Solid-phase synthesis of an N-(phenylalkyl)cinnamide library via Horner-Wadsworth-Emmons reaction

A readily automated solid-phase approach to the synthesis of diverse N-(phenylalkyl)cinnamides, analogues of the NR2B antagonist 2, is described. The procedure utilizes polymer supported N-(phenylalkyl)amines, (diethylphosphono)acetic acid and a wide range of commercially available hydroxybenzaldehydes. The key step, a Horner-Wadsworth-Emmons reaction is achieved under mild conditions and was found to be general for a large number of benzaldehydes. A 225-member focused library was synthesized using a Tecan Combitec synthesizer.     

A simple fold with variations: the pacifastin inhibitor family

Members of the pacifastin family are small, approximately 35-residue serine protease inhibitors isolated from arthropod species. Several locust inhibitors exhibit intriguing taxon specificity while others do not. The structural basis of this phenomenon may lie in the different dynamical properties of the proteins originating from different stabilizing interactions. In this study, we identify new members of the family to confirm the universal role of these interactions in the family. Structural investigations show that both the disulfide pattern and the stabilizing interactions are unique among small all-beta proteins.     

Synthesis and neuropharmacological evaluation of R(-)-N-alkyl-11-hydroxynoraporphines and their esters

We synthesized several N-substituted-11-hydroxynoraporphines and their esters of varying chain length, evaluated their binding affinity at dopamine (DA) receptor sites in rat caudate-putamen membranes, and quantified their effects on motor activity in normal adult male rats. The 11-hydroxyaporphines showed similar neuropharmacological properties to the corresponding 10,11-catecholaporphines. At moderate doses, their esters proved to have more prolonged behavioral actions and superior oral bioavailability.     

Servers for sequence-structure relationship analysis and prediction

We describe several algorithms and public servers that were developed to analyze and predict various features of protein structures. These servers provide information about the covalent state of cysteine (CYSREDOX), as well as about residues involved in non-covalent cross links that play an important role in the structural stability of proteins (SCIDE and SCPRED). We also discuss methods and servers developed to identify helical transmembrane proteins from large databases and rough genomic data, including two of the most popular transmembrane prediction methods, DAS and HMMTOP. Several biologically interesting applications of these servers are also presented. The servers are available through http://www.enzim.hu/servers.html.     

NHE blockade inhibits chemokine production and NF-kappaB activation in immunostimulated endothelial cells

Na⁺/H⁺exchanger (NHE) activation has been documented to contribute toendothelial cell injury caused by inflammatory states. However, therole of NHEs in regulation of the endothelial cell inflammatoryresponse has not been investigated. The present study tested thehypothesis that NHEs contribute to endothelial cell inflammationinduced by endotoxin or interleukin (IL)-1. NHE inhibition usingamiloride, 5-(N-ethyl-N-isopropyl)-amiloride, and5-(N-methyl-N-isobutyl)amiloride as well as thenon-amiloride NHE inhibitors cimetidine, clonidine, and harmalinesuppressed endotoxin-induced IL-8 and monocyte chemoattractant protein(MCP)-1 production by human umbilical endothelial vein cells (HUVECs). The suppressive effect of amiloride on endotoxin-induced IL-8 production was associated with a decreased accumulation of IL-8 mRNA.NHE inhibitors suppressed both inhibitory (I)B degradation andnuclear factor (NF)-B DNA binding, suggesting that a decrease inactivation of the IB-NF-B system contributed to the suppression of HUVEC inflammatory response by NHE blockade. NHE inhibition decreased also the IL-1-induced HUVEC inflammatory response, becauseamiloride suppressed IL-1-induced E-selectin expression on HUVECs.These results demonstrate that maximal activation of the HUVECinflammatory response requires a functional NHE.

     

Microarray Analysis of Gene Expression in Rat Hippocampus After Chronic Ethanol Treatment

It is thought that changes in gene expression in the brain mediate chronic ethanol-induced complex behaviors such as tolerance, dependence, and sensitization, and also relate to ethanol-induced brain toxicity. Using high-density filter-based cDNA microarrays (GeneFilters), we analyzed the expression of over 5000 genes in the dorsal hippocampus of rats treated with 12% ethanol or tap water for 15 months. Ethanol-induced changes in gene expression were particularly prominent in two groups of genes. One group consisted of oxidoreductases, including ceruloplasmin, uricase, branched-chain alpha-keto acid dehydrogenase, NADH ubiquinone oxidoreductase, P450, NAD⁺-isocitrate dehydrogenase, and cytochrome c oxidase, which may be related to ethanol-induced oxidative stress. The other group of genes included ADP-ribosylation factor, RAS related protein rab10, phosphatidylinositol 4-kinase, dynein-associated polypeptides, and dynamin-1, which seem to be involved in membrane trafficking. The results may reveal some of the pathways involved in ethanol-induced pathophysiological changes.     

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.