Patricia S. Reisert

In vitro characterization of response to stimulus (wounding) with regard to ageing in human skin fibroblasts.

Confluent cultured normal human skin fibroblasts from neonatal, adult and aged donors have been stimulated to respond to wounding of the cell sheet. The latent period prior to initial migration of cells from the leading edge of the monolayer is correlated with in vitro population doubling level and in vivo donor age. Time-lapse photography of areas along the edge of the cell sheet reveals a specific pattern of migration by which the cells reestablish a confluent monolayer.

Anoxia-induced changes in purine nucleoside metabolism of in vitro aged human fibroblasts.

Inosine deriving from the metabolism of adenosine or inosine monophosphate (IMP) in the fibroblast provides the substrate for xanthine oxidase and is, therefore, an important source of toxic oxygen free radicals. With well-oxygenated medium, adenosine release appears to be greater for aged than young fibroblasts. In that the adenosine release by young cells is enhanced by reduced oxygenation, the effect anoxic stress on the release of the purine nucleosides adenosine and inosine by low-passage (PDL 23-26; young) vs. high-passage (PDL 43-51; aged) human lung fibroblasts (IMR-90) was studied. Cultures of confluent fibroblasts were incubated for 16 hr under normoxic (NF) or anoxic (AF) atmospheres. The release of adenosine and inosine was determined by HPLC at 0, 3, 6 and 24 hr after termination of the 16-hr period. Immediately following anoxia (time 0), adenosine release by young AF was 29% greater than for young NF, whereas both the youn

Synthetic peptides to identify antigenic determinants on Epstein-Barr virus gp350/220.

We synthesized three peptides, MA1 - Thr19-Val28(+Tyr) -, MA2 - Ser807-Ala816-, and MA3-Ser718-Glu729(+Tyr) from the sequence of Epstein-Barr virus gp350/220 and immunized rabbits with these peptides. Rabbit antisera to the peptides had antipeptide radioimmunoassay titers of 1:400 for anti-MA1, 1:200 for anti-MA2, and 1:1600 for anti-MA3. The anti-MA1 serum recognized gp350/220 in Western blotting to SDS-electrophoresed proteins from 12-O-tetradecanoylphorbol-13-acetate- and n-butyrate-treated B95-8 cells, but anti-MA2 and MA3 sera did not. None of the sera reacted with gp350/220 by membrane or cytoplasmic immunofluorescence or by immunoprecipitation of Triton X-100 solubilized proteins.

Roles of Accessory Molecules in Processing and Presentation of Foreign Antigens

The Ii sequence Phe146-Val164 was hypothesized to coil as an amphipathic, α helix in the desetope of class II MHC antigens until release in an acidic, foreign antigen-containing endosome to catalyze charging of the desetope with a structurally similar foreign peptide (1). A serum from one of four rabbits injected with a KLH-conjugated, synthetic peptide of Ii sequence 146–169, substituting Tyr for Phe146, immunoprecipitated a 67-kD protein from Raji cells after 3 hours [35S]methionine labeling and 69- and 67 kD-proteins after 5 or 10 hours of labeling, respectively. The 67-kD protein was not sensitive to endoglycosidase F treatment or tunicamycin and had a pI about 5.5. p67 was not surface expressed as judged by immunofluorescence analyses with the antiserum and by immunoprecipitation of surfacebiotinylated proteins. These human molecules might correspond to the murine proteins p72/74, described by Lakey et al. (2) to have a potential role in antigen presentation.

Structures of Class II MHC Molecules and Accessory Proteins During Trafficking in Subcellular Compartments

Synthetic rates of class II major histocompatibility complex (MHC) and associated molecules increased appreciably after 4 to 8 hours of polyclonal activation without any perceptible change in forms of those molecules. Ii was cleaved in intracellular vesicles to release a COOH-terminal p25 fragment at 20 to 40 minutes after [35S] methionine labeling. The association of Ii to class II MHC antigen could protect the desetope of class II MHC molecules from ambient peptides until reaching a foreign peptide containing endosome, there catalyzing the charging of digested foreign peptides onto the desetope in a concerted mechanism of Ii release and association of the foreign peptide. Ii could also retain class II MHC antigens in an endosome until charged for transport to the cell surface.