Meredith N. Runner

Histological and fine structural changes during chondrogenesis in micromelia induced by 6-aminonicotinamide☆

Abstract Investigations of drug-induced congenital deformity can lead to better understanding of developmental mechanisms. An analog of nicotinamide, 6-aminonicotinamide (6-AN), given in a dose of 10 μg to chick embryos on day 4 of incubation, resulted in micromelia as diagnosed by reduced cartilage and bone in 100% of embryos. Histologically, chondrogenic cells at day 5 were compactly arranged and the extracellular matrix showed reduced metachromatic staining with toluidine blue. Electron microscopy of these cells showed a deficiency of rough endoplasmic reticulum on days 5 and 6 with dilated cisternae by day 8. In the Golgi apparatus at day 5, vacuoles, which normally collect and transport mucopolysaccharides to the extracellular matrix, were small or absent. Degenerative changes occurred in the core of the chondrogenic rudiment on days 5 to 8, whereas cells and matrix subjacent to the developing perichondrium by day 7 appeared normal in staining and ultrastructural properties. The Golgi apparatus of chondrogenic cells was atypical by virtue of reduced numbers of collecting vacuoles. Necrosis of the central region of the chondrogenic rudiment was transitory; the damage to the rudiment was incompletely repaired by synthesis of matrix by cells near the perichondrial layer. These observations suggest that the limb malformation induced by 6-AN is brought about by inhibition of special synthesis of matrix by chondrogenic cells in addition to necrosis of core cells of the chondrogenic rudiment.

Coenzyme competition and precursor specificity during teratogenesis induced by 6-aminonicotinamide

Abstract The teratogenic mechanism of the nicotinamide analog 6-aminonicotinamide (6-AN), is thought to be inhibition of energy exchange reactions which in turn selectively interfere with synthesis of mucopolysaccharides. Nicotinamide and 6-AN were administered singly or in combination for studying micromelia in the embryonic chick limb. These coenzyme competition, protection, and reversibility experiments suggest that cells of 4-day embryos readily accept either nicotinamide or 6-AN. Preloading with nicotinamide 1–24 hours prior to exposure to 6-AN prevented teratogenicity of 6-AN. An excess of nicotinamide up to 2 times the maximal protective dose given 2 hours or more after 6-AN did not offset the teratogenicity of 6-AN. Once exposed either to 6-AN or to nicotinamide the cells do not readily accept either alternate molecule. This mutual exclusivity enabled demonstration that 6-AN is 80% effective as a teratogen in 4-day chick embryos within 2 hours after its administration. Duration of the effectiveness of a standard dose of 6-AN given to a 4-day embryo was studied by a 30-minute terminal label with 35S-sulfate. Maximum inhibition was seen 3 days after treatment and recovery to control values occurred in about 4 1 2 days. Because limb teratogenicity is a result of atypical chondrogenesis and because the experiments above were performed on mixed tissues of whole embryos or whole limbs, early rudiments of tibia at 9, 10, and 11 days were isolated and tested with 6-AN and labeled precursors. It was found that 2 hours of exposure of bone rudiments to 6-AN was sufficient to demonstrate selective inhibition of 35S incorporation while amino acid incorporation remained similar to controls. The effect of 6-AN in the limb of the chick embryo is prompt, demonstrable in 2 hours and temporarily irreversible. Recovery is seen in 4 to 5 days, but distortion of the cartilage model results in micromelia.

Differential permeability of murine visceral yolk sac to thymidine and to hydroxyurea.

Abstract Mouse embryos at the 10–12-somite stage of development were excised from the uterus either with or without the encapsulating visceral yolk sac and were incubated in vitro in 3 × 10 −7 M thymidine (methyl-T, 5 μCi/ml) for 30 min or in 4 × 10 −3 M hydroxyurea for 45 min with [ 3 H]thymidine present for the last 30 min. Radioautograms of nuclei of neural epithelium enabled an estimate of the effectiveness of the barrier imposed by the visceral yolk sac membrane to the passage of thymidine and hydroxyurea. Labeling of nuclei in the neural epithelium showed that the visceral yolk sac caused a 44% decrease in frequency and a 51% decrease in intensity of label. Hydroxyurea inhibited labeling by 15% in frequency and 37% in intensity irrespective of the presence or absence of visceral yolk sac. These results show that hydroxyurea and the presence of visceral yolk sac independently interfered with labeling of the neural epithelium by thymidine and that visceral yolk sac caused a proportionally greater retardation of label than did hydroxyurea. Nuclei of the endodermal epithelium of the intervening yolk sac, following exposure to hydroxyurea, showed a labeling decrease of 44% in frequency and 77% in intensity. The inhibitory effect of hydroxyurea on yolk sac labeling, however, did not alter yolk sac permeability to hydroxyurea. The results indicate that the visceral yolk sac, by offering no detectable barrier to hydroxyurea, permits prompt teratogenic action of hydroxyurea directly upon the embryo and suggest that the visceral yolk sac is a likely candidate to account for reports that the 8.5-day mouse embryo in situ fails to label with radioisotopic thymidine.

Transfer RNA changes in HeLa cells after vaccinia virus infection

Abstract Reversed phase cochromatography of aminoacyl-tRNAs prepared from uninfected and 9 h vaccinia virus-infected HeLa cells has revealed a quantitative alteration in aspartyl-tRNA and both a quantitative and qualitative change in phenylalanyl-tRNA. The chromatographic change in aspartyl-tRNA can be correlated with an increased acceptor activity for aspartic acid by tRNA preparations from infected cells, whereas phenylalanine acceptance is not significantly altered after infection.

Factors Associated with the Incidence of Infantile Diarrhea in Mice.

Summary 1. Observations on 900s C3H mice born to 76 parents showed that 30% of the young contracted diarrhea severe enough to make it necessary that they be discarded. The 46 litters so affected comprised 50% of the offspring from 39 pairs of mice. 2. Observations on the 39 pairs of parents which had one or more litters afflicted with diarrhea showed that the litters born before and after clean boxes were supplied were not differentially susceptible to diarrhea. 3. Correlations between the incidence of diarrhea and age of parents, and seriation of litters and size of litters, showed that each of these had some influence on the numbers of litters showing symptoms of diarrhea. Litters from young parents having early parities and large litters showed highest incidences of the disease. The physiological condition of the parents common to these three factors seems to be an important consideration. 4. A higher incidence of the disease was observed in December than in either October or November. Part of the increased incidence in December was due to a greater number of large litters, early parities and younger parents. Superimposed upon physiological condition of the parents were other factors which were tentatively called seasonal influences.