Structural requirements for the procoagulant activity of nucleic acids.

Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects

Detection of the ABCB11930_1931del TC Mutation in Two Suspected Ivermectin-Sensitive Cats and Their Relatives by a Novel TaqMan Allelic Discrimination Assay

The multidrug resistance gene MDR1 (syn. ABCB1) encodes for the multidrug efflux transporter P-glycoprotein (P-gp), which is highly expressed at the blood-brain barrier and protects the brain from potentially neurotoxic compounds, such as ivermectin. MDR1 mutation in dogs is known to be linked to dramatically increased brain accumulation of ivermectin and life-threatening neurological toxicity. The present report describes two suspected ivermectin-sensitive Maine Coon cats, which exhibited neurological toxicity following subcutaneous application of therapeutic doses of ivermectin. Both cats showed a homozygous 2-bp deletion in the MDR1/ABCB1 coding sequence (ABCB11930_1931del TC, syn. MDR1 nt1930(del2)) that had previously been associated with a drug-sensitive phenotype in cats. For cat MDR1 genotyping, a novel TaqMan allelic discrimination assay was established and validated. This assay was used for ABCB11930_1931del TC genotyping of the drug-sensitive cats as well as of more than 50 relatives. About half of them had the heterozygous MDR1(+/-) genotype, while none of these related cats with former ivermectin treatment had a history of drug-sensitivity. In conclusion: The present study supports previous findings on drug-sensitivity in cats with homozygous ABCB11930_1931del TC mutation. The newly established TaqMan allelic discrimination assay provides a useful and reliable method for routine MDR1 genotyping in cats in order to identify drug-sensitive cats prior to treatment with established P-gp substrates such as ivermectin and other macrocyclic lactones and thus to improve therapeutic safety

Self-extracellular RNA acts in synergy with exogenous danger signals to promote inflammation

Self-extracellular RNA (eRNA), released from stressed or injured cells upon various pathological situations such as ischemia-reperfusion-injury, has been shown to act as an alarmin by inducing procoagulatory and proinflammatory responses. In particular, M1-polarization of macrophages by eRNA resulted in the expression and release of a variety of cytokines, including tumor necrosis factor (TNF)-α or interleukin-6 (IL-6). The present study now investigates in which way self-eRNA may influence the response of macrophages towards various Toll-like receptor (TLR)-agonists. Isolated agonists of TLR2 (Pam2CSK4), TLR3 (PolyIC), TLR4 (LPS), or TLR7 (R848) induced the release of TNF-α in a concentration-dependent manner in murine macrophages, differentiated from bone marrow-derived stem cells by mouse colony stimulating factor. Here, the presence of eRNA shifted the dose-response curve for Pam2CSK4 (Pam) considerably to the left, indicating that eRNA synergistically enhanced the cytokine liberation from macrophages even at very low Pam-levels. The synergistic activation of TLR2 by eRNA/Pam was duplicated by other TLR2-agonists such as FSL-1 or Pam3CSK4. In contrast, for TLR4-agonists such as LPS a synergistic effect of eRNA was much weaker, and was not existent for TLR3-, or TLR7-agonists. The synergistic eRNA/Pam action was dependent on the NFκB-signaling pathway as well as on p38MAP- and MEK1/ERK-kinases and was prevented by predigestion of eRNA with RNase1 or by antibodies against TLR2. Thus, the presence of self-eRNA as alarming molecule sensitizes innate immune responses towards pathogen-associated molecular patterns (PAMPs) in a synergistic way and may thereby contribute to the differentiated outcome of inflammatory responses

Tyrosine 146 of the Human Na⁺/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes

Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) is a hepatic bile acid uptake carrier participating in the enterohepatic circulation of bile acids. Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with Km values of 57.3–112.4 µM and Vmax values of 6683–7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes

Influences of DNA-aptamers on the intrinsic coagulation pathway.

<p>(A) The activation of prekallikrein was followed in the presence of increasing doses of the DNA-aptamers 15mer-thrombin (open circles, interrupted line), 44mer-APC (closed squares), 26mer-AS1411 (closed circles) or 25mer-VEGF (closed triangles, dotted line). (B) Turbidity clot-lysis assays were performed in the absence (black bars) or presence of 1.25 µg/mL (white bars) and 10 µg/mL (striped bars) of the DNA-aptamers 15mer-thrombin, 44mer-APC, 26mer-AS1411 or 25mer-VEGF, respectively. Coagulation was initiated by recalcification, clotting times were defined as respective time points of maximal absorbance. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n = 3; *p<0.05; 1.25 µg/mL or 10 µg/mL vs. control). (C) The activation of prekallikrein was followed in the presence of increasing doses of the oligonucleotide 21mer-H1 (closed circles) and 21mer-H1-HEG (closed squares). All data represent mean ± SEM (n = 6).</p

Stability of DNA- and RNA-oligonucleotides in human plasma.

<p>(A) Integrity of 21mer-L1 and (B) 21mer-H1 DNA- and RNA-oligonucleotides was confirmed by polyacrylamide gel electrophoresis without or after preincubation in pooled human plasma for 1 or 5 min, respectively. Each panel represents one representative experiment out of three independent ones.</p

Sequences and secondary structures of DNA- and RNA-oligonucleotides.

<p>Described are the secondary structures of (A) DNA- and (B) RNA-oligonucleotides as predicted by the mFold DNA or RNA database. Delta G (ΔG) values represent changes in free enthalpy representative for the stability of the compounds with negative (exergonic) or positive values (endergonic).</p

Binding of biotinylated DNA-oligonucleotides to different coagulation factors of the intrinsic coagulation pathway.

<p>Microtiter plate wells were coated with 10 µg/mL each of (A) kininogen or (B) prekallikrein and binding of increasing concentrations of the biotinylated DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H3 (open triangles), 21mer-L1 (closed squares) was assessed. All data represent mean ± SD (n = 3; *p<0.05; 21mer-L1 and 21mer-H3 vs. 21mer-H1) of one representative experiment out of three independent ones. (C) Microtiter plate wells were coated with 10 µg/mL kininogen, factor XI (FXI) or factor XII (FXII) each and incubated with 25 µg/mL each of different biotinylated DNA-oligonucleotides: 21mer-H1 (black bars), 21mer-H3 (white bars) or 21mer-L (hatched bars). All data represent mean ± SD (n = 3) of one representative experiment out of three independent ones. (D) Increasing concentrations of the biotinylated DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H3 (open triangles) or 21mer-L1 (closed squares) were analyzed for prekallikrein auto-activation. All data represent mean ± SEM (n≥3; *p<0.05; 21mer-H1 vs. 21mer-H3).</p