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In a series of experiments first published in 1961, we reported that antibodies prepared against rat kidney were potent teratogenic agents when administered to pregnant rats (Brent 1961). It was determined that antisera which were teratogenic localized in the developing yolk sac (Slotnick and Brent, 1966). Antisera prepared against VYS proved to be the most potent teratogens, and these teratogenic antisera reduced the endocytic uptake of proteins by the VYS (Brent et al, 1972, Freedman et al, 1983). Utilizing isolectric focusing techniques, various proteins were isolated from the VYS and antisera were prepared against these individual proteins. A large number of antisera were not teratogenic and did not localize in the developing yolk sac. A smaller group of antisera were found to localize in the VYS These antisera were studied with several techniques: Determination of the endocytic index utilizing (14C) sucrose, in vivo yolk sac localization utilizing fluorescent labelled antibody and teratogenic potency. It was found that yolk sac localization of antibody specific for yolk sac antigens does not prove that the antibodies have teratogenic potency. On the other hand, if the antibodies also reduce the endocytic index, then the antibodies will invariably have teratogenic potency. Thus, the reduction of endocytosis is the first method of accurately predicting teratogenic potential without having to perform time-consuming in vivo bioassays.

Polyclonal antisera against rat visceral yolk sac (VYS) produces severe congenital malformations when injected into pregnant rats on the 9th day of gestation. To define and characterize the teratogen-stimulating antigen(s) from the VYS, monoclonal antibodies (MCA) against rat VYS were prepared. Spleen cells from BALB/c mice, hyperimmunized with VYS antigens purified by IEF and gel filtration were fused with two myeloma cell lines SP2/0-Ag 14 and P3x63 Ag. 8.653. Hybrids specific for VYS by indirect immunofluorescence were selected and cloned by limiting dilution and further expanded as ascitic tumor in BALB/c mice. More than 25 immunofluorescent hybrids for VYS have been selected and their biological activity defined. The first 12 clones were not teratogenic but since last year (1986) ascitic fluid from B-3 clone (IgG 2b type) induced CNS malformations and the clone D-4 (IgG 2a type) induced growtn retardations when injected into pregnant rats on the 9th day of gestation. Each teratogenic MCA stained the apical portion of VYS endodermal cell, tubular kidney brush border and failed to react with kidney glomeruli, Reicherts membrane, long, lens capsule and liver tissue by immunofluorescence. Both teratogenic MCA's failed to recognize the VYS antigens by Western blots. Further studies will be directed toward using the teratogenic MAC's to study the mechanism of teratogenesis and the quantitative aspects of embryonic nutrition during early organogenesis. (Supported by NIH)

In 1961 we reported that heterologous antisera prepared in the rabbit against rat kidney proved to be teratogenic when injected into pregnant rats (Brent et al, Proc. Soc. Exp. Biol. Med.). The teratogenic antiserum localized in the yolk sac and yolk sac antisera proved to be a potent rat teratogen. Metabolic studies have indicated that the yolk sac dysfunction is due to the antiserum's ability to interfere with the endocytosis of macromolecules, thus depleting the embryo and yolk sac of required quantities of amino acids. Two important questions were asked. At what stage of rat gestation does teratogenic antiserum no longer have an embryotoxic effect on the developing embryo or fetus arid does the teratogenic antiserum affect pinocytosis late in rat development. Pregnant rats were exposed to teratogenic antiserum and while the most dramatic effects were produced during early organogenesis, embryonic and fetal effects were observed during the early fetal period several days after trypan blue no longer affected the embryo. Mortality or growth retardation were not observed when pregnant rats were administered teratogenic antiserum the last 5 days of rat gestation, in spite of the fact that the antisera does depress pinocytosis in vitro in the yolk sac late in gestation. (Supported by NIH)

Rat embryo culture was initiated on the 10th day of gestation to study embryonic site protein synthesis in normal and pathologic states. Three culture media were employedsrat serum, serum plus teratogenic sheep anti-rat visceral yolk sac (VYS) serum, or rat serum plus normal sheep serum. Two studies were performed: a) 3H-leucine was added to produce incubation periods of 20 min. to 24 hr.; and after an initial period of culture in the presence of 3H-leucine to obtain a constant precursor specific radioactivity, culture was continued in the absence of 3H-leucine for a total of 1-30 hr. Embryos, VYS and culture media were analysed for total radioactivity and 3H-leucine specific radioactivity in the protein-bound and free amino acid fractions. The results suggest that: a) anti-VYS serum inhibited protein synthesis by the embryo and VYS but had no detectable effect on protein degradation; b) anti-VYS serum increased intracellular labeled leucine concentration. The teratogenic mechanism of anti-VYS serum appears to involve both the suppression yolk sac amino acid nutritive support of the embryo and the inhibition of the utilization of amino acids by the embryo for protein synthesis. These techniques provide us with the ability to determine the amino acid requirements which will support normal organogenesis and to quantitate the amino acid deprivation which will result in embryopathology. (Supported by NIH)

A nucleoside phosphorylase specific for 6-amino purine nucleosides has been purified from extracts of promastigotes of the pathogenic protozoan Leishmania donovani. Unlike the three nucleoside hydrolases previously isolated from this organism (Koszalka and Krenitsky, JBC, 254, 8185-8193, 1979), this enzyme was capable of nucleoside synthesis from pentose-1-phosphate and adenine or selected analogues. The purified enzyme had a pl value of 6.2, a molecular weight of 86,000 daltons, and a pH optimum for adenosine phosphorolysis between 5.6 and 6.5. The energy of activation for the synthetic direction with adenine and Rib-1-P was 8600 cal/mole, while that for the phosphorolytic direction was 11,300 cal/mole.Substrates for the phosphorolytic direction in decreasing order of substrate efficiency (Rel Vmax/Km) were: 5'-methylthioadenosine (26), 2'-deoxyadenosine (12.5), 5'-deoxyadenosine (11.8) and adenosine (0.7). The Kmvalues ranged from 1-46 µM. Adenine arabinoside, inosine, 2'-deoxyinosine, guanosine, and 2'-deoxyguanosine were neither substrates nor potent inhibitors. Adenine and various 2-substituted adenine analogues were substrates with Rib-1-P, whereas 4-amino-pyrazolo(3,4-d)pyrimidine and the 3-deaza-, 7-deaza-, and 8-aza-adenine analogues were all non-substrate competitive inhibitors. This enzyme catalyzed nucleoside synthesis from adenine and Rib-1-P, 2-'dRib-1-P or 5'-methylthio-Rib-1-P. A marked difference between this protozoal MTA phosphorylase and the mammalian counterpart is its ability to synthesize and cleave 2'-deoxyadenosine.