Paul Christner

Immunofluorescent localization of amelogenins in developing bovine teeth

Abstract Antiserum was prepared to the proteins (amelogenins) isolated from fetal bovine enamel matrix. This antiserum was used to localize the amelogenins in the developing bovine molar by immunofluorescent microscopy. Amelogenins could be identified in the preameloblasts before enamel matrix deposition had begun as well as in the secretory ameloblasts. The closely adherent layer of stratum intermedium cells also contained some immunoreactive material, suggesting that they may contribute protein to the enamel matrix. The newly deposited enamel matrix consisted of brightly fluorescent particles. Mature enamel matrix did not contain the immunoreactive protein except in a thin layer along the dentino-enamel junction and adjacent to the ameloblasts. No other portion of the tooth bud or other tissues reacted with the specific antiserum.

Secretion of Elastin in the Embryonic Chick Aorta as Visualized by Immunoelectron Microscopy

Recently, significant advances have been made in characterizing the pathway of elastin biosynthesis from the biochemical point of view and a 70,000 dalton protein, designated tropoelastin, appears to be the primary translation product and soluble intermediate of the insoluble elastin. However, relatively little is known concerning the intracellular secretory pathway of tropoelastin. We previously developed an electron microscopic technique using elastin-specific antibody and ferritin-conjugated secondary antibody to identify intracellular elastin and to identify, provisionally, intracellular vesicles containing elastin ( Damiano et al., Conn. Tiss . Res. 8: 185-188, 1981). However, the method did not permit localization of elastin in other intracellular organelles. We now describe an improved post-embedding technique using the peroxidase-antiperoxidase method to detect the primary elastin antibody and have localized elastin in both the endothelial and medial cells of the embryonic chick aorta. Specific staining was visualized in the cisternae of the endoplasmic reticulum, in the Golgi apparatus, and in vesicles forming on the trans side of the Golgi. Some of these smaller vesicles appeared to fuse, forming larger vesicles which may have a storage function. Both types of vesicles were seen fusing with the cell plasma membrane, suggesting that elastin is secreted by an exocytotic process. These results suggest that tropoelastin follows the classical pathway for protein secretion.

An Analysis of the Organ and Species Immunospecificity of Elastin

The connective tissue protein elastin is largely responsible for maintaining the elasticity of major blood vessels and lung tissue. Comparatively few studies have been made of the immunologic properties of elastin because of its high degree of insolubility and because of its apparent limited capacity to elicit precipitating antibodies. Recent studies have focused on the possible role of elastolytic enzymes, particularly leukocyte elastase, in the pathogenesis of chronic obstructive lung disease, but there are no reports in the literature concerning the antigenic properties of peptides released by digestion of insoluble elastin with leukocyte elastase. In the present study, we have obtained antibodies in rabbits to peptides prepared by digestion of dog and human lung and aortic elastin with either oxalic acid or leukocyte elastase. By hemagglutination assays, we have shown generally that: (1) within a given species the peptides from aorta and lung cross-react strongly with one another, (2) there was rather poor cross-reactivity between peptides from different species (3) the peptides obtained by oxalic acid digestion cross-react poorly with those obtained by elastase digestion, (4) the antibodies to the peptides cross-reacted with the insoluble elastin from which the peptides were derived. These elastin-specific antibodies may be useful for ultrastructural localization of elastin and identification of fragments derived from elastin in the sera of humans and experimental animals.

A comparison of transfer RNA isoaccepting species between collagenous and noncollagenous tissues in the embryonic chick.

Abstract Transfer RNA was isolated from different organs of 17-day-old chick embryos and the acceptor activity for each of the 20 amino acids was determined. The most abundant acceptor activities found in tRNA from tendon cells were for glycine, arginine, proline and alanine. When compared to the average acceptor activity found in brain, liver and heart, the tendon tRNA showed an increase in acceptor activity of 33% in glycine, 40% in arginine and 83% in proline. Reversed phase chromatography of the tRNA charged with glycine demonstrated that the increase in glycyl-tRNA in tendon could be accounted for by an increase in one of four major isoaccepting species. Such an increase in a single species was also observed in tRNA isolated from calvaria. The codon response of this species was shown to differ from that of the other glycyl-tRNA species. No major differences in the relative proportions of isoaccepting species could be demonstrated for any other amino acid. These results suggest that a characteristic complement of tRNA species may be associated with collagen synthesis.

Degradation of tropoelastin by proteases

Abstract A simple assay is described which is capable of detecting single breaks in purified radioactively labeled tropoelastin after incubation with dilute solutions of proteases. Using this assay we show that tropoelastin is rapidly cleaved by a variety of proteases, including leukocyte and pancreatic clastase, at physiologic pH and ionic strength. The results suggest the possibility that degradation of tropoelastin or other related biosynthetic intermediates may play a role in the pathogenesis of emphysema.

Effect of various aminoacid analogues on chick tendon procollagen synthesis and secretion: Selective inhibition by S-2-aminoethyl cysteine

Abstract Matrix-free chick embryo tendon cells were incubated with [ 14 C]-proline in the presence of various aminoacid analogues and the effects of the analogues on [ 14 C]-proline incorporation, [ 14 C]-hydroxyproline synthesis and secretion of labeled molecules were examined. It was found that the structural lysine analogue S-2-aminoethylcysteine was a potent inhibitor of procollagen synthesis and secretion. At a concentration of 1 mM it produced a 75% decrease in [ 14 C]-hydroxyproline synthesis and a 90% decrease in the secretion of [ 14 C]-hydroxyproline-containing macromolecules.

Immunofluorescent Evidence for the Similarity of Amelogenins in Calf, Mouse and Pig Teeth

Antiserum was prepared to fetal bovine enamel matrix and was used to localize the amelogenins in developing bovine molars by immunofluorescent microscopy. Amelogenins could be identified to preameloblasts, secretory ameloblasts, stratum intermedium cells, and the newly deposited enamel matrix. Mature enamel matrix did not fluoresce except in a thin line along the DEJ and adjacent to the ameloblasts. Immature enamel matrix of murine and porcine teeth fluoresced when treated with antiserum to bovine enamel matrix. No other portions of tooth buds or other tissues reacted with the specific antiserum.

The fate of mevalonate in the crab: The case for CO2 and t-RNA containing isopentenyl-adenosine

1. 1. The Maryland Blue Crab (Callinectes sapidus Rathbun) does not synthesize cholesterol; sterol ingestion yields its cholesterol. 2. 2. This crab was selected to study the fate of mevalonate, a required intermediary in cholesterol biosynthesis. 3. 3. Mevalonate yielded 82–94% of its label into CO2; but, <1% into isopentenyl-adenosine t-RNA (ia-t-RNA) and only a trace into dolichols. 4. 4. Identical labeling patterns to those used in mevalonate were found in ia-t-RNA; isopentenyl alcohol was recovered labeled; these facts suggest that mevalonate was utilized intact. 5. 5. The above results and the recovery of hydroxyl-methylglutarate labeled, suggests an active non-steroidal pathway for mevalonate.