Identification of candidate genes in the type 2 diabetes modifier locus using expression QTL

To identify new genetic determinants relevant to type 2 diabetes (T2D), diabetic F2 progeny were generated by intercrossing F1 mice obtained from a cross of BKS.Cg-Lepr(db)+/+m and DBA/2, and T2D-related phenotypes were measured. In the F2 population, increased susceptibility to diabetes and obesity was observed. We also detected the major quantitative trait loci (QTL) modifying the severity of diabetes on chromosome 9, where peaks of logarithm of odds (LOD) overlapped for three traits. To identify candidate genes in the QTL intervals, we combined "expression QTL" (eQTL), taking mRNA levels as quantitative traits, and "interstrain sequence variations, including cSNPs." As a result, four genes were identified from cosegregation of clinical QTL with eQTL and 13 genes were found from interstrain cSNPs as candidates in the T2D modifier QTL. Our combined approach shows the acceleration of the discovery of candidate genes in the QTL of interest, spanning several megabases.     

Ly-5: A new T-lymphocyte antigen system

Ly-5 is a third genetic locus of the type so far represented in the mouse byLy-1 andLy-2/Ly-3; it specifies antithetical alloantigens, one of which is present exclusively on T lymphocytes of every mouse. The chromosomal locus ofLy-5 has not been established, but it is not closely linked toLy-1 orLy-2/Ly-3. Like other T-lymphocyte surface markers, expression of Ly-5 antigens on T-lymphocyte precursor cells can be initiated in vitro by inducers of T-cell differentiation.Recipient of a fellowship from the New York Cancer Research Institute, Inc.     

Characteristic glycopeptides associated with extreme human longevity identified through plasma glycoproteomics

Background

Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105?years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity.

Molecular cloning and characterization of the murine gnathodiaphyseal dysplasia gene GDD1

Mutations in the GDD1 gene cause gnathodiaphyseal dysplasia, a rare human skeletal syndrome with autosomal dominant inheritance. The biochemical function(s) of GDD1 protein and the molecular pathophysiology of GDD1 mutations leading to GDD have not yet been elucidated. In this study, we characterized the complete cDNA sequence and genomic organization of the mouse GDD1 gene. Analysis of GDD1 mRNA revealed a complex alternative splicing pattern, involving five exons of the GDD1 gene. GDD1 isoforms lacking conserved amino acids at the N-terminus cytoplasmic tails, and with changes in transmembrane topology, are presumably associated with changes in protein functions and subcellular localizations of GDD1. We found GDD1 expression to be up-regulated during the course of myogenic differentiation in the murine pluripotent mesenchymal precursor cell line C2C12, whereas its expression was diminished during osteoblastic differentiation. These observations suggest diverse cellular roles of GDD1 protein.     

Specific induction of macrophage inflammatory protein 1-alpha in glial cells of Sandhoff disease model mice associated with accumulation of N-acetylhexosaminyl glycoconjugates

Sandhoff disease is a lysosomal storage disease caused by simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), due to a primary defect of the beta-subunit gene (HEXB) associated with excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylhexosamine residues at their non-reducing termini, and with neurosomatic manifestations. To elucidate the neuroinflammatory mechanisms involved in its pathogenesis, we analyzed the expression of chemokines in Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex beta-subunit gene allele (Hexb-/-). We demonstrated that chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) was induced in brain regions, including the cerebral cortex, brain stem and cerebellum, of SD mice from an early stage of the pathogenesis but not in other systemic organs. On the other hand, little changes in other chemokine mRNAs, including those of RANTES (regulated upon activation, normal T expressed and secreted), MCP-1 (monocyte chemotactic protein-1), SLC (secondary lymphoid-tissue chemokine), fractalkine and SDF-1 (stromal derived factor-1), were detected. Significant up-regulation of MIP-1alpha mRNA and protein in the above-mentioned brain regions was observed in parallel with the accumulation of natural substrates of HexA and HexB. Immunohistochemical analysis revealed that MIP-1alpha-immunoreactivity (IR) in the above-mentioned brain regions of SD mice was co-localized in Iba1-IR-positive microglial cells and partly in glial fibrillary acidic protein (GFAP)-IR-positive astrocytes, in which marked accumulation of N-acetylglucosaminyl (GlcNAc)-oligosaccharides was observed from the presymptomatic stage of the disease. In contrast, little MIP-1alpha-IR was observed in neurons in which GM2 accumulated predominantly. These results suggest that specific induction of MIP-1alpha might coincide with the accumulation of GlcNAc-oligosaccharides due to a HexB deficiency in resident microglia and astrocytes in the brains of SD mice causing their activation and acceleration of the progressive neurodegeneration in SD mice.     

Metabolic correction in microglia derived from Sandhoff disease model mice

Sandhoff disease is an autosomal recessive lysosomal storage disease caused by a defect of the beta-subunit gene (HEXB) associated with simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), and excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylglucosamine (GlcNAc) residues at their non-reducing termini. Recent studies have shown the involvement of microglial activation in neuroinflammation and neurodegeneration of this disease. We isolated primary microglial cells from the neonatal brains of Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex beta-subunit gene allele (Hexb-/-). The cells expressed microglial cell-specific ionized calcium binding adaptor molecule 1 (Iba1)-immunoreactivity (IR) and antigen recognized by Ricinus communis agglutinin lectin-120 (RCA120), but not glial fibrillary acidic protein (GFAP)-IR specific for astrocytes. They also demonstrated significant intracellular accumulation of GM2 and GlcNAc-oligosaccharides. We produced a lentiviral vector encoding for the murine Hex beta-subunit and transduced it into the microglia from SD mice with the recombinant lentivirus, causing elimination of the intracellularly accumulated GM2 and GlcNAc-oligosaccharides and secretion of Hex isozyme activities from the transduced SD microglial cells. Recomibinant HexA isozyme isolated from the conditioned medium of a Chinese hamster ovary (CHO) cell line simultaneously expressing the human HEXA (alpha-subunit) and HEXB genes was also found to be incorporated into the SD microglia via cell surface cation-independent mannose 6-phosphate receptor and mannose receptor to degrade the intracellularly accumulated GM2 and GlcNAc-oligosaccharides. These results suggest the therapeutic potential of recombinant lentivirus encoding the murine Hex beta-subunit and the human HexA isozyme (alphabeta heterodimer) for metabolic cross-correction in microglial cells involved in progressive neurodegeneration in SD mice.     

Molecular cloning and expression analysis of a putative nuclear protein, SR-25

We cloned a full-length mouse cDNA and its human homologue encoding a novel protein designated as "SR-25." In Northern blot analysis, SR-25 mRNA was expressed in all organs tested, and relatively abundant in testis and thymus. Deduced amino acid sequences of mouse SR-25 and human SR-25 showed 77.7% identity. SR-25 has a serine-arginine repeat (SR repeat) and two types of amino acid clusters: a serine cluster and a highly basic cluster. Based on the presence of many nuclear localizing signals and a similarity to RNA splicing proteins, SR-25 is strongly suggested to be a nuclear protein and may contribute to RNA splicing.     

NMR studies of the stable mismatch purine-thymine in the self-complementary d(CGPuAATTTCG) duplex in solution

One- and two-dimensional nuclear Overhauser effect experiments demonstrate that a single hydrogen bond between a T imino proton and purine N3 is sufficient to hold the base pair dPu.dT in d(CGPuAATTTCG) by a Watson-Crick fashion rather than a Hoogsteen type. In addition, the dPu.dT base pair is well stacked with neighboring base pairs. The spin-lattice relaxation measurements at 30 and 35 degrees C of two decamers, d(CGPuAATTTCG) and d(CGAAATTTCG), reveal that the elimination of two single hydrogen bonds of dA.dT base pairs (due to the substitution of adenine for purine) in the sequence results in an increase in the overall imino proton exchange rate from 7 to 36 s-1 at the site of mismatch.     

Ultrastructural insights into pathogen clearance by autophagy

Autophagy defends cells against proliferation of bacteria such as Salmonella in the cytosol. After escape from a damaged Salmonella‐containing vacuole (SCV) exposing luminal glycans that bind to Galectin‐8, the host cell ubiquitination machinery deposits a dense layer of ubiquitin around the cytosolic bacteria. The nature and spatial distribution of this ubiquitin coat in relation to other autophagy‐related membranes are unknown. Using transmission electron microscopy, we determined the exact localisation of ubiquitin, the ruptured SCV membrane and phagophores around cytosolic Salmonella. Ubiquitin was not predominantly present on the Salmonella surface, but enriched on the fragmented SCV. Cytosolic bacteria without SCVs were less efficiently targeted by phagophores. Single bacteria were contained in single phagophores but multiple bacteria could be within large autophagic vacuoles reaching 30 μm in circumference. These large phagophores followed the contour of the engulfed bacteria, they were frequently in close association with endoplasmic reticulum membranes and, within them, remnants of the SCV were seen associated with each engulfed particle. Our data suggest that the Salmonella SCV has a major role in the formation of autophagic phagophores and highlight evolutionary conserved parallel mechanisms between xenophagy and mitophagy with the fragmented SCV and the damaged outer mitochondrial membrane serving similar functions.     

Induction of Two Forms of Eel Cytochrome P450 1A Genes by 3-Methylcholanthrene

In eel (Anguilla japonica), exposure to polyaromatic hydrocarbons such as 3-methylcholanthrene leads to induction of two CYP1A enzymes, CYP1A1 and CYP1A6. We studied the time course and tissue specificity of induction of messenger RNAs for CYP1A1 and CYP1A6 in eel by administering 3-methylcholanthrene intraperitoneally. In both cases, the drug induced a rapid increase of mRNAs and biphasic expression. In the liver, mRNA levels of CYP1A1 and CYP1A6 increased 22-fold at 3 hours and 27-fold at 6 hours after the administration, respectively, showing initial peaks in the induction. After the initial inductions, mRNA levels decreased unexpectedly. Following these temporary decreases, the mRNA levels again increased and reached levels that were 35 and 41 times the basal levels at 24 hours after administration, respectively. CYP1A1 and CYP1A6 resembled each other also in the tissue specificity of gene expression; the expression levels were liver ≫ gill > intestine > kidney. The rapid induction, the biphasic expression, and the tissue-specific expression were common features of gene expression in CYP1A1 and CYP1A6 and may come from common structures of the regulatory regions of the two genes. Received December 7, 1998; accepted February 15, 1999