A genome scan for loci linked to quantitative insulin traits in persons without diabetes: the Framingham Offspring Study.

AIMS/HYPOTHESIS: Hyperinsulinaemia and insulin resistance usually precede clinical hyperglycaemia and Type 2 diabetes. Thus, plasma insulin concentrations and insulin resistance are important quantitative traits associated with risk of Type 2 diabetes, and represent key measures for genetic analysis of the syndrome. METHODS: We carried out a genome-wide search for loci related to plasma insulin concentrations and insulin resistance in 330 extended, community-based pedigrees from the Framingham Heart Study. Normalized deviates of the standardized residuals of plasma insulin concentrations in the fasting state, 2 h after oral glucose challenge and as a measure of insulin resistance were used in linkage analysis with the variance components model implemented in the computer program SOLAR. RESULTS: The results suggest susceptibility loci influencing plasma concentrations of fasting insulin and insulin resistance on chromosomes 11 (LOD 2.43 at 85 cM close to D11S2002) and 17 (LOD 1.8 at 60 cM, close to D17S784); and susceptibility loci influencing 2-h plasma insulin concentrations on chromosomes 9 (LOD 2.8 at 80 cM, close to D9S922) and 19 (LOD 1.8 at 66 cM, close to D19S245). The results of the analysis of 1000 simulations of the trait and an unlinked marker suggest that in a genome scan of 401 markers fewer than one LOD score over 1 would be due to Type 1 error, and be a false positive. CONCLUSION/INTERPRETATION: We conclude that these suggestive regions for quantitative pre-diabetic insulin traits could contain major loci in the pathogenesis of Type 2 diabetes.

A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide.

An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.

Z-DNA binding protein from chicken blood nuclei.

A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

T-cell ontogeny: the role of a stimulator - suppressor cell.

The thymus presents two major problems in cellular differentiation. How is self-non-self discrimination achieved in developing thymocytes? What determines the development of T-cell classes? In this discussion, Alan Herbert and James Watson propose a mechanism for regulating T-cell differentiation which involves the alternative pathway of T-cell activation. They postulate that T cells with a stimulator-suppressor phenotype stimulate resting helper T cells (Th) to produce interleukin 2 (IL-2) and suppress T cells which have bound antigen through antigen-specific receptors by preventing induction of IL-2 receptors. Stimulator-suppressor T cells therefore suppress the clonal expansion of T cells in an antigen-specific manner, yet promote their own clonal expansion in a manner independent of antigen. They further suggest that the molecule responsible for suppression is the product of the γ genes known to rearrange in γ cells.

The effects of sodium butyrate on lymphokine production.

The effect of sodium butyrate on lymphokine production in WEHI-3, LBRM-33, and JURKAT cells, as well as an IL-2-dependent T cell line (I) was studied. Constitutive production of IL-3 by WEHI-3 cells was enhanced by sodium butyrate and was accompanied by the growth arrest of WEHI-3 cells in the G1 phase of the cell cycle. Treatment of I cells with ConA and sodium butyrate led to an increase in IL-3 production and the initiation of IL-2 production. In contrast, the synthesis of IL-2 and IL-3 by the ConA-activated T lymphoma line LBRM-33 was coordinately inhibited by sodium butyrate. Similarly, the synthesis of IL-2 by the human T cell lymphoma JURKAT was inhibited by sodium butyrate. No cell cycle-specific effects of sodium butyrate were observed in LBRM-33 or JURKAT cells.

In vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. III. Spontaneous and natural cytotoxic effector cells.

Cytotoxic effector cell populations in murine spleen can be characterized by the phenotype of the cytotoxic cells or the nature of target cells. Lytic events can be antigen-specific, MHC-restricted and clonal, or target cell-specific but apparently non-MHC-restricted. Two cytotoxic effectors of this latter category are spontaneous and natural killers. Normal spleen cells from (BALB/c X DBA/2J)F1 mice (CDF1) cultured without added antigen develop a population of Thy-1+ spontaneous cytotoxic lymphocytes (SCTL) that lyse the DBA/2J mastocytoma P815, as well as the BALB/c-derived plasmacytomas MOPC-11 and SP2/0. Cold target competition experiments reveal the BALB/c-derived plasmacytomas MOPC-11, SP2/0, J558 and the A strain-derived T cell lymphoma YAC-1, but not normal lymphoblasts, block the lysis of P815 target cells. Thus, while these tumour cells appear to express common antigens which are recognized by SCTL cells, plasmacytomas such as J558 are not susceptible to lysis by SCTL. The relationship of SCTL to natural killer (NK) cells was examined. In-vivo treatment of mice with monoclonal anti-Thy-1 antibody leads to a rapid loss of SCTL and precursors from the spleen, but there is a concomitant increase in NK cell activity.

In vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. II. Separation of natural killer cells and cytotoxic T cells.

Treatment of mice with ascitic fluid containing high titres of T24-31.7 monoclonal antibody (rat anti-mouse Thy-1) lead to a rapid loss of T cells from peripheral lymphoid organs. Spleen and lymph node tissue lost all detectable Thy-1+ and mitogen-responsive T cells within 72 hr. These tissues were completely T cell-depleted for more than a week before repopulation with T cells began. Lectin-induced splenic T cell cytoxicity in culture was lost within 72 hr after treatment of mice in vivo. In contrast, treatment of mice with T24-31.7 ascitic fluid was followed by an immediate increase in natural killer (NK) cell-mediated cytolytic activity. After 96 hr, NK activity began to decrease and did not reappear in the T cell-depleted spleens. While purified T24-31.7 antibody was responsible for T cell depletion in vivo, a non-immunoglobulin component of the ascitic fluid stimulated splenic NK cell activity. The presence of phytohaemagglutinin (PHA) on target cells in the lytic assay was shown to inhibit NK activity but enhance T cell-mediated cytotoxicity. The relationship of NK cells and cytotoxic T lymphocytes (CTL) to the T cell lineage is discussed.