Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. I. Correlation of graft survival with antidonor IgG antibody subclasses

We have recently demonstrated that cardiac allograft rejection in the PVG.R8-to-PVG.1U rat strain combination involves the recognition of a isolated class I (RT1.Aa) molecules as peptides in the context of the recipient MHC molecules. Three synthetic peptides (P1, P2, and P3) corresponding to the alpha-helices of the RT1.Aa molecule served as T-cell epitopes for graft rejection. In this study, we demonstrate that two of these peptides (P2 and P3) are sufficient to induce immune nonresponsiveness (median survival time >237 days) to cardiac allografts when presented to the recipient immune system in the thymus 7 days before transplantation. This effect was time dependent, as intrathymic inoculation 60 days before transplantation did not prolong graft survival (median survival time=12 days). Previous studies have demonstrated a critical role for alloantibody responses in mediating graft rejection in this rat strain combination. We, therefore, studied the role alloantibody responses may play in the observed immune nonresponsiveness. The titers of alloantibody in serum samples harvested from graft recipients at different times after transplantation were measured. We used recipient primary aortic endothelial cells genetically manipulated to express the donor RT1.Aa molecule as targets in an enzyme-linked immunosorbent assay. High titers of anti-RT1.Aa IgM antibody were detected in unmanipulated controls at the time of graft rejection. The IgM antibody switched to high IgG titers in intrathymically inoculated rats with accelerated or delayed rejection. Graft rejection in intrathymically manipulated recipients that had achieved a transient state of immunological nonresponsiveness correlated with higher titers of the IgG2b alloantibody. In marked contrast, the long-term graft survivors expressed undetectable or low levels of the IgG2b antibody and moderate to high levels of the IgG1 and IgG2a subclasses. These data suggest that the IgG2b alloantibody may contribute to the rejection reaction, whereas IgG1 and IgG2a may be involved in active enhancement of graft survival.     

On the structure of the cofactor in the complex formed with thymidylate synthetase, 5,10-methylenetetrahy-drofolate and 5-fluoro-2'-deoxyuridylate

A study was undertaken to ascertain whether dihydrofolic acid is produced in the complex formed with 5,10-methylenetetrahydrofolate, 5-fluorodeoxyuridylate and thymidylate synthetase, as suggested by ultraviolet difference spectral studies. The complex was formed using the cofactor specifically labeled with tritium at the 6-position. After dissociation by equilibration with unlabeled cofactor, it was demonstrated that the tritium remained exclusively at the 6-position. Had oxidation of the cofactor occurred within the complex to give a methylated enzyme form, tritium should have been transferred to the one-carbon unit of the cofactor. It was also found that the difference spectrum of the ternary complex which resembles that of dihydrofolic acid can also be produced by substituting an analog of the cofactor which is not susceptible to oxidation. The results described here demonstrate that oxidation of the cofactor does not occur in the ternary complex and suggest that the unusual ultraviolet spectrum results from perturbations of a chromophore of the bound cofactor.     

Cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage SPO1

2'-Deoxyuridylate hydroxymethylase (dUMP-hmase) from phage SPO1 has been cloned and expressed in Escherichia coli. In crude extracts, the enzyme represents about 25% of the soluble protein and has a higher specific activity than the most purified preparation yet reported. The enzyme was purified to homogeneity by ion-exchange and hydrophobic chromatography. The subunits of dUMP-hmase are 45 kDa by SDS-PAGE and form dimers with a molecular mass of 89.2 kDa by analytical centrifugation. In addition to the normal reaction, dUMP-hmase catalyzes the 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate)-independent tritium exchange of [5-3H]dUMP for protons of water and dehalogenation of 5-bromo-2'-deoxy-uridine-5'-monophosphate; the enzyme also forms a covalent binary adduct with pyridoxal 5'-monophosphate and a covalent ternary complex with 5-fluoro-2'-deoxyuridine-5'-monophosphate and CH2H4folate. Folic acid inhibits the tritium release catalyzed by dUMP-hmase in the presence of cofactor but has no effect on the catalysis of cofactor-independent tritium exchange.     

Detection and analysis of Tetrahymena pyriformis 26S ribosomal DNA domain sequences, differing in degree of evolutionary conservation

Domains of different evolutionary conservatism were defined in the 26S rDNA sequence of T. pyriformis. The fragment of studied DNA (1212 bp) showing high evolutionary conservatism was cloned. It was shown this fragment of DNA may be used to a probe for blot-hybridization analysis of the structure of rDNA from various taxa, protists to mammals. Superconservative and hypervariable domains were defined. The first are good for the primers for PCR analysis of rDNA from various taxa, the second--for species specific primers.     

Study of methods of inactivating a concentrated, purified cultured antirabies vaccine]

Two methods of inactivation, with ultraviolet (UV) and gamma (gamma) rays, of the concentrated purified tissue culture rabies vaccine from the Vnukovo-32 strain were studied. Under the optimal conditions of treatment of 100-fold concentrated purified virus-containing suspension, a completely inactivated vaccine with high immunogenicity indices (4.1 to 78) was obtained. Both the inactivation methods were found to be of equal value.