Background
The synthesis, secretion, and fate of matrix proteins released by ameloblasts during enamel formation was studied in continuously erupting rat incisors.

Methods
Computerized image processing was used to quantify silver grain distribution in radioautographs of sections prepared from rats injected with 3H-methionine, and this was correlated with fluorographs defining radiolabeling patterns of proteins in enamel organ cell and enamel homogenates prepared from freeze-dried teeth of rats injected with 35S-methionine and other radioactive amino acids and precursors such as sugar, sulfate, and phosphate. Some rats were also treated with brefeldin A to characterize newly formed proteins blocked from being secreted from ameloblasts.

Results
The results indicate that ameloblasts rapidly synthesize and secrete (minutes) at least five primary enamel matrix proteins, including a 65 kDa sugar-containing sulfated enamel protein and four nonsulfated proteins with molecular weights near 31, 29, 27, and 23 kDa as estimated by SDS-PAGE. The 27 kDa protein appears to correspond to the primary amelogenin described in many species. The cells also appear to release at least one phosphoprotein with molecular weight near 27 kDa, which may be an amelogenin, and up to five cysteine-containing proteins with molecular weights near 94, 90, 72, 55, and 27 kDa. The proteins collectively are released at interrod and rod growth sites where they appear to remain close to their point of release from ameloblasts. The 65 kDa sulfated protein and 31 kDa nonsulfated protein are rapidly converted into lower molecular weight forms (hours), whereas nonsulfated proteins near 29, 27, and 23 kDa are more slowly transformed into fragments near 20, 18, and 10 kDa in molecular weight (days). These fragments do not accumulate but appear to be removed from the enamel layer as they are created.

Conclusions
Enamel proteins seen by Coomassie blue (or silver) staining of one-dimensional polyacrylamide gels, therefore, represent a composite image of newly secreted and derived forms of sulfated and nonsulfated proteins that sometimes have similar molecular weights. © 1996 Wiley-Liss, Inc.

Protein dynamics of amelogenesis.

Autoradiographic study with [35S]-sodium sulphate of loss of sulphated glycosaminoglycans during amelogenesis in the guinea pig.

After injection of labeled precursors such as 3H-proline or 3H-tyrosine into rats, the incisor dentin contains a continuous and stable record of precursor incorporation into labeled proteins. This record was visualized and quantitated with radioautography in order to evaluate the quantitative changes in enamel where newly secreted proteins randomize with older proteins and both are eventually lost. Up to 4 hours after injection, the pulse-dose was incorporated as a highly labeled band of predentin. The band was entirely within calcified dentin at 2 days and was further removed from new predentin by 4 and 8 days. Dentin which formed proximal to the heavily labeled band contained an amount of radioactivity reflecting the level of labeled precursor available at that time. A standardizing factor for experimental error was obtained by quantitating the reaction in the heavily labeled band, and a post-pulse incorporation factor was determined from the amount of radioactivity added per day as weakly labeled dentin. The variation within the heavily labeled band was assumed to reflect experimental error. The number of grains in the bands were averaged from 4 hours to 8 days to give the standardizing factor. This was multiplied by the ratio of enamel to dentin counts in the same section to obtain a corrected enamel count. In this way the coefficient of variation was improved from a high of 17.2% in uncorrected enamel counts to 2.4% in corrected counts. The post-pulse incorporation factor was higher with tyrosine than with proline. With proline it amounted to 5% increase per day from 1 to 4 days and 2.5% per day from 4 to 8 days after injection. In addition, with 3H-proline the incorporation into predentin increased from 30 minutes to 4 hours. With tyrosine, the counts increased from 30 minutes to 1 hour, but decreased by nearly one third from 1 to 4 hours. This was interpreted as a loss of short-lived matrix proteins including procollagen peptides produced during conversion from procollagen to tropocollagen in the predentin.

Radioautography of rat incisor dentin as a continuous record of the incorporation of a single dose of 3H-labeled proline and tyrosine.

Epigenetic Regulation of Enamel Protein Synthesis during Epithelial-Mesenchymal Interactions

It has long been recognized that histochemistry and cytochemistry offer the only ways of gathering information about the biochemical composition of tissues and cells without disrupting their microscopic architecture. A variety of methods have been put forward for studying nuclei acids, proteins, carbohydrates, lipids, enzymes and other components of intact tissues and cells. By now, many of these have only a historical interest. Some do, however, survive in microscopic and ultramicroscopic applications, and have become incorporated in the most refined and precise techniques that are currently available. Histochemical reactions range from the classic procedures carried out on histological sections to yield final stained products recognizable under the light microscope, to those which are applied on ultrathin sections, using heavy metals or other electron-dense compounds to reveal specific components under the electron microscope; others range from procedures based on the antigen-antibody reaction that are capable of revealing the presence of specific biological molecules, to the biophysical techniques which permit the qualitative and quantitative analysis of elements; lastly, there are the recently proposed ultra-high resolution methods that allow nanoparticles to be recognized. This brief review, which is based on personal experience and on the data in the literature, will discuss the most important methods now being used.

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Calcified tissue histochemistry: from microstructures to nanoparticles.

La formacion de la matrix organica del esmalte fue estudiada, a traves de la radioautografia, en molares de cobayas que recibieron Timidina-3H y Prolina-3H. Los resultados parecen indicar que la remocion de material organico de la matriz, una de las etapas del proceso de maduracion del esmalte, estan en la dependencia de los ameloblastos secretores y no de los ameloblastos reduzidos como ha sido propuesto.

J. Merzel

Papers

The decrease in the concentration of organic material in the course of formation of the enamel matrix