Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6.

CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6 in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs.

Application of LC-TOF MS to analysis of hemoglobin acetaldehyde adducts in alcohol detoxification patients.

Acetaldehyde adducts of hemoglobin have been regarded as potential biochemical markers of alcohol exposure. In this study a novel sensitive method using liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF MS) has been used to investigate changes in adduct levels in alcohol detoxification patients. Hemoglobin and authentic blood samples from 66 adults with an alcohol-dependence syndrome and from 12 children were analyzed for acetaldehyde modifications with and without trypsin digestion using LC-TOF MS. After in-vitro incubation of hemoglobin with increasing concentrations of acetaldehyde, followed by tryptic digestion, 21 modified peptide fragments could be identified from their accurate mass and retention time shift. Eight of these could also be detected in authentic human blood samples. Trace amounts in children's blood were indicative of an endogenous source. Modified peptide levels in patients' samples with and without ethanol were significantly different, as also were levels in samples from admission and from five days later. Samples obtained 5, 10, or 15 days after admission did not differ in adduct levels. The LC-TOF MS method was sensitive enough to detect acetaldehyde-modified hemoglobin peptides in blood samples from patients with an alcohol-dependence syndrome. However, elevated levels were only observed after recent ethanol consumption and decreased during five days of abstinence, suggesting that acetaldehyde-modified tryptic peptides of hemoglobin are potential biomarkers only for short-term ethanol ingestion.

Lack of bufadienolides in the skin secretion of red bellied toads, Melanophryniscus spp. (Anura, Bufonidae), from Uruguay.

The South-American red bellied toads (Melanophryniscus spp.) belonging to the Bufonidae family contain toxic alkaloids in their skin, predominantly of the pumiliotoxin group. Whole animal methanolic extracts of individual specimens of three species (Melanophryniscus atroluteus, M. devincenzii, and M. montevidensis) were analyzed for the presence of toad specific bufadienolides and indolalkylamines (serotonin derivatives) by HPLC-electrospray (ESI)-MS-TOF. No bufadienolides, but few bufotenines, mainly dehydrobufotenine, were detected in the extracts in variable amounts. The concentration of the dehydrobufotenine in the extracts seems to be species specific. Whereas M. atroluteus and M. montevidensis contain very low or trace amounts, M. devincenzii specimens exhibit high concentrations of this indolalkylamine. In comparison, analysis of extracts from Bufo arenarum (Uruguay) and from B. bufo (Germany) confirmed the presence of bufadienolides as well as of bufotenine derivatives. Tadpoles of both species exhibited a different pattern: extracts from B. arenarum tadpoles contained only dehydrobufotenine, but those from B. bufo tadpoles bufotoxin and two alkylamines. Melanophryniscus toads appear not to be able to compensate the high variability of toxic skin alkaloids by producing defensive bufadienolides.