Increased incidence of aberrant DNA methylation within diverse imprinted gene loci outside of IGF2/H19 in Silver-Russell syndrome.

Almost half of the patients with Silver-Russell syndrome (SRS) are affected by DNA hypomethylation of the Imprinting Center Region 1 (ICR1) at the IGF2/H19 locus on 11p15. We searched genome-wide for additional aberrant DNA methylation in such SRS patients that could account for the clinical variability of the disorder. For this purpose, 18 children with SRS (11 with ICR1 hypomethylation) and 9 children small for gestational age (SGA), serving as controls, were recruited. Genomic DNA from whole blood was subjected to microarray analysis with the HumanMethylation27 BeadChip. This array allows investigating 27,500 CpG sites mostly located in the promoter regions of 14,000 genes. Data were validated by the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technique for the 11p15 region. SRS patients with ICR1 hypomethylation were significantly more frequently affected by DNA hypo- and hypermethylation of CpG sites from diverse imprinted loci than the SGA controls (p = 0.0048). There was no recurrent specific methylation defect outside of IGF2/H19. These findings suggest as causative in SRS a defective mechanism necessary for establishment or maintenance of imprinting marks, which affects imprinted loci in general with low specificity and the IGF2/H19 locus with high specificity, implying the existence of some structural peculiarities at the IGF2/H19 locus.

Mutant and misfolded human growth hormone is rapidly degraded through the proteasomal degradation pathway in a cellular model for isolated growth hormone deficiency type II.

Autosomal dominant isolated growth hormone deficiency type II (IGHD II) is mainly caused by splice site mutations of the GH-1 gene, leading to deletion of amino acids 32-71 of the human growth hormone (hGH). The severe hGH deficit in IGHD II suggests a dominant negative effect of the partially deleted del(32-71)-hGH on the production, storage or secretion of normal wild-type (wt)-hGH in somatotrophic cells of the pituitary. To shed more light on the cellular and molecular basis of IGHD II, we established and analysed diverse clones of the rat somatotrophic cell line GH(4)C(1) stably expressing either wt-hGH, del(32-71)-hGH, or both proteins concomitantly. The cellular morphology of all transfected GH(4)C(1) cell clones showed moderate differences to untransfected GH(4)C(1) cells. On the molecular level, both cDNA-constructs induced transcription but, under normal culture conditions, only wt-hGH protein was found to be synthesised and secreted in readily detectable amounts. By contrast, only after inhibition of proteasomes did high amounts of del(32-71)-hGH show up. The solubility of del(32-71)-hGH in nondenaturing buffer was poor compared to wt-hGH, hinting at molecular aggregation, and several epitopes recognised by monoclonal hGH antibodies were not present on del(32-71)-hGH, confirming the assumption that del(32-71)-hGH must be severely misfolded. Expression of both proteins in Escherichia coli mirrored the findings from the GH(4)C(1) cell clones in terms of solubility and immunological reactivity. The results of the present study indicate that, in IGHD II, somatotrophs continuously have to remove misfolded del(32-71)-hGH via the proteasomal degradation pathway, suggesting a mechanism that may result in chronic cellular stress.

Treatment-Associated Anxiety among Pregnant Women and their Partners: What is the Influence of Sex, Parity, Age and Education?

BACKGROUND: Anxiety during pregnancy can influence outcomes negatively. The aim of this study was to assess the fears of expectant parents in the setting of antenatal and obstetric care according to their sex, age, parity and education. METHODS: 259 pregnant women and 183 partners, who had presented for antenatal investigation, routine antenatal care or for delivery in the UKSH women's hospital, Lübeck campus, completed a self-assessment questionnaire of fears and the German version of the STAI (Laux et al.). ANOVA and t-tests were used for significance testing. RESULTS: Pregnant women had higher levels of trait anxiety and state anxiety than their partners. Level of education had a significant, inverse effect on trait anxiety. Age had no influence. Fears for the child's health ranked highest among pregnancy-specific fears. Expectant fathers had the same level of anxiety for the birth irrespective of parity; for women fear of the birth decreased with increasing parity. Anxiety only increased significantly for expectant fathers from the 20th week of gestation onwards. CONCLUSIONS: Pregnant women and their partners have different levels of anxiety dependant on their age, education, parity and the stage of pregnancy. These findings could contribute towards improving support of couples during pregnancy. The fears of expectant fathers require particular attention.

Cellular prion protein and GABAA receptors: no physical association?

The so-called prion diseases are probably caused by the conformational conversion of the cellular prion protein (PrPc) into an abnormal, pathological form (PrPsc). PrPc is widely expressed in neuronal tissues, but its function is not known. From electrophysiological measurements in prion-less mice it was proposed that PrPc may contribute to the structural integrity of central synapses containing gamma-aminobutyric acid type A (GABAA) receptors. We tried to substantiate this hypothesis by obtaining evidence for a structural link between the GABAA receptor and PrPc. Preparations of PrPc and GABAA receptors, respectively, from cow brain were analysed for PrPc-GABAA receptor complexes. No evidence for such complexes could be obtained in our experiments, although the protein purification schemes used should favour the preservation of intermolecular linkages. We conclude that further data concerning interactions of PrPc with other proteins are needed to obtain insight into its normal functional role.

IGF-I sensitivity in Silver-Russell syndrome with IGF2/H19 hypomethylation.

BACKGROUND: Silver-Russell syndrome (SRS) is characterized by intrauterine and postnatal growth retardation, typical facial appearance and body asymmetry. The mechanism of growth retardation is unclear. 50% of the patients have a paternal chromosome 11 epimutation-DNA hypomethylation of the imprinting center region 1 (ICR1) of the insulin-like growth factor 2 (IGF2)/H19 locus. SRS children who carry such an epimutation have increased levels of IGF-I and IGFBP-3 in relation to their stature and body weight, suggesting IGF-I resistance. No IGF-I receptor (IGF-1R) defect has been discovered. Therefore, another mechanism, probably an IGF-I post-receptor signaling defect, might be present. OBJECTIVE: The aim of this in-vitro study was to examine: 1) if IGF-I- and IGF-II-induced fibroblast growth is different in SRS children with IGF2/H19 hypomethylation compared to controls; and 2) whether there is IGF-I insensitivity in this subgroup of SRS children due to IGF-I post-receptor signaling defects. DESIGN: Four SRS patients (two males, two females; 9.2 to 16.6 years of age) with an IGF2/H19 hypomethylation defect and three age-matched healthy controls were included in the in-vitro study. Cultivated skin fibroblasts from the patients and the healthy controls were used for the experiments. Proliferation rates of fibroblasts were measured in the presence or absence of recombinant human IGF-I and IGF-II using the commercially available 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test. PI3K (phosphoinositide 3-kinase) assay and NF-κB transcription factor assay were performed using ELISA in order to estimate the IGF-I-stimulated Akt phosphorylation and IκB phosphorylation, respectively. RESULTS: Fibroblasts from SRS patients and fibroblasts from control individuals showed a comparable potential to proliferate in serum-free medium when stimulated with IGFs. No significant differences were found between both groups concerning Akt phosphorylation and IκB phosphorylation rates. CONCLUSIONS: The results of the in-vitro study do not support the hypothesis that IGF-I/IGF-II resistance is a major pathogenetic mechanism responsible for the growth failure in the subgroup of SRS children with IGF2/H19 hypomethylation.

Proteins associated with alpha 1-subunit-containing GABAA receptors from bovine brain.

In contrast to some other ion channels, there are at present no proteins known that bind specifically to mature GABAA receptor channels. Such proteins may be important for the structural organization and cytoskeletal anchoring of GABAA receptors and could also be expected to have channel modulatory effects. To identify proteins that are associated with naturally occurring GABAA receptors we immunoprecipitated these receptors from detergent-solubilized bovine brain membranes by an antibody directed against the alpha 1-subunit. Tubulin and actin were observed to coprecipitate specifically with the receptors. Nine additional proteins were detected, hinting at a complex protein network associated with alpha 1-subunit-containing GABAA receptors. Results of a biochemical characterization of these GABAA receptor-tubulin complex-associated proteins (GTAPs) are presented here. Peptide mass fingerprinting analysis and microsequencing of tryptic peptides indicated that at least three GTAPs have not been described until the present.

Clusters of GABAA receptors on cultured hippocampal cells correlate only partially with functional synapses.

We describe a method to label gamma-aminobutyric acid (GABA)A receptors on the surface of living hippocampal neurons in primary culture, and we compare the distribution of receptors with that of active synapses. To visualize GABAA receptors, the affinity-purified antibody beta3(1-13), recognizing the extracellular N-termini of the GABAA receptor beta2- and beta3-subunits, was used in combination with fluorescent secondary antibodies. The beta2- and beta3-subunits belong to the predominant GABAA receptor subunits in the hippocampus. As expected for aggregates of GABAA receptors in the somato-dendritic plasma membrane, a patchy staining pattern similar to that seen by labelling neurons after fixation was obtained. An antiserum recognizing an intracellular epitope of GABAA receptor beta3-subunits did not label the receptors in living neurons. Whole-cell recordings of GABA-evoked Cl - currents were not affected after decorating GABAA receptors with antibody beta3(1-13). Combining the staining of GABAA receptors with the labelling of active presynaptic terminals with the fluorescent dyes FM1-43 or FM4-64, consistently resulted in the detection of GABAA receptor clusters that were not located at active synapses. These amounted to approximately 50% of all labelled GABAA receptor clusters. GABAA receptor clusters that were not associated with active presynaptic terminals partially colocalized with the synaptic vesicle marker protein sv2, while another fraction had no presynaptic counterpart at all. These findings suggest the presence of presynaptically silent GABAergic synapses in cultured hippocampal neurons. They also indicate that for the maintenance of GABAA receptor aggregates, the release of GABA from an opposing active terminal is not essential.

Interaction between GABA(A) receptor beta subunits and the multifunctional protein gC1q-R.

gamma-Aminobutyric acid type A (GABA(A)) receptors were immunopurified from bovine brain using a monoclonal antibody directed against the alpha1 subunit. Of the several proteins that copurified, a 34-kDa protein was analyzed further. After enrichment and tryptic proteolysis, the resulting fragments were sequenced, and the protein was identified as gC1q-R. Using anti-gC1q-R and anti-GABA(A) receptor antibodies, mutual coimmunoprecipitation could be demonstrated from solubilized rat brain membranes. The stability of this interaction was estimated to be very high. Using the yeast two-hybrid system, various GABA(A) receptor subunit intracellular loop constructs were tested for an interaction with gC1q-R. All beta subunits, but not alpha 1 and gamma 2 subunits, were found to bind to gC1q-R. NH(2)- and COOH-terminally truncated beta 2 subunit loops were used to find the region responsible for the interaction with gC1q-R. A stretch of 15 amino acids containing 7 positively charged residues was identified (amino acids 399--413). This region contains residue Ser-410, which is a protein kinase substrate, and it is known that phosphorylation of this residue leads to an alteration in receptor activity. Localization studies suggested a predominantly intracellular localization. Our observations therefore suggest a tight interaction between gC1q-R and the GABA(A) receptor which might be involved in receptor biosynthesis or modulation of the mature function.

A novel serine kinase with specificity for beta3-subunits is tightly associated with GABA(A) receptors.

Tuning of gamma-aminobutyric acid type A (GABA(A)) receptor function via phosphorylation of the receptor potentially allows neurons to modulate their inhibitory input. Several kinases, both of the serine-threonine kinase and the tyrosine kinase families, have been proposed as candidates for such a modulatory role in vivo. However, no GABA(A) receptor-phosphorylating kinase physically associated with the receptor has been identified so far on a molecular level. In this study, we demonstrate a GABA(A) receptor-associated protein serine kinase phosphorylating specifically beta3-subunits of native GABA(A) receptors. The characteristics of this novel kinase clearly distinguish it from enzymatic activities that have been shown so far to phosphorylate the GABA(A) receptor. We putatively identify this protein kinase as the previously described GTAP34 (GABA(A) receptor-tubulin complex-associated protein of molecular mass 34 kDa). Using expressed recombinant fusion proteins, we identify serine 408 as a major target of the phosphorylation reaction, whereas serine 407 is not phosphorylated. This demonstrates the high specificity of the kinase. Phosphorylation of serine 408 is known to result in a decreased receptor function. The direct association of this kinase with the receptor indicates an important physiological role.