Separation and determination of alpha-amino acids by boroxazolidone formation

Reaction of an alpha-amino acid (alpha-AA) with 1,1-diphenylborinic acid (DPBA) leads to the formation of a kinetically stable adduct at pH 2-5 in which both the alpha-amino and the alpha-carboxyl groups are bound to boron forming a cyclic mixed anhydride termed a boroxazolidone. In this adduct, the greater than N:B bond is coordinate, involving the free electron pair of nitrogen, thereby satisfying the octet rule for the second electron shell of boron (Group IIIA). Consequently, the alpha-amino function of the boroxazolidone can be primary, secondary, or tertiary, as demonstrated by boroxazolidone formation with glycine, N-methylglycine, and N,N-dimethylglycine. On reaction with DPBA, the alpha-AA moiety of N-terminal gamma-glutamyl peptides is also derivatized as demonstrated by the formation of a glutathione boroxazolidone. The 1,1-diphenylboroxazolidone adducts of alpha-AA may be separated by reversed-phase (RP)-HPLC (AA-DPBA/RP-HPLC) enabling the derivatization procedure to be used as a precolumn reaction for alpha-AA analysis. Under the conditions we describe here, DPBA is not stably reactive with the epsilon-amino group of lysine. Furthermore, it does not complex with amide bonds of the peptide backbone or to any side chains of the common amino acids. Reaction of an alpha-AA mixture with DPBA, followed by RP-HPLC (AA-DPBA/RP-HPLC) is then a simple method by which to analyze alpha-AA in a mixture with peptides and amines. Precolumn reaction with DPBA may be used to separate peptides from alpha-AA and from those peptides which contain an alpha-AA moiety. Unreacted peptides are bound only weakly to the HPLC column and thus are separated from reacted alpha-amino acids which are retained as 1,1-diphenylboroxazolidones until their selective elution. This method is particularly suited for the analysis of alpha-amino acids that are derived from post-translational modification of protein side chains.     

Effect of Taurine on Calcium Accumulation in Resting and Depolarised Insect Synaptosomes

The effect of taurine (2-aminoethanesulphonic acid) on 45Ca2+ accumulation in resting and depolarised synaptosomes obtained from the locust Schistocerca americana gregaria was studied. Taurine reduced 45Ca2+ accumulation in resting synaptosomes, and this effect was more pronounced when synaptosomes were depolarised with either high [K+] or veratridine. Veratridine-induced 45Ca2+ accumulation was not affected by either gamma-aminobutyric acid or leucine, but was reduced by both verapamil and tetrodotoxin.     

Structures of monosialyl oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Lea+b-) patient suffering from chronic bronchitis. Characterization of a novel type of mucin carbohydrate core structure

Mucin glycopeptides were prepared from the respiratory mucusof a non-secretor, chronic bronchitis patient with blood groupO, Le^a+b–. Oligosaccharides were released by alkalineborohydride treatment and purified by anionexchange chromatography,size-exclusion chromatography and high-performance liquid chromatographyon a silica-bonded alkylamine column. Structural studies employed500-MHz ¹H-NMR spectroscopy and fast atom bombardment-mass spectrometry.Twenty-six monosialyl oligosaccharides ranging in size fromdi- to octasaccharide, were fully characterized in this study.The sialic acid occurs either     

C1s-induced vascular permeability in C2-deficient guinea pigs

Normal guinea pigs that have been intradermally injected with C1s exhibit increased vascular permeability at the injection site. Guinea pigs that are genetically deficient in complement component C2 do not exhibit increased vascular permeability when given a similar injection. The C2-deficient guinea pigs respond normally to injections of bradykinin and kallikrein, suggesting that these animals can respond to kinins and have a normal kininogen pathway. When the C2-deficient guinea pigs are given guinea pig C2 before C1s injection, increased vascular permeability is observed. These results demonstrate a definite requirement for complement component C2 in the generation of C1s-induced vascular permeability.     

Predicting the points of interaction of small molecules in the NF-κB pathway

Background  

The similarity property principle has been used extensively in drug discovery to identify small compounds that interact with specific drug targets. Here we show it can be applied to identify the interactions of small molecules within the NF-κB signalling pathway.     

Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells

Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα.