Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl- cotransporter in the nervous system: evidence for a scaffolding role of the kinase

Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K+ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activity-dependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress.     

Sequence-specific peptide aptamers,interacting with the intracellular domain of the epidermal growth factor receptor,interfere with Stat3 activation and inhibit the growth of tumor cells

Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1.EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.     

Identification of differentially expressed genes like cofilin2 in growing collateral arteries

Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbits by occlusion of the femoral artery. In order to analyze the differential gene expression in arteriogenesis, cDNA of collateral arteries 24h after femoral occlusion or sham operation was subjected to suppression subtractive hybridization (SSH). We demonstrated an upregulation of the U6 snRNA binding protein Lsm5, cytochrome b, an expressed sequence tag, and the actin-depolymerizing factor cofilin2 mRNA in collateral arteries 24h after femoral ligation. For cofilin2, we also detected an increase in the protein level and a localization predominantly in smooth muscle cells of collaterals. Simultaneously with the upregulation of cofilin2 we found a downregulation of the alpha-smooth muscle actin mRNA in growing collateral arteries. In summary, our data showed an augmented expression level of genes contributing to different fundamental processes of arteriogenesis.     

Trisomy 4q syndrome: presentation of a new case and review of the literature

We describe the 11th case of a de novo partial trisomy of the long arm of chromosome 4, with the extra segment spanning from 4q27 to 4q35. The aberration resulted from an unbalanced translocation of material from 4q to the short arm of chromosome 7, as evident from fluorescent in situ hybridization. Microsatellite analysis revealed the extra material to originate from the father. The karyotype was interpreted as 46,XX,der(7)t(4;7)(q27;p22). The patient is a 13-year-old girl with severe mental retardation, growth retardation, hearing impairment as well as minor foot, thumb and facial anomalies. Although the extent of the aberration varies between the reported patients, there are nevertheless features in common, suggestive of a trisomy 4q syndrome. The clinical findings most frequently reported are: mental retardation, seizures, microcephaly, hearing impairment and growth retardation, as well as epicanthic folds, high/broad/depressed nasal bridge, malformed ears, tooth and thumb anomalies. Almost the entire long arm of chromosome 4, except band q11, has been involved in trisomies/duplications, but 4q27 and 4q31 seem to be preferentially engaged in the trisomy 4q syndrome.     

Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils

Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.     

Cation chloride cotransporters interact with the stress-related kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1)

Cells respond to stress stimuli by mounting specific responses. During osmotic and oxidative stress, cation chloride cotransporters, e.g. Na-K-2Cl and K-Cl cotransporters, are activated to maintain fluid/ion homeostasis. Here we report the interaction of the stress-related serine-threonine kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) with the cotransporters KCC3, NKCC1, and NKCC2 but not KCC1 and KCC4. The interaction was identified using yeast two-hybrid assays and confirmed via glutathione S-transferase pull-down experiments. Evidence for in vivo interaction was established by co-immunoprecipitation of SPAK from mouse brain with anti-NKCC1 antibody. The interacting region of both kinases comprises the last 100 amino acids of the protein. The SPAK/OSR1 binding motif on the cotransporters consists of nine residues, starting with an (R/K)FX(V/I) sequence followed by five additional residues that are essential for binding but for which no consensus was found. Immunohistochemical analysis of choroid plexus epithelium revealed co-expression of NKCC1 and SPAK on the apical membrane. In contrast, in choroid plexus epithelium from NKCC1 null mice, SPAK immunostaining was found in the cytoplasm. We conclude that several cation chloride co-transporters interact with SPAK and/or OSR1, and we hypothesize that this interaction might play a role during the initiation of the cellular stress response.     

Selecting neural networks for a committee decision

To improve recognition results, decisions of multiple neural networks can be aggregated into a committee decision. In contrast to the ordinary approach of utilizing all neural networks available to make a committee decision, we propose creating adaptive committees, which are specific for each input data point. A prediction network is used to identify classification neural networks to be fused for making a committee decision about a given input data point. The jth output value of the prediction network expresses the expectation level that the jth classification neural network will make a correct decision about the class label of a given input data point. The proposed technique is tested in three aggregation schemes, namely majority vote, averaging, and aggregation by the median rule and compared with the ordinary neural networks fusion approach. The effectiveness of the approach is demonstrated on two artificial and three real data sets.     

Two distinctly HLA-associated contiguous linear epitopes uniquely expressed within the islet antigen 2 molecule are major autoantibody epitopes of the diabetes-specific tyrosine phosphatase-like protein autoantigens

The related tyrosine phosphatase-like proteins islet Ag (IA)-2 and IA-2beta are autoantigens of type 1 diabetes in humans. Autoantibodies are predominantly against IA-2, and IA-2-specific epitopes are major autoantibody targets. We used the close homology of IA-2 and IA-2beta to design chimeras and mutants to identify humoral IA-2-specific epitopes. Two major IA-2 epitopes that are absent from the related autoantigens IA-2beta and IA-2Delta 13 splice variant ICA512.bdc were found contiguous to each other within IA-2 juxtamembrane amino acids 611-620 (epitope JM1) and 621-630 (epitope JM2). JM1 and JM2 are recognized by sera from 67% of patients with IA-2 Abs, and relatives of patients with type 1 diabetes having Abs to either JM epitope had a >50% risk for developing type 1 diabetes within 6 years, even in the absence of diabetes-associated HLA genotypes. Remarkably, the presence of Abs to one of these two epitopes was mutually exclusive of the other; JM2 Abs and not JM1 Abs were found in relatives with HLA DR3/4, DR4/13, or DR1/4 genotypes; and the binding of autoantibodies to the JM2 epitope, but not the JM1 epitope, markedly affected proteolysis of IA-2. This is a unique demonstration of HLA-associated B cell responses to epitopes within a single autoantigen in humans and is consistent with modification of Ag processing by specific Ab-influencing peptide presentation by HLA molecules.     

Low frequency noise enhances cortisol among noise sensitive subjects during work performance

Salivary free cortisol concentration, rated stress and annoyance were determined in 32 subjects before, during and after carrying out a battery of performance tasks for 2 hours during exposure to ventilation noise, with dominant low frequencies (low frequency noise) or a flat frequency spectrum (reference noise). Both noises had a level of 40 dBA. All subjects were studied on two occasions and were exposed to both noises in strict rotation. Subjects were categorised as high- or low-sensitive to noise in general and low frequency noise in particular on the basis of questionnaires. Cortisol concentrations during the task were not significantly modulated by the noises or related to noise sensitivity alone. The normal circadian decline in cortisol concentration was however significantly attenuated in subjects high-sensitive to noise in general, when they were exposed to the low frequency noise. This noise was rated as more annoying and more disruptive to working capacity than the reference noise. The study showed physiological evidence of increased stress related to noise sensitivity and noise exposure during work. This is the first study to demonstrate an effect of moderate levels of noise on neuroendocrine activity. The impact of long-term exposure to moderate noise levels, and particularly low frequency noise, in the workplace deserves further investigation.