Noriko Yamada

Developmental features of cerebellar hypoplasia and brain bilirubin levels in a mutant (Gunn) rat with hereditary hyperbilirubinaemia.

Abstract— The marked cerebellar hypoplasia found in the homozygous (jj) Gunn rat with hereditary unconjugated hyperbilirubinaemia may provide an explanation of bilirubin neurotoxicity in vivo. In the jj Gunn rat. Purkinje cells were nearly selectively affected in the cerebellar cortex, and the cerebellar weight showed no increase after 10 days of age. The development‐dependency of the cerebellar lesion was supported by the observation that the cerebellar lobuli which developed earlier were less affected. Brain bilirubin in the developing jj Gunn rat was determined by a spectrophotometric method, and was found to be extremely low (1–3 μg). The level of brain bilirubin decreased after birth, and showed little correlation with the level of bilirubin free of albumin which correlated clearly with total serum bilirubin level even in the neonate. These findings suggest that there is an affinity of brain tissue for bilirubin associated with the blood‐brain barrier to bilirubin. No significant difference was found between the levels of bilirubin in the cerebellum and those of other brain regions in jj Gunn rat.

Impairment of DNA synthesis in Gunn rat cerebellum

Brain DNA synthesis was developmentally investigated in Gunn rat with marked cerebellar hypoplasia due to hereditary hyperbilirubinemia. In this mutant rat, the Purkinje cell was nearly selectively affected in the cerebellar cortex by bilirubin. The impaired DNA synthesis was observed in homozygous (jj) Gunn rat cerebellum, in which the DNA content and [3H]thymidine incorporation rate into DNA decreased after 10 days of age compared to those in the heterozygous (Jj)littermate. In contrast, these impairments were not found in the non-cerebellar parts of the brain and liver of jj Gunn rat. The activity of cerebellar thymidine kinase in jj Gunn rat decreased from a very early stae, being 80% of Jj rat at 6 days, and 50% at 10 days of age. The enzyme activity was not affected in the non-cerebellar parts of the brain. Although bilirubin competitively inhibited cerebellar thymidine kinase activity in vitro (15% at 10(-5) M), such bilirubin level was found to be about 1000-fold that in vivo. Moreover, photo-degradation of bilirubin in jj cerebellum exhibited no improvement in thymidine kinase activity, and the presence of an enzyme inactivator was not suggested in jj cerebellum. These results seem to indicate that the induction of thymidine kinase might be affected in jj Gunn rat cerebellum. The possibility that the impaired DNA synthesis in the external granular cells in jj cerebellum may be due to Purkinje cell damage is discussed.

Effects of estrogen and progesterone on thymidine kinase activity in the immature rat uterus

The effects of progesterone and/or 17 beta-estradiol on thymidine kinase activity and autoradiograms were investigated in immature rats. Thymidine kinase activity increased more than thirtyfold above the control level 30 hours after 17 beta-estradiol injection. The enzyme activity induced by 17 beta-estradiol was suppressed by progesterone, the dose of which was approximately 1,000-fold that of 17 beta-estradiol. The specific thymidine kinase isozyme, which was separated from 17 beta-estradiol-induced uterine thymidine kinase by diethylaminoethyl (DEAE) cellulose column chromatography and not affected by deoxycytidine triphosphate, was involved in the DNA replication and inhibited by progesterone. The autoradiogram revealed many grains due to 3H-thymidine in the endometrial epithelium, stroma, and the myometrium in the immature rat 30 hours after 17 beta-estradiol injection, whereas progesterone reduced remarkably the number of grains induced by 17 beta-estradiol in the epithelium. Progesterone seems to inhibit the increment of the specific thymidine kinase isozyme induced by 17 beta-estradiol in the endometrial epithelium.

Differences in the induction of thymidine kinase isozymes in estrogen-treated immature and adult rats

Thymidine kinase activity in immature and castrated adult rat uterus has been examined in response to estrogen treatment. Following estrogen administration, it was found that immature uterine thymidine kinase activity was increased 30-fold after 24 h, but almost no effect was produced on castrated or non-castrated adult uterus. Uterine thymidine kinase activity was separated into three peaks (peak 1, 2 and 3) by means of DEAE-cellulose column chromatography. In response to estrogen, the thymidine kinase isozymes differed in adult and immature uteri. In immature uteri, marked and selective increase of the activity was found in peak I, whereas in adult only a slight increase in peak 2 activity was observed. The thymidine kinase activity in peak 1 and peak 2 were found to have different enzymatic properties and molecular weight, as determined by gel filtration of 125 000 for peak 1 and 100 000 for peak 2. From these results, it is suggested that estrogen induces specific thymidine kinase isozyme in immature uterus and that the isozyme may be involved in DNA synthesis. Such a induction mechanism seems to be lost during the development.

Synergistic effect of estrogen and androgen on induction of uterine thymidine kinase activities in immature rats.

Estradiol-17 beta induced a significant increase in the uterine thymidine kinase activity with a characteristic isozyme pattern 30 h after injection into immature rats. Testosterone propionate also revealed a similar increase. Following combined injection of estradiol-17 beta and testosterone propionate, the overall and separate isozyme activities of thymidine kinase increased to nearly the total amount of those when each hormone was injected separately.