Tetraalkylammonium derivatives as real-time PCR enhancers and stabilizers of the qPCR mixtures containing SYBR Green I

Tetraalkylammonium (TAA) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (PCR) detected by end-point analysis. In this study, we compared the ability of various TAA derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time PCR based on fluorescence detection from intercalating dye SYBR Green I (SGI). Our data indicate that TAA chlorides and some other TAA derivatives serve as potent enhancers of SGI-monitored real-time PCR. Optimal results were obtained with 10-16 mM tetrapropylammonium chloride. The effect of TAA compounds was dependent on the nature of counter ions present and composition of the reaction mixtures used. Based on measurements of SGI-generated fluorescence signal in the presence of PCR-amplified DNA fragments, oligonucleotide primers and/or various additives, we propose that TAA-derivatives reduce the binding of SGI to oligonucleotide primers and thus enhance primer-template interactions during annealing phase. Furthermore, these compounds serve as stabilizers of SGI-containing PCR mixtures. The combined data indicate that TAA derivatives might be a new class of additives contributing to robustness of real-time PCR monitored by asymmetrical cyanine dye SGI.     

Complement mediates the binding of HIV to erythrocytes

A fraction of HIV is associated with erythrocytes even when the virus becomes undetectable in plasma under antiretroviral therapy. The aim of the present work was to further characterize this association in vitro. We developed an in vitro model to study the factors involved in the adherence of HIV-1 to erythrocytes. Radiolabeled HIV-1 (HIV) and preformed HIV-1/anti-HIV immune complexes (HIV-IC) were opsonized in various human sera, purified using sucrose density gradient ultracentrifugation, and incubated with human erythrocytes. We observed that, when opsonized in normal human serum, not only HIV-IC, but also HIV, bound to erythrocytes, although the adherence of HIV was lower than that of HIV-IC. The adherence was abolished when the complement system was blocked, but was maintained in hypogammaglobulinemic sera. Complement-deficient sera indicated that both pathways of complement were important for optimal adherence. No adherence was seen in C1q-deficient serum, and the adherence of HIV was reduced when the alternative pathway was blocked using anti-factor D Abs. The adherence could be inhibited by an mAb against complement receptor 1. At supraphysiological concentrations, purified C1q mediated the binding of a small fraction of HIV and HIV-IC to erythrocytes. In conclusion, HIV-IC bound to erythrocytes as other types of IC do when exposed to complement. Of particular interest was that HIV alone bound also to erythrocytes in a complement/complement receptor 1-dependent manner. Thus, erythrocytes may not only deliver HIV-IC to organs susceptible to infection, but free HIV as well. This may play a crucial role in the progression of the primary infection.     

X-ray structure of two crystalline forms of a streptomycete ribonuclease with cytotoxic activity

Ribonuclease (RNase) Sa3 is secreted by the Gram-positive bacterium Streptomyces aureofaciens. The enzyme catalyzes the cleavage of RNA on the 3' side of guanosine residues. Here, x-ray diffraction analysis was used to determine the three-dimensional structure of two distinct crystalline forms of RNase Sa3 to a resolution of 2.0 and 1.7 A. These two structures are similar to each other as well as to that of a homolog, RNase Sa. All of the key active-site residues of RNase Sa (Asn(42), Glu(44), Glu(57), Arg(72), and His(88)) are located in the putative active site of RNase Sa3. Also herein, RNase Sa3 is shown to be toxic to human erythroleukemia cells in culture. Like onconase, which is an amphibian ribonuclease in Phase III clinical trials as a cancer chemotherapeutic, RNase Sa3 is not inhibited by the cytosolic ribonuclease inhibitor protein. Thus, a prokaryotic ribonuclease can be toxic to mammalian cells.