Filling accuracy and imprecision of commercial evacuated sodium citrate coagulation tubes.

Current recommendations advocate that blood tubes for coagulation testing should be filled not less than 90% of their nominal filling volume, since under- or over-filling >10% may generate unreliable results of some hemostasis assays. This study was hence aimed to explore filling accuracy and precision of commercial blood tubes. Between-lot variations of 3 different lots (20 tubes per lot) of 3.2% citrate blood tubes manufactured by Becton Dickinson, Greiner and Kima were studied. One additional lot from each manufacturer was assessed in triplicate (three series of 20 tubes), to assess within-lot variation. All tubes were first weighed empty and then filled with distilled water by a syringe, under ideal filling conditions. Filled tubes were weighed again, in duplicate. For each 20 tubes series, mean bias (deviation from the ideal tube filling volume) and imprecision (coefficient of variation; CV%) were calculated. All biases were within ±10%. Within-lot and between-lot variation in filling volume was acceptable, and comprised between 0.4 and 2.4%. Greiner tubes were the most accurate (bias, -1.0 to 2.4%), followed by Kima (bias, -7.8 to -5.9%) and Becton Dickinson (bias, -9.6 to 3.3%) tubes. The highest between-lot difference was noted for Becton Dickinson tubes (up to 12.9%), followed by Greiner and Kima tubes (up to 3.4 and 1.8%, respectively). Although coagulation tubes filling accuracy was within ±10% for all three tested manufacturers, the overall bias was found to be variable among manufacturers and lots. Major effort shall be made by blood tube manufacturers for improving standardization of their products.

Alanine-glyoxylate aminotransferase 2 (AGXT2) polymorphisms have considerable impact on methylarginine and ß-aminoisobutyrate metabolism in healthy volunteers.

Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common AGXT2 single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as ß-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding AGXT2 SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the AGXT2 SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p<0.001). ADMA concentrations were not affected by both SNPs. A haplotype analysis revealed that the second investigated AGXT2 SNP rs16899974, which was not significantly linked to the other AGXT2 SNP, further aggravates the effect of rs37369 with respect to BAIB concentrations in plasma and urine. To investigate the impact of the amino acid exchange p.Val140Ile, we established human embryonic kidney cell lines stably overexpressing wild-type or mutant (p.Val140Ile) AGXT2 protein and assessed enzyme activity using BAIB and stable-isotope labeled [²H6]-SDMA as substrate. In vitro, the amino acid exchange of the mutant protein resulted in a significantly lower enzyme activity compared to wild-type AGXT2 (p<0.05). In silico modeling of the SNPs indicated reduced enzyme stability and substrate binding. In conclusion, SNPs of AGXT2 affect plasma as well as urinary BAIB and SDMA concentrations linking methylarginine metabolism to the common genetic trait of hyper-ß-aminoisobutyric aciduria.

Synthesis and biological evaluation of biphenyl amides that modulate the US28 receptor.

To prepare and biologically evaluate 38 new potential US28 allosteric modulators, we employed a straightforward synthetic route involving radical arylation. The study was based on a former lead structure but with the dihydroisoquinolinone moiety replaced by substituted biphenyls. The investigation of structure-activity relationships among the new biphenyl-derived ligands led to a preliminary pharmacophore model and the discovery of four promising candidates with full inverse agonist properties.

Development of flavonoid-based inverse agonists of the key signaling receptor US28 of human cytomegalovirus.

A series of 31 chalcone- and flavonoid-based derivatives were synthesized in good overall yields and screened for their inverse agonist activity on the US28 receptor of human cytomegalovirus (HCMV). With one exception (e.g., 2-(5-bromo-2-methoxyphenyl)-3-hydroxy-4H-chromen-4-one), halogen-substituted flavonoids were typically more potent inverse agonists than their related hydro derivatives. While toxicity could be used to partially explain the inverse agonist activity of some members of the series, 5-(benzyloxy)-2-(5-bromo-2-methoxyphenyl)-4H-chromen-4-one (11b) acted on the US28 receptor as a nontoxic, inverse agonist. The full inverse agonism (efficacy, -89%) and potency (EC50 = 3.5 µM) observed with flavonoid 11b is especially important as it provides both a new tool to study US28 signaling and a potential platform for the future development of HCMV-targeting drugs.

Identification of novel allosteric modulators for the G-protein coupled US28 receptor of human cytomegalovirus.

The highly constitutively active G-protein coupled receptor US28 of human cytomegalovirus (HCMV) is an interesting pharmacological target because of its implication on viral dissemination, cardiovascular diseases and tumorigenesis. We found that dihydroisoquinolinone and tetrahydroisoquinoline scaffolds may be promising lead structures for novel US28 allosteric inverse agonists. These scaffolds were rapidly synthesized by radical carboamination reactions followed by non-radical transformations. Our novel US28 allosteric modulators provide valuable scaffolds for further ligand optimization and may be helpful chemical tools to investigate molecular mechanisms of US28 constitutive signaling and its role in pathogenesis.

Trichothecenes induce accumulation of glucosylceramide in neural cells by interfering with lactosylceramide synthase activity.

Trichothecenes are sesquiterpenoid metabolites produced by several fungal strains that impair human and animal health. Since sphingolipids were connected with fungal toxicity the aim of the present study was to test the influence of fungal metabolites on sphingolipid metabolism in neural cells. The crude extract of fungal strain Spicellum roseum induced accumulation of glucosylceramide (GlcCer), and simultaneous reduction of the formation of lactosylceramide (LacCer) and complex gangliosides in primary cultured neurons. Following a bioassay-guided fractionation of the respective fungal extract we could demonstrate that the two isolated trichothecene derivatives, 8-deoxy-trichothecin (8-dT) and trichodermol (Td-ol) were responsible for this effect. Thus, incubation of primary cultured neurons as well as of neuroblastoma B104 cells for 24 h with 30 microM of either of the two fungal metabolites resulted in uncoupling of sphingolipid biosynthesis at the level of LacCer. For the observed reduction of LacCer synthase activity by about 90% cell integrity was crucial in both cell types. In neuroblastoma cells the amount of LacCer synthase mRNA was reduced in the presence of trichothecenes, whereas in primary cultured neurons this was not the case, suggesting a post-transcriptional mechanism of action in the latter cell type. The data also show that the compounds did not interfere with the translocation of GlcCer in neuroblastoma cells. Collectively, our results demonstrate that trichodermol and 8-deoxy-trichothecin inhibit LacCer synthase activity in a cell-type-specific manner.

Two new depsipeptides from the marine fungus Spicellum roseum.

Investigation of the secondary metabolites of the marine-derived fungus Spicellum roseum yielded two new cyclohexadepsipeptides, spicellamide A (1) and spicellamide B (2). The structures of 1 and 2 were determined based on extensive evaluations of NMR and MS data. The absolute configuration was deduced after hydrolysis using Marfey's method, chiral chromatography, as well as NOESY and modeling data.

Arugosins G and H: prenylated polyketides from the marine-derived fungus Emericellanidulans var. acristata.

The fungus Emericella nidulans var. acristata was isolated as an endophyte from a Mediterranean green alga. Cultivation of this fungus yielded two new compounds, arugosins G (1) and H (2), together with the known metabolites 3-9. Arugosins (1-4) are benzophenone derivatives, biosynthetically related to the xanthones 5, 6, and 9. The indole alkaloid 7 displayed antitumor activity in a panel of 36 human tumor cell lines, exhibiting a mean IC(50) value of 5.5 microg/mL in an in vitro survival and proliferation assay. Furthermore, compounds 3 and 4 showed moderate antitumor activity toward individual tumor cell lines. None of compounds 1-8 exhibited any immunostimulatory activity assessed as the capacity to induce cytokines in PBMCs from healthy donors.