Molecular characterization, enzymatic analysis, and purification of murine proprotein convertase-1/3 (PC1/PC3) secreted from recombinant baculovirus-infected insect cells.

A cDNA coding for the murine proprotein convertase-1 (mPC1 also known as mPC3 or mSPC3) was inserted into the Autographa californica nuclear polyhedrosis virus. Following infection of Spodoptera frugiperda cells, the recombinant N-glycosylated protein is secreted into the cell culture medium from which it can be purified to homogeneity as a fully enzymatically active enzyme. Two major secreted molecular forms of mPC1 with apparent molecular weights of 85 and 71 kDa, respectively, and a minor one of 75 kDa are immunodetected in the medium. Automated NH2-terminal sequencing reveals that all three forms result from processing at the predicted zymogen activation site whereas both the 75- and the 71-kDa forms are truncated at their COOH-terminus. Labeling by an active-site titrant demonstrates that the 85-kDa form is optimally labeled at near neutral pH whereas the COOH-truncated forms are optimally labeled at acidic pH. Additionally it is shown that the 85-kDa mPC1 is transformed into the COOH-truncated forms following in vitro incubation at acidic pH levels and in presence of calcium. Concomitantly, the transformation from 85 to 71 kDa is accompanied by a 10- to 40-fold increase in enzymatic activity upon assaying at pH 6.0. The 71-kDa form can be recovered after purification at a level of 1 to 1.5 mg per liter of cell culture medium and is enzymatically stable only in the pH range from 5.0 to 6.5. Cells treated with tunicamycin show a drastically reduced secretion of the convertase in the medium but are not affected by swainsonine and deoxymannojirimycin. Finally, the 85-kDa secreted mPC1 is shown to be sulfated.

Live influenza vaccine: screening of attenuated virus strains by a 50% ciliary activity inhibition test in organ cultures of ferret trachea.

This study of three live attenuated inhibitor-resistant influenza vaccines showed that these preparations are usually antigenic and that they caused no significant reactions when characterized by an index of attenuation equal to or slightly better than 1.0 arbitrarily attributed to the 'reference' attenuated A/Hong Kong/68 strain of Beare and Bynoe. This index, measured in vitro on ferret tracheal rings, is expressed as the ratio of the time required for ciliary activity inhibition of 50% of the rings by the tested candidate vaccine strain and the 'reference' attenuated strain. Induction of heterologous antibodies was also observed. Oral administration of underattenuated perparations did not cause the severe reactions which were observed when the same vaccine was administered intranasally.

Lesions of the central nervous system induced in nonhuman primates by live influenza viruses.

Intracerebral and intraspinal inoculations of non-neuropathic and neuropathic strains of influenza virus into rhesus, patas and cercopithecus monkeys resulted in an acute focal ependymitis, choroiditis and meningitis followed by focal ependymal denuding without parenchymal involvement. Aqueductal stenosis and moderate hydrocephalus developed in two animals as sequelae of ependymal cell loss.