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Background Increasingly, genetic specimens are collected to expand the value of clinical trials through study of genetic effects on disease incidence, progression or response to interventions. Purpose and methods We describe the experience obtaining IRB-approved DNA consent forms across the 19 institutions in the Action for Health in Diabetes (Look AHEAD), a clinical trial examining the effect of a lifestyle intervention for weight loss on the risk of serious cardiovascular events among individuals with type 2 diabetes. We document the rates participants provided consent for DNA research, identify participant characteristics associated with consent, and discuss implications for genetics research. Results IRB approval to participate was obtained from 17 of 19 institutions. The overall rate of consent was 89.6% among the 15 institutions that had completed consenting at the time of our analysis, which was higher than reported for other types of cohort studies. Consent rates were associated with factors expected to be associated with weight loss and cardiovascular disease and to affect the distribution of candidate genes. Non-consent occurred more frequently among participants grouped as African-American, Hispanic, female, more highly educated or not dyslipidemic. Limitations The generalizabilty of results is limited by the inclusion/exclusion criteria of the trial. Conclusions Barriers to obtaining consent to participate in genetic studies may differ from other recruitment settings. Because of the potentially complex associations between personal characteristics related to adherence, outcomes and gene distributions, differential rates of consent may introduce biases in estimates of genetic relationships.

The role of cationic amino acids in the Na(+)/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased K(m) for succinate of 1.8 mM, compared with 0.3 mM in the wild-type NaDC-1. The R349A mutant had Na(+) and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

Modern mass spectrometry (MS) streamlined with two-dimensional gel electrophoresis, in-gel digestion and HPLC-interfaced electrospray ionization quadrupole MS or matrix-assisted laser desorption ionization time-of-flight MS enables us to analyse proteins at a minuscule scale. We present here two examples of MS applications in which (1) we identified the in vivo phosphorylation site of Drosophila arrestin, phosrestin I (PRI), and (2) we revealed the identity of an 80 kDa phosphoprotein (80K) in Drosophila eyes to be the InaD gene product, a member of the PDZ domain proteins. Available evidence suggests that PRI quenches the activation of rhodopsin and that the InaD protein adjusts photoreceptor responsiveness by assembling/disassembling components involved in photoreceptor transduction in flies. PRI undergoes a reversible phosphorylation at a single site, and 80K at multiple sites. The phosphorylation states of PRI and 80K depend on the intensity and/or duration of light stimuli. From these results we postulate that these proteins function as a molecular switch adjusting the signalling cascade through phosphorylation. The combination of two-dimensional gel electrophoresis with MS will be a powerful tool for detailed investigation of such complex switching processes. The techniques described here can be applied also to other complex signalling systems.

Research over the past decade indicates that aspects of maternal nutrition may play a greater role in immediate and long-term health of offspring than was thought previously. The effects of nutritional exposures on outcomes likely depend primarily on the timing of insults during pregnancy and may not have to be severe to have a long-term impact on health. If the rate of change in knowledge of the effects of maternal nutritional exposures on outcomes continues at its current pace, work initiated in the past decade may well be looked back upon as representing the renaissance of maternal nutrition research.

The Atherosclerosis Risk in Communities Study investigators examined nearly 4000 postmenopausal women from 1987 through 1989 and 3 years later to determine changes in plasma lipids occurring with the starting or stopping of hormone replacement therapy. Women who started estrogen plus progestin therapy (n = 74) had decreases of 9.8 mg/dl in low-density lipoprotein (LDL) cholesterol and 5.8 mg/dl in apolipoprotein B and increases of 1.2 mg/dl in high-density lipoprotein (HDL) cholesterol (HDL change not significant), 13.5 mg/dl in apolipoprotein A-I, and 14.0 mg/dl in triglycerides. Women who started estrogen alone (n = 149) had similar changes, except for a much larger increase in HDL cholesterol (5.8 mg/dl), principally in HDL-2. Women who stopped hormone therapy (n = 138) had lipid changes opposite to those who started therapy, but smaller in magnitude. These results confirm those of the Postmenopausal Estrogen/Progestin Interventions Trial in a community-based longitudinal cohort: women initiating estrogen plus progestin therapy have decreases in LDL cholesterol, but the increase in HDL cholesterol is less than that for starting estrogen alone. In addition, the current study extends findings to apolipoproteins and HDL subfractions.

Differences in the generation of anti-H-2Kb wild-type specific cytotoxic T lymphocytes (CTL) and specific suppressor T cells (SSTC) were investigated in H-2Kbm mutant mouse strains. To this end, optimal conditions for in vivo induction of highly active CTL in this mutant system were found and the T-cell origin of CTL and SSTC was confirmed using the anti-Thy-1.2 monoclonal antibody G4. Unlike the CTL, which were generated in vivo by any of the mutant strains tested (bm1, bm3, and bm4), the SSTC were only produced by bm3, whose H-2Kbm3 antigen, in contrast to the other H-2Kbm molecules, differs from wild type by serologically defined determinants. The high activity of anti-wild type bm4 CTL induced in vivo contrasted with a low activity of such CTL induced in mixed lymphocyte culture (MLC). This appeared to be the property of bm4 only, but not of bm1 or bm3, and it was reproduced in the reciprocal system B10 anti-bm4. CTL generation could be restored in the MLC by the addition of concanavalin A supernate or a mixture of bm4 and bm12 stimulator cells. Three of the six in vivo induced and in vitro propagated bm3 anti-B6 CTL clones demonstrated selective cross-reactivity to only one of the third-party H-2K molecules used, either Kk, Kd, or Kbm4. The present results indicate that (a) the SSTC and antibody recognize similar H-2Kb epitopes; (b) the H-2Kb epitopes recognized by the CTL and SSTC are not identical; (c) the genetic control of CTL generation in vivo is distinct from that in MLC, and (d) the affinities of antigen-specific receptors on T-cell clones of the same specificity may be different, leading to their individual cross-reactivity patterns.