Achiral-chiral LC/LC-MS/MS coupling for determination of chiral discrimination effects in phenprocoumon metabolism

Many physiological processes show a high degree of stereoselectivity, including the metabolism of xenobiotics as catalyzed by cytochrome P450 enzymes. An analysis of these chiral discrimination effects in drug metabolism is essential for an in-depth understanding of metabolic pathways that differ between enantiomers of a given chiral drug or metabolite thereof. Achiral chromatographic separation and structural identification followed by chiral analysis of metabolites from blood specimens usually requires a time-consuming multistage analytical technique. In an effort to optimize such a complicated analytical scheme, a novel two-dimensional online achiral-chiral liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) coupling method was developed by using a peak parking technique in combination with a makeup flow system. Metabolites were separated in the first dimension using a C18 reversed-phase system. A makeup eluent of water/methanol (95/5) was split into the flow before storing the metabolites separately on chiral cartridges. Subsequently, the metabolite enantiomers were eluted backward onto the analytical chiral column and separated, and the ratio of enantiomers was determined. The method was successfully validated with respect to limit of detection, linearity, intra- and interday accuracy, and precision. In the course of a human volunteer study investigating the influence of CYP (cytochrome) 2C9 genetic polymorphism on phenprocoumon (PPC) metabolism, we used this new two-dimensional online analytical technique for the analysis of PPC metabolites in plasma. The enantiomeric forms of 4'-, 6-, and 7-hydroxy-PPC metabolites as well as two novel metabolites were identified, and the ratio of the enantiomers was calculated. We found that the enantiomeric ratio for the different metabolites in the plasma sample of each measured individual differs markedly from a nearly 100% chiral discrimination for the two new putative metabolites. This new analytical coupling method possesses general utility in the analysis of chiral discrimination effects, particularly as it relates to pharmacokinetics and dynamics, a scientific field that is rapidly becoming an area of concern and interest.     

Carcinoembryonic antigen-related cell adhesion molecule 1 on murine dendritic cells is a potent regulator of T cell stimulation

Dendritic cells (DC) are important APCs that play a key role in the induction of an immune response. The signaling molecules that govern early events in DC activation are not well understood. We therefore investigated whether DC express carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1, also known as BGP or CD66a), a well-characterized signal-regulating cell-cell adhesion molecule that is expressed on granulocytes, monocytes, and activated T cells and B cells. We found that murine DC express in vitro as well as in vivo both major isoforms of CEACAM1, CEACAM1-L (having a long cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs) and CEACAM1-S (having a short cytoplasmic domain lacking phosphorylatable tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the chemokines macrophage inflammatory protein 1alpha, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 and induced migration of granulocytes, monocytes, T cells, and immature DC. Furthermore, the surface expression of the costimulatory molecules CD40, CD54, CD80, and CD86 was increased, indicating that CEACAM1-induced signaling regulates early maturation and activation of dendritic cells. In addition, signaling via CEACAM1 induced release of the cytokines IL-6, IL-12 p40, and IL-12 p70 and facilitated priming of naive MHC II-restricted CD4(+) T cells with a Th1-like effector phenotype. Hence, our results show that CEACAM1 is a signal-transducing receptor that can regulate early maturation and activation of DC, thereby facilitating priming and polarization of T cell responses.     

Stabilization of short collagen-like triple helices by protein engineering

Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions.     

The laminin-binding domain of agrin is structurally related to N-TIMP-1

Agrin is the key organizer of postsynaptic differentiation at the neuromuscular junction. This organization activity requires the binding of agrin to the synaptic basal lamina. Binding is conferred by the N-terminal agrin (NtA) domain, which mediates a high-affinity interaction with the coiled coil domain of laminins. Here, we report the crystal structure of chicken NtA at 1.6 A resolution. The structure reveals that NtA harbors an oligosaccharide/oligonucleotide-binding fold with several possible sites for the interaction with different ligands. A high structural similarity of NtA with the protease inhibition domain in tissue inhibitor of metalloproteinases-1 (TIMP-1) supports the idea of additional functions of agrin besides synaptogenic activity.     

Heterodimerization of a functional GABAB receptor is mediated by parallel coiled-coil alpha-helices.

A detailed understanding of GABAB receptor assembly is an important issue in view of its role as attractive target for treatment of epilepsy, anxiety, depression, cognitive defects, and nociceptive disorders. Heteromerization of GABAB-R1 and GABAB-R2 subunits is a prerequisite for the formation of a functional GABAB receptor. Each individual subunit contains one stretch of approximately 30 amino acid residues within its intracellular C-terminal domain that mediates heteromer formation. To investigate the mechanism of the GABAB-R1/GABAB-R2 interaction and to assess the subunit stoichiometry of the complex, recombinant polypeptide chain fragments containing the heteromerization site were produced by heterologous gene expression in Escherichia coli. When mixed in equimolar amounts, these peptides preferentially formed parallel coiled-coil heterodimers under physiological buffer conditions. This demonstrates that the short C-terminal regions are sufficient to determine the specificity of interaction between GABAB receptor subunits. In contrast, isolated GABAB-R1 peptides folded into relatively unstable homodimers, whereas GABAB-R2 peptides were largely unstructured. Together with the data reported in the literature, the results presented here indicate that the functional GABAB receptor is a heterodimer assembled by parallel coiled-coil alpha-helices.     

Pedigree analysis of HDL cholesterol concentration in baboons on two diets.

Using complex segregation analysis, we examined the effects of genetic factors and diet on serum concentrations of high-density-lipoprotein cholesterol (HDL-C) in baboons. In analyses of 710 baboons in 23 sire families, we found evidence for a major gene as well as a polygenic contribution to HDL-C concentration in baboons fed a basal (chow) diet and also in the same animals after challenge with a high-cholesterol, saturated-fat diet. There was evidence for a polygenic contribution to the change in HDL-C concentration in response to the dietary challenge, but there was no evidence for a major gene effect.     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.