A low-toxicity method for the separation of lanosterol and dihydrolanosterol from commercial mixtures

We describe an inexpensive, low-toxicity and high-yielding method for the production of pure lanosterol and dihydrolanosterol from the commercially available mixture. Optimum conditions are presented for the one-pot production of the intermediate 24,25 vicinal diol of lanosterol acetate (via either epoxidation or hydroxyhalogenation) which is readily separated from the unreacted dihydrolanosterol acetate. The lanosterol diol can then be converted to pure (>97%) lanosterol. Hypophosphorous acid was used for both the conversion of the epoxide to the diol, and as a catalyst for the hydroxyhalogenation by N-halosuccinimides of the olefinic bond.     

Discovery of novel aspartyl ketone dipeptides as potent and selective caspase-3 inhibitors

The discovery of a series of potent, selective and reversible dipeptidyl caspase-3 inhibitors are reported. The iterative discovery process of using combinatorial chemistry, parallel synthesis, moleculare modelling and structural biology will be discussed.     

Catalytic activity of caspase-3 is required for its degradation: stabilization of the active complex by synthetic inhibitors

The activation of caspase-3 represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Upon induction of apoptosis, the large (p17) and small (p12) subunits, comprising active caspase-3, are generated via proteolytic processing of a latent proenzyme dimer. Two copies of each individual subunit are generated to form an active heterotetramer. The tetrameric form of caspase-3 cleaves specific protein substrates within the cell, thereby producing the apoptotic phenotype. In contrast to the proenzyme, once activated in HeLa cells, caspase-3 is difficult to detect due to its rapid degradation. Interestingly, however, enzyme stability and therefore detection of active caspase-3 by immunoblot analysis can be restored by treatment of cells with a peptide-based caspase-3 selective inhibitor, suggesting that the active form can be stabilized through protein-inhibitor interaction. The heteromeric active enzyme complex is necessary for its stabilization by inhibitors, as expression of the large subunit alone is not stabilized by the presence of inhibitors. Our results show for the first time, that synthetic caspase inhibitors not only block caspase activity, but may also increase the stability of otherwise rapidly degraded mature caspase complexes. Consistent with these findings, experiments with a catalytically inactive mutant of caspase-3 show that rapid turnover is dependent on the activity of the mature enzyme. Furthermore, turnover of otherwise stable active site mutants of capase-3 is rescued by the presence of the active enzyme suggesting that turnover can be mediated in trans.     

Microarray-based genomic surveying of gene polymorphisms in <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>

By comparing two fully sequenced genomes of Chlamydia trachomatis using competitive hybridization on DNA microarrays, a logarithmic correlation was demonstrated between the signal ratio of the arrays and the 75-99% range of nucleotide identities of the genes. Variable genes within 14 uncharacterized strains of C. trachomatis were identified by array analysis and verified by DNA sequencing. These genes may be crucial for understanding chlamydial virulence and pathogenesis.     

Adipogenic differentiating agents regulate expression of fatty acid binding protein and CD36 in the J744 macrophage cell line

Adipocyte fatty acid binding protein (aP2) is a key mediator of intracellular transport and metabolism of fatty acids. Its expression during adipocyte differentiation is regulated through the actions of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer binding protein alpha (C/EBPalpha). Macrophages also express aP2, and the lack of macrophage aP2 significantly reduces atherosclerotic lesion size in hypercholesterolemic mice. We investigated the regulation of expression of macrophage aP2 and CD36, a fatty acid membrane binding protein and scavenger receptor, in response to the adipogenic agents isobutylmethylxanthine (IBMX), insulin, and dexamethasone, a combination of agents shown to induce fibroblast-to-adipocyte differentiation. Treatment of J774 macrophages with adipogenic agents significantly induced aP2 mRNA expression, while CD36 expression was inhibited. Dexamethasone was essential and sufficient to induce aP2 expression, and insulin had a synergistic effect. However, IBMX antagonized induced-aP2 expression. aP2 protein expression and [14C]oleic acid uptake by macrophages were also increased by dexamethasone. Unlike what occurs in adipocytes, adipogenic agents had mixed effects on the expression of PPARgamma and C/EBPalpha in macrophages. Our data demonstrate differences in the regulation of aP2 in adipocytes and macrophages and show that macrophage aP2 expression by adipogenic agents is independent of the PPARgamma and/or C/EBPalpha signaling pathway.     

The cell adhesion molecule CEACAM1-L is a substrate of caspase-3-mediated cleavage in apoptotic mouse intestinal cells

The CEACAM1 cell adhesion molecule is a member of the carcinoembryonic antigen family. In the mouse, four distinct isoforms are generated by alternative splicing. These encode either two or four immunoglobulin domains linked through a transmembrane domain to a cytoplasmic domain that encompasses either a short 10-amino acid tail or a longer one of 73 amino acids. Inclusion of exon 7, well conserved in evolution, generates the long cytoplasmic domain. A potential caspase recognition site in mouse, rat, and human CEACAM1-L also becomes available within the peptide encoded by exon 7. We used CEACAM1-L-transfected mouse colon carcinoma CT51 cells treated with three different apoptotic agents to study its fate during cell death. We found that CEACAM1-L is cleaved resulting in rapid degradation of most of its 8-kDa cytoplasmic domain. Caspase-mediated cleavage was demonstrated using purified recombinant caspases. The long cytoplasmic domain was cleaved specifically by caspase-3 in vitro but not by caspase-7 or -8. Moreover cleavage of CEACAM1-L in apoptotic cells was blocked by addition of a selective caspase-3 inhibitor to the cultures. Using point and deletion mutants, the conserved DQRD motif in the membrane-proximal cytoplasmic domain was identified as a caspase cleavage site. We also show that once CEACAM1-L is caspase-cleaved it becomes a stronger adhesion molecule than both the shorter and the longer expressing isoforms.     

Metal complexes of sporidesmin D and dimethylgliotoxin, investigated by electrospray ionisation mass spectrometry

Electrospray ionisation mass spectrometry (ES-MS) has been used to probe the coordination chemistry of metabolites such as sporidesmin D (spdD), found in the saprophytic fungus Pithomyces chartarum, and the related bisdethiobis(methylthio)gliotoxin (dimethylgliotoxin, Megtx). SpdD forms complexes of the type [spdD+M(MeCN)] and [2spdD+M]+ (M=Cu, Ag) and, at higher cone voltages, [spdD+M]+. The bis(ligand) ion [2spdD+M]+ was observed at very high cone voltages, indicating it has appreciable stability; the proposed structure of this species has a four-coordinate metal ion with two bidentate spdD ligands, coordinated through their SMe groups. 1H NMR titrations of spdD with K+, Ag+ and Cu+ provided additional evidence for complex formation with the soft metals. SpdD forms only relatively weak complexes with Zn2+, Cd2+, Co2+ and Mn2+, in keeping with the known reduced tendency of these metals to form stable thioether complexes. ES-MS studies of Megtx showed similar results to spdD, with stable adducts formed with Cu+ and Ag+ ions. The X-ray crystal structure of spdD is also reported.     

The comparison of plasma deproteinization methods for the detection of low-molecular-weight metabolites by (1)H nuclear magnetic resonance spectroscopy

Blood plasma is the major vehicle by which metabolites are transported around the body in mammalian species, and chemical analysis of plasma can provide a wealth of information relating to the biochemical status of an individual and is important for diagnostic purposes. However, plasma is very complex in physicochemical terms because it is composed of a range of organic and inorganic constituents with a wide range of molecular weights and chemical classes and this makes analysis non-trivial. It is now well established that high-resolution (1)H NMR spectroscopy of blood plasma provides useful qualitative and quantitative biochemical information relating to metabolic disorders. However, one of the problems encountered in NMR spectroscopic analysis of blood plasma is the extensive peak overlap or presence of broad macromolecule peaks in the (1)H NMR spectrum, which can severely limit the amount of obtainable information. Even with spectroscopic editing, information relating to low-molecular-weight (MW) metabolites is frequently lost. Therefore, the efficiency of a range of conventional protein removal methods, in combination with the use of one- and two-dimensional NMR spectroscopic methods for evaluation, have been compared for the extraction of NMR-observable low-MW metabolites. It has been shown that these "deproteinization" methods vary considerably in recovery of low MW metabolites and a judicious choice is crucial for optimal extraction of a given analyte. The results presented here show that while ultrafiltration provides the "safest" method of plasma deproteinization, the signal-to-noise ratio of the resultant (1)H NMR spectra is poor. On the other hand, acetonitrile precipitation at physiological pH allows the detection of more low-MW metabolites and at higher concentrations than any other method and provides the further advantages of being a rapid and simple procedure.