Segregation analysis of serum uric acid in the NHLBI Family Heart Study

Segregation analysis was performed on the serum uric acid measurements from 523 randomly ascertained Caucasian families from the NHLBI Family Heart Study. Gender-specific standardized residuals were used as the phenotypic variable in both familial correlation and segregation analysis. Uric acid residuals were adjusted for age, age2, age3, body mass index (kg/m2), creatinine level, aspirin use (yes/no), total drinks (per week), HOMA insulin resistance index [(glucose * insulin)/22.5], diuretic use (yes/no), and triglyceride level. Sibling correlations (r=0.193) and parent-offspring correlations (r=0.217) were significantly different from zero, but these two familial correlations were not significantly different from one another. After adjustment for covariates, the heritability estimate for serum uric acid was 0.399. Segregation analysis rejected the "no major gene" model but was unable to discriminate between an "environmental" and a "Mendelian major gene" model. These results support the hypothesis that uric acid is a multifactorial trait possibly influenced by more than one major gene, modifying genes, and environmental factors.     

Region-specific DNA damage by AT-specific DNA-reactive drugs is predicted by drug binding specificity

Bizelesin and adozelesin are DNA-reactive antitumor drugs that alkylate adenines at the 3' ends of their preferred binding sites [5'T(A/T)(4)A3'and 5'(A/T)(3)(-4)A3', respectively]. We used these drugs to examine the determinants for region-specific damage of human genomic DNA. The distribution of bizelesin binding motifs in several regions analyzed "in silico" correlated well with the experimentally determined lesions in these regions assessed by quantitative polymerase chain reaction (QPCR) stop assay. In contrast to the typically low motif density, clusters of potential bizelesin binding sites were found in the matrix-associated regions (MAR domains) of the c-myc and apolipoprotein B (apoB) genes. Accordingly, lesions induced by bizelesin in these domains (2.13 and 7.06 lesions kbp(-1) microM(-1), respectively) markedly exceeded lesions in bulk DNA (0.87 lesions kbp(-1) microM(-1)) or in regions with typically low motif density (e.g., 0.75 and 0.87 lesions kbp(-1) microM(-1) in a beta-globin gene and c-myc origin of replication regions, respectively). Consistent with the more frequent, less localized adozelesin motif, actual lesions induced by adozelesin exceeded by severalfold lesions by bizelesin in four selected regions (within the c-myc and HPRT loci). Whereas adozelesin is likely to affect similar regions as bizelesin, adozelesin's more promiscuous binding probably compromises its relative specificity for such targets. In contrast, findings for bizelesin provide for the first time a proof of principle that a small molecular weight drug can preferentially damage specific regions in cellular DNA. Targeting of critical repetitive sequences, such as AT-rich MAR domains, which allow for clustering of drug binding motif, can be the paradigm for region specificity of small molecular weight agents.     

pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis

We examined the effects of HCO(3)(-) and CO(2) acidosis on osteoclast-mediated Ca(2+) release from 3-day cultures of neonatal mouse calvaria. Ca(2+) release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H(+) to culture medium (HCO(3)(-) acidosis). For example, addition of 4 meq/l H(+) reduced pH from 7.12 to 7.03 and increased Ca(2+) release 3.8-fold. The largest stimulatory effects (8- to 11-fold), observed with 15-16 meq/l added H(+), were comparable to the maximal Ca(2+) release elicited by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3); 10 nM], parathyroid hormone (10 nM), or prostaglandin E(2) (1 microM); the action of these osteolytic agents was attenuated strongly when ambient pH was increased from approximately 7.1 to approximately 7.3. CO(2) acidosis was a less effective stimulator of Ca(2+) release than HCO(3)(-) acidosis over a similar pH range. Ca(2+) release stimulated by HCO(3)(-) acidosis was almost completely blocked by salmon calcitonin (20 ng/ml), implying osteoclast involvement. In whole mount preparations of control half-calvaria, approximately 400 inactive osteoclast-like multinucleate cells were present; in calvaria exposed to HCO(3)(-) acidosis and to the other osteolytic agents studied, extensive osteoclastic resorption, with perforation of bones, was visible. HCO(3)(-) acidosis, however, reduced numbers of osteoclast-like cells by approximately 50%, whereas 1,25(OH)(2)D(3) treatment caused increases of approximately 75%. The results suggest that HCO(3)(-) acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones, rather than by inducing formation of new osteoclasts, and provide further support for the critical role of acid-base balance in controlling osteoclast function.     

Inheritance of the Waist‐to‐Hip Ratio in the National Heart,Lung, and Blood Institute Family Heart Study

Objective: Considering that waist‐to‐hip ratio (WHR) is a simple anthropometric measure of obesity and is a better predictor of coronary heart disease than body mass index (BMI), the genetic underpinnings of WHR are of interest. The inheritance pattern of WHR, before and after adjustment for BMI (WHR‐BMI), was investigated in 2713 individuals from 1038 nuclear families in the National Heart, Lung, and Blood Institute Family Heart Study (NHLBI‐FHS). Research Methods and Procedures: Waist and hip measurements were taken twice, and the means of the measurements were used to calculate the WHR. Adjustments for age were carried out separately by sex, using stepwise multiple regression procedures for WHR and WHR‐BMI phenotypes. Segregation analysis was applied using the unified model as implemented in the computer program POINTER. Results: For age‐adjusted WHR, the segregation results suggested an additive major gene that accounts for 35% of the phenotypic variance, and approximately 30% of the sample are homozygous for the “high” genotype. The results for age‐ and BMI‐adjusted WHR were also compatible with a major gene; however, the multifactorial model provided the most parsimonious fit to the data. Discussion: Although the genetic mechanisms for several obesity traits have been studied, tests of Mendelian segregation on this simple anthropometric measure (WHR) have not been reported previously. This study provides evidence for the presence of a major gene for age‐adjusted WHR, suggesting that it is an appropriate trait for further genetic analysis, especially because it has strong predictive value and probably relates biologically to cardiovascular risk.     

Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry,Transparency, and Mechanical Stiffness of the Mouse Lens

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α₂β₂-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.     

18F]-N-Methylspiroperidol: the radioligand of choice for PETT studies of the dopamine receptor in human brain

N-Methylspiroperidol, the amide N-methyl analogue of the neuroleptic spiroperidol, was radiolabeled with fluorine-18, and its distribution in the baboon brain was studied using positron emission transaxial tomography. Stereospecific binding was demonstrated in the striatum (but not in the cerebellum) by pretreatment with (-)- or (+)-butaclamol. The kinetic distribution was similar to that of [18F]spiroperidol, but the absolute striatal uptake (in percent of administered dose) was at least two-fold higher. Analysis of baboon blood at 10 min after injection indicated that less than half of the radioactivity in the plasma was due to unchanged radioligand. Analysis of the metabolic stability of [18F]-N-methylspiroperidol in rat brain for 4 hr indicated that, like [18F]spiroperidol, it is very stable to metabolic transformation in the rat central nervous system. Striatal uptake and retention in the rat was five-fold higher for [18F]-N-methylspiroperidol than for [18F]spiroperidol. These results suggest that [18F]-N-methylspiroperidol is an ideal choice for studies of the dopamine receptor in humans.     

A rheumatoid factor paradox: inhibition of rituximab effector function

Introduction

Rituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC).

Methods

We developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated.

Results

Using human sera, addition of RTX resulted in rapid and profound (> 50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)'' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis.

Conclusions

Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.     

Quantitative-Trait Loci Influencing Body-Mass Index Reside on Chromosomes 7 and 13: The National Heart, Lung, and Blood Institute Family Heart Study

Obesity is a risk factor for many chronic diseases, including glucose intolerance, lipid disorders, hypertension, and coronary heart disease. Even though the body-mass index (BMI) is a heterogeneous phenotype reflecting the amount of fat, lean mass, and body build, several studies have provided evidence of one or two major loci contributing to the variation in this complex trait. We sought to identify loci with potential influence on BMI in the data obtained from National Heart, Lung, and Blood Institute Family Heart Study. Two complementary samples were studied: (a) 1,184 subjects in 317 sibships, with 243 markers typed by the Utah Molecular Genetics Laboratory (UMGL) and (b) 3,027 subjects distributed among 401 three-generation families, with 404 markers typed by the Mammalian Genotyping Service (MGS). A genome scan using a variance-components-based linkage approach was performed for each sample, as well as for the combined sample, in which the markers from each analysis were placed on a common genetic map. There was strong evidence for linkage on chromosome 7q32.3 in each sample: the maximum multipoint LOD scores were 4.7 (P<10-5) at marker GATA43C11 and 3.2 (P=.00007) at marker D7S1804, for the MGS and UMGL samples, respectively. The linkage result is replicated by the consistent evidence from these two complementary subsets. Furthermore, the evidence for linkage was maintained in the combined sample, with a LOD score of 4.9 (P<10-5) for both markers, which map to the same location. This signal is very near the published location for the leptin gene, which is the most prominent candidate gene in this region. For the combined-sample analysis, evidence of linkage was also found on chromosome 13q14, with D13S257 (LOD score 3.2, P=.00006), and other, weaker signals (LOD scores 1.5-1.9) were found on chromosomes 1, 2, 3, 5, 6, 14, and 15.     

Transcriptional response to alcohol exposure in Drosophila melanogaster

Background  

Alcoholism presents widespread social and human health problems. Alcohol sensitivity, the development of tolerance to alcohol and susceptibility to addiction vary in the population. Genetic factors that predispose to alcoholism remain largely unknown due to extensive genetic and environmental variation in human populations. Drosophila, however, allows studies on genetically identical individuals in controlled environments. Although addiction to alcohol has not been demonstrated in Drosophila, flies show responses to alcohol exposure that resemble human intoxication, including hyperactivity, loss of postural control, sedation, and exposure-dependent development of tolerance.