Imaging Single Retrovirus Entry through Alternative Receptor Isoforms and Intermediates of Virus-Endosome Fusion

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes

Intermittent on line (vivo) measurement of pO/sub 2/, pCO/sub 2/, pH and K/sup +/ by means of automatic sampling of blood and automatic calibration of electrodes and amplifiers

ABGA (Automated Blood Gas Analyser) may be connected by sample catheter to either an artery or vein. At desired intervals ABGA draws a blood sample automatically and performs an analysis. The measuring part consists of a special cuvette with conventional electrodes. Before a blood sample is drawn the four signal amplifiers are calibrated automatically by means of two calibration solutions equilibrated with gas mixtures of known composition. After completion of the analysis the blood within the cuvette is rinsed away but the blood within the catheter is returned to the patient with the aid of a saline infusion. The measuring cycle time including calibration is 8 minutes; without calibration 3 minutes. Blood loss per cycle amounts to 2 cm/sup 3/. With electrical grounding the patient current is less than 1 mu

Clinical imaging of the vulnerable plaque in the coronary arteries: new intracoronary diagnostic methods.

Rupture of a vulnerable plaque is the main cause of acute coronary syndromes and myocardial infarction. The features of rupture-prone atherosclerotic plaques have been previously described by pathologists. However, identification of vulnerable plaques in vivo is essential to study their natural history and to evaluate potential treatment modalities. Coronary angiography is the gold standard for the diagnosis of coronary artery disease, but it is unable to distinguish between stable and unstable plaques and to accurately predict future cardiac events. This current perspective describes the recently developed invasive imaging techniques to detect atherosclerotic vulnerable plaques in the coronary tre