The significance of a differential distribution of phosphomonoesterases on bone surfaces after prolonged demineralization.

An observed differential distribution of alkaline and acid phosphatase on the surfaces of growing bones may serve to describe transformative processes of bone growth. This conclusion has been reached by comparing the distribution of the two enzymes on the surfaces of fibulae from young rats with the patterns of apposition and resorption on the periosteal surfaces of this bone, revealed by in vivo staining with alizarin red S. Presence of reaction to acid phosphatase is, as shown before, an indication of resorptive surfaces, while the presence of reaction to alkaline phosphatase is an indication of depository surfaces.     

Patterns of distribution of phosphomono-esterases on surfaces of demineralized bone

Summary Decalcification over short periods (5 days) with MnNa₂ EDTA, MgNa₂ EDTA and EGTA according to a method described in the present paper, creates sections of high quality with simultaneous good preservation of phosphomonoesterases on bone surfaces. In fact, the enzyme distribution seems to be comparable to that obtained by using undecalcified sections.Na₂ EDTA creates, on the other hand, poor preservation of alkaline phosphatase probably due to the fact that this chelate contrary to the other chelates removes the essential metal from the protein, leaving an unstable enzyme molecule which undergoes denaturation.Decalcification over longer periods (15 days) does not influence the pattern of distribution of acid phosphatase, whereas the alkaline phosphatase reaction becomes depressed in certain surface areas. The significance of this differential distribution is discussed. It might be an indication of differential processes of bone transformations in such a way that bone surfaces corresponding to areas of enzyme reactions are depository whereas bone surfaces corresponding to areas of lack of enzyme reaction are resorptive. New experimental designs are, however, necessary before the phenomenon is fully perceived. Two different coupling agents were used in connexion with the demonstration of acid phosphatase reaction. When HPR was used as the coupler the final enzyme distribution coincided with that usually described in the literature, i.e., strong reaction of cells adjacent to resorptive surfaces and weak reaction of cells adjacent to depository surfaces. When, however, Fast dark blue R was used all surface cells reacted markedly. This method also revealed certain cell types with nuclear reaction.     

Patterns of distribution of phosphomono-esterases on surfaces of demineralized bone.

Decalcification over short periods (5 days) with MnNa2 EDTA, MgNa2 EDTA and EGTA according to a method described in the present paper, creates sections of high quality with simultaneous good preservation of phosphomonoesterases on bone surfaces. In fact, the enzyme distribution seems to be comparable to that obtained by using undecalcified sections. Na2 EDTA creates, on the other hand, poor preservation of alkaline phosphatase probably due to the fact that this chelate contrary to the other chelates removes the essential metal from the protein, leaving an unstable enzyme molecule which undergoes denaturation. Decalcification over longer periods (15 days) does not influence the pattern of distribution of acid phosphatase, whereas the alkaline phosphatase reaction becomes depressed in certain surface areas. The significance of this differential distribution is discussed. It might be an indication of differential processes of bone transformations in such a way that bone surfaces corresponding to areas of enzyme reactions are depository whereas bone surfaces corresponding to areas of lack of enzyme reaction are resorptive. New experimental designs are, however, necessary before the phenomenon is fully perceived. Two different coupling agents were used in connexion with the demonstration of acid phosphatase reaction. When HPR was used as the coupler the final enzyme distribution coincided with that usually described in the literature, i.e., strong reaction of cells adjacent to resorptive surfaces and weak reaction of cells adjacent to depository surfaces. When, however, Fast dark blue R was used all surface cells reacted markedly. This method also revealed certain cell types with nuclear reaction.     

Alkaline and acid phosphatase activity in murine femoral bone marrow following X-irradiation, or X-irradiation and repopulation with syngenic or allogeneic bone marrow or marrow stroma cells

The activity of alkaline and acid phosphatases in the bone marrow from the femoral cavity was investigated in the following groups of mice: (1) normal (non-irradiated); (2) irradiated with 600 R; (3) irradiated and repopulated with syngeneic bone marrow; (4) irradiated and repopulated with syngeneic marrow stroma; (5) non-irradiated, infused with allogeneic bone marrow (host versus graft reaction, HvG); (6) irradiated and repopulated with allogeneic bone marrow (graft versus host reaction, GvH). In addition, the activity of alkaline and acid phosphatases was examined in bone marrow stromal cultures. In irradiated animals the activity of both enzymes was lower than in non-irradiated ones, repopulation with syngeneic bone marrow restoring it to normal. Repopulation with allogeneic marrow (GvH) resulted in a very deep reduction of alkaline, but not acid, phosphatase. It is postulated that the decrease in bone marrow alkaline phosphatase activity can be a sensitive test for the early GvH reaction, preceding such parameters as splenomegaly. Marrow stroma cultured in vitro also showed very low alkaline phosphatase activity.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

Subcellular localization and properties of pyridoxal phosphate phosphatases of human polymorphonuclear leukocytes and their relationship to acid and alkaline phosphatase

Using a novel fluorimetric assay for pyridoxal phosphate phosphatase, human polymorphonuclear leucocytes were found to exhibit both acid an alkaline activities. The neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrigfugation. The alkaline pyridoxal phosphate phosphatase showed a very similar distribution to alkaline phosphatase an was located solely to the phosphasome granules. Fractionation experiments on neutrophils treated with isotonic sucrose containing digitonin and inhibitor studies with diazotised sulphanilic acid and levamisole further confirmed that both enzyme activities had similar locations and properties. Acid pyridoxal phosphate phosphatase activity was located primarily to the tertiary granule with a partial azurophil distribution. Fractionation studies on neutrophils homogenised in isotonic sucrose containing digitonin and specific inhibitor studies showed that acid pyridoxal phosphate phosphatase and acid phosphatase were not the result of a single enzyme activity, Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (munits/mg protein) of alkaline pyridoxal phosphate phosphatase an alkaline phosphatase varied widely in the three groups and the alterations occurred in a parallel manner. The specific activities of acid pyridoxal phosphate phosphatase and of acid phosphatase were similar in the three groups. These results, together with the fractionation experiments and inhibition studies strongly suggest that pyridoxal phosphate is a physiological substrate for neutrophil alkaline phosphatase.     

The os penis of the rat. I. Phosphomonoesterases in its proximal growth cartilage

The distribution of reaction for acid and alkaline phosphatases in the proximal cartilage of the os penis and the mandibular condylar cartilage has been compared. The distribution of acid phosphatase in the two structures seems to be identical, whereas the distribution of alkaline phosphatase in the os penis cartilage seems to differ from that in the mandibular condylar cartilage and, by this, from all other studied growth cartilages.     

Genetic transformation between strains of Listeria monocytogenes

Acid and alkaline phosphatase activities of microbial films colonizing glass surfaces were studied. Films developed in water with a high organic content were characterized by a high ratio of alkaline to acid phosphatase activity. Alkaline phosphatase activity of these films was enhanced by a period of prior heating at 60°C for 10 min. Microbial films developed in poorer water exhibited higher proportions of acid phosphatases and heat treatment had a less favourable effect on the alkaline phosphatase activity.     

A modified wire-loop method for quantitative electron microscopic radioautography

Summary The distribution of reaction for acid and alkaline phosphatases in the proximal cartilage of the os penis and the mandibular condylar cartilage has been compared. The distribution of acid phosphatase in the two structures seems to be identical, whereas the distribution of alkaline phosphatase in the os penis cartilage seems to differ from that in the mandibular condylar cartilage and, by this, from all other studied growth cartilages.     

The significance of a differential distribution of alkaline phosphatase in the perichondrium of the mandibular condylar cartilage in the wistar albino rat

Summary The aim of the present investigation has been to further study an incidentally observed rare distribution of alkaline phosphatase in the covering of the mandibular condyle. It was felt that this phenomenon might be related to the necessary interaction between the bony and the cartilaginous condylar head during the transformative growth movements of the condylar process.The study has been based on histomorphological and histochemical observations on frontal and sagittal sections of mandibular condyles from rats between 10 and 21 days of age. As regards the bony condylar head which is oval with its long axis in the antero-posterior direction the observations showed that this structure during growth is transformed in a superior, posterior and medial direction. This involves differential resorption on the surfaces in the anterior part and differential apposition on the surfaces in the posterior part.As regards the cartilaginous condylar head, the observations showed that its shape in the frontal plane changes from triangular in the anterior part to rectangular in the posterior part. Alkaline phosphatase reaction in its perichondrium always reaches a higher level medially than laterally.General observations of perichondrial alkaline phosphatase reaction were applied to the distribution of the enzyme in the perichondrium of the mandibular condyle. These data suggest that as the condylar cartilage grows medially, it becomes narrower anteriorly and broader posteriorly.     

Cytochemical localization of alkaline and acid phosphatase in human vanishing bone disease

This report is the first cytochemical investigation of vanishing bone disease "Gorham's Disease" (Gorham and Stout 1955). The ultrastructural localization of non-specific alkaline phosphatase and of specific and non-specific acid phosphatase activity was studied in slices of tissue removed from a patient with this rare disorder. Sodium beta-glycerophosphate and phosphorylcholine chloride were used as substrates. Alkaline phosphatase was present around the plasma membranes of osteoblasts and associated with extracellular matrix vesicles in new woven bone. This is consistent with the proposed role for this enzyme (Robison 1923) and for matrix vesicles (Bonucci 1967) in the mineralization of bone (Bernard and Marvaso 1981). Concentrations of specific secretory acid phosphatase reaction product in the cytoplasm of degenerating osteoblasts may contribute to the imbalance between bone formation and resorption. Osteoclasts, while few in number, showed non-specific and specific acid phosphatase activity. The Golgi apparatus and heterophagic lysosomes of mononuclear phagocytes were rich in non-specific acid phosphatase. This was also present in the Golgi lamellae and lysosomes of endothelial cells. Acid phosphatase cytochemistry suggests that mononuclear phagocytes, multinuclear osteoclasts and the vascular endothelium are involved in bone resorption in this disease.     

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : I. Histochemical Staining of Bone Marrow Smears

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, β-galactosidase, β-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.     

An in vitro production of bone specific alkaline phosphatase

Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadaveric bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented.     

Ultrastructural localization of phosphohydrolases in gametes, zygotes and zoospores of Ulva mutabilis Foyn.

Gametes, zoospores, and zygotes of the multicellular, green alga Ulva mutabilis showed acid phosphatase reaction product in Golgi vesicles and on the membrane lining the vacuole. In addition gametes and zoospores showed enzyme reaction product on the entire surface membrane including the flagellar membrane. The surface membrane enzyme activity disappears from the zygote shortly after copulation and at the same time lysosome-like bodies start to appear in the cytoplasm. No alkaline phosphatase activity could be detected. The distribution of acid phosphatase is discussed in relation to the events taking place during and shortly after fertilization.     

Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.     

Histochemical studies on the localization and activity of acid phosphatase in calcifying tissues

Summary Acid phosphatase activity has been studied in cold microtome sections and using simultaneous azo coupling method in developing teeth and bone, and serial sections were made for the demonstrations of alkaline phosphatase.1. In developing teeth, strongest activity of acid phosphatase was found in the distal portion of high columnar ameloblasts associated with heavy calcification in the rodent incisor, and ameloblasts and odontoblasts in adjacent occlusal surface in molar teeth. However, the activity of immatured ameloblast and crevicular aspects of molar were weaker.2. In the epiphyseal bone trabeculae a striking acid phosphatase reaction was found.3. As regards to the effects of decalcifying solutions to the enzymatic activity, the use of EDTA decalcifying agent (10% and pH 7 to 4) showed the best results. That is, a decrease of decalcifying time and a greater preservation of acid phosphatase activity.With 11 Figures in the Text     

Lactoperoxidase-125I localization of salt-extractable alkaline phosphatase on the cytoplasmic membrane of Bacillus licheniformis.

Previous histochemical and biochemical localizations of alkaline phosphatase in Bacillus licheniformis MC14 have shown that the membrane-associated form of the enzyme is located on the inner surface of the cytoplasmic membrane, and soluble forms are located in the periplasmic space and in the growth medium. The distribution of salt-extractable alkaline phosphatase on the surfaces of the cytoplasmic membrane of B. licheniformis MC14 was determined by using lactoperoxidase-125I labeling techniques. Cells harvested during rapid alkaline phosphatase production were converted to protoplasts or lysed protoplasts and labeled. Analysis of the data obtained indicated that 30% of the salt-extractable, membrane-associated alkaline phosphatase was located on the outer surface of the cytoplasmic membrane, whereas 70% of the membrane-associated enzyme was localized on the inner surface. Controls for protoplast integrity (release of tritiated thymidine or examination of cytoplasmic proteins for label content) indicated excellent protoplast stability. Controls indicated that chemical labeling was not a factor in the apparent distribution of alkaline phosphatase on the membrane. These results support the previously reported histochemical localization of alkaline phosphatase on the membrane inner surface. The presence of alkaline phosphatase on the membrane outer surface is reasonable, considering the soluble forms of the enzyme found in the periplasmic region and in the culture medium.     

Ultrahistochemistry of matrix vesicles in elastic cartilage.

Matrix vesicles in the elastic cartilage of epiglottis were negative for acid phosphatase, alkaline phosphatase, and ATPase. This is in agreement with the very rare occurrence of mineralization of elastic cartilage. Only the lysosomes of the chondrocytes showed a positive reaction for acid phosphatase, and a positive reaction for alkaline phosphatase and ATPase was found in relation to the cells of the perichondrium.     

Histochemical aspects of the differentiation of adventitious cartilage on the membrane bones of the embryo chick

Summary The histochemistry of the adventitious cartilage of the chick has been studied and compared with both primary cartilage and the bone on which the adventitious cartilage develops. The distribution of DNA, RNA, collagen, acid mucopolysaccharide, mucoprotein, glycogen, lipid, alkaline phosphatase and inorganic phosphate has been studied. Adventitious cartilage was found to have the histochemistry of primary hypertrophic cartilage and to calcify. The appearance of lipid and alkaline phosphatase activity coincided with the onset of calcification.The proliferating osteogenic and chondrogenic cells of the chick embryo have been classified and compared histochemically. Collagen synthesis was found to be high in the osteogenic cells and acid mucopolysaccharide and mucoprotein synthesis high in the chondrogenic cells.It has been postulated that the morphogenetic switch from osteogenesis to adventitious chondrogenesis most probably involves a change in the rate of collagen and acid mucopolysaccharide synthesis by the germinal cells of the membrane bones.     

Paget's Disease in a 5-year-old: Acute Response to Human Calcitonin

A five-year-old boy presented with a three-and-a-half-year history of repeated bone fractures and progressive bone deformity. The excretion of hydroxyproline in the urine was greatly increased, and serum alkaline phosphatase and acid phosphatase levels were very high. These abnormalities together with the findings on bone histology and radiology suggested a diagnosis of juvenile Paget''s disease. Human calcitonin reduced the bone turnover as evidenced by an immediate and sustained fall in urine hydroxyproline excretion, while calcium and phosphate balance became more positive. This treatment is therefore being continued on an outpatient basis.