Identification of longevity-associated genes in long-lived Snell and Ames dwarf mice

Abstract Recent landmark molecular genetic studies have identified an evolutionarily conserved insulin/IGF-1 signal transduction pathway that regulates lifespan. In C. elegans, Drosophila, and rodents, attenuated insulin/IGF-1 signaling appears to regulate lifespan and enhance resistance to environmental stress. The Ames (Prop1 df/df ) and Snell (Pit1 dw/dw ) hypopituitary dwarf mice with growth hormone (GH), thyroidstimulating hormone (TSH), and prolactin deficiencies live 40-60% longer than control mice. Both mutants are resistant to multiple forms of environmental stress in vitro. Taken collectively, these genetic models indicate that diminished insulin/IGF-l signaling may play a central role in the determination of mammalian lifespan by conferring resistance to exogenous and endogenous stressors. These pleiotropic endocrine pathways control diverse programs of gene expression that appear to orchestrate the development of a biological phenotype that promotes longevity. With the ability to investigate thousands of genes simultaneously, several microarray surveys have identified potential longevity assurance genes and provided information on the mechanism(s) by which the dwarf genotypes (dw/dw) and (df/df), and caloric restriction may lead to longevity. We propose that a comparison of specific changes in gene expression shared between Snell and Ames dwarf mice may provide a deeper understanding of the transcriptional mechanisms of longevity determination. Furthermore, we propose that a comparison of the physiological consequences of the Pit1dw and Prop1df mutations may reveal transcriptional profiles similar to those reported for the C. elegans and Drosophila mutants. In this study we have identified classes of genes whose expression is similarly affected in both Snell and Ames dwarf mice. Our comparative microarray data suggest that specific detoxification enzymes of the P 450 (CYP) family as well as oxidative and steroid metabolism may play a key role in longevity assurance of the Snell and Ames dwarf mouse mutants. We propose that the altered expression of these genes defines a biochemical phenotype which may promote longevity in Snell and Ames dwarf mice

Analysis of Fcγ receptor haplotypes in rheumatoid arthritis: FCGR3A remains a major susceptibility gene at this locus, with an additional contribution from FCGR3B

The Fcγ receptors play important roles in the initiation and regulation of many immunological and inflammatory processes, and genetic variants (FCGR) have been associated with numerous autoimmune and infectious diseases. The data in rheumatoid arthritis (RA) are conflicting and we previously demonstrated an association between FCGR3A and RA. In view of the close molecular proximity with FCGR2A, FCGR2B and FCGR3B, additional polymorphisms within these genes and FCGR haplotypes were examined to refine the extent of association with RA. Biallelic polymorphisms in FCGR2A, FCGR2B and FCGR3B were examined for association with RA in two well characterized UK Caucasian and North Indian/Pakistani cohorts, in which FCGR3A genotyping had previously been undertaken. Haplotype frequencies and linkage disequilibrium were estimated across the FCGR locus and a model-free analysis was performed to determine association with RA. This was followed by regression analysis, allowing for phase uncertainty, to identify the particular haplotype(s) that influences disease risk. Our results reveal that FCGR2A, FCGR2B and FCGR3B were not associated with RA. The haplotype with the strongest association with RA susceptibility was the FCGR3A–FCGR3B 158V-NA2 haplotype (odds ratio 3.18, 95% confidence interval 1.13–8.92 [P = 0.03] for homozygotes compared with all genotypes). The association was stronger in the presence of nodules (odds ratio 5.03, 95% confidence interval 1.44–17.56; P = 0.01). This haplotype was also more common in North Indian/Pakistani RA patients than in control individuals, but not significantly so. Logistic regression analyses suggested that FCGR3A remained the most significant gene at this locus. The increased association with an FCGR3A–FCGR3B haplotype suggests that other polymorphic variants within FCGR3A or FCGR3B, or in linkage disequilibrium with this haplotype, may additionally contribute to disease pathogenesis

Human T cell lines differing in phenotype and specificity are reactive with the same anti-idiotypic antibody

3D6, a monoclonal antibody selected for reactivity with the T cell antigen receptor on the T leukemic cell line HPB-ALL, was found to react with 3 to 13% of peripheral blood T lymphocytes of 10 out of 15 normal donors. Peripheral T cells of two donors were stimulated with allogeneic cells, and the 3D6+ cells were enriched by rosetting 3D6-coated cells with goat anti-mouse-coupled human red blood cells and were expanded in interleukin 2-containing medium. In this way, 90 to 100% 3D6+ cell lines were obtained that were cytotoxic for the allogeneic stimulator cells. 3D6 antibody could block antigen-specific cytotoxicity, as well as induce nonspecific cytotoxicity toward target cells that could not be killed in the absence of the 3D6 antibody. The 3D6+ cell populations contained T4+, as well as T8+ cells, indicating that 3D6 antibody defined a T cell receptor population that might harbor various antigenic specificities. One 3D6+ cell line was separated into T4+ T8- and T4- T8+ populations. 3D6 reactive T cell receptors isolated from HPB-ALL and normal cell lines were analyzed biochemically by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and V8 protease peptide mapping. Isoelectric focusing analysis provided additional evidence for the idea that 3D6 antibody detected a number of structurally distinct T cell receptors, because the T cell receptor alpha-chain was homogeneous in charge after desialation on the clonal tumor line HPB-ALL, but remained heterogeneous in charge on the 3D6+ normal cell lines. No great differences in charge were found between T cell receptors isolated from T4+ and T8+ 3D6+ lines, but their isoelectric focusing patterns were not identical. V8 protease peptide mapping revealed structural differences between the T cell receptor alpha-chain isolated from HPB-ALL on one hand and from the normal 3D6+ lines on the other, whereas the beta-chains did not differ greatly in primary structure according to this analysis. In addition, the peptide mapping suggested differences in primary structure between T cell receptors present on the T4+ population vs those present on the T8+ population

Properties of a panel of monoclonal antibodies which react with the human T cell antigen receptor on the leukemic line HPB-ALL and a subset of normal peripheral blood T lymphocytes

Five Mab raised against the T cell antigen receptor of the human T cell line HPB-ALL which react with a subpopulation of normal peripheral blood T cells are described. Three Mab, 3D6, 1C1, and 1C2, react with 3 to 5% of normal PBL and stimulate proliferation of the cells with which they react. An increase in the number of cells which react with all five Mab occurs. Two Mab, 2D4 and 65, react with subsets of the cells which bind 1C1, 1C2, and 3D6 and divide the family into four subgroups, 2D4+ 65+, 2D4+ 65-, 2D4- 65+, and 2D4- 65-. Functional T cell clones in all four subfamilies have been observed. Cytolytic function can be correlated with the TcR phenotype expressed because all of the Mab which react with a particular clone inhibit its ability to lyse a specific target. The epitopes recognized by the panel are closely related because all five block each other's binding to HPB-ALL. In addition, the determinants recognized by 3D6, 1C1, and 1C2 on normal lymphocytes are probably very closely related because all clones examined react with all three Ma

Altered oxidative stress response of the long-lived Snell dwarf mouse.

Several single gene mutations in mice that increase the murine life span have been identified, including the Pit-1 mutation which results in the Snell dwarf (Pit1(dw/dw)), however, the biological mechanism of this life-span extension is still unclear. Based on studies that show oxidative stress plays an important role in the aging process, we hypothesized that the increased longevity seen in Snell dwarf mice may result from a resistance to oxidative stress. We report that Snell dwarf mice respond to oxidative stress induced by 3-NPA differently than their wild type littermates. This altered response results in diminished activation of the MEK-ERK kinase cascade and virtually no phosphorylation of c-Jun at Ser63 in dwarf mice after 3-NPA treatment, despite a robust phosphorylation of Ser63 in wild type mice. We propose that this altered management of oxidative stress in dwarf mice is partially responsible for the increased longevity in Snell dwarf mice

Altered cholesterologenic and lipogenic transcriptional profile in livers of aging Snell dwarf (Pit1dw/dwJ) mice.

Several murine models demonstrate that mammalian longevity can be increased by single gene mutations affecting endocrine signalling, particularly via the GH/IGF-1 axis. In this study, we identify age-independent patterns of hepatic gene expression characteristic of long-lived Snell (Pit1(dw/dwJ)) dwarf mice. Comparative microarray analysis of young and aged male livers was performed to discover specific genes differentially expressed between Pit1(dw/dwJ) and control mice. Further examination by real-time RT-PCR confirmed that transcripts encoding HMG-CoA synthase-1, HMG-CoA reductase, farnesyl diphosphate synthase, isopentenyl pyrophosphate isomerase, mevalonate decarboxylase, squalene epoxidase, lanosterol demethylase, malic enzyme and apolipoprotein A-IV were significantly decreased in both male and female Pit1(dw/dwJ) livers at 3-5 and 24-28 months of age. In contrast, transcripts encoding the beta(3)-adrenergic receptor, lipoprotein lipase, PPAR gamma and a very low-density lipoprotein receptor homologue were increased significantly in dwarf livers relative to age-matched controls. These studies reveal enduring transcriptional changes characteristic of Pit1(dw/dwJ) dwarf mice that involve genes regulating cholesterol biosynthesis, fatty acid metabolism and lipoprotein homeostasis. Linked to global energy metabolism, this stable shift in hepatic gene expression may contribute to longevity determination by influencing particular metabolic functions often compartmentalized within the mitochondrion and peroxisome; further this metabolic shift may also parallel many transcriptional changes induced by caloric restriction

Complexity of T cell receptor recognition sites for defined alloantigens

Three monoclonal antibodies react with the T cell receptor on the tumor line HPB-ALL and in addition with 3 to 13% of human peripheral blood T cells of normal donors. These antibodies are shown to react with an epitope encoded by the V beta 5 family of T cell receptor beta-chain variable region gene segments. Cells expressing V beta 5 gene segments can have cytotoxic or helper function, be of the T4+ or T8+ phenotype, and have specificity for either class I or class II major histocompatibility complex alloantigens. Seven T cell clones were generated, which express V beta 5 and are specific for the HLA-A2 molecule. With the use of these clones, we illustrate how isoelectric focusing can be used to analyze T cell receptor alpha- and beta-chain structure. The seven clones recognize five distinct conformational determinants on HLA-A2. They procure different binding sites by the use of different alpha-chains, J beta sequences, or bot