Sea buckthorn berry oil inhibits platelet aggregation

A small-scale preliminary cross-over study was conducted to investigate the effects of supercritical CO(2)-extracted sea buckthorn berry oil (SBO) on some risk factors of cardiovascular disease. Special features of the oil are high proportions of palmitic (16:0), oleic (18:1n-9), palmitoleic (16:1n-7), linoleic (18:2n-6), and alpha-linolenic (18:3n-3) acids as well as vitamin E, carotenoids, and sterols. Twelve healthy normolipidemic men were recruited and each volunteer consumed SBO and fractionated coconut oil (control) 5 g per day for a period of 4 weeks in a random order (wash-out 4-8 weeks). Phospholipid fatty acids, plasma lipids, and glucose were unaffected by SBO supplementation. Instead, a clear decrease in the rate of adenosine-5'-diphosphate-induced platelet aggregation and maximum aggregation were found. This suggested the beneficial effects of SBO on blood clotting, but further studies on the dose-response effects are needed to assess the practical use of SBO supplements.     

Endomorphins interact with the substance P (SP) aminoterminal SP(1-7) binding in the ventral tegmental area of the rat brain

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP(1-7) in the rat spinal cord. This site appeared very specific for SP(1-7) as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP(1-7) from this site. In the present work using a [(3)H]-labeled derivative of the heptapeptide we have identified and characterized [(3)H]-SP(1-7) binding in the rat ventral tegmental area (VTA). Similarly to the [(3)H]-SP(1-7) binding in the spinal cord the affinity of unlabeled SP(1-7) to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the mu-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP(1-7) site was 4-5 times weaker than that for SP(1-7) but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. It was concluded that the specific site identified for SP(1-7) binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.     

Rapid PCR amplification of DNA utilizing Coriolis effects

A novel polymerase chain reaction (PCR) method is presented that utilizes Coriolis and centrifugal effects, produced by rotation of the sample disc, in order to increase internal circulatory rates, and with them temperature homogenization and mixing speeds. A proof of concept has been presented by testing a rapid 45-cycle PCR DNA amplification protocol. During the repeated heating and cooling that constitutes a PCR process, the 100 μL samples were rotated at a speed equivalent to an effective acceleration of gravity of 7,000 g. A cycle time of 20.5 s gave a total process time of 15 min to complete the 45 cycles. A theoretical and numerical analysis of the resulting flow, which describes the increased mixing and temperature homogenization, is presented. The device gives excellent reaction speed efficiency, which is beneficial for rapid PCR.     

Developmental origin of smooth muscle cells in the descending aorta in mice

Aortic smooth muscle cells (SMCs) have been proposed to derive from lateral plate mesoderm. It has further been suggested that induction of SMC differentiation is confined to the ventral side of the aorta, and that SMCs later migrate to the dorsal side. In this study, we investigate the origin of SMCs in the descending aorta using recombination-based lineage tracing in mice. Hoxb6-cre transgenic mice were crossed with Rosa 26 reporter mice to track cells of lateral plate mesoderm origin. The contribution of lateral plate mesoderm to SMCs in the descending aorta was determined at different stages of development. SMC differentiation was induced in lateral plate mesoderm-derived cells on the ventral side of the aorta at embryonic day (E) 9.0-9.5, as indicated by expression of the SMC-specific reporter gene SM22alpha-lacZ. There was, however, no migration of SMCs from the ventral to the dorsal side of the vessel. Moreover, the lateral plate mesoderm-derived cells in the ventral wall of the aorta were replaced by somite-derived cells at E10.5, as indicated by reporter gene expression in Meox1-cre/Rosa 26 double transgenic mice. Examination of reporter gene expression in adult aortas from Hoxb6-cre/Rosa 26 and Meox1-cre/Rosa 26 double transgenic mice suggested that all SMCs in the adult descending aorta derive from the somites, whereas no contribution was recorded from lateral plate mesoderm.     

Large-scale occurrence patterns of red-listed lichens and fungi on old oaks are influenced both by current and historical habitat density

Current occurrence patterns of species associated with ancient trees may reflect higher historical habitat densities, because the dynamics of the habitat and the colonisation-extinction processes for many inhabiting species are expected to be slow. We tested this hypothesis in southeast Sweden by analysing species occurrence per parish for twelve redlisted lichen species and nine redlisted fungus species in relation with current density of big oaks, the density of oaks in the 1830s and connectivity with parishes with the species present. For most species, the occurrence was positively related with current density of habitat (for 18 species out of 21) and parish area (for 16 species). Historical habitat density was positively related with occurrence for 11 species, while connectivity with current occurrences in the surroundings was positive for the occurrence of 12 species and negative for the occurrence of two. For lichen species the connectivity measure that best explained the variation was at a larger spatial scale as compared to fungus species. Even if the density of old oaks remains in the future, inhabiting species will most likely decline because their distribution patterns are not in equilibrium with the current habitat density. Therefore, to allow long-term persistence of inhabiting species the number of old oaks should be increased. Areas where such an increase is most urgent could be identified based on species occurrence data and current habitat density, but because species data will always be incomplete data on the historical habitat distribution is valuable.     

Tick-borne encephalitis virus NS5 associates with membrane protein scribble and impairs interferon-stimulated JAK-STAT signalling

Tick-borne encephalitis virus (TBEV) NS5 protein is a multifunctional RNA-dependent RNA polymerase that is indispensable for viral replication. TBEV is considered to be highly neurovirulent and can cause lethal encephalitis. In this study, we demonstrate a novel interaction between TBEV NS5 and the PDZ protein scribble (hScrib) affecting interferon (IFN) type I and II mediated JAK-STAT signalling. The sequence of TBEV NS5 interacting with hScrib was identified using extensive site-directed mutagenesis analysis. Two consecutive mutations in the methyltransferase (MTase) domain of NS5 were found to disrupt binding to hScrib. Colocalization studies with hScrib demonstrated that TBEV NS5 was present at the plasma membrane of mammalian cells. To address the role of viral interference with the IFN response, NS5 proteins were expressed in IFN-stimulated cells. While TBEV NS5 substantially blocked phosphorylation of STAT1, a mutated NS5 protein defective in hScrib binding failed to inhibit JAK-STAT signalling correctly. Furthermore, hScrib knock-down resulted in re-localization of NS5 to intracellular locations and abrogated the impaired STAT1 phosphorylation. These results define the TBEV NS5 protein in concert with hScrib as an antagonist of the IFN response, by demonstrating a correlation between the association and JAK-STAT interference.     

Cyclobutane pyrimidine dimers do not fully explain the mutagenicity induced by UVA in Chinese hamster cells

UVA generates low levels of cyclobutane pyrimidine dimers (CPDs). Here we asked the question whether CPDs could fully explain the level of mutations induced by UVA. Relative mutagenicities of UVA and UVC were calculated at equal levels of CPDs in cell lines, deficient in different aspects of repair. Survival and gene mutations in the hprt locus were analyzed in a set of Chinese hamster ovary (CHO) cell lines, i.e., wild-type, Cockayne syndrome B protein-deficient (CSB), XRCC3-deficient and XRCC1-deficient adjusted to the same level of CPDs which was analyzed as strand breaks as a result of DNA cleavage by T4 endonuclease V at CPD sites. Induced mutagenicity of UVA was approximately 2 times higher than the mutagenicity of UVC in both wild-type and XRCC1-deficient cells when calculated at equal level of CPDs. Since this discrepancy could be explained by the fact that the TT-dimers, induced by UVA, might be more mutagenic than C-containing CPDs induced by UVC, we applied acetophenone, a photosensitizer previously shown to generate enhanced levels of TT-CPDs upon UVB exposure. The results suggested that the TT-CPDs were actually less mutagenic than the C-containing CPDs. We also found that the mutagenic effect of UVA was not significantly enhanced in a cell line deficient in the repair of CPDs. Altogether this suggests that neither base excision- nor nucleotide excision-repair was involved. We further challenge the possibility that the lesion responsible for the mutations induced by UVA was of a more complex nature and which possibly is repaired by homologous recombination (HR). The results indicated that UVA was more recombinogenic than UVC at equal levels of CPDs. We therefore suggest that UVA induces a complex type of lesion, which might be an obstruction during replication fork progression that requires HR repair to be further processed.     

Quantitative analysis of root and ectomycorrhizal exudates as a response to Pb, Cd and As stress

We examined exudation of low molecular weight (LMW) organic compounds of ectomycorrhizal (ECM) and non-mycorrhizal (NM) seedlings in relation to metals. Scots pine seedlings, either colonized by one of six different ECM fungi or NM, were grown in Petri dishes containing glass beads and liquid growth medium and exposed to elevated concentrations of Pb, Cd and As. Exudation of LMW organic compounds (LMW organic acids (LMWOAs), amino acids and dissolved monosaccharides) and dissolved organic carbon (DOC) was determined qualitatively and quantitatively and exudation rates were calculated. Metals had a significant impact on exudation, especially of oxalate. For Pb and Cd treatments, exudation of oxalate and total LMWOAs generally increased by 15–45% compared to nutrient controls. Production of amino acids, dissolved monosaccharides and DOC was not significantly stimulated by exposure to metals; however, there were non-significant trends towards increased exudation. Finally, exudation generally increased in the presence of mycorrhizal seedlings compared to NM seedlings. The results suggest that ECM fungi may reduce the toxicity of metals to plants through significant increases in the production of organic chelators. Axenic conditions are required to assess the full potential for production of these molecules but their overall significance in soil ecosystems needs to be determined using additional experiments under more ecologically realistic conditions.     

Evaluation of electron spin resonance for studies of superoxide anion production by human neutrophils interacting with Staphylococcus aureus and Staphylococcus epidermidis

The present study evaluates electron spin resonance (ESR) and the spin trapper 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) for analysis of superoxide radical production by human neutrophils interacting with viable Staphylococcus aureus and Staphylococcus epidermidis bacteria. To avoid auto-activation due to interaction with glass surfaces, neutrophils were preincubated in plastic tubes until the peak response was reached, and then transferred to a quartz flat cell to record the ESR spectra. The time point for peak response was identified by parallel analysis of the bacteria–neutrophil interaction using luminol amplified chemiluminescence. We found detectable ESR spectra from neutrophils interacting with as few as five bacteria of the weak activating S. epidermidis per neutrophil. Addition of the NADPH oxidase inhibitor diphenylene iodonium totally abolished spectra. Catalase, DMSO or an iron chelator had no impact on the produced spectra and ionomycin, a selective activator of intracellular NADPH oxidase, gave significant ESR spectra. Taken together, our results indicate that DEPMPO is cell permeable and detects NADPH oxidase derived superoxide anions formed in phagosomes or released by human neutrophils phagocytosing viable S. aureus and S. epidermidis. The technique may be used as a sensitive tool to evaluate superoxide anion production in human neutrophils.