Column purification of adenovirus cores

A simple and efficient technique for purifying adenoviral chromatin (nucleoprotein cores) with Sephacryl S-1000 is described. This method is significantly faster than previous methods and gives a higher degree of purity with an increased recovery of the nucleoprotein. In addition, the structural integrity of the cores is maintained.     

Stability of acetylated and superguanidinated chymotrypsinogens

Conversion of lysine residues to homoarginine led to protein stabilization as determined earlier by hydrogen isotope exchange (P. Cupo W. El-Deiry, P. L. Whitney and W. M. Awad, Jr., 1980, J. Biol. Chem.255, 10828–10833). In order to see if neutralization of charges on lysine residues affected stability, a homogeneous derivative of chymotrypsinogen was prepared wherein all amino groups were acetylated. Hydrogen isotope exchange studies indicated that the derivative was less stable than the native protein. In addition, highly guanidinated chymotrypsinogen was prepared by first coupling ethylenediamine to carboxyl groups of guanidinated chymotrypsinogen. Thereafter the protein was treated with O-methylisourea to form guanidinoethylamido groups at the ends of carboxyl residues. Acrylamide gel electrophoresis indicated that two products were formed. Hydrogen isotope exchange studies demonstrated that superguanidinated chymotrypsinogen is even less stable than the acetylated derivative. Thus guanidination of residues in addition to lysine does not lead to protein stabilization. The possibility is that such a highly cationic protein causes backbone fluctuations because of repulsion of surface charges.     

Detection of dermorphin-like immunoreactivity in immune tissues

Site-directed antibodies against synthetic relateddermorphin peptides have previously been produced andcharacterized. One of them, specifically recognizingthe crucial opioid message (the N-terminal part ofthe molecule Tyr-D-Ala-Phe-Gly), was used in the presentstudy in order to detect and localize endogenousdermorphin-like molecules in immune tissues.Dermorphin-like peptides were found to be present inspleen and thymus of rat and mouse. The HPLCprofile of the immunoreactive material showeda major peak at a retention time of 32±1 min.Purification of immune cells by panning proceduresshowed that both B and T cells contained thisimmunoreactive material. Biochemical characterizationof the dermorphin-like immunoreactivity indicated thatthis material is a peptide resistant toaminopeptidase hydrolysis, suggesting the presence ofa putative D-amino acid residue or a residueconferring resistance to a proteolytic process.     

Sequential and Simultaneous Immunization of Rabbits with HIV-1 Envelope Glycoprotein SOSIP.664 Trimers from Clades A,B and C

We have investigated the immunogenicity in rabbits of native-like, soluble, recombinant SOSIP.664 trimers based on the env genes of four isolates of human immunodeficiency virus type 1 (HIV-1); specifically BG505 (clade A), B41 (clade B), CZA97 (clade C) and DU422 (clade C). The various trimers were delivered either simultaneously (as a mixture of clade A + B trimers) or sequentially over a 73-week period. Autologous, Tier-2 neutralizing antibody (NAb) responses were generated to the clade A and clade B trimers in the bivalent mixture. When delivered as boosting immunogens to rabbits immunized with the clade A and/or clade B trimers, the clade C trimers also generated autologous Tier-2 NAb responses, the CZA97 trimers doing so more strongly and consistently than the DU422 trimers. The clade C trimers also cross-boosted the pre-existing NAb responses to clade A and B trimers. We observed heterologous Tier-2 NAb responses albeit inconsistently, and with limited overall breath. However, cross-neutralization of the clade A BG505.T332N virus was consistently observed in rabbits immunized only with clade B trimers and then boosted with clade C trimers. The autologous NAbs induced by the BG505, B41 and CZA97 trimers predominantly recognized specific holes in the glycan shields of the cognate virus. The shared location of some of these holes may account for the observed cross-boosting effects and the heterologous neutralization of the BG505.T332N virus. These findings will guide the design of further experiments to determine whether and how multiple Env trimers can together induce more broadly neutralizing antibody responses.     

Effects of indomethacin on moloney sarcoma virus-induced tumors

Chronic administration of indomethacin to weanling (20 day old) BALB/c mice inoculated with Moloney sarcoma virus (MSV) delayed the onset of the tumor and suppressed the tumor growth. As expected, the prostaglandin (PG) levels associated with the MSV-injected leg muscle of the indomethacin treated mice were greatly depressed when compared to the elevated PG content associated with the tumors of MSV injected control mice. There was no effect of indomethacin on the cyclic AMP or cyclic GMP levels.The elevated levels of PG and cyclic AMP associated with the tumor were found to parallel the tumor growth.Administration of antilymphocyte serum (ALS) to the mice at the time of MSV inoculation resulted in accelerated and enhanced tumor growth. Indomethacin treatment of these mice similarly suppressed the tumor growth, but less dramatically.     

Synthesis and post-translational processing of proenkephalin in light- and dark- adapted chicken retinas

Synthesis and post-translational processing of retinal proenkephalin in response to the light or darkness were studied in newly hatched chickens using chromatography, enzymatic digestion (trypsin-carboxypeptidase B) and radioimmunoassay. We found that the concentration of free [Met⁵]-enkephalin in crude retinal extracts increased with the time in the light and decreased in the dark. This effect was directly dependent on illumination, rather than the consequence of an endogenous circadian rhythm. In contrast, the total amount of cryptic [Met⁵]-enkephalin (in larger enkephalin-containing polypeptides) remained constant in all stages of the light/dark cycle. We also showed that the relative amounts of cryptic [Met⁵]-enkephalin stored at different molecular weights remained constant, and that the concentrations of three identified proenkephalin-derived peptides, [Met⁵]-enkephalin, [Leu⁵]-enkephalin and [Met⁵]-enkephalin-Arg⁶-Phe⁷, were higher in the light-adapted than in the dark-adapted retina; but the relative amounts (ratios) of the three proenkephalin-derived peptides stored in the light and in the dark were equal. These data are consistent with the hypothesis that synthesis and processing of proenkephalin proceed at a constant rate and with similar pattern independent of illumination or adaptation, whereas the processed enkephalins are released preferentially in the darkness and accumulated in the light.