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     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum an nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

Presence of lysosomal enzymes in the normal glomerular basement membrane matrix

Summary The question posed in the present study was: are there hydrolytic enzymes, including proteases, present in the extracellular matrix of the glomerular basement membrane? If these enzymes are present they may play a role in the catabolism of the glomerular basement membrane (GBM) and removal of macromolecular debris resulting from ultrafiltration. Enzymes, acid phosphatase - the marker for lysosomal enzymes - β-galactosidase, β-glucuronidase and acid protease (using albumin as substrate) were biochemically assayed in purified basement membrane preparations. It was found that all enzymes were present in significant amounts in the basement membrane. Compared to other enzymes, acid protease activity was present in much higher amounts. The pH optima of these enzymes were variable but all had significant activity at neutral pH. A method was developed to localize the marker enzyme, acid phosphatase, ultrastructurally in the basement membrane in order to substantiate the biochemical findings. Activity was shown by the presence of dense deposits of lead phosphate. Staining for acid phosphatase could also be shown on isolated, purified basement membrane. The demonstration of acid hydrolases in the GBM matrix argues for their role in (i) the extracellular turnover of basement membrane macromolecules, and (ii) clearance of debris of ultrafiltration which tend to clog the membrane pores.     

Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus

Flanagan, John F. (Duke University School of Medicine, Durham. N.C.). Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus. J. Bacteriol. 91:789-797. 1966.-The effect of poliovirus and herpes simplex virus infection on the activity of five hydrolytic enzymes was studied in tissue culture cells of KB type. During the course of poliovirus infection, the activity of beta-glucuronidase, acid protease, acid ribonuclease, acid deoxyribonuclease, and acid phosphatase in the cytoplasm rose to levels two- to fourfold greater than the activity present in the cytoplasm of uninfected cells. The rise in cytoplasmic activity was accompanied by a concomitant decrease in enzymatic activity bound to cell particles. Shift of enzymatic activity from the particulate to soluble state was first detected at 6 hr after poliovirus infection, coinciding with the appearance of new infectious particles and virus cytopathic effect. No net synthesis of these enzymes after poliovirus infection was found. Hydrocortisone added to the culture medium failed to affect either the titer of virus produced in the cells or the release of hydrolytic enzymes from the particulate state. Herpes simplex infection produced minimal alterations in the state of these enzymes in KB cells. It is hypothesized that the breakdown of lysosomes and release of hydrolytic enzymes accompanying poliovirus infection is produced by alterations in cell membrane permeability during the course of virus replication and by the consequent change in the ionic content of the cell sap.     

Ultracytochemical studies of capillary endothelial cells in the rat central nervous system

The ultracytochemical localization of eight hydrolytic enzymes (TMPase, 5'-NPase, TPPase, TTPase, Mg++-ATPase, Ca++-ATPase, ALPase and K+-NPPase) and one oxidative enzyme (MAO) was determined in rat brain capillary endothelial cells. In the somal plasma membrane, the enzymatic activity was mainly located in the antiluminal plasma membrane. This finding was appropriate for enzymes possessing the optimal pH at alkaline ranges, except for alkaline phosphatase. Most enzymes investigated showed a positive reaction on the pinocytotic vesicles of capillary endothelial cells. Differences in the intensity of the enzyme activities of the luminal and antiluminal plasma membranes may reflect the polarity in the capillary endothelial cells and relate to blood-brain barrier mechanisms.     

Differentiation of odontoblasts is negatively regulated by MEPE via its C-terminal fragment

Matrix extracellular phosphoglycoprotein (MEPE) is an extracellular matrix protein that is mainly expressed in mineralizing tissues, including the dental pulp. The purposes of this study were to clarify the localization of MEPE in the tooth germ and to investigate the roles of MEPE in the differentiation of odontoblasts. The immunohistochemical staining in the tooth germ of the upper first molars of male Wistar rats (postnatal day 3) revealed that MEPE was mainly localized in odontoblasts during dentinogenesis. Stable MEPE-overexpressing and MEPE-knockdown cell lines, which were established in odontoblast-lineage cells (OLCs), showed lower and higher differentiation capabilities, respectively. Eukaryotic proteins of the N-terminal fragment of MEPE produced in HEK cells had no effect on the differentiation of OLCs, whereas the C-terminal fragment containing an RGD sequence inhibited their differentiation. These results indicated that the C-terminal fragment of MEPE containing an RGD sequence, cleaved in odontoblasts, appeared to be the active form of MEPE, which may play important roles in dentinogenesis and pulpal homeostasis by keeping the odontoblasts in immature condition.     

Comparative enzyme histochemical studies on the development of teeth in man and mouse

Summary On the basis of a material consisting of primordia of deciduous teeth from 17 human foetuses and tooth germs from 20 newborn mice and 4 mouse foetuses, the histochemical distribution of succinic dehydrogenase, LDH, BDH, glucose-6-phosphate-dehydrogenase, NADH₂ cytochrome C reductase, and NADPH₂ diaphorase was studied.The investigation comprised partly mapping of the above-mentioned oxidative enzymes in human tooth germs during the period from bud stage to bell stage with incipient hard-tissue formation, and partly a comparative enzyme histochemical investigation of the dental development in man and in the mouse.In both materials there was an increase of all the demonstrated enzyme systems in the ameloblasts in early amelogenesis. On the other hand, the ameloblasts of the two materials showed differences during the period of matrix production: In the human ameloblasts there was activity of succinic dehydrogenase basally as well as apically, while the non-human ameloblasts showed such activity only basally.The odontoblasts of both materials showed a definite reaction for glucose-6-phosphatedehydrogenase, LDH, and BDH, but slight activity of succinic dehydrogenase. This indicates the existence of the pentose shunt as the source of energy in dentinogenesis. On the basis of the enzyme reactions studied, neither material showed a subodontoblastic layer or any reaction in the hard tissues.Material supplied by the Association for the Aid of the Crippled Children, New York. Aided by grants from the Danish State Research Foundation and the Rask-Ørsted Foundation.     

Changes in droplet fractions from rat kidney cells after intraperitoneal injection of egg white

1. Kidney homogenates from rats injected with egg white and from control rats were fractionated simultaneously into six fractions and the content of acid phosphatase, ribonuclease, desoxyribonuclease, cathepsin, and beta-glucuronidase in corresponding fractions from treated and untreated animals was compared. These observations were correlated with the amount of dark brown bottom sediments in fractions NDrI, DrII, and DrIII, and with the number of droplets in fraction NDrI. 2. It was found that after injection of egg white the amount of small droplets decreased as indicated by the decrease of the dark brown bottom layer in the sediment of fraction DrIII and by the concomitant decrease of hydrolytic enzymes in the same fraction, and that the number of large droplets increased as indicated by the increase of brown sediment in fraction NDrI and the increase in the number of droplets counted in a bacterial counting chamber in the same fraction. It was concluded that the treatment with egg white induced the transformation of small droplets into large droplets. 3. The decrease of hydrolytic enzymes in the fractions containing the small droplets was accompanied by a marked increase of these enzymes in the supernatant fluid. The enzyme content of fraction NDrI was not increased after treatment, although it contained greatly increased numbers of large droplets. Counting of the droplets in this fraction showed decreased enzymatic activity of the average large droplet after treatment with egg white. It was suggested that during the transformation of small into large droplets, a portion of the hydrolytic enzymes was released into the surrounding cytoplasm, and that this was partly responsible for the increased enzyme content of the supernatant fluid after fractionation of the kidney homogenate. In contrast to the four other hydrolytic enzymes, beta-glucuronidase was not increased in the supernatant fluid. 4. Eighteen hours after intraperitoneal injection of egg white, the specific enzymatic activities of kidney homogenates showed a 25 to 35 per cent increase for cathepsin, ribonuclease, and desoxyribonuclease, no change for acid phosphatase and beta-glucuronidase, and approximately a 7 per cent decrease for cytochrome oxidase. The increase of cathepsin, ribonuclease, and desoxyribonuclease in the total homogenate was interpreted as an indication of the formation of new enzymes, and it was suggested that this partly accounted for the increase of these enzymes in the supernatant fluid. 5. The activation of the enzymes by osmotic effects was investigated in vitro by incubation of droplet fractions in the presence of different concentrations of sucrose.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Summary Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum and nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

Decalcification for histochemical demonstration of hydrolytic and oxidative enzymes

Summary A method for histochemical demonstration of hydrolytic and oxidative enzymes following decalcification was described in mature bone and tooth by neutral EDTA decalcifying solution.The decalcifying solution, 0.5 M EDTA tetrasodium salt was adjusted to neutral pH with 5 M citric acid, obtained the most sufficient results for demonstration of enzymes in decalcified tissue. The hard tissue decalcified in 30 to 40 days at 4° C exhibited a good preservation of hydrolytic and oxidative enzymes histochemically, but a few structural destruction in a long term decalcification was found in soft tissues and certain organs.     

CHANGES IN "DROPLET" FRACTIONS FROM RAT KIDNEY CELLS AFTER INTRAPERITONEAL INJECTION OF EGG WHITE

1. Kidney homogenates from rats injected with egg white and from control rats were fractionated simultaneously into six fractions and the content of acid phosphatase, ribonuclease, desoxyribonuclease, cathepsin, and β-glucuronidase in corresponding fractions from treated and untreated animals was compared. These observations were correlated with the amount of dark brown bottom sediments in fractions NDrI, DrII, and DrIII, and with the number of droplets in fraction NDrI. 2. It was found that after injection of egg white the amount of small droplets decreased as indicated by the decrease of the dark brown bottom layer in the sediment of fraction DrIII and by the concomitant decrease of hydrolytic enzymes in the same fraction, and that the number of large droplets increased as indicated by the increase of brown sediment in fraction NDrI and the increase in the number of droplets counted in a bacterial counting chamber in the same fraction. It was concluded that the treatment with egg white induced the transformation of small droplets into large droplets. 3. The decrease of hydrolytic enzymes in the fractions containing the small droplets was accompanied by a marked increase of these enzymes in the supernatant fluid. The enzyme content of fraction NDrI was not increased after treatment, although it contained greatly increased numbers of large droplets. Counting of the droplets in this fraction showed decreased enzymatic activity of the average large droplet after treatment with egg white. It was suggested that during the transformation of small into large droplets, a portion of the hydrolytic enzymes was released into the surrounding cytoplasm, and that this was partly responsible for the increased enzyme content of the supernatant fluid after fractionation of the kidney homogenate. In contrast to the four other hydrolytic enzymes, β-glucuronidase was not increased in the supernatant fluid. 4. Eighteen hours after intraperitoneal injection of egg white, the specific enzymatic activities of kidney homogenates showed a 25 to 35 per cent increase for cathepsin, ribonuclease, and desoxyribonuclease, no change for acid phosphatase and β-glucuronidase, and approximately a 7 per cent decrease for cytochrome oxidase. The increase of cathepsin, ribonuclease, and desoxyribonuclease in the total homogenate was interpreted as an indication of the formation of new enzymes, and it was suggested that this partly accounted for the increase of these enzymes in the supernatant fluid. 5. The activation of the enzymes by osmotic effects was investigated in vitro by incubation of droplet fractions in the presence of different concentrations of sucrose.     

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.     

Ca-ATPase and ALPase activities at the initial calcification sites of dentin and enamel in the rat incisor

Summary Enzymatic activities of calcium-magnesium dependent adenosine triphosphatase (Ca-ATPase) and nonspecific alkaline phosphatase (ALPase) were localized at the initial calcification sites of dentin and enamel of rat incisor teeth using electron-microscopic cytochemistry.Ca-ATPase was localized in the Golgi cisternae, cytoplasmic vesicles and along the outer surface of the presecretory and secretory ameloblasts, whereas it was totally absent from the odontoblasts in the pulp. Inversely, ALPase reaction was localized along the outer surface of the odontoblasts, but almost completely absent from the ameloblasts.Diffuse extracellular reactions of both enzymes were distributed throughout the unmineralized fibrous matrix of mantle dentin in which a large number of matrix vesicles were scattered. Both Ca-ATPase and ALPase reactions, which appeared in the matrix vesicles in the process of formation of mantle dentin, became most conspicuous at the site of initial dentin calcification. At this stage, an intense Ca-ATPase reaction also appeared along some of the collagen fibrils adjacent to the reactive matrix vesicles. No ALPase reaction was localized along these Ca-ATPase reactive collagen fibrils.Our observations suggest strongly that Ca-ATPase in the matrix vesicles originates from the inner enamel epithelium and/or preameloblasts whereas ALPase originates from the odontoblasts in the pulp. The importance of the coexistence of both enzymes for the control of initial calcification of dental hard tissues is suggested.     

Matrix vesicle heterogeneity: possible morphogenetic functions for matrix vesicles.

Extracellular membranous matrix vesicles were localized and described using electronmicroscopy during chondrogenesis, osteogenesis, and dentinogenesis. Evidence indicates that matrix vesicles in each of these specific tissue types function to concentrate and transport ions and enzymes which serve as nucleation sites for the mineralization of hydroxylapatite. We have examined different developmental stages of Meckel's cartilage, alveolar bone and epithelial-mesenchymal interactions associated with tooth formation in newborn mice. These ultrastructural studies indicate matrix vesicle heterogeneity. Whereas most matrix vesicles contain alkaline phosphatase activity during cartilage, bone and dentine mineralization, in earlier developmental stages matrix vesicles contain acid phosphatase activities and little, if any, alkaline phosphatase. Tissue type, specific developmental stage, and ultrastructural criteria indicate various "classes" of matrix vesicles. During epithelial-mesenchymal interactions in tooth development, mesenchymal cells (preodontoblasts) appear to be the source of matrix vesicles as indicated by the complementarity between H-2 histocompatibility alloantigen specificity on the cell surface and that of the matrix vesicle outer surface; matrix vesicles are limited by a trilaminar membrane derived from the mesenchymal cells. Some of the vesicles located adjacent to dividing inner enamel epithelial cells contain RNA's as determined by electron microscopic autoradiography in situ, as well as by direct biochemical assays. We postulate that matrix vesicles have many different and important biological functions, one of which may be to mediate developmental information from mesenchyme to epithelia during "instructive" stages of tooth development.     

Differential expression of decorin and biglycan genes during mouse tooth development.

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.     

Cytochemical response of kidney, liver and nervous system of fluoride ions in drinking water.

Morphological and cytochemical studies on the squirrel monkey have been made after maintaining the sujects on pure distilled water and fluoridated distilled water for 18 months with the objective of determining the effect of fluoride on the activity of some hydrolytic and oxidative enzymes in the kidney, liver and nervous system. Daily water intake by individual animals was measured over the final 10 months of the animal's exposure to 0,1 and 5 ppm fluoride. Water consumption was considerably higher in the animals on higher fluoride intake. Whereas the nervous system remained totally unaffected by this experimental procedure, the liver showed a slightly enhanced acivity of Krebs citric acid cycle enzymes. The kidneys, however, showed significant cytochemical changes, especially in the animals on 5 ppm fluoride intake in their drinking water. In these animals, the glomeruli showed an increase in the activity of acid phosphatase and the enzymes belonging to the citric acid cycle and the pentose shunt, whereas lactate dehydrogenase, a resentative of the anaerobic glyoclytic pathway, remained unchanged or only slightly changed. These observations suggest that fluoride in concentrations as low as 5 ppm interferes to some extent with the intracellular metabolism of the excretory system.     

Effect of quinacrine treatment on the activity of acid hydrolases (phosphatase, N-acetyl-beta-glucosaminidase, glucosidase, galactosidase and esterase) in Tetrahymena.

The effect of phospholipase A2 (PLA2) inhibitor, quinacrine, on the activity of hydrolytic enzymes in Tetrahymena pyriformis homogenate, was investigated. The activity of all of the enzymes studied (acid phosphatase, N-acetyl-beta-glusosaminidase, glucosidase, galactosidase and esterase) was significantly reduced in the presence of quinacrine. Since there are no data on the inhibitory effect of PLA2 and PLA2 influenced metabolic pathways to the hydrolytic enzymes, the direct effect of quinacrine on the hydrolytic enzymes (of Tetrahymena) can be supposed. This is supported by the fact that the other PLA2 inhibitor, 4-bromophenacyl bromide, did not influence phosphatase activity.     

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.     

Enzyme histochemistry of the choroid plexus in rat and squirrel monkey

Summary The reactions given for various oxidative and hydrolytic enzymes by the choroid plexus of the squirrel monkey and the rat brain have been studied in detail. The lining cells show strong activity for citric acid cycle and glycolytic pathways enzymes. The stroma shows strong activity for adenosine triphosphatase, alkaline phosphatase, adenosine monophosphatase and glucose-6-phosphatase. The peripheral part or luminal borders of the cytoplasm of the choroidal cells show strong activity for alkaline phosphatase, adenosine monophosphatase and adenosine triphosphatase, and a well developed thiamine pyrophosphatase positive Golgi complex, indicating their participation in the formation and transport of secretory material. The nucleoli of the lining cells give a positive reaction for glucose-6-phosphatase and adenosine triphosphatase. Acid phosphatase like the thiamine pyrophosphatase positive Golgi material is found all over the cytoplasm. The functional significance of these findings is briefly discussed.This work has been carried out with the aid of Grant No. FR-00165 from the Animal Resources Branch, National Institutes of Health and NASA Grant NGR-11-001-016. T. R. Shanthaveerappa in previous publications.     

Odontoclastic resorption of the superficial nonmineralized layer of predentine in the shedding of human deciduous teeth

Resorption by odontoclasts of a superficial nonmineralized layer of predentine that occurs in prior to the shedding of human deciduous teeth was studied by light and electron microscopy. As resorption of the tooth roots neared completion, multinucleate cells appeared on the predentine surface of the coronal dentine between the degenerated odontoblasts, excavated characteristic resorption lacunae in the nonmineralized predentine. These multinucleate cells had the same ultrastructural characteristics as odontoclasts and histochemical demonstration of tartrate-resistant acid phosphatase activity in the multinucleate cells revealed intense staining in numerous small granules identified as lysosomes. Occasionally, the multinucleate cells simultaneously resorbed both nonmineralized and calcospherite-mineralized matrix in the predentine. The study demonstrates that multinucleate odontoclasts can resorb nonmineralized predentine matrix in vivo, probably in the same way as they resorb demineralized organic matrix in the resorption zone underlying their ruffled border.