High prevalence of aspartylglycosaminuria among school-age children in eastern Finland

Summary A high prevalence of the lysosomal storage disease aspartylglycosaminuria was found in a study of four birth cohorts of 12882 children in eastern Finland. Using school achievement tests and registers of mentally retarded individuals, 178 mentally retarded children were identified. Randomized urine samples from 151 of the 178 retarded children and from 101 healthy children were analyzed quantitatively for aspartylglucosamine by high-performance liquid chromatography. The results identified three affected individuals in the retarded group indicating an exceptionally high prevalence of aspartylglycosaminuria (1:3643) in the study population, consistent with a carrier frequency of 1:30. The 95% confidence limits for the prevalence are 1:4 352-1:16389. This is the highest prevalence described for any glycoproteinosis in any population and comparable to the incidence figures of the most common lysosomal storage diseases, Gaucher disease type I and Tay-Sachs disease among Ashkenazi Jews. In the study group, aspartylglycosaminuria was, after trisomy 21 (n = 19) and the fragile X syndrome (n = 6), the most common genetic cause for mental retardation.     

Employing a Monte Carlo Algorithm in Newton-Type Methods for Restricted Maximum Likelihood Estimation of Genetic Parameters

Estimation of variance components by Monte Carlo (MC) expectation maximization (EM) restricted maximum likelihood (REML) is computationally efficient for large data sets and complex linear mixed effects models. However, efficiency may be lost due to the need for a large number of iterations of the EM algorithm. To decrease the computing time we explored the use of faster converging Newton-type algorithms within MC REML implementations. The implemented algorithms were: MC Newton-Raphson (NR), where the information matrix was generated via sampling; MC average information(AI), where the information was computed as an average of observed and expected information; and MC Broyden''s method, where the zero of the gradient was searched using a quasi-Newton-type algorithm. Performance of these algorithms was evaluated using simulated data. The final estimates were in good agreement with corresponding analytical ones. MC NR REML and MC AI REML enhanced convergence compared to MC EM REML and gave standard errors for the estimates as a by-product. MC NR REML required a larger number of MC samples, while each MC AI REML iteration demanded extra solving of mixed model equations by the number of parameters to be estimated. MC Broyden''s method required the largest number of MC samples with our small data and did not give standard errors for the parameters directly. We studied the performance of three different convergence criteria for the MC AI REML algorithm. Our results indicate the importance of defining a suitable convergence criterion and critical value in order to obtain an efficient Newton-type method utilizing a MC algorithm. Overall, use of a MC algorithm with Newton-type methods proved feasible and the results encourage testing of these methods with different kinds of large-scale problem settings.     

Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated

Twinfilin is a highly conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. In addition to the previously characterized mammalian twinfilin-1, a second protein with approximately 65% sequence identity to twinfilin-1 exists in mouse and humans. However, previous studies failed to identify any actin binding activity in this protein (Rohwer, A., Kittstein, W., Marks, F., and Gschwendt, M. (1999) Eur. J. Biochem. 263, 518-525). Here we show that this protein, which we named twinfilin-2, is indeed an actin monomer-binding protein. Similar to twinfilin-1, mouse twinfilin-2 binds ADP-G-actin with a higher affinity (KD = 0.12 microM) than ATP-G-actin (KD = 1.96 microM) and efficiently inhibits actin filament assembly in vitro. Both mouse twinfilins inhibit the nucleotide exchange on actin monomers and directly interact with capping protein. Furthermore, the actin interactions of mouse twinfilin-1 and twinfilin-2 are inhibited by phosphatidylinositol (4,5)-bisphosphate. Although biochemically very similar, our Northern blots and in situ hybridizations show that these two proteins display distinct expression patterns. Twinfilin-1 is the major isoform in embryos and in most adult mouse non-muscle cell-types, whereas twinfilin-2 is the predominant isoform of adult heart and skeletal muscles. Studies with isoform-specific antibodies demonstrated that although the two proteins show similar localizations in unstimulated cells, they are regulated by different mechanisms. The small GTPases Rac1 and Cdc42 induce the redistribution of twinfilin-1 to membrane ruffles and cell-cell contacts, respectively, but do not affect the localization of twinfilin-2. Taken together, these data show that mammals have two twinfilin isoforms, which are differentially expressed and regulated through distinct cellular signaling pathways.     

Analysis of Netrin 1 receptors during inner ear development

Netrin 1 plays key roles in axon guidance and neuronal migration during central nervous system (CNS) development. Outside the CNS, Netrin 1 has been shown to be involved in epithelial morphogenesis of various organs. We have shown that Netrin 1 is essential for inner ear semicircular duct formation, but the involvement of Netrin 1 receptors in this process has remained unknown. Netrin 1 receptors include members of the Deleted in colorectal cancer (Dcc), Unc5-homologue and integrin families. Here we have analysed the expression of these receptor genes during inner ear development and verified the inner ear phenotypes of several receptor mutant mice. Special interest was directed to receptors that could cooperate with Netrin 1 during semicircular duct formation. We show that Neogenin (Neo1), Unc5c as well as integrin b1 (Itgb1) are expressed in periotic mesenchyme, while Dcc, Unc5b, Unc5c, Itga3, Itga6 and Itgb1 are expressed in different parts of the otic epithelium. In spite of the broad and strong expression of several receptors in ear region, none of the analysed receptor mutant embryos showed any defects in inner ear development.     

Structural determinants of the agonist-independent association of human peroxisome proliferator-activated receptors with coactivators

Lipid homeostasis is controlled by various nuclear receptors (NRs), including the peroxisome proliferator-activated receptors (PPARalpha, delta, and gamma), which sense lipid levels and regulate their metabolism. Here we demonstrate that human PPARs have a high basal activity and show ligand-independent coactivator (CoA) association comparable with the NR constitutive androstane receptor. Using PPARgamma as an example, we found that four different amino acid groups contribute to the ligand-independent stabilization of helix 12 of the PPAR ligand-binding domain. These are: (i) Lys329 and Glu499, mediating a charge clamp-type stabilization of helix 12 via a CoA bridge; (ii) Glu352, Arg425, and Tyr505, directly stabilizing the helix via salt bridges and hydrogen bonds; (iii) Lys347 and Asp503, interacting with each other as well as contacting the CoA; and (iv) His351, Tyr(355), His477, and Tyr501, forming a hydrogen bond network. These amino acids are highly conserved within the PPAR subfamily, suggesting that the same mechanism may apply for all three PPARs. Phylogenetic trees of helix 12 amino acid and nucleotide sequences of all crystallized NRs and all human NRs, respectively, indicated a close relationship of PPARs with constitutive androstane receptor and other constitutive active members of the NR superfamily. Taking together, the ligand-independent tight control of the position of the PPAR helix 12 provides an effective alternative for establishing an interaction with CoA proteins. This leads to high basal activity of PPARs and provides an additional view on PPAR signaling.     

Prevalence of the fragile X syndrome in four birth cohorts of children of school age

Summary The prevalence of the fragile X syndrome among 12,882 children (6594 boys and 6288 girls) born during the years 1969–1972 in Kuopio province in eastern central Finland has been studied retrospectively. Mentally retarded children were selected from normal schools by using school achievement tests and from registers of mentally retarded individuals. In the present study fragile X syndrome was found in 6/111 mentally retarded children (5.4%), in 4/61 boys and in 2/50 girls, respectively. It was not detected in the control group of 85 healthy children. The corrected prevalence of fragile X syndrome among boys in four successive birth cohorts was estimated to the 1 in 1210 or 0.8/1000, and that among girls, 1 in 2418 or 0.4/1000. The overall prevalence was calculated to be 1 in 1612 or 0.6/1000 children.     

Synthesis, cannabinoid receptor activity, and enzymatic stability of reversed amide derivatives of arachidonoyl ethanolamide

Retroanandamide (2f) and its 10 analogues (1a-e, 2a-e) were synthesized and evaluated for the cannabinoid receptor activation by a [35S]GTPgammaS binding assay using rat cerebellar membranes, and Chinese hamster ovary cell membranes expressing human CB2 receptors. The primary goal of the study was to develop cannabinoid receptor agonists having improved enzymatic stability compared to endogenous N-arachidonoyl ethanolamide (AEA). Furthermore, by reversing the amide bond of AEA, the formation of arachidonic acid would be prevented. Finally, an effect of the carbonyl carbon position on the cannabinoid receptor activity was explored by synthesizing retroanandamide analogues having different chain lengths (1a-e, C19; 2a-f, C20). All the synthesized compounds, except 2c, behaved as partial agonists for the both cannabinoid receptors. In rat brain homogenate, the reversed amides possessed significantly higher stability against FAAH induced degradation than AEA. Therefore, the reversed amide analogues of AEA may serve as enzymatically stable structural basis for the drug design based on the endogenous cannabinoids.