in both PAOpt and the PAOpt-AB complex. All three structures have an oxygen channel that is open to the surface of the protein from the flavin ring. These results suggest that structural changes were induced by proteolysis; that is, the proteolysis of proPAO removes the prosequence and closes the funnel to keep the active site hydrophobic but keeps the oxygen channel open. The possibility that an interaction of the hydrophobic side chain of substrate with the residues of the vestibule region may open the funnel as a putative amino acid channel is discussed.     

Structure-activity relationship studies of Bz amide-containing α-GalCer derivatives as natural killer T cell modulators

Abstract

CD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.