Development of catecholaminergic phenotypic characters in the mouse locus coeruleus in vivo and in culture

While abundant studies have begun to elucidate ontogeny of the peripheral nervous system, molecular mechanisms underlying brain development remain obscure. To approach this problem, we initiated parallel in vivo and in vitro studies of the mouse locus coeruleus (l.c.), a brainstem noradrenergic nucleus. The catecholaminergic enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) were used to monitor phenotype expression and development. TH catalytic activity and immunocytochemical reactivity were initially detectable on gestational Day 13 (E-13) in vivo, and adult levels of activity were approximately by the third postnatal week. Immunotitration studies indicated that the developmental increase was due to accumulation of enzyme molecules and not enzyme activation. The in vivo developmental profile of DBH approximated that of TH. To begin defining regulatory mechanisms, explants of embryonic brainstem were placed in culture. Explantation on E-12, prior to expression of TH or DBH, resulted in the de novo appearance of these phenotypic characters after 4 days. Explantation on E-18, after the enzymes are already expressed, was followed by a striking sixfold rise in TH activity. Immunotitration studies revealed that the increase in TH activity in E-18 cultures was attributable to increased molecule number, reproducing the in vivo results. Moreover, the E-18 explants, cultured for 3 weeks, attained higher plateau levels of TH activity than E-12 cultures, and this differences was due to increased molecule number. Morphometric analysis indicated that 3-week E-12 cultures actually had more l.c. cells than E-18 cultures, indicating that differences in TH were not due to increased cells in the E-18 l.c. Finally, systemic study revealed that the development of TH activity in culture increased progressively from E-11 to E-12 to E-13, suggesting that critical regulatory events occur at this time. Our studies suggest that the l.c. is an excellent model for the study of brain development in vivo and in vitro. Initial phenotypic expression and dramatic development occur in culture in the absence of normal targets, normal afferent innervation and, presumably, normal humoural milieu.     

The abortive synthesis of new DNA chains in Ehrlich ascites tumour cells treated with nitrogen mustard (HN2)

Nitrogen mustard (HN2)-sensitive Ehrlich ascites tumour cells, exposed to HN2 in vivo, showed an inhibition of DNA synthesis which increased with dosage. The effects of alkylation involved at least three distinct components: (1) interference with new 9S DNA chain formation, (2) slowing of the rate of chain growth and (3) loss of newly formed short chains. The dominant effect seemed to be abortive synthesis of new 9S chains; this effect could account for most of the inhibition of DNA synthesis if an initial rapid synthesis of 9S DNA were accompanied by an initial rapid rate of destruction of these chains. By relating the known frequencies of guaninyl alkylations to the postulated ‘replicon’ size observed in control experiments, it appears that only difunctional alkylation frequencies can be directly correlated with the inhibition of discontinuous DNA synthesis by HN2, Mechanistically, this could reflect interference of di-guaninyl alkylations with the integration of 9S chains into 30S, 44S and higher molecular weight species of DNA by ligases. The resulting obstructed short chains, ≦ 9S, might be exposed and so be vulnerable to destruction by the increased nuclease activities expected after alkylation.     

CHANGES IN HEPATIC STRUCTURE IN RATS PRODUCED BY BREATHING PURE OXYGEN

The livers of rats exposed to pure oxygen were examined electron microscopically to study toxic effects of oxygen in a metabolically sensitive organ. Pressures of 1/3 (258 mm Hg), 1 (760 mm Hg), and 3 (2280 mm Hg) atmospheres were used, with exposures up to 90 days with the lowest pressures. The first changes in the hepatocytes were loss of glycogen and enlargement of mitochondria with development of mitochondria with bizarre shapes which were seen after 3 days at 258 mm, 1 day at 760 mm, and 3 hours at 2280 mm. These changes were followed by formation of increased numbers of cristae, membranes surrounding mitochondria, autophagic vacuoles, and polyribosome clusters. After 2 weeks at 258 mm, which is the pressure of the atmosphere of space cabins, numerous mitochondrial myelin figures appeared but the mitochondrial enlargement had begun to regress. After 90 days at 258 mm, the liver cells appeared almost normal except that many pigment granules had accumulated in the pericanalicular zones. The changes were non-specific and seemed to parallel biochemical alterations recorded elsewhere. They are not considered the result of toxicity but rather of adaptation. These atmospheres, which are used in clinical medicine and in space travel, appear to have no permanent deleterious effects on the liver in rats under the conditions of this experiment.     

Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins

Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.