Effects of dideoxyforskolin on proteoglycan synthesis and structure in embryonic chick chondrocyte cultures

1,9-Dideoxyforskolin inhibits proteoglycan synthesis and xyloside-initiated glycosaminoglycan (GAG) synthesis in chick embryo chondrocytes. Dideoxyforskolin does not affect the length of xyloside-initiated GAG chains secreted into the medium but chains from the dense proteoglycan secreted into the medium appear slightly longer. Incorporation of labeled serine into the dense proteoglycan and subsequent digestion with Pronase revealed a dramatic decrease in percent of total radioactivity associated with GAG chains in the proteoglyean from cultures treated with forskolin or dideoxyforskolin. These observations suggest that these diterpenes have a specific inhibitory effect on chain initiation reactions and thus may be useful tools in the study of proteoglycan synthesis and processing.     

Target syntheses of saturated Keggin polyoxometalate-based extended solids

By the approach of target synthesis, three infinitely extended hybrid compounds based on the saturated Keggin polyoxoanions have been synthesized under hydrothermal conditions: , and (phen = 1,10-phenanthroline, trea = triethylamine). The isostructural compounds 1 and 2 belong to the monoclinic space group P2₁/c and both contain neutral 2D layers and discrete polyoxometalate clusters decorated by transitional metal complexes. They represent an important example of the family of intercalated solids, in which both the 2D layers and the intercalated molecules are polyoxometalates with covalently linked transitional metal complex fragments. Compound 3, crystallizing in the monoclinic space group C2/c, consists of 1D zigzag chains constructed from alternating polyoxoanions and [Ni(phen)₂]²⁺ fragments. More interestingly, these three compounds are constructed directly from saturated polyoxometalates, in which the intact skeletons of Keggin clusters are maintained under hydrothermal conditions. Variable-temperature magnetic susceptibility measurements of compounds 1 and 2 reveal the feature of antiferromagnetic exchange interaction in these compounds.     

Synthesis of full length PB1-F2 influenza A virus proteins from 'Spanish flu' and 'bird flu'.

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian isolates. Previous synthetic protocols have been improved to provide a synthetic full length H1N1 type PB1-F2 protein that is encoded by the 'Spanish flu' isolate and an equivalent protein from an avian host that is representative of a highly pathogenic H5N1 'bird flu' isolate, termed SF2 and BF2, respectively. Full length SF2, different mutants of BF2 and a number of fragments of these peptides have been synthesized by either the standard solid-phase peptide synthesis method or by native chemical ligation of unprotected N- and C-terminal peptide fragments. For SF2 chemical ligation made use of the histidine and the cysteine residues located in positions 41 and 42 of the native sequence, respectively, to afford a highly efficient synthesis of SF2 compared to the standard SPPS elongation method. By-product formation at the aspartic acid residue in position 23 was prevented by specific modifications of the SPPS protocol. As the native sequence of BF2 does not contain a cysteine residue two different mutants of BF2 (Y42C) and BF2 (S47C) with appropriate cysteine exchanges were produced. In addition to the full length molecules, fragments of the native sequences were synthesized for comparison of their physical characteristics with those from the H1N1 human isolate A/Puerto Rico/8/34 (H1N1). All peptides were analyzed by mass spectrometry, (1)H NMR spectroscopy, and SDS-PAGE. The protocols allow the synthesis of significant amounts of PB1-F2 and its related peptides. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Evidence of feminization in wild Elliptio complanata mussels in the receiving waters downstream of a municipal effluent outfall

The endocrine-disrupting activity of municipal effluents has the potential to alter the reproductive system and induce feminization to aquatic organisms. The purpose of this study was to examine the sex ratio, vitellogenin (Vtg)-like proteins, serotonin, arachidonate cyclooxygenase (COX) activity and dopamine status in wild mussels living at sites upstream and downstream of two municipal effluent outfalls in the Mille-Îles River (Quebec, Canada). Gonad integrity was also studied by monitoring the gonado-somatic index (GSI), the activity of the rate-limiting enzyme aspartate transcarbamoylase (ATC) for purine synthesis, and changes in lipid peroxidation (LPO). The results showed that the proportion of females was dramatically increased from 30% at the upstream sites to 80% at the downstream sites. The levels of Vtg-like proteins were significantly elevated in the male mussels only. Male mussels downstream of the municipal effluent plumes expressed female-specific protein bands (Vtg-like), as determined by high-resolution gel electrophoresis and silver staining. The serotonin/dopamine ratio was significantly decreased in the downstream mussels, indicating that the gonad was in a state of early vitellogenesis. However, this change was not accompanied by changes in ATC, suggesting no significant egg production was underway; this was confirmed by the observation that the downstream mussels displayed significantly low GSIs. GSIs were rather dependent on the serotonin/dopamine ratio (r = 0.44; p < 0.001), while Vtg-like proteins were dependent on dopamine levels (r = 0.50; p < 0.001). The increase in COX activity at the downstream sites and its close relationship with increased serotonin levels suggest a concomitant serotonergic signalling in addition to VTG production. The production of Vtg-like proteins combined with the serotonergic effects of the municipal effluents was associated with oxidative damage (LPO) in the gonad. This study provides the first evidence of feminization in wild mussel populations and the disruption in gonad physiology by exposure to municipal effluents.     

Regulation of Histamine Release and Synthesis in the Brain by Muscarinic Receptors

The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with L-[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+-induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1-receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with Ki values of 1-6 X 10(-8) M. 11-[(2-[(Diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116), considered a preferential M2-receptor antagonist, reversed the carbachol effect with a mean Ki of approximately 2 X 10(-7) M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+-induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H- labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1-receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF-DX 116.     

Protein synthesis is not required for the inhibitory effect of selenite on cell colony formation and RNA synthesis

Selenite has been shown to undergo intracellular metabolism that results in its conversion to other low molecular weight Secontaining species and also to its incorporation into a selenocysteine residue in selenoprotein. In order to investigate whether the incorporation into protein is required for the cytotoxic effects of selenite, we have examined whether inhibition of protein synthesis prevents the inhibitory effect of selenite on the ability of cells to form colonies or to synthesize RNA. We have found that treatment of HeLa cells with cycloheximide inhibited protein synthesis by >90% but had no effect on the inhibitory effect of selenite on cell colony formation or RNA synthesis. Since protein synthesis is not necessary for these cytotoxic effects of selenite they are unlikely to result from an increase in the synthesis of selenoproteins.     

Synthesis and interaction of bovine serum albumin with p‐hydroxybenzoic acid derivatives

Three novel p‐hydroxybenzoic acid derivatives (HSOP, HSOX, HSCP) were synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfamonomethoxine sodium, sulfamethoxazole and sulfachloropyridazine sodium) and characterized by elemental analysis, HNMR and MS. Interactions between derivatives and bovine serum albumin (BSA) were studied by fluorescence quenching spectra, UV–vis absorption spectra and time‐resolved fluorescence spectra. Based on fluorescence quenching calculation and Förster's non‐radioactive energy transfer theory, the values of the binding constants, basic thermodynamic parameters and binding distances were obtained. Experimental results indicated that the three derivatives had a strong ability to quench fluorescence from BSA and that the binding reactions of the derivatives with BSA were a static quenching process. Thermodynamic parameters showed that binding reactions were spontaneous and exothermic and hydrogen bond and van der Waals force were predominant intermolecular forces between the derivatives and BSA. Synchronous fluorescence spectra suggested that HSOX and HSCP had little effect on the microenvironment and conformation of BSA in the binding reactions but the microenvironments around tyrosine residues were disturbed and polarity around tyrosine residues increased in the presence of HSOP. Copyright © 2012 John Wiley & Sons, Ltd.     

Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

Synthesis of linear and cyclic phosphopeptides as ligands for the N-terminal SH2-domain of protein tyrosine phosphatase SHP-1.

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.