RDH10-mediated retinol metabolism and RARα-mediated retinoic acid signaling are required for submandibular salivary gland initiation.

In mammals, the epithelial tissues of major salivary glands generate saliva and drain it into the oral cavity. For submandibular salivary glands (SMGs), the epithelial tissues arise during embryogenesis from naïve oral ectoderm adjacent to the base of the tongue, which begins to thicken, express SOX9 and invaginate into underlying mesenchyme. The developmental mechanisms initiating salivary gland development remain unexplored. In this study, we show that retinoic acid (RA) signaling activity at the site of gland initiation is colocalized with expression of retinol metabolic genes Rdh10 and Aldh1a2 in the underlying SMG mesenchyme. Utilizing a novel ex vivo assay for SMG initiation developed for this study, we show that RDH10 and RA are required for salivary gland initiation. Moreover, we show that the requirement for RA in gland initiation involves canonical signaling through retinoic acid receptors (RAR). Finally, we show that RA signaling essential for gland initiation is transduced specifically through RARα, with no contribution from other RAR isoforms. This is the first study to identify a molecular signal regulating mammalian salivary gland initiation.

Repair of oxidative DNA damage by amino acids.

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

Radioprotection of plasmid DNA by oligolysines.

PURPOSE: To define ionic conditions under which oligolysines condense DNA as assayed by radioprotection of a plasmid substrate. And to compare these conditions with those required by the well-characterized ligands spermidine and hexammine cobalt (III). This will enable a reversible compaction model for plasmid DNA to be devised that models more closely mammalian chromatin than those based on polyamines. MATERIALS AND METHODS: Aqueous solutions containing plasmid DNA, sodium perchlorate and one of the five ligands trilysine, tetralysine, pentalysine, spermidine, or hexammine cobalt (III) were subjected to gamma-irradiation. The yields of the resulting single-strand breaks were quantified by gel electrophoresis. The effects of tetralysine and pentalysine were also examined by light scattering. RESULTS: The combination of low concentrations of the ligand and high concentrations of sodium perchlorate produced a relatively high yield of single-strand breaks. In contrast, the combination of high concentrations of the ligand and low concentrations of sodium perchlorate resulted in an approximately 25-fold lower single-strand break yield. The transition between these two break yields took place over very narrow concentration ranges of the ligand. A large change in light scattering occurred at the same concentration. The radioprotective ability of the ligands decreased in the order pentalysine > tetralysine > hexammine cobalt (III) > spermidine > trilysine. CONCLUSIONS: The effect of the oligolysines is qualitatively very similar to the previously reported radioprotection produced under similar conditions by the polyamines spermidine and spermine. It is caused by condensation of the DNA into a highly compacted form. As peptides, oligolysines are structurally more closely related than other ligands to naturally occurring DNA condensing agents such as histone proteins. Therefore, they may form the basis of a model system suitable for studying DNA damage produced by the direct effect of ionizing radiation (ionization of the DNA itself).

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II.

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.

Overexpression of human apolipoprotein A-II in mice induces hypertriglyceridemia due to defective very low density lipoprotein hydrolysis.

Two lines of transgenic mice, hAIItg-delta and hAIItg-lambda, expressing human apolipoprotein (apo)A-II at 2 and 4 times the normal concentration, respectively, displayed on standard chow postprandial chylomicronemia, large quantities of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) but greatly reduced high density lipoprotein (HDL). Hypertriglyceridemia may result from increased VLDL production, decreased VLDL catabolism, or both. Post-Triton VLDL production was comparable in transgenic and control mice. Postheparin lipoprotein lipase (LPL) and hepatic lipase activities decreased at most by 30% in transgenic mice, whereas adipose tissue and muscle LPL activities were unaffected, indicating normal LPL synthesis. However, VLDL-triglyceride hydrolysis by exogenous LPL was considerably slower in transgenic compared with control mice, with the apparent Vmax of the reaction decreasing proportionately to human apoA-II expression. Human apoA-II was present in appreciable amounts in the VLDL of transgenic mice, which also carried apoC-II. The addition of purified apoA-II in postheparin plasma from control mice induced a dose-dependent decrease in LPL and hepatic lipase activities. In conclusion, overexpression of human apoA-II in transgenic mice induced the proatherogenic lipoprotein profile of low plasma HDL and postprandial hypertriglyceridemia because of decreased VLDL catabolism by LPL.