A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The "defect" in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating. Remarkably, replacement of the homologous HCN tripeptide with the CNGA4 sequence transformed cAMP into an inverse agonist that inhibits HCN channel opening. Using analytical ultracentrifugation, we identified the structural basis for this gating switch: whereas cAMP normally enhances the assembly of HCN C-terminal domains into a tetrameric gating ring, inclusion of the CNGA4 tripeptide reversed this action so that cAMP now causes gating ring disassembly. Thus, ligand gating depends on the dynamic oligomerization of C-terminal binding domains.     

Synthesis and SAR of potent EGFR/erbB2 dual inhibitors

A series of 6-alkoxy-4-anilinoquinazoline compounds was prepared and evaluated for in vitro inhibition of the erbB2 and EGFR kinase activity. The IC(50) values of the best compounds were below 0.10 uM. Further, several of these compounds inhibit the growth of erbB2 and EGFR over-expressing tumor cell lines at concentrations below 1 uM.     

A comparison of scoring functions for protein sequence profile alignment

MOTIVATION: In recent years, several methods have been proposed for aligning two protein sequence profiles, with reported improvements in alignment accuracy and homolog discrimination versus sequence-sequence methods (e.g. BLAST) and profile-sequence methods (e.g. PSI-BLAST). Profile-profile alignment is also the iterated step in progressive multiple sequence alignment algorithms such as CLUSTALW. However, little is known about the relative performance of different profile-profile scoring functions. In this work, we evaluate the alignment accuracy of 23 different profile-profile scoring functions by comparing alignments of 488 pairs of sequences with identity < or =30% against structural alignments. We optimize parameters for all scoring functions on the same training set and use profiles of alignments from both PSI-BLAST and SAM-T99. Structural alignments are constructed from a consensus between the FSSP database and CE structural aligner. We compare the results with sequence-sequence and sequence-profile methods, including BLAST and PSI-BLAST. RESULTS: We find that profile-profile alignment gives an average improvement over our test set of typically 2-3% over profile-sequence alignment and approximately 40% over sequence-sequence alignment. No statistically significant difference is seen in the relative performance of most of the scoring functions tested. Significantly better results are obtained with profiles constructed from SAM-T99 alignments than from PSI-BLAST alignments. AVAILABILITY: Source code, reference alignments and more detailed results are freely available at http://phylogenomics.berkeley.edu/profilealignment/     

Efficient two-sample designs for microarray experiments with biological replications

In the last years, biostatistical research has begun to apply linear models and design theory to develop efficient experimental designs and analysis tools for gene expression microarray data. With two-colour microarrays, direct comparisons of RNA-targets are possible and lead to incomplete block designs. In this setting, efficient designs for simple and factorial microarray experiments have mainly been proposed for technical replicates. But for biological replicates, which are crucial to obtain inference that can be generalised to a biological population, this question has only been discussed recently and is not fully solved yet. In this paper, we propose efficient designs for independent two-sample experiments using two-colour microarrays enabling biologists to measure their biological random samples in an efficient manner to draw generalisable conclusions. We give advice for experimental situations with differing group sizes and show the impact of different designs on the variance and degrees of freedom of the test statistics. The designs proposed in this paper can be evaluated using SAS PROC MIXED or S+/R lme.     

Interleukin-1 beta (IL-1beta) induces tumor necrosis factor alpha (TNF-alpha) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation

Interleukin-1 alpha (IL-1alpha) and beta (IL-1beta) are well known factors that stimulate hematopoiesis, nevertheless there are reports that show that they can also inhibit this activity. While both IL-1alpha and IL-1beta induce the expression of hematopoietic cytokines, such as growth factors and their receptors on myeloid cells, helping thus to regulate hematopoiesis, it is not known if their inhibitory activity is also mediated through the induction of other specific cytokines. In this work we show that recombinant human IL-1beta (rhIL-1beta) inhibits the proliferation of a mouse IL-3-dependent myeloid multipotent cell line (32D cl3), without inducing its differentiation. We show that rhIL-1beta induces in 32D cl3 cells the expression of the tumor necrosis factor alpha (TNF-alpha) gene, a well known growth inhibitor, and that the rhIL-1beta growth inhibition property on 32D cl3 cells is partially due to this secreted TNF-alpha, hinting thus that the inhibition of hematopoiesis by IL-1 is mediated through other induced cytokines.     

Depletion of host Langerhans cells before transplantation of donor alloreactive T cells prevents skin graft-versus-host disease

Skin is the most commonly affected organ in graft-versus-host disease (GVHD). To explore the role of Langerhans cells in GVHD, the principal dendritic cells of the skin, we studied the fate of these cells in mice transplanted with allogeneic bone marrow. In contrast to other dendritic cells, host Langerhans cells were replaced by donor Langerhans cells only when donor T cells were administered along with bone marrow, and the extent of Langerhans cell chimerism correlated with the dose of donor T cells injected. Donor T cells depleted host Langerhans cells through a Fas-dependent pathway and induced the production in skin of CCL20, which was required for the recruitment of donor Langerhans cells. Administration of donor T cells to bone marrow-chimeric mice with persistent host Langerhans cells, but not to mice whose Langerhans cells had been replaced, resulted in marked skin GVHD. These findings indicate a crucial role for donor T cells in host Langerhans cell replacement, and show that host dendritic cells can persist in nonlymphoid tissue for the duration of an animal's life and can trigger GVHD despite complete blood chimerism.     

Protein Phosphorylation and APP Metabolism

Numerous lines of evidence place signal transduction cascades at the core of many processes having a direct role in neurodegeneration and associated disorders. Key players include neurotransmitters, growth factors, cytokines, hormones, and even binding and targeting proteins. Indeed, abnormal phosphorylation of key control proteins has been detected in many cases and is thought to underlie the associated cellular dysfunctions. Several signaling cascades have been implicated, affecting processes as varied as protein processing, protein expression, and subcellular protein localization, among others. The Alzheimer's amyloid precursor protein (APP) is a phosphoprotein, with well-defined phosphorylation sites but whose function is not clearly understood. The factors and pathways regulating the processing of APP have been particularly elusive, both in normal ageing and the Alzheimer's disease (AD) condition. Not surprisingly, the physiological function(s) of the protein remain(s) to be elucidated, although many hypotheses have been advanced. Nonetheless, considerable data has accumulated over the last decade, placing APP in key positions to be modulated both directly and indirectly by phosphorylation and phosphorylation-dependent events. The pathological end product of APP processing is the main proteinaceous component of the hallmark senile plaques found in the brains of AD patients, that is, a toxic peptide termed A. In this minireview we address the importance of phosphorylation and signal transduction cascades in relation to APP processing and A production. The possible use of the identified molecular alterations as therapeutic targets is also addressed.