Acid Phosphatase Reaction Research Articles

Histoenzymology of the developing rat spinal cord.

Using histochemical methods the activity of acetycholinesterase, acid phosphatase and various dehydrogenases was investigated in the developing rat spinal cord. At the ninth embryonic day (E9) only the activity of the lactate dehydrogenase slow-moving isoenzymes was prominent in spinal neurons. Between E13 and E15 an increase was observed in the activity of most of the dehydrogenases and of acid phosphatase in motoneurons and posterior root ganglion cells. Between E15 and E17 acetylcholinesterase activity increased markedly. On E17 and E20, this enzyme was also detectable in anterior and posterior roots and in neurons of the intermediate grey matter. On E20, although all grey matter neurons were cytologically fully differentiated, their enzymatic content was found to be still incomplete. The prominent acid phosphatase reaction within laminae I and II, which is characteristic of the adult rat, was absent in the fetal spinal cord. These findings indicate that the spinal cord metabolism is predominantly anaerobic during the first two-thirds of gestation. The histoenzymological maturation of grey-matter neurons is delayed in comparison to their cytological differentiation. Furthermore, the ontogenesis of motoneuronal acetylcholinesterase activity correlates well with the development of motor activities in the rat fetus.

Cytochemical studies of acid phosphatase in ovarian follicles: a suggested role for lysosomes in steroidogenesis.

Lysosomal activity in follides of ovaries of untreated and gonadotropic-treated immature rats was investigated using cytochemical techniques for acid phosphatase localization. The major site of acid phosphatase reaction product in ovaries of untreated immature rats was in autophagic vacuoles associated with numerous atretic follicles. Little reaction product was found in cells of intact preantral follicles. In contrast, antral follicles of rats treated with PMSG (20 IV of pregnant mare serum gonadotropin 54 h before killing) displayed more acid phosphatase reaction product than did preantral follicles, especially within their thecal steroidogenic cells. Craafian follicles of rats treated with PMSG-hCG (20 IV of PMSG plus 40 IV of human chorionic gonadotropin 54 h later and killed 16 h later) were characterized by large quantities of reaction product within both granulosa and thecal cells. However, follicular cells undergoing luteinization had more acid phosphatase reaction product than did any other cell type. The reaction product in all ovarian steroidogenic cells was associated predominantly with the active Golgi complex and the extensive agranular endoplasmic reticulum, with some reaction product within vesicles (<0.15 Mm). It is concluded that gonadotropic treatment stimulates the development of an extensive lysosomal system within steroidogenic cells of ovarian follicles. Since there were no morphologic indications of degeneration within these cells, these observations suggest that the lysosomal system may play a role in ovarian steroidogenesis.

Relationship between the Golgi apparatus, GERL, and secretory granules in acinar cells of the rat exorbital lacrimal gland.

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.

Reassessment of a cytochemical test for differential diagnosis of leukemic reticuloendotheliosis.

Reports of negative tartrate-resistant acid phosphatase reactions in a few cases of leukemic reticuloendotheliosis prompted the authors to re-evaluate the diagnostic specificity of this test. As a result, they modified the test by (1) incorporating a dual-control system for excluding a false-negative test due to technical errors, and (2) instituting an objective grading system for assuring consistent interpretation of the test on blood smears. When these modifications were applied to materials of patients suspected to have leukemic reticuloendotheliosis, there was an excellent, although not specific, correlation between the positive test and the diagnosis of leukemic reticuloendotheliosis. Tartrate-resistant acid phosphatase reactions were positive, intermediate, and negative for 76, 21, and 3% of 29 patients who had leukemic reticuloendotheliosis, whereas the figures were 3, 32, and 65%, respectively, for 37 patients who had chronic lymphocytic leukemia and other hematologic disorders.

Phosphomonoesterases in growth cartilages of the rat

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.

Cytochemical reactions of normal and neoplastic lymphocytes.

Rosetting and non-rosetting lymphocytes collected from normal individuals were stained for the presence of beta-glucuronidase, periodic-acid Schiff activity, gamma glutamyl transpeptidase, acid phosphatase, and alpha-naphthyl butyrate esterase. Lymphocytes which formed rosettes with sheep erythrocytes and non-rosette forming lymphocytes contained cytochemical reaction products for all five stains. Beta-glucuronidase (P less than 0-02) and acid phosphatase (P less than 0-01) were more frequently found in rosette forming lymphocytes. However, non-rosetting cells were more frequently periodic-acid Schiff positive (P less than 0-001). Gamma-glutamyl transpeptidase and alpha-naphthyl butyrate esterase were present equally in rosette and non-rosette forming lymphocytes. In addition, 33 non-Hodgkin's lymphomas were studied for cell surface markers and cytochemical reactions. In 17 of 19 B cell lymphomas, there was a paucity of lymphocytes containing beta-glucuronidase. However, in three of four T cell proliferations, there were numerous lymphoid cells positive for beta-glucuronidase. The periodic-acid Schiff and acid phosphatase reactions varied greatly within B, T, and null cell lymphomas and thus were of little diagnostic value in determining the cell of origin of these neoplastic lymphoid cells.

Cytophotometric studies on the reaction kinetics of acid phosphatase in motoneurons marked by injection of Evans blue into the soleus muscle of the mouse.

Evans blue was injected into the soleus muscle of albino mice in order to mark retrogradely the corresponding motoneurons of the spinal cord. Subsequently, reaction kinetics of acid phosphatase were studied in the marked nerve cells. 41-51 motoneurons per animal were counted. They are located in the dorsolateral portion of the Rexed zone IX where they form motor cell columns approximately 1.5 mm in length. After identification of the motoneurons, the enzyme reaction for acid phosphatase was performed by covering the section with a gel film containing naphthol-AS-BI-phosphate-hexazonium-pararosanilin. The formation of the azo dye was measured cytophotometrically at 520 nm. During the first 10 min of registration, a linear decrease in transmission of 0.4 per cent/min was shown. Because of the unimodal distribution of changes in transmission, the motoneurons of the soleus muscle could not be characterized as fast or slow types on the basis of the reaction kinetics of acid phosphatase.

ULTRASTRUCTURAL STUDIES IN THE BROWN ALGA ECTOCARPUS IN CULTURE: AUTOLYSIS*

SUMMARYDuring autolysis of senescent cells of the brown alga Ectocarpus the disintegration of nuclei, endoplasmic reticulum, and Golgi apparatus is completed. Degenerating mitochondria and plastids remain as the only recognizable organelles. Results from ultrastructural study in correlation with cytochemical localization of acid phosphatase indicate that lysosomal compartmentation no longer exists because the acid phosphatase reaction becomes distributed throughout the remainder of the cytoplasm and is also shown by the disintegrating plasmodesmata. Aspects of cell wall breakdown are described and related to knowledge of the chemistry of the cell wall.

Lipofuscin formation in the developing nervous system of squirrel monkeys consequent to maternal dietary protein deficiency during gestation

The formation of lipofuscin pigment under the extrinsic influence of maternal protein deprivation during most of the gestation period has been demonstrated in the cerebellum and motor cortex of the developing fetuses and neonates in squirrel monkeys. The controls maintained on high protein diets failed to show lipofuscin pigment in appreciable quantities. The dirty yellow to dark brown pigment bodies exist as homogeneous particulates as well as duplex structures, each with a thin, irregular, light brown cortex, studded with one or more darker granules, and a medulla filled with sharp yellow substance. The malnourished neonates, especially in the third and fifth laminae of motor cortex, show loose aggregations of lipofuscin bodies in the apical dendrite and occasionally in the body of the dendritic branches but not in the axon hillock or the body of the axon. Histochemically, the pigment is a complex of lipids, carbohydrates and proteins, showing strong reaction for non-specific esterase and acid phosphatase and mild activity of succinate and lactate dehydrogenase and monoamine oxidase, which puts the pigment observed in the same category as the typical aging pigment described in literature. It is clear that maternal protein deficiency during the critical period of the fetal brain development has an adverse effect on its biochemical maturation. The accumulation of lipofuscin pigment is probably one manifestation of the metabolic changes in the neurons, which may have the impact of hastening the aging process, if protein deprivation is continued into the postnatal period.

Structural alterations of rabbit ovarian follicles after mating with special reference to the overlying surface epithelium.

Rabbit ovarian preovulatory follicles and in particular the overlying surface epithelium were studied by morphological and ultrahistochemical means at different times after mating. By light microscopy an increase of cytoplasmic granules was found in the surface epithelium at the follicle apex 4 h after mating. The granules increased in amount and showed maximal accumulation 8-9 h after mating. They then disappeared at the same time as the connective tissue elements in the underlying tunica albuginea and theca externa disintegrated. Transmission electron microscopy showed that the membrane-bounded granules or dense bodies fused with one another and by 8 h after mating they often changed character and appeared more electron lucent. Furthermore, open communications were found between altered granules and vacuoles and between vacuoles and the extracellular space below the epithelium. Acid phosphatase reaction product was localized to the granules and Golgi cisternae. Not all the dense bodies were enzyme positive. At later stages, close to the time of follicle rupture, the epithelial cells were attenuated and thin, with only a few granules. By scanning electron microscopy it was found that the epithelial cells at the follicle apex increased in size approaching the time of follicle rupture and that their microvilli decreased in number and in size. At 8 h and later, the contours of intracellular granules could be visualized. The results of this study were similar to those found when rabbits were induced to ovulate by HCG-stimulation. This further strengthens the hypothesis that the surface epithelium contributes porteolytic enzymes which help to disintegrate the follicle apex prior to rupture.

Fine structure and cytochemistry of specific granules in the lamprey atrium.

Fine structural and cytochemical studies were performed to examine the nature of three types of specific granules found in the atrium of lamprey; specific granules of the atrial muscle cell (ASG), interstitial cell granules (ICG) and endocardial endothelial granules (ESG). Ultrastructurally, ASG and ICG appeared quite similar in size, shape and electron opacity, while ESG were much larger and less dense in opacity than the other two. None of the granules showed positive DAB reaction or acid phosphatase reaction. Only ICG revealed positive chromaffin reaction, which agreed with formaldehyde induced green fluorescence along the atrial lumen. Phosphotungstic acid at low pH stained ICG and ASG strongly positive, and ESG weakly positive. Pronase treatment in Epon sections for 24 h digested ASG alone, whereas in glycol-methacrylate embedded sections, ESG were digested first, ASG were digested thoroughly after 30 min, but ICG were not digested completely after 90 min. From these results it can be concluded that the three types of specific granules have different constituents. ESG consist of protein with some polysaccharides; ASG are composed of protein carbohydrate complexes and lack catecholamines; ICG contain catecholamine as well as protein carbohydrate complexes.

Demonstration of the stimulation of the reticulohistiocytic system (RHS) of mice by treatment with BCG by means of biometric and histochemical techniques

The efficiency of different intravenously-given BCG doses on the RHS was investigated in the present paper. A time- and dose-dependent increase in the weight of liver, spleen, and lungs, was demonstrated in BCG-treated animals. It was shown, by means of histochemical methods, that the weight increase is due to an enhanced proliferation of cells with acid phosphatase reaction. There is a good correlation between the results obtained with biometric and histochemical methods. The question as to whether there is a direct connection between the proliferation of phosphatase-positive cells of the RHS and an increase in tumour defense especially in the model of metastasizing is under discussion.

Observations on the human corpus luteum: Histochemical changes during development and involution

Morphologic studies of the developing, gestational, and involuting corpus luteum show that rapid structural changes occur and that involution is not associated with an appropriate inflammatory response. Histochemical techniques were used to demonstrate several patterns of enzyme activity. The luteunized granulosa cells stained with increasing intensity for lactic dehydrogenase, succinic dehydrogenase, cytochrome oxidase, glucose-6-phosphatase, and 3β-hydroxysteroid dehydrogenase, but reactivity for these enzymes dropped markedly with early involution, and staining was never conspicuous in the organizing cavity. Reactions for acid phosphatase, glucosaminidase, galactosidase, glucuronidase, and nonspecific esterase were also present in the developing corpus luteum, but staining decreased more slowly during involution and was prominent in the occasional macrophages in the granulosa and the granulation tissue in the cavity. Staining was moderately intense for all of the enzymes in the corpus luteum of pregnancy. The decrease in activity for these metabolic enzymes confirms the histologic impression of degeneration, but the loss of staining for lysosomal enzymes was more rapid than expected. The latter finding complicates the hypothesis of involution of the corpus luteum as an example of programmed cell death.

The subcellular distribution and properties of Crithidia sp. hydrolases with particular reference to pyrophosphate and orthophosphate monoester phosphohydrolases.

A cell fractionation scheme was developed for studying the distribution of certain hydrolases, especially phosphohydrolases in a Crithidia sp. (Trypanosomatidae). Whilst between 26-56% of the total cellular hydrolase activities were soluble (probably of flagellar pocket origin), a certain percentage, 5-40%, was sedimentable. A particulate fraction obtained after isopycnic density gradient centrifugation (p = 1.187-1.241), designated fraction FA/FB, was enriched in various acid hydrolases (relative specific activities 1.33-6.24) and displayed latent phosphohydrolase activities. The density gradient distributions of this hydrolytic enzymes were compared with reference to one another and malate dehydrogenase (mitochondrial marker). From the results obtained it appears that the sedimentable acid hydrolases of Crithidia are associated with a heterogeneous population of subcellular particles. Cytochemical observations on the FA/FB fraction supported this finding and revealed the association of acid phosphatase reaction product with subcellular elements resembling multivesicular bodies.

Lymphoblastic lymphoma of convoluted or acid phosphatase type-a tumor of T precursor cells.

Five lymphatic neoplasms with strong focal acid phosphatase reactivity were selected from a group of acute lymphocytic leukemias and lymphoblastic lymphomas. All five cases showed an anterior mediastinal mass and exhibited identical morphology. This type of lymphoma has been described by Lukes under the term "malignant lymphoma of convoluted lymphocytes". Analysis of surface membrane receptors revealed that the tumor cells lacked surface immunoglobulin and receptors for Fc-fragment, but possessed receptors for complement (C3), untreated SRBC (ES) and SRBC treated with neuraminidase (ESN). By applying a mixed rosette assay using nucleated chicken erythrocytes coated with antibodies and C3, and denucleated ESN, it was found that a considerable number of tumor cells in all five cases formed mixed rosettes, i.e. that they bore the C3 receptor characteristic of B cells and simultaneously the E receptor characteristic of T cells. Thus the tumor cells resembled immature thymocytes of 10-15 weeks' gestation, which also show focal acid phosphatase reactivity and simultaneous expression of C3 and E receptors.

Serum acid phosphatase levels following prostatic massage (author's transl)

We find an elevation of serum acid phosphatase levels 5 min after prostatic massage in only 10% of patients with prostatic adenoma. This increase is caused by stored prostatic secretion being pressed into the blood vessels. 60 min later these serum levels decrease. With some other patients a slower increase of phosphatase levels occurs; this increase, however, lasts for hours. This kind of increase is caused by prostatic fluid being forced into the interstitium where it is slowly absorbed. A combination of both kinds leads to a curve with two peaks; this could be demonstrated in two cases. No difference was seen in reaction of total acid phosphatase and prostatic phosphatase levels. The increase of phosphatase levels following prostatic massage was no sign of prostatic carcinoma.

Lokalisation und verhalten von hydrolasen und transmitterenzymen im dorsalen Corpus geniculatum laterale der ratte nach unilateraler enucleation

In the Corpus geniculatum laterale [pars dorsalis] (Cgl d) of the rat the distribution of the following enzymes is described on the light microscopical level: acid phosphatase, nonspecific esterases, Thiaminepyrophosphatase (TPPase), cholinesterases (AchE and ChE), GABA-alpha-ketoglutarat-transaminase (GABA-T), monoaminooxidase (MAO) and leucin-aminopeptidase. The acid phosphatase and TPPase are localized in the pericarya of different cell types of the Cgl d. The nonspecific esterases are present intracellularly and in the neuropil. With the GABA-T-reaction single cells of Cgl d are stained. The leucine aminopeptidase could not be demonstrated in the Cgl. The nonspecific cholinesterase is localized in the vessel walls. AChE and MAO are present in different fiber structures in the Cgl. Electron microscopical investigations of AChE showed a localization of the enzym in the extracellular room, intraaxonal and in the synaptic cleft. Separation of ChE in polyacrylamid gel resulted in 2 bands for the AChE and 1 band for the nonspecific ChE. After left side enucleation the reaction of the enzymes acid phosphatase, nonspecific esterases cholinesterases and TPPase was proofed topochemically at the section and biochemically at the homogenate. A decrease of enzyme activity in the enzymes acid phosphatase, nonspecific esterases and AchE is shown. The alterations are discussed in connection with the transneuronal degeneration.

The effects of ductal ligation on the parenchyma of salivary glands of cat studied by enzyme histochemical methods.

Submandibular, sublingual and parotid glands of cat have been studied following periods of ductal ligation ranging from 1 day to 1 year. An increased prominence of granules of acid phosphatase, beta-glucuronidase and E600-resistant esterase reaction products was sometimes seen in acinar cells and atropic striated ductal cells, and probably represents increased lysosomal enzymic activity, which may be of importance in the adapation of the parenchyma to the altered environment. Other reaction products often appeared to be at normal levels in parenchymal structures that were not very atrophic, and at reduced levels in those that were very atrophic, suggesting a reduction of functional activity in these structures.

The skin of the palms and soles of the marmosets (Callithrix jacchus and Callithrix penicillata).

The structure and histochemistry of the palmar and plantar skin were studied in four adult male marmosets (two Callithrix jacchus and two Callithrix penicillata). In this skin there exist well-developed epidermal ridges, to which are attached one or two ducts of sweat glands. A thick stratum corneum can be seen in the epidermis, while a distinct stratum lucidum cannot be isolated from the other layers. The stratum granulosum is constituted by one or three layers of cells containing keratohyalin granules. Melanin granulations are mainly concentrated in the basal cells of the epidermal ridges. Dendritic melanocytes and amelanotic melanocytes containing alkaline phosphatase are found among the epidermal cells. Glycogen, UDPG-GT and phosphorylases are mainly present in the middle and lower Malpighian cells of the epidermal ridges. Alkaline phosphatase, ATPase, alanyl amino-peptidase and leucine aminopeptidase were absent in the epidermal cells. SDH, cytochrome oxidase, MAO and a certain number of NAD-dependent dehydrogenases (LDH, ADH, MDH, alpha-GPDH, beta-OHBDH and GDH) showed a stronger reactivity in the basal cells and Malpighian layer. The NADP-dependent enzymes (G-6-PDH, 6-PGDH, cis-aconistase and ICDH) were more reactive in the upper Malpighian layer and stratum granulosum. The stratum corneum showed some acid phosphatase and nonspecific esterase reactivity. The collagenous fibers intertwined with a small number of very thin elastic ones and a larger amount of reticular fibers run almost parallel to the epidermal ridges in the papillary body. In the reticular dermis some fibers are disposed transversely to the epidermal ridges. Meissner corpuscles reactive to butyrylcholinesterase, acetylcholinesterase, nonspecific esterase and G-6-PA are disposed at regular intervals and frequently at each side of the epidermal ridges. Pacinian corpuscles were found only in the hypodermis. The eccrine sweat glands contain glycogen, UDPG-GT and phosphorylase in their secretory, ductal and myoepithelial cells. The secretory part shows a uniform reactivity for every dehydrogenase because it contains only one type of cells (clear cells). The intraepidermal segment of the ducts shows a stronger reactivity to nonspecific esterase and NADP-dependent dehydrogenases than the epithelial cells around it.

CYTOCHEMICAL STUDY OF ACID PHOSPHATASE IN ORAL EXFOLIATED CELLS

Cytochemical studies of acid phosphatase activity of oral mucosa cells have been carried on the frozen-dried smears employing the method of standard naphthol AS phosphate devised by Burstone. The purpose is to observe the possible interrelation between the intensity of acid phosphatase activity and some pathologic changes. The specimens were obtained from various areas of oral cavity of 71 persons. In the oral mucosa from healthy individuals, acid phosphatase activity was found to be positive in all the cells from various areas. Intense acid phosphatase reaction was seen in the cells of palate and the gingiva. Activity of the enzyme decreased in inflammatory conditions. The mechanism of enhancement of acid phosphatase activity in inflammatory condition was discussed in relation to keratinization.