Lucien A. Bavetta

Collagen defect induced by penicillamine.

Collagen synthesis, as judged by the accumulation of collagen in a subcutaneous, induced granuloma, was significantly decreased by penicillamine. Penicillamine also caused a marked increase in the amount of soluble collagen in skin and a sharp drop in insoluble material. These findings, which reflect an abnormal pattern of collagen metdbolism, are accompanied by an inhibition of wound-healing and by skin fragility.

Epithelial-mesenchymal interactions during odontogenesis. I. Isolation of several intercellular matrix low molecular weight methylated RNAs.

Abstract The present results and those previously reported (Slavkin et al., 1969a) show that embryonic tooth primordia synthesize in organ culture four discrete methylated RNA species (7 S, 5 S, 4 S, and 2 S) isolated from the intercellular matrix interposed between epithelia and mesenchyme. These RNA species were first separated by gel exclusion chromatography and subsequently resolved with polyacrylamide gel disc electrophoresis. Resolution required employment of a variety of nucleic acid precursors, labeled methyl-methionine, liquid scintillation spectrometry, and autoradiography. The labeled precursors are initially incorporated into both epithelial and mesenchymal cells, primarily in the germinative or cervical region of the tooth primordia. Subsequently, small amounts of RNase labile material(s) are observed over the intercellular matrix; presumably transferred from both cell types. Tritiated thymidine is not found as a component of the intercellular matrix; deoxyribose is not found in the isolated matrices. Various labels cannot be incorporated into the intercellular matrix in the absence of associated cell populations. The kinetics of labeling within the components, both cell populations and matrix, have been investigated using autoradiography (Slavkin et al., 1969a). On the basis of grain counts, only 2% of the total grain density incorporated within this epithelial-mesenchymal system is transferred into the intercellular matrix; Dactinomycin inhibits this process. This specific intercellular matrix has recently been shown to enhance homotypic cellular differentiation in vitro (Slavkin et al., 1969b). Ultrastructural findings indicate the presence of membrane-bound, electron dense material (500–3000 A diameter) within the matrix prior to phenol extraction; this material is not present in phenol-extracted matrices. Whether the extracted intercellular RNAs are to be found within the membrane-bound material observed within the matrix in situ (Slavkin et al., 1969b,c) is not as yet certain. Further studies designed to evaluate this biological activity (Slavkin et al., to be published) suggest that the activity is RNase sensitive. As to the mechanism(s) for the transfer of RNA or possibly ribonucleoprotein during epithelial-mesenchymal interactions, the present experiments do not provide adequate evidence with which to understand the developmental specificity of the isolated RNA species.

Odontogenesis: Cell–Cell Interactions in vitro

EMBRYONIC interactions of epithelial and mesenchymal tissue before organogenesis have been observed in several organ systems1–4. These phenomena have been tested by isolating embryonic tissues from their in vivo environment and transplanting them in an artificial environment from which no morphogenetic signals can be received. The chorio-allantoic membrane (CAM) of chick provides a versatile site for the maintenance and further differentiation of embryonic organs5,6. One such organ, the tooth primordium, arises as a result of interactions between epithelial and mesenchymal tissues and offers a model system for developmental studies.

Collagen Synthesis and Turnover in the Growing Rat Under the Influence of Methyl Prednisolone.

Summary At different time intervals, over a 115 day period skin biopsies were performed in growing rats which had received an initial tracer dose of C14-glycine. Specific activity values for the different fractions of skin collagen isolated (0.15 M NaCl, 0.5 M NaCl, 0.5 M citrate pH = 3.6 and insoluble) were corrected for growth taking into account changes in surface area, skin thickness and pool size of the particular fraction. The 0.15 M and 0.5 M NaCl fractions in the normally growing rats decayed almost exponentially with T½ of 17 and 20 days. The curve for the insoluble collagen did not reflect the presence of a single component but seemed to indicate a progressive insolubilization. A T½ of 28 days was evident during the period of rapid growth whereas a value close to 300 days could be extrapolated towards the end of the experiment. Corticoid treatment (0.11 and 0.36 mg/day) decreased the turnover rate of all fractions, and caused a decrease on the amount of collagen extractable by neutral salt.

Changes in collagen metabolism associated with the administration of penicillamine and various amino and thiol compounds

Abstract D-Penicillamine and BAPN (β-amino propionitrile) were found to be the most active agents able to cause an accumulation of neutral salt-soluble collagen in the skin of rats. Most analogues of penicillamine had very little or no effects. Blocking the α-amino group ( N -acetyl-penicillamine) or removal of the sulfhydryl group (valine) render inactive compounds. A higher homologue (mercaptoisoleucine) was ineffective. It would seem that the structure of D-penicillamine meets all the criteria for effectiveness, such as extracellular distribution, lack of metabolic deamination, sufficient amount of steric hindrance of the sulfhydryl group to retard its oxidation to disulfide, but yet not enough to prevent completely its reactivity. D-Penicillamine totally blocks the crosslinking of newly formed tropocollagen and is able to degrade a certain fraction of the more recently sythesized process insoluble collagen which has not yet completed its maturation D-Penicillamine affects equally both sexes, is more effective in young rats than in older ones and is not antagonized by copper if this metal is administered by a different route. If given simultaneously, mixed in the diet, the effect on collagen is inhibited and the toxicity of copper reduced, probably due to the marked affinity of both compounds and possible inactivation in the gastrointestinal tract. The urinary excretion of hydroxyproline in the D-penicillamine-treated rats falls within normal limits. Only the BAPN-treated animals showed an increase in urinary hydroxyproline. Since BAPN causes severe osseous abnormalities, whereas penicillamine does not, these findings would tend to support the view that an increase in urinary hydroxyproline is primarily a manifestation of abnormal metabolism of bone collagen.

Epithelial-mesenchymal interactions during odontogenesis II. Intercellular matrix vesicles

Abstract The present study has demonstrated morphologically the probable sequential formation of matrix vesicles during embryonic rabbit incisor tooth formation. The observations presented suggest that these vesicles may have been selectively formed within each cell type and then exported into the matrix, or that matrix vesicles may have been formed by a pinocytotic mechanism. It is uncertain on the basis of our morphological data whether one or both of these explanations is adequate. Matrix vesicles were increasingly more concentrated in the matrix region adjacent to dividing inner enamel epithelia and preodontoblast mesenchyme. Thereafter, the concentration of matrix vesicles diminished with the cessation of cell division within each cell type. The vesicles varied considerably in size (500Ato 0.1 μm), shape and the electron density of their contents. They were limited by a unit trilaminar membrane, often coated with a filamentous mat, and contained materials of varying electron density. Morphological data indicate that vesicles observed in the developmentally more advanced dentine organic matrix (in association with nondividing cells) function in the initiation of calcification during dentinogenesis. On the basis of morphological information we anticipate that additional developmental events within dissimilar cell types and within the forming organic matrix may be mediated through matrix vesicles. The interpretation of vesicle functions on the basis of matrix vesicle ultrastructure and location with respect to each cell type must be highly qualified until additional criteria can be employed. However, with regard to possible informational and structural macromolecular transmission and/or ion transport by matrix vesicles, the morphological evidence presented may be highly significant.

Turnover and age distribution of a collagen fraction extractable from rat skin by mercaptoethylamine

Abstract Thiol compounds such as 0.2 M cysteamine at neutral pH and at 4 ° are able to solubilize significant amounts of dermal insoluble collagen. In young rats (2 months old or younger) 90% of the insoluble collagen can be extracted by 0.2 M cysteamine. In older animals the proportion of insoluble residue increases. Two successive extractions of insoluble collagen with 0.2 M cysteamine yielded 33% β and 50% β components, respectively, an indication that the material which is more resistant to solubilization may be more crosslinked. This increased crosslinking seems to be related to the biological age of the material, a conclusion substantiated by the slower turnover rate of the less readily soluble fraction. The native collagen solubilized by cysteamine shows the same sedimentation properties and electrophoretic mobility as salt soluble collagen, but yields different proportions of α and β components Following thermal denaturation.

Odontogenic Epithelial-Mesenchymal Interactions in Vitro

Epithelial or mesenchymal isolates cultivated on the chicken chorioallantoic membrane singly do not possess the potential for tooth morphogenesis. However, when randomly recombined in vitro, their histogenic interaction relates to tooth formation. After 16 days of continuous culture, surviving recombinants formed an advanced tooth germ. Such structures did not, however, demonstrate uniformity in odontogenesis or in developmental cusp patterns.

An unusual property of chromatin isolated from mammalian salivary glands

Abstract Chromatin from mammalian salivary glands has been isolated and found to exhibit an absolute requirement for NaCl or KCl for its template activity. By chemical analysis this chromatin shows no difference in its basic protein complements as compared with liver chromatin. The absolute requirement of monovalent ions for the template activity of a chromatin has not been observed previously.

Effect of testosterone propionate on tissue protein synthesis in the castrated male rat.

Summary Injection of testosterone propionate to castrated male rats stimulated protein synthesis at the level of the sex linked organs, skin, and in subcutaneously implanted polyvinyl sponge. Other tissues studied showed no effect of testosterone on rate of protein labeling after receiving 1-C14-glycine. The unique behavior of the perineal muscle in contrast to other skeletal muscles points to its unsuitability for myotrophic assay of anabolic hormones. The tissues where anabolic activity was stimulated also showed a concomitant increase of radioactivity in their non-protein fractions.