Probing the gamma2 calcium-binding site: studies with gammaD298,301A fibrinogen reveal changes in the gamma294-301 loop that alter the integrity of the "a" polymerization site

To determine the significance of the gamma2 calcium-binding site in fibrin polymerization, we synthesized the fibrinogen variant, gammaD298,301A. We expected these two alanine substitutions to prevent calcium binding in the gamma2 site. We examined the influence of calcium on the polymerization of gammaD298,301A fibrinogen, evaluated its plasmin susceptibility, and solved 2.7 and 2.4 A crystal structures of the variant with the peptide ligands Gly-Pro-Arg-Pro-amide (GPRP) and Gly-His-Arg-Pro-amide (GHRP), respectively. We found that thrombin-catalyzed polymerization of gammaD298,301A fibrinogen was modestly impaired, whereas batroxobin-catalyzed polymerization was significantly impaired relative to normal fibrinogen. Notably, the influence of calcium on polymerization was the same for the variant and for normal fibrinogen. Fibrinogen gammaD298,301A was more susceptible to plasmin proteolysis in the presence of GPRP. This finding suggests structural changes in the near-by "a" polymerization site. Comparisons of the structures revealed minor conformational changes in the gamma294-301 loop that are likely responsible for the weakened "a" site. When considered altogether, the data suggest that the gamma2 calcium-binding site does not significantly modulate polymerization. We cannot, however, rule out the possibility that the weakened "a" polymerization site masks an important role for the gamma2 calcium-binding site in normal polymerization. Somewhat unexpectedly, the structure data showed that GPRP bound to the "b" site and induced the same local conformational changes as GHRP to this site. This structure shows that "A:b" interactions can occur and suggests that these may participate in normal polymerization.     

The oxidative stress metabolite 4-hydroxynonenal promotes Alzheimer protofibril formation

4-Hydroxynonenal (4-HNE), formed as a consequence of oxidative stress, exists at increased concentrations in Alzheimer's disease (AD) patients and is found in amyloid beta peptide (Abeta) plaques associated with AD. Although it remains an open question as to whether oxidative stress is a causative factor or a consequence of AD, we show here that 4-HNE, putatively resulting from the peroxidation of lipids, covalently modifies Abeta, triggering its aggregation. These Abeta modifications result from 1,4 conjugate addition and/or Schiff base formation, they occur at multiple locations on a single Abeta peptide, and they result in covalent cross-linking of Abeta peptides. The consequence of these reactions is that 4-HNE accelerates the formation of Abeta protofibrils while inhibiting the production of straight, mature fibrils. Recent studies implicating Abeta oligomers and protofibrils in the neurotoxic process that ultimately leads to AD suggest that the Abeta aggregates induced by 4-HNE may be important in the pathogenesis of AD. These results provide further incentive to understand the role of oxidative stress and small-molecule Abeta modifications in sporadic AD.     

Heritability of brachydactyly type A3 in children, adolescents, and young adults from an endogamous population in eastern Nepal

Brachymesophalangia-V (BMP-V), a short and broad middle phalanx of the fifth digit, is the most common of all skeletal anomalies of the hand. When this feature appears alone, it is clinically known as brachydactyly type A3 (BDA3). A high prevalence of BDA3 has been observed among the children of the Jirel ethnic group in eastern Nepal. As part of the Jiri Growth Study, a hand-wrist radiograph is taken annually of each child to assess skeletal development. For this study the most recent radiographs of 1,357 Jirel children, adolescents, and young adults (676 boys, 681 girls), age 3-20 years, were examined for the presence or absence of BDA3, to report the prevalence and estimate the heritability of BDA3 in the Jirel population. The overall prevalence of BDA3 in this sample was 10.5% (12.9% of the males and 8.9% of the females were classified as BDA3 affected). The additive genetic heritability of BDA3 was statistically significant in this sample (h2 +/- SE = 0.87 +/- 0.16, p < 0.0001). This study is the first to estimate the prevalence and heritability of BDA3 in a large South Asian family-based sample.     

Epithelial Na+ channel stimulation by n-3 fatty acids requires proximity to a membrane-bound A-kinase-anchoring protein complexed with protein kinase A and phosphodiesterase

Essential polyunsatured fatty acids have been shown to modulate enzymes, channels and transporters, to interact with lipid bilayers and to affect metabolic pathways. We have previously shown that eicosapentanoic acid (EPA, C20:5, n-3) activates epithelial sodium channels (ENaCs) in a cAMP-dependent manner involving stimulation of cAMP-dependent protein kinase (PKA). In the present study, we explored further the mechanism of EPA stimulation of ENaC in A6 cells. Fluorescence resonance energy transfer experiments confirmed activation of PKA by EPA. Consistent with our previous studies, EPA had no further stimulatory effect on amiloride-sensitive transepithelial current (INa) in the presence of CPT-cAMP. Thus, we investigated the effect of EPA on cellular pathways which produce cAMP. EPA did not stimulate adenylate cyclase activity or total cellular cAMP accumulation. However, membrane-bound phosphodiesterase activity was inhibited by EPA from 2.46 pmol/mg of protein/min to 1.3 pmol/mg of protein/min. To investigate the potential role of an A-kinase-anchoring protein (AKAP), we used HT31, an inhibitor of the binding between PKA and AKAPs as well as cerulenin, an inhibitor of myristoylation and palmitoylation. Both agents prevented the stimulatory effect of EPA and CPT-cAMP on INa and drastically decreased the amount of PKA in the apical membrane. Colocalization experiments in A6 cells cotransfected with fluorescently labeled ENaC beta subunit and PKA regulatory subunit confirmed the close proximity of the two proteins and the membrane anchorage of PKA. Last, in A6 cells transfected with a dead mutant of Sgk, an enzyme which up-regulates ENaCs, EPA did not stimulate Na+ current. Our results suggest that stimulation of ENaCs by EPA occurs via SGK in membrane-bound compartments containing an AKAP, activated PKA, and a phosphodiesterase.     

Mutations in autism susceptibility candidate 2 (AUTS2) in patients with mental retardation

We report on three unrelated mentally disabled patients, each carrying a de novo balanced translocation that truncates the autism susceptibility candidate 2 (AUTS2) gene at 7q11.2. One of our patients shows relatively mild mental retardation; the other two display more profound disorders. One patient is also physically disabled, exhibiting urogenital and limb malformations in addition to severe mental retardation. The function of AUTS2 is presently unknown, but it has been shown to be disrupted in monozygotic twins with autism and mental retardation, both carrying a translocation t(7;20)(q11.2;p11.2) (de la Barra et al. in Rev Chil Pediatr 57:549–554, 1986; Sultana et al. in Genomics 80:129–134, 2002). Given the overlap of this autism/mental retardation (MR) phenotype and the MR-associated disorders in our patients, together with the fact that mapping of the additional autosomal breakpoints involved did not disclose obvious candidate disease genes, we ascertain with this study that AUTS2 mutations are clearly linked to autosomal dominant mental retardation.     

Linkage and association analysis of candidate genes for TB and TNFα cytokine expression: evidence for association with IFNGR1, IL-10, and TNF receptor 1 genes

Tuberculosis (TB) is a growing public health threat globally and several studies suggest a role of host genetic susceptibility in increased TB risk. As part of a household contact study in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB by developing an intermediate phenotype model for TB susceptibility, analyzing levels of tumor necrosis factor-α (TNFα) in response to culture filtrate as the phenotype. In the present study, we analyzed candidate genes related to TNFα regulation and found that interleukin (IL)-10, interferon-gamma receptor 1 (IFNGR1), and TNFα receptor 1 (TNFR1) genes were linked and associated to both TB and TNFα. We also show that these associations are with progression to active disease and not susceptibility to latent infection. This is the first report of an association between TB and TNFR1 in a human population and our findings for IL-10 and IFNGR1 replicate previous findings. By observing pleiotropic effects on both phenotypes, we show construct validity of our intermediate phenotype model, which enables the characterization of the role of these genetic polymorphisms on TB pathogenesis. This study further illustrates the utility of such a model for disentangling complex traits. C. C. Whalen and S. K. Iyengar contributed equally as senior authors of this work.     

Sex-based differences in myocardial contractile reserve

Recent studies have identified sex differences in heart function that may affect the risk of developing heart failure. We hypothesized that there are fundamental differences in calcium (Ca) regulation in cardiac myocytes of males and premenopausal females. Isometric force transients (n = 45) were measured at various stimulation frequencies to define the force frequency responses (FFR) (0.5, 1.0, 1.5, and 2.0 Hz) during either changes in bath Ca ([Ca]o) (1.0, 1.75, 3.5, and 7.0 mM) or length-tension (20, 40, 60, 80, and 100% L(max)) in right ventricle trabeculae from normal male (MT) and premenopausal female (FT) cats. Force-Ca measurements were also obtained in chemically skinned trabeculae. Under basal conditions (0.5 Hz, 1.75 mM Ca, 80% L(max)) both MT and FT achieved similar developed forces (DF) (MT 11 +/- 1, FT = 10 +/- 1 mN/mm2). At low rates and lengths, there is no sex difference. At higher preloads and rates, there is a separation in DF in MT and FT. At basal [Ca]o both MT and FT exhibited positive FFR (2.0 Hz, 1.75 mM Ca: MT 38 +/- 3, FT 21 +/- 4 mN/mm2); however, at higher [Ca]o, MT achieved greater DF (2.0 Hz, 7.0 mM Ca: MT 40 +/- 3 and FT = 24 +/- 4 mN/mm2). We detected no sex difference in myofilament Ca sensitivity at a sarcomere length of 2.1 mum. However, rapid cooling contractures indicated greater sarcoplasmic reticulum (SR) Ca load in MT at higher frequencies. Despite virtually identical contractile performance under basal conditions, significant sex differences emerge under conditions of increased physiological stress. Given the lack of sex differences in myofilament Ca sensitivity, these studies suggest fundamental sex differences in cellular Ca regulation to achieve contractile reserve, with myocardium from males exhibiting higher SR Ca load.