Benedetto de Bernard

Isolation of a soluble Ca2+ binding glycoprotein from ox liver mitochondria

Abstract A soluble glycoprotein has been extracted from ox liver mitochondria and purified to a very considerable degree by polyacrylamide gel electrophoresis. Its protein moiety has a molecular weight of about 42,000. It contains about 5 per cent total carbohydrates, including some sialic acid, and up to 30 per cent phospholipids. It binds Ca 2+ to 2 classes of sites having different affinity. The binding of Ca 2+ is sensitive to some of the inhibitors of Ca 2+ binding and transport in mitochondria, among them La 3+ and ruthenium red.

Transformation of fetal secondary cartilage into embryonic bone in organ cultures of human mandibular condyles

Mandibular condyles of human fetuses, 14–21 weeks in utero, were kept in an organ culture system for up to 60 days. After 6 days in culture, the cartilage of the mandibular condyle appeared to have maintained its inherent structural characteristics, including all its various layers: chondroprogenitor, chondroblastic, and hypertrophic. After 12 days in culture, no chondroblasts could be seen; instead, the entire cartilage was occupied by hypertrophic chondrocytes. At the same time, the mesenchymal cells in the vicinity of the chondroprogenitor zone differentiated into osteoblast-like cells that produced type I collagen. The progenitor cells were still actively incorporating 3H-thymidine. The newly formed osteoid-like tissue lacked both metachromatic reactivity and a response to antibodies against chondroitin sulfate. Instead, the tissue reacted positively for osteocalcin (bone gla-protein). The process of new bone formation further progressed and, by the 20th day in culture, the new bone reacted positively for type I collagen, osteonectin, and to a lesser extent for chondroitin sulfate. The osteoid also underwent mineralization as revealed by both the von Kossa stain and vital staining with tetracycline. The above feature appeared even more intense in 40-day-old cultures. After 60 days, the newly formed bone contained osteoblasts and osteocytes, whereas the extracellular matrix revealed a high degree of matrix polarization. The results of the present study recapitulate findings reported for organ cultures of mice mandibular condyles. However, the in vitro process of de novo bone formation in human specimens requires a 6-fold longer culture time than that needed for mice condyles.

Energy state of chondrocytes assessed by 31P-NMR studies of preosseous cartilage.

The energy state of resting and hypertrophic chondrocytes from growth plate was studied by 31P-NMR spectroscopy of superfused cartilage slices. The presence of phosphocreatine was demonstrated in both cell types, using a repetition time of 3 s. By comparing the decline in the nucleoside triphosphate level after adding blockers of the glycolysis or of the mitochondrial respiration, it was deduced that resting and hypertrophic chondrocytes use both metabolic pathways for energy production, but the glycolysis dominates. Hypertrophic cells rely more on the mitochondrial respiration than the resting cells.

Solubility properties of alkaline phosphatase from matrix vesicles

Alkaline phosphatase has been extracted from matrix vesicles of a calcifying cartilage with 0.15 M KCl, 0.4 M guanidinium chloride and 0.05 M deoxycholate/50% butanol mixture. The catalytic properties of the three extracts have been compared. Although the highest amount of enzyme activity is extracted with the latter reagent (55%), some of it is also extracted with KCl (11%) and guanidinium (7%). By submitting isolated matrix vesicles to a short time sonication the distribution pattern of the alkaline phosphatase activity in the extracts is clearly modified, as the amount extracted with KCl increases from 14 to 50% and the portion extracted with deoxycholate decreases from 55 to 27% of the total enzyme activity of matrix vesicles. The enzymatic preparations were comparable on the basis of specific activities, affinity for the substrates (p-nitrophenylphosphate, ATP), thermostability, sensitivity to inhibitors and activators. By electrofocusing a value of pI = 4.15 was found for the alkaline phosphatase of matrix vesicles independently of the extraction medium. These results contradict the concept that alkaline phosphatase is exclusively an intrinsic membrane protein.

Evidence in vitro for an enzymatic synthesis of phosphocitrate

A biological synthesis of phosphocitrate is described from precursors, citrate and adenosinetriphosphate reacting in the presence of rat liver homogenate. Identity of the newly formed product was examined by enzymatic digestion of reactions mixtures, HPLC chromatography and 1H-NMR spectra. Authenticity of product was established by comparison to chemically synthesized phosphocitrate. Recognition of the existence of a biologically synthetic pathway adds credence to the known presence of phosphocitrate in mitochondria and a postulated role to control calcium phosphate deposition in that organelle.

High-performance liquid chromatographic preparation of galactosyl-hydroxylsine, a specific bone collagen marker

Galactosyl-hydroxylysine, a specific bone collagen marker, has been prepared directly from human urine samples by high-performance liquid chromatography (HPLC) on a preparative column. The compound is the didansyl derivative, as proved by HPLC and mass spectrometry under fast atom bombardment conditions. Since this compound is not commercially available, the procedure reported appears to be the simplest way to prepare it, which is necessary to measure the urinary excretion of this collagen metabolite by HPLC.

Cartilage proteoglycans and calcification

Calcifiable matrices of scapula cartilage and nasal septum, a non-mineralizing tissue, have been analyzed in parallel to compare their composition and some biochemical properties. The results obtained have shown that the two tissues present the following differences: 1. (i) the guanidinium extract from nasal septum has a higher content of uronic acid than the extract from scapula cartilage; 2. (ii) the distribution of nasal proteoglycans in the fractions obtained by spinning the extract in CsCl density gradients is different from that of scapula proteoglycans; 3. (iii) a glycoprotein endowed with Ca2+ affinity is present in the two tissues. However, the glycoprotein extracted from nasal septum lacks the high-affinity binding sites of the scapula cartilage glycoprotein; 4. (iv) proteoglycans from nasal septum show higher inhibition of the rate of formation of crystalline calcium phosphate in vitro than those from scapula cartilage.

Biochemistry of the Intercellular Matrix in Cartilage Calcification

Calcification is a process which takes place in the extracellular matrix, although strictly dependent on the biochemical activity of cells.