Orexin signaling in recombinant neuron-like cells

Recently, we cloned several fluorescent proteins of different colors homologous to Aequorea victoria green fluorescent protein, which have great biotechnological potential as in vivo markers of gene expression. However, later investigations revealed severe drawbacks in the use of novel fluorescent proteins (FPs), in particular, the formation of tetramers (tetramerization) and high molecular weight aggregates (aggregation). In this report, we employ a mutagenic approach to resolve the problem of aggregation. The elimination of basic residues located near the N-termini of FPs results in the generation of non-aggregating versions of several FPs, specifically, drFP583 (DsRed), DsRed-Timer, ds/drFP616, zFP506, zFP538, amFP486, and asFP595.     

XPak3 promotes cell cycle withdrawal during primary neurogenesis in Xenopus laevis

We have isolated the Xenopus p21-activated kinase 3 (XPak3) by virtue of its expression in the territory of primary neurogenesis in the developing embryo. XPak3, but not the other Pak variants, responds positively to X-Ngnr-1 and negatively to X-Notch-1. A constitutively active form of XPak3, generated by fusing a myristylation signal to the N-terminus (XPak3-myr), induces early cell cycle arrest at high concentrations, while ectopic expression of low amounts induces premature neuronal differentiation. Conversely, XPak3 loss of function achieved by use of an antisense morpholino oligonucleotide increases cell proliferation and inhibits neuronal differentiation; this phenotype is rescued by co-injection of XPak3-myr. We conclude that XPak3 is a novel member of the proneural pathway, functioning downstream of neurogenin to withdraw neuronally programmed cells from the mitotic cell cycle, thus allowing for their differentiation.     

Uptake and intracellular transportation of a bacterial surface protein in lymphoid cells

Some strains of the human pathogen Streptococcus pyogenes express a surface protein called protein H, which is released from the streptococcal surface by a cysteine proteinase produced by the bacteria. Here, we find that soluble protein H binds to the surface of lymphocytes and granulocytes, and that the molecule is taken up by lymphocytes and transported to the perinuclear region. The translocation over the cell membrane is rapid, and the uptake and intracellular transportation is not dependent on actin polymerization. Protein H could be immunoprecipitated from cell extracts and nuclear preparations of lymphocytes, and analysis of molecular interactions between protein H and proteins of different cellular compartments demonstrated a binding to nucleophosmin/ B23, a protein known to shuttle between the cytoplasm and the nucleus, and to the nuclear proteins SET and hnRNP A2/B1. Nucleophosmin/B23 was co-immunoprecipitated with protein H from cell and nuclear extracts, and binding experiments, including kinetic analyses, suggest that protein H dissociating from nucleophosmin/B23 complexes in the perinuclear region or in the nucleus binds to proteins SET and hnRNP A2/B1. Finally, the uptake and intracellular transportation of protein H was found to result in a cytostatic effect on B and T lymphocytes.     

The mu2 subunit of the clathrin adaptor AP-2 binds to FDNPVY and YppØ sorting signals at distinct sites

The endocytic sorting signal on the low-density lipoprotein receptor for clathrin-mediated internalization is the sequence FDNPVY in the receptor's cytosolic tail. We have used a combination of surface plasmon resonance and crosslinking with a photoactivated peptide probe to demonstrate the interaction between FDNPVY-containing peptides and the μ2 chain of purified AP-2 clathrin adaptors (the complexes responsible for plasma membrane sorting). We show that recognition of the FDNPVY signal is mediated by a binding site in the μ2-subunit that is distinct from the site for the more general YppØ sorting signal, another tyrosine-based sequence also recognized by μ2-adaptin. These results suggest the possibility that low-density lipoprotein receptor uptake may be modulated specifically and independently of other proteins in the clathrin pathway.     

Glitazones regulate glutamine metabolism by inducing a cellular acidosis in MDCK cells

We studied the effect of the antihyperglycemic glitazones, ciglitazone, troglitazone, and rosiglitazone, on glutamine metabolism in renal tubule-derived Madin-Darby canine kidney (MDCK) cells. Troglitazone (25 microM) enhanced glucose uptake and lactate production by 108 and 92% (both P < 0.001). Glutamine utilization was not inhibited, but alanine formation decreased and ammonium formation increased (both P < 0.005). The decrease in net alanine formation occurred with a change in alanine aminotransferase (ALT) reactants, from close to equilibrium to away from equilibrium, consistent with inhibition of ALT activity. A shift of glutamine's amino nitrogen from alanine into ammonium was confirmed by using L-[2-(15)N]glutamine and measuring the [(15)N]alanine and [(15)N]ammonium production. The glitazone-induced shift from alanine to ammonium in glutamate metabolism was dose dependent, with troglitazone being twofold more potent than rosiglitazone and ciglitazone. All three glitazones induced a spontaneous cellular acidosis, reflecting impaired acid extrusion in responding to both an exogenous (NH) and an endogenous (lactic acid) load. Our findings are consistent with glitazones inducing a spontaneous cellular acidosis associated with a shift in glutamine amino nitrogen metabolism from predominantly anabolic into a catabolic pathway.     

Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.     

Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi

Phosphatidylinositol 4 phosphate [PI(4)P] is essential for secretion in yeast, but its role in mammalian cells is unclear. Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator. We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions. This AP-1 binding defect is rescued by adding back PI(4)P. In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.     

The role of antimicrobial peptides in innate immunity

Production of antimicrobial peptides and proteins is an importantmeans of host defense in eukaryotes. The larger antimicrobialproteins, containing more than 100 amino acids, are often lyticenzymes, nutrient-binding proteins or contain sites that targetspecific microbial macromolecules. The smaller antimicrobialpeptides act largely by disrupting the structure or functionof microbial cell membranes. Hundreds of antimicrobial peptideshave been found in the epithelial layers, phagocytic cells andbody fluids of multicellular animals, from mollusks to humans.Some antimicrobial peptides are produced constitutively, othersare induced in response to infection or inflammation. Studiesof the regulation of antimicrobial peptide synthesis in Drosophilahave been particularly fruitful, and have provided a new paradigmfor the analysis of mammalian host defense responses. It nowappears that the general patterns of antimicrobial responsesof invertebrates have been preserved in vertebrates ("innateimmunity") where they contribute to host defense both independentlyand in complex interplay with adaptive immunity.     

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.