Species-scanning mutagenesis of the serotonin transporter reveals residues essential in selective, high-affinity recognition of antidepressants

The serotonin transporter (SERT) is a high-affinity sodium/chloride-dependent neurotransmitter transporter responsible for reuptake of serotonin from the extracellular space. SERT is a selective target of several clinically important antidepressants. In a cross-species analysis comparing human and bovine SERTs, the kinetic parameters for serotonin uptake were found to be similar, however, the pharmacological profiles of the two transporters differ. Following transient expression in COS-1 cells, IC(50) values were determined for several antidepressants and psychostimulants. The potencies of the antidepressants citalopram, fluoxetine, paroxetine and imipramine were several-fold higher at hSERT compared with bSERT. No species selectivity was observed for the antidepressants fluvoxamine, and sertraline or for the psychostimulants cocaine, the cocaine analogue beta-carbomethoxy-3beta-(4-iodophenyl)tropane, or for 3,4-methylenedioxymethamphetamine (MDMA). Analysis of six hSERT/bSERT chimeras and subsequent species-scanning mutagenesis of each isoform revealed methionine-180, tyrosine-495, and phenylalanine-513 to be responsible for the increase in citalopram and paroxetine potencies at hSERT and methionine-180 and phenylalanine-513 to confer species selectivity at hSERT for fluoxetine and imipramine. Results were obtained by doing the forward, bovine to human, mutations and confirmed by doing the reverse mutations. Citalopram analogues were used to define the roles of methionine-180, tyrosine-495, and phenylalanine-513 and to reveal molecular interactions with individual functional groups of citalopram. We suggest that methionine-180 interacts with the heterocyclic nucleus of citalopram or stabilizes the binding pocket and phenylalanine-513 to be a steric blocker of antidepressant recognition.     

Identification of Escherichia coli Recovered from Milk of Sows with Coliform Mastitis by Random Amplified Polymorphic DNA (RAPD) Using Standardized Reagents

A standardized-reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis was used for typing 58 Escherichia coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swineherd in Sweden. Previously, the 58 E. coli strains were characterized serologically and profiled biochemically. They were also evaluated for their serum resistance and their ability to adhere to fibronectin and bovine fetal fibroblasts. The RAPD analysis was fast, easily performed, and required only a nanogram of DNA. The indistinguishable banding patterns obtained with repeated analyses of 2 isolates from each strain demonstrated that RAPD analysis using standardized beads is a technique that provides reproducible results for typing E. coli strains that cause mastitis in sows. The results of the RAPD analyses demonstrated that E. coli sow mastitis strains are highly variable in serotype, biochemical profiles, virulence factors, and RAPD type, and that all 58 strains can be differentiated by means of the RAPD technique. The strains grouped into 24 RAPD types by combining the results of 2 primers, and into 38 groups by combining the results of serotype and RAPD type. No relationship between serotypes, virulence factors and RAPD types was found.     

Acylguanidine inhibitors of beta-secretase: optimization of the pyrrole ring substituents extending into the S1 and S3 substrate binding pockets

Proteolytic cleavage of amyloid precursor protein by beta-secretase (BACE-1) and gamma-secretase leads to formation of beta-amyloid (A beta) a key component of amyloid plaques, which are considered the hallmark of Alzheimer's disease. Small molecule inhibitors of BACE-1 may reduce levels of A beta and thus have therapeutic potential for treating Alzheimer's disease. We recently reported the identification of a novel small molecule BACE-1 inhibitor N-[2-(2,5-diphenyl-pyrrol-1-yl)-acetyl]guanidine (3.a.1). We report here the initial hit-to-lead optimization of this hit and the SAR around the aryl groups occupying the S(1) and S(2') pockets leading to submicromolar BACE-1 inhibitors.     

Pex19p Interacts with Pex3p and Pex10p and Is Essential for Peroxisome Biogenesis in Pichia pastoris

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Delta cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.     

A new rapid and simple method for large-scale purification of mycobacterial lipoarabinomannan

Lipoarabinomannan (LAM) is a major and structurally important outer cell wall component of all mycobacteria. LAM is also generally regarded as an important immunomodulating substance affecting several immunologic networks and hence important in the pathogenesis of mycobacterial infections. We here describe a new method for large-scale purification of mycobacterial LAM. A crude cell wall preparation was prepared from batch-grown Mycobacterium tuberculosis H37Rv. From this cell wall preparation LAM was purified by sequential extractions and chromatographic steps. From 20 g dry weight cell wall preparation 313 mg of highly purified (> 98%) LAM was obtained in only 3 days. The LAM content of the final purification step was quantified by ELISA using reference LAM as standard. The identity and purity of the LAM preparation was further confirmed by comparison with reference LAM preparation from M. tuberculosis strain Erdman in polyacrylamide gel electrophoresis and Western blots, using reference anti-LAM monoclonals CS-35 and CS-40.     

Electroporation of primary neural cultures: a simple method for directed gene transfer in vitro

Gene transfer into cells of the nervous system is an important method to analyze tissue-specific gene functions. Although highest transfection efficiencies are generally obtained by viral gene transfer, non-viral methods are attractive because they are less labor intensive and more suitable for high throughput screening approaches. Here we describe an approach for electroporation-based gene transfer into primary neural cells isolated from dissociated murine cerebella. Using GFP as reporter molecule, we show that electroporation allows for efficient gene transfer into embryonic and postnatal neural cells under highly controlled experimental conditions. Furthermore we show that adaptation of electroporation parameters allowed for the preferential transfection of subsets of neural cells within the mixed primary culture. Using electroporation settings of high voltage and low capacitance (500 V/50 microF) we achieved a transfection efficiency of about 10% of small neural cells which were identified as granule cells by the expression of the granule cell-specific marker NeuN. At electroporation settings of 220 V/975 microF, large and stellate-shaped cells that comprised about 10% of the GFAP-positive population of astrocytes were preferentially transfected. We conclude that electroporation of primary neural cells can be used to target gene transfer to subsets of neural cells.     

Detection of autoantibodies to cytokines

Autoantibodies to various cytokines have been reported in normal individuals and in patients with various infectious and immunoinflammatory disorders, and similar antibodies (Ab) may be induced in patients receiving human recombinant cytokines. The clinical relevance of these Ab is often difficult to evaluate. Not only are in vitro neutralizing cytokine Ab not necessarily neutralizing in vivo, but assays for binding and neutralizing Ab to cytokines are often difficult to interpret. For example, denaturation of immobilized cytokines in immunoblotting techniques and immunometric assays may leave Ab to the native forms of the mediators unrecognized. On the other hand, Ab may bind nonspecifically and/or with biologically irrelevant low affinities, leading to erroneous interpretations. This article describes in detail the use of radioimmunoassays that we have optimized and used successfully for the detection of high-affinity (auto)Ab to IL-1α, IL-6, GM-CSF, and IFNα.