Ezetimibe stimulates faecal neutral sterol excretion depending on abcg8 function in mice

Ezetimibe stimulates faecal neutral sterol (FNS) excretion in mice, which cannot be explained by cholesterol absorption inhibition alone. We investigated whether these effects are mediated via the sterol exporter ATP binding cassette transporter G8 (abcg8). Ezetimibe increased FNS excretion 2.7-fold in WT mice and 1.5-fold in abcg8^−/− mice, without affecting biliary cholesterol secretion. Daily FNS excretion exceeded the sum of dietary cholesterol intake and biliary secretion by about 60%. Ezetimibe enhanced this ‘extra’ FNS excretion by 3.5-fold and 1.5-fold in wildtype (WT) and abcg8^−/− mice, respectively. Ezetimibe stimulates fecal sterol excretion of non-biliary and non-dietary origin, probably through stimulation of trans-intestinal cholesterol excretion. We show that this effect depends on intact abcg8 function.     

RACK1 associates with muscarinic receptors and regulates M(2) receptor trafficking

Receptor internalization from the cell surface occurs through several mechanisms. Some of these mechanisms, such as clathrin coated pits, are well understood. The M(2) muscarinic acetylcholine receptor undergoes internalization via a poorly-defined clathrin-independent mechanism. We used isotope coded affinity tagging and mass spectrometry to identify the scaffolding protein, receptor for activated C kinase (RACK1) as a protein enriched in M(2)-immunoprecipitates from M(2)-expressing cells over those of non-M(2) expressing cells. Treatment of cells with the agonist carbachol disrupted the interaction of RACK1 with M(2). We further found that RACK1 overexpression inhibits the internalization and subsequent down regulation of the M(2) receptor in a receptor subtype-specific manner. Decreased RACK1 expression increases the rate of agonist internalization of the M(2) receptor, but decreases the extent of subsequent down-regulation. These results suggest that RACK1 may both interfere with agonist-induced sequestration and be required for subsequent targeting of internalized M(2) receptors to the degradative pathway.     

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca²⁺-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.Open in a separate windowClick here to view.^{(43M, flv)}     

Role of A β and the α 7 nicotinic acetylcholine receptor in regulating synaptic plasticity in Alzheimer's disease

Alzheimer's disease (AD) is caused by the accumulation of β-amyloid protein (Aβ) in the brain. The aggregation of β-amyloid protein to higher molecular weight fibrillar forms is also considered to be an important step in the pathogenesis of the disease. The memory problems associated with AD are likely to be caused by changes in synaptic plasticity. Recent studies suggest that Aβ binds to the α 7 nicotinic acetylcholine receptor (α 7 nAChR), which plays an important role in synaptic plasticity and memory. A loop domain localized towards the C-terminus of the extracellular region of the receptor has been identified as forming part of a putative Aβ-binding site. In cell culture experiments, the binding of Aβ to the α 7 nAChR has been found to cause an increase in the level of acetylcholinesterase, which is also increased around amyloid plaques in the AD brain. These studies indicate that the Aβ-binding site on the α 7 nAChR receptor is an important new target for therapeutic development in AD.     

Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing

Distinct classes of small RNAs, 20-32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable the identification of new members or new classes of small RNAs, and they are also suitable to obtain miRNA expression profiles based on clone count frequencies. This protocol includes the use of new deep sequencing methods (454/Roche and Solexa) to facilitate the characterization of diverse sequence pools of small RNAs.     

Rational protein engineering in action: the first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.     

cDNA expression library screening and identification of two novel antigens: ubiquitin and receptor for activated C kinase (RACK) homologue, of the fish parasite Trypanosoma carassii

Trypanosoma carassii is a kinetoplastid parasite infecting cyprinid fish with a high prevalence in nature. Antibodies have been shown to play a protective role in the immune response against this parasite in common carp, Cyprinus carpio. To identify immunogenic and putative protective T. carassii antigens we constructed a lambdaTriplEx2 expression library of the parasite and screened this with pooled carp immune serum collected 6 weeks post-infection. Screening of the library not only revealed ribosomal proteins but identified ubiquitin and a homologue of the receptor for activated C kinase (RACK) as immunogenic proteins. Equivalents of all these proteins have been identified as immunogenic in expression library screenings of other Trypanosomatida, suggesting an evolutionary conservation of their immunogenicity. The possibility that ubiquitin and/or the homologue of RACK could represent protective antigens and be targets for the design of novel therapies is discussed.     

Characterization of seed nuclei in glucagon aggregation using light scattering methods and field-flow fractionation

Background  

Glucagon is a peptide hormone with many uses as a therapeutic agent, including the emergency treatment of hypoglycemia. Physical instability of glucagon in solution leads to problems with the manufacture, formulation, and delivery of this pharmaceutical product. Glucagon has been shown to aggregate and form fibrils and gels in vitro. Small oligomeric precursors serve to initiate and nucleate the aggregation process. In this study, these initial aggregates, or seed nuclei, are characterized in bulk solution using light scattering methods and field-flow fractionation.     

Evaluation of microarray surfaces and arraying parameters for autoantibody profiling

Autoantigen microarrays are being used increasingly to study autoimmunity. Significant variation has been observed when comparing microarray surfaces, printing methods, and probing conditions. In the present study, 24 surfaces and several arraying parameters were analyzed using >500 feature autoantigen microarrays printed with quill pins. A small subset of slides, including FAST, PATH, and SuperEpoxy2, performed well while maintaining the sensitivity and specificity of autoantigen microarrays previously demonstrated by our laboratory. By optimizing the major variables in our autoantigen microarray platform, subtle differences in serum samples can be identified that will shed light on disease pathogenesis.