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.     

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.     

Presence of ATPase and alkaline phosphatase activities in the starfish sperm acrosome

ATPase activity was cytochemically detected in the peripheral acrosomal component of ionophore-reacted sperm, while alkaline phosphatase activity was demonstrated in the upper and central components of the acrosome and, at fertilization, at the site of sperm-oocyte binding. Supernatants of ionophore treated sperm suspensions were assayed for ATPase, alkaline and acid phosphatase activities. Results suggest that alkaline phosphatase may be involved both in the acrosomal reaction and oocyte jelly lysis but the function of the acrosomal ATPase remains unknown.     

Phosphomonoesterases in growth cartilages of the rat

Summary Epiphyseal plate cartilage, epiphyseal cartilage, synchondroseal cartilage and mandibular condylar cartilage were studied morphologically and histochemically in 14 days old rats. Ordinary decalcified paraffin sections were stained with hematoxylin & eosin, van Giesons connective tissue stain, or toluidine blue, and used for morphological studies of the different cartilaginous structures. Undecalcified cryostat sections were used for demonstration of acid and alkaline phosphatase. The enzyme activity was tested for at regular intervals during incubation from 15 sec to 120 min.The morphologic study revealed that a marked similarity of construction exists between epiphyseal plate cartilage and synchrondroseal cartilage. The construction of epiphyseal and condylar cartilage differ from that of the other two structures and also differ mutually.With small variations the reaction for both alkaline and acid phosphatase was found to be identical in the zones of erosion, hypertrophy and maturation of the four structures. Intercellularly, acid phosphatase is present in all zones in the synchondroseal and the epiphyseal plate cartilage, while in the epiphyseal and condylar cartilages it is only present in the zones of erosion, hypertrophy and maturation.The identical reaction for acid phosphatase in the epiphyseal and the condylar cartilage is thought, in all likelihood, to be accidental. When kinetic conditions are taken into account, epiphyseal cartilage seems to react like epiphyseal plate and synchondroseal cartilage, while the condylar cartilage takes up an exceptional position among growth cartilages.     

牛蛙消化道黏膜5种酶的分布

目的研究牛蛙(Rana catesbeinana)消化道黏膜碱性磷酸酶(ALP)、酸性磷酸酶(ACP)、ATP酶、酯酶和脂酶的分布。方法在消化道的8个部位取材,采用冰冻切片技术和酶的组织化学方法。结果 ALP主要分布于十二指肠、空肠和回肠,胃中酶反应呈弱阳性。ACP主要分布于胃中,食道和肠道酶反应呈弱阳性。ATP酶在消化道各部位均有较多分布,十二指肠、空肠和回肠显著较多,胃各部位其次。酯酶和脂酶均主要分布于肠道,胃各部位其次。结论牛蛙消化道黏膜酶的分布同其它动物有相似之处,也有其自身特点。十二指肠、空肠和回肠是牛蛙的主要消化吸收部位。     

Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase.

Fractions composed primarily of cells (Fraction I), membrane fragments (Fraction II) and matrix vesicles (Fraction III) were isolated from chick epiphyseal cartilage. The characteristics of the alkaline phosphatase (EC 3.1.3.1), pyrophosphatase (EC 3.6.1.1) and ATPase (EC 3.6.1.3) activities in the matrix vesicle fraction were studied in detail. Mg-2-+ was not absolutely essential to any of the activities, but at low levels was stimulatory in all cases. Higher concentrations inhibited both pyrophosphatase and ATPase activities. Both the stimulatory and inhibitory effects were pH-dependent. Ca-2-+ stimulated all activities weakly in the absence of Mg-2-+. However, when Mg-2-+ was present, Ca-2-+ was slightly inhibitory. Thus, none of the activities appear to have a requirement for Ca-2-+, and hence would not seem to be involved with active Ca-2-+ transport in the typical manner. The distribution of alkaline phosphatase, pyrophosphatase, and Mg-2-+ ATPase activities among the various cartilage fractions was identical, and concentrated primarily in the matrix vesicles. Conversely, the highest level of (Na-+ + K-+)-ATPase activity was found in the cell fraction. All activites showed nearly identical sensitivities to levamisole (4 - 10-3 M) which caused nearly complete inhibition of alkaline phosphatase and pyrophosphatase. About 10-15% of the ATPase activity was levamisole-insensitive. The data are consistent with the concept that the Mg-2-+-ATPase and pyrophosphatase activities of matrix vesicles stem from one enzyme, namely, alkaline phosphatase.     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Differential expression of alkaline phosphatase and ATPase activities in human colon carcinoma cell line HT-29.18 during differentiation

The expression and cytochemical localization of alkaline phosphatase and Na+-pump sites were investigated in the human adenocarcinoma cell line HT-29.18 during differentiation. In the undifferentiated state, HT-29.18 cells expressed ATPase activity on plasma membrane whereas they displayed no alkaline phosphatase activity. In differentiated HT-29.18 cells, strong alkaline phosphatase activity was present on the apical membrane, whereas ATPase activity was restricted to the basolateral membrane. Intra- and intercellular lumina (cysts) observed in undifferentiated cells were devoid of both enzyme activities. In differentiated cells, cysts bearing well developed microvilli were strongly positive for alkaline phosphatase activity, while this activity seemed to be lacking in cysts without microvilli. ATPase activity was not found in either type of structure. Finally, HT-29.18 differentiated cells expressed, at pH 9.0, a p-nitrophenylphosphatase activity six-fold greater than that of undifferentiated cells.     

Metabolic and genetic control of isoenzyme spectrum of alkaline phosphatase inEscherichia coli

Three proteins possessing alkaline phosphatase activity were detected in a fraction of periplasmic material ofEscherichia coli K-10 and its mutants with constitutive synthesis of alkaline phosphatase. They also showed acid phosphatase, pyrophosphatase and ATPase activities. Through the use of phosphatase-negative mutants it was shown that these proteins were the products of a single structural gene and therefore represented alkaline phosphatase isozymes. The numbers of enzyme isoforms and possibly the spectrum of their phosphohydrolase activities were controlled by exogenous orthophosphate and depended on the integrity of regulator genes for alkaline phosphatase.     

Similarities between alkaline phosphatase and Ca2+ ATPase activities in HeLa cells

Parallel changes in the enzyme activities of CA2+ATPase and alkaline phosphatase were observed in HeLa cells. Both enzymes were inhibited to a similar degree by L-phenylalanine, L-tryptophan, and L-leucine, while being relatively resistant to L-homoarginine. Exposure to heat (56 degrees C, 60 degrees C, and 65 degrees C) resulted in a loss of both enzyme activities. Both alkaline phosphatase and Ca2+ ATPase, when treated with EGTA, required Ca2+ for the restoration of activity. Cells grown in the presence of agents that affect alkaline phosphatase (dexamethasone, butyric acid, and hyperosmolar NaCl) showed similar changes in the activities of both enzymes.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.     

Androgen receptors and gender-specific distribution of alkaline phosphatase in human thyroid cartilage

The degree of mineralization in human thyroid cartilage is gender specific. Until now, laryngeal tissue was tested for sexual hormone receptors by the use of radiolabelled hormones only without exact localization of the receptors. In this study immediately frozen cartilage specimens from seven male and one female patient who underwent laryngectomy were used for immunolocalization of sexual hormone receptors. Additionally, serum sexual hormone levels were measured by means of radioimmunoassay. Alkaline phosphatase was localized enzymohistochemically in another cohort of six male and four female cartilage specimens from laryngectomies and autopsies. Chondrocytes in thyroid cartilage from both sexes reacted with antibodies to the androgen receptor. The low serum testosterone levels, which varied between 1.5 and 3.9 ng/ml, did not correlate with insufficient mineralization of thyroid cartilage in men (r=0.363, P=0.432). Chondrocytes did not react with antibodies to the estrogen receptor α and the progesterone receptor in both sexes. Expression of alkaline phosphatase started about the middle of the second decade. Some chondrocytes near the mineralization front were positive for androgen receptor and alkaline phosphatase, other chondrocytes were negative for both. Our results suggest the involvement of androgen receptor positive chondrocytes in thyroid cartilage mineralization, probably by a testosterone-linked stimulation of alkaline phosphatase.     

Inhibition and reversion of chondrogenesis by retinoic acid in rat limb bud cell cultures

Summary Mesenchyme cells derived from embryonic rat limb buds cultured at high density differentiated into chondrocytes. The degree of chondrogenesis was assessed by alcian blue staining, a stain specific for cartilage matrix. The addition of retinoic acid on day 1 of culture inhibited chondrogenesis in a dose-dependent fashion. When retinoic acid was added to the cultures on day 5, the cartilage nodules, consisting of newly differentiated cartilage cells, disappeared during the following 6 days. Coinciding with this process the histochemically demonstrable alkaline phosphatase activity, localized in the internodular areas, also disappeared. This indicated that retinoic acid not only inhibited chondrogenesis but also induced resorption of cartilage cells and that at least two cell types were affected, the cartilage cells and the cells bearing alkaline phosphatase.Actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, suppressed the retinoic acid effect in day 5 limb bud cell cultures. This result indicated that the effect of retinoic acid required RNA and protein synthesis and is compatible with the view that vitamin A may act in a hormone-like way.     

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.     

Effect of mutations in regulatory genes for alkaline phosphatase on the phosphohydrolase spectrum of E. coli periplasm]

The isoform spectra of alkaline and acid phosphatase, pyrophosphataes, and ATPase in periplasm of E. coli were studied using electrophoresis in polyacrylamide gel with subsequent development of zimograms directly in the gel. Wild strains and mutants on 4 regulatory genes of alkaline phosphatase were analyzed. Mutations in regulatory genes were shown to influence the amount of each of the 3 isoforms of alkaline phosphatase and also the spectra of other phosphohydrolases.     

Alkaline phosphatase and phosphoamino acid phosphatases in normal and cancerous tissues of the human larynx

The activities of alkaline phosphatase and phosphoamino acid phosphatases were measured in normal and cancerous regions of the human larynx. For each larynx, alkaline phosphatase and phosphotyrosine phosphatase activities were higher in the tumor than in the corresponding normal tissue. Phosphothreonine and phosphoserine phosphatase activities were relatively low and there were no consistent trends. The increased alkaline phosphatase activity in the tumors supports histological observations that ossification of cartilage seems to occur at the site of invasion; the phosphatase acting on phosphotyrosine could serve as a regulator of cell differentiation during tumorigenesis.     

Calcification of in vitro developed hypertrophic cartilage

We have recently reported that dedifferentiated cells derived from stage 28-30 chick embryo tibiae, when transferred in suspension culture in the presence of ascorbic acid, develop in a tissue closely resembling hypertrophic cartilage. Ultrastructural examination of this in vitro formed cartilage showed numerous matrix vesicles associated with the extracellular matrix (C. Tacchetti, R. Quarto, L. Nitsch, D. J. Hartmann, and R. Cancedda, 1987, J. Cell Biol. 105, 999-1006). In the present article we report that the in vitro developed hypertrophic cartilage undergoes calcification. We indicate a correlation between the levels of alkaline phosphatase activity and calcium deposition at different times of development. Following the transfer of cells into suspension culture and an initial lag phase, the level of alkaline phosphatase activity rapidly increased. In most experiments the maximum of activity was reached after 5 days of culture. When alkaline phosphatase activity and 45Ca deposition were measured in the same experiment, we observed that the increase in alkaline phosphatase preceded the deposition of nonwashable calcium deposits in the cartilage.     

The calcified-noncalcified cartilage interface: the tidemark

Tidemark is an interface which may better be defined by biochemical methods than by morphology. It originates, by chondrocyte activity, between calcified and noncalcified cartilage layers of any kind, hyaline or fibrous, in areas exposed to either loading (joint) or pulling (insertion). In the articular cartilage it appears with skeletal maturation, in other localizations it is age-independent. It should be regarded as a special instance of a broader phenomenon of the calcification/mineralization front. Inside the joint cartilage its changes reflect the slow remodelling of the calcified layer and its inapparent shift towards the surface of the articular cartilage. In the marginal transitional zone of the joint, tidemark smoothly passes into the periosteum. Chondrocytes on both sides of the tidemark are positive for alkaline phosphatase and the positive reaction continuously goes on to the periosteum.     

Alkaline and Acid Phosphatase Demonstration in Human Bone and Cartilage: Effects of Fixation Interval and Methacrylate Embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.     

Alkaline and acid phosphatase demonstration in human bone and cartilage: effects of fixation interval and methacrylate embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.