LC-MS/MS-analysis of sphingosine-1-phosphate and related compounds in plasma samples

Sphingosine-1-phosphate (S1P) and related compounds are important signaling molecules and are normal constituents of human plasma. So far, only a few methods exist for their determination specifically in plasma demanding radioactive agents, more or less time consuming extraction or derivatization procedures. Here, we describe a very simple, reliable, sensitive standard-addition method for the simultaneous determination of S1P, sphingosine (SPH), sphinganine (SAPH) and sphinganine-1-phosphate (SA1P) in human and rat plasma samples. After methanol precipitation of plasma samples the supernatants were directly assessed by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). HPLC analysis was done under gradient conditions using a C18 reversed phase column. The lower limit of quantification (LLOQ) was <10.2, <4.6, <1.9 and 0.57ng/ml for S1P, SPH, SAPH and SA1P, respectively. Variations in accuracy and intraday and interday precision were <15% over the range of calibration. All analytes were normal constituents both in human and rat plasma although the SA1P concentrations in a few rat plasma samples were below the lower limit of quantification. This validated method is suitable to generate new pharmacological findings by monitoring plasma concentrations of S1P and related compounds especially when low amounts of plasma samples are present (e.g. plasma samples from rodents).     

Genome-wide mapping of susceptibility to coronary artery disease identifies a novel replicated locus on chromosome 17

Coronary artery disease (CAD) is a leading cause of death world-wide, and most cases have a complex, multifactorial aetiology that includes a substantial heritable component. Identification of new genes involved in CAD may inform pathogenesis and provide new therapeutic targets. The PROCARDIS study recruited 2,658 affected sibling pairs (ASPs) with onset of CAD before age 66 y from four European countries to map susceptibility loci for CAD. ASPs were defined as having CAD phenotype if both had CAD, or myocardial infarction (MI) phenotype if both had a MI. In a first study, involving a genome-wide linkage screen, tentative loci were mapped to Chromosomes 3 and 11 with the CAD phenotype (1,464 ASPs), and to Chromosome 17 with the MI phenotype (739 ASPs). In a second study, these loci were examined with a dense panel of grid-tightening markers in an independent set of families (1,194 CAD and 344 MI ASPs). This replication study showed a significant result on Chromosome 17 (MI phenotype; p = 0.009 after adjustment for three independent replication tests). An exclusion analysis suggests that further genes of effect size λ_sib > 1.24 are unlikely to exist in these populations of European ancestry. To our knowledge, this is the first genome-wide linkage analysis to map, and replicate, a CAD locus. The region on Chromosome 17 provides a compelling target within which to identify novel genes underlying CAD. Understanding the genetic aetiology of CAD may lead to novel preventative and/or therapeutic strategies.     

The crystal structure of Aspergillus fumigatus cyclophilin reveals 3D domain swapping of a central element

The crystal structure of Aspergillus fumigatus cyclophilin (Asp f 11) was solved by the multiwavelength anomalous dispersion method and was refined to a resolution of 1.85 A with R and R(free) values of 18.9% and 21.4%, respectively. Many cyclophilin structures have been solved to date, all showing the same monomeric conformation. In contrast, the structure of A. fumigatus cyclophilin reveals dimerization by 3D domain swapping and represents one of the first proteins with a swapped central domain. The domain-swapped element consists of two beta strands and a subsequent loop carrying a conserved tryptophan. The tryptophan binds into the active site, inactivating cis-trans isomerization. This might be a means of biological regulation. The two hinge loops leave the protein prone to misfolding. In this context, alternative forms of 3D domain swapping that can lead to N- or C-terminally swapped dimers, oligomers, and aggregates are discussed.     

Method for preparing single-stranded DNA templates for Pyrosequencing using vector ligation and universal biotinylated primers

In Pyrosequencing, the addition of nucleotides to a primer-template hybrid is monitored by enzymatic conversion of chemical energy into detectable light. The technique yields both qualitative and quantitative sequence information because the chemical energy is released by a stoichiometric split off of pyrophosphates from incorporated deoxynucleotide triphosphates and a defined nucleotide dispensation order is given. Because Pyrosequencing works best if single-stranded DNA templates are used, template generation usually requires PCR with a target-specific biotinylated primer and a subsequent purification involving interaction of the biotin label with immobilized streptavidin. To circumvent the need for numerous and expensive template-specific biotinylated primers, we developed a method that uses the ligation of amplified DNA fragments into a plasmid vector, thereby facilitating subsequent PCR using a universal vector-specific biotinylated primer. This approach allows easy and straightforward isolation of single-stranded templates of any PCR product. As a proof of principle, we used the method for genotyping two single-nucleotide polymorphisms in the human genes CARD15 and A2M and for characterization of four multisite variations in the human DEFB104 gene.     

Transport of multidrug resistance substrates by the Streptococcus agalactiae hemolysin transporter

Streptococcus agalactiae (group B streptococcus [GBS]) causes neonatal sepsis, pneumonia, and meningitis, as well as infections of the bovine udder. The S. agalactiae hemolysin is regarded as an important virulence factor, and hemolysin expression is dependent on the cyl gene cluster. cylA and cylB encode the ATP binding and transmembrane domains of a typical ATP binding cassette (ABC) transporter. The deduced proteins contain the signature sequence of a multidrug resistance (MDR) transporter, and mutation of the genes results in a nonhemolytic and nonpigmented phenotype. To further elucidate the function of the putative transporter, nonpolar deletion mutants of cylA were constructed. These mutants are nonhemolytic and can be complemented by the transporter genes. Wild-type strain and nonhemolytic cylA and cylK deletion mutants were exposed to known substrates of MDR transporters. Mutation of cylA significantly impaired growth in the presence of daunorubicin, doxorubicin, and rhodamine 6G and resulted in a decreased export of doxorubicin from the cells. The mutation of cylK, a gene of unknown function located downstream from cylA, caused a loss of hemolysis but had no effect on the transport of MDR substrates. Furthermore, the hemolytic activity of the wild-type strain was inhibited by reserpine in a dose-dependent manner. We conclude that CylAB closely resembles an ABC-type MDR transporter and propose that the GBS hemolysin molecule represents a natural substrate of the transporter.     

Influence of CD4+CD25+ regulatory T cells on low/high-avidity CD4+ T cells following peptide vaccination

We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1(157-170) peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1(157-170) peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1-specific pre-existing naive CD4+CD25- T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1(157-170) peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors.     

Human IgG1 Hinge Fragmentation as the Result of H2O2-mediated Radical Cleavage

Hinge cleavage of a recombinant human IgG1 antibody, generated during production in a Chinese hamster ovary cell culture, was observed in the purified material. The cleavage products could be reproduced by incubation of the antibody with H₂O₂ and featured complementary ladders of the C- and N-terminal residues (Asp²²⁶–Lys²²⁷–Thr²²⁸–His²²⁹–Thr²³⁰) in the heavy chain of the Fab domain and the upper hinge of one of the Fc domains, respectively. Two adducts of +45 and +71 Da were also observed at the N-terminal residues of some Fc fragments and were identified as isocyanate and α-ketoacyl derivatives generated by radical cleavage at the α-carbon position through the diamide and α-amidation pathways. We determined that the hinge cleavage was initiated by radical-induced breakage of the disulfide bond between the two hinge cysteines at position 231 (Cys²³¹-Pro-Pro-Cys-Pro), followed by the formation of a thiyl radical (Cys²³¹-S^•) on one cysteine and sulfenic acid (Cys²³¹-SOH) on the other. The location of the initial radical attack and the critical role of Cys²³¹ were demonstrated by the observation that 5,5-dimethyl-1-pyrroline N-oxide only reacted with the Cys²³¹ radical and completely blocked hinge cleavage, suggesting the necessity of an electron/radical transfer from the Cys²³¹ radical to the hinge residues where cleavage was observed. As a precursor of hydroxyl radicals, H₂O₂ is widely produced in healthy cells and tissues and therefore could be the source for the radical-induced fragmentation of human IgG1 antibodies in vivo.     

Structural Substrate Conditions Required for Neutral Endopeptidase-Mediated Natriuretic Peptide Degradation

Natriuretic peptides are cyclic vasoactive peptide hormones with great diagnostic and therapeutic relevance. The main catabolic pathway postulated for natriuretic peptides is the degradation by neutral endopeptidase (NEP). However, B-type natriuretic peptide has been found to be resistant to NEP. Here, we compared the degradation of various mature, truncated, and recombinant natriuretic peptides by NEP. The degradation was clearly dependent on the length of the N- or C-terminus as well as on distinct sequence differences within the essential loop structure of the natriuretic peptides. Based on these findings, we developed a model for the interaction of NEP and natriuretic peptides that enables new insights into the mode of action and prediction of substrates of NEP, a peptidase that plays a key role in crucial (patho-) physiological processes.     

Mechanical load-dependent cardiac ER stress in vitro and in vivo: effects of preload and afterload

Proteins are folded in the endoplasmic reticulum (ER). ER stress initially leads to compensatory upregulation of ER chaperones and later to apoptosis, but the contribution of biomechanical load vs. neurohumoral stress to myocardial ER stress is unknown. We show that the ER chaperones Grp78 and calreticulin (CRT) are upregulated by afterload, but not by preload in vitro and in vivo. Angiotensin II upregulated ER chaperones in unloaded muscle strips, but the angiotensin receptor-1 antagonist irbesartan did not significantly blunt the induction of ER chaperones by afterload. In monocrotaline-treated rats, Grp78 and CRT were upregulated in the afterloaded right ventricle, but not in the only neurohumorally stressed left ventricle. These findings suggest that afterload but not preload induces myocardial ER stress, largely independent of angiotensin II signaling.