Demonstration Of Acid Phosphatase Activity Research Articles

Histochemical Characterization of Rain-Forest Strain of Onchocerca Volvulus Microfilariae Isolated in Akamkpa, Cross River State, Nigeria.

Abstract: The histochemical characterization of rain-forest strain of Onchocerca volvulus isolated in Akamkpa of Cross River State, Nigeria was studied. In a preliminary survey of 350 persons from eight villages, 75(21.4%) were found to be positive for the parasite. Males (23.6%) were more infected than the females but there was no statistically significant difference in the presence of infection by gender (p=0.506). Staining pattern of the parasite using Lead nitrate method for the demonstration of acid phosphatase activity revealed that of the six patterns, four corresponded to the enzyme types already described by some authors in West African rain-forest, and Sudan. One corresponded to the type found in Guinea savannas while the remaining did not correspond to any of the strains described elsewhere. The multiple enzyme staining patterns of Onchocerca volvulus microfilariae in the study area is indicative of polymorphic strain than that of West African strains. This variation in strain might contribute to the degree of pathogenicity and clinical manifestation of onchocerciasis in this endemic zone.

Cryptosporidium parvum: identification and characterization of an acid phosphatase

Acid phosphatases are putative virulence factors in different pathogenic microorganisms. Acid phosphatases can also inhibit the respiratory burst of human neutrophils. In Cryptosporidium parvum, a protozoan parasitic, the study of enzymes is limited. In this paper, we report the presence of a membrane-bound acid phosphatase activity in C. parvum oocysts. The enzymatic activity was inhibited by protein tyrosine phosphatase inhibitors such as sodium orthovanadate, ammonium molybdate, and sodium tungstate and was not affected by protein serine/threonine phosphatase inhibitors such as okadaic acid and calyculin. Antibodies against the catalytic domain of human placental PTPase 1B cross-reacted with two molecules of 30 and 31 kDa present in membrane fraction of a Cryptosporidium oocyst homogenate. This is the first demonstration of acid phosphatase activity in Cryptosporidium.

The effect of sublethal lead exposure on the ultrastructure and on the distribution of acid phosphatase activity in chloragocytes of earthworms (Annelida, Oligochaeta)

Laboratory experiments were conducted to study the effects of the exposure to a sublethal concentration (500 p.p.m.) of lead on the ultrastructure and acid phosphatase compartmentalization of the chloragogenous tissue of earthworms, Eisenia foetida. For the cytochemical demonstration of acid phosphatase activity, lead and cerium were used as capturing agents. In both cases there was a change in the compartmentalization of acid phosphatase, the enzyme activity being localized within the chloragosomes in controls, but distributed throughout the cytosol in treated animals. In addition, acid phosphatase activity increased following lead exposure. At the ultrastructural level, disruption of the chloragosomal membranes, an increase in chloragosomal fusion processes and vesiculation of the cytoplasm were evident. Moreover, an enhanced release of chloragosomes to the extracellular space was found in lead-exposed worms.

Lysosomal origin of the chloragosomes in the chloragogenous tissue of the earthworm Eisenia foetida: cytochemical demonstration of acid phosphatase activity

The cytochemical localization of the lysosomal marker enzyme acid phosphatase was studied in the chloragogenous tissue of earthworms. The Gomori lead technique and the cerium capture technique were utilized. Both techniques demonstrated the chloragosomal location of this enzyme. Only a small proportion of chloragosomes presented reactivity, which suggests that these organelles are distinctly heterogeneous. The reaction product was localized in the periphery of chloragosomes, suggesting a membrane-bound compartmentalization of acid phosphatase. In addition, degenerating mitochondria and membrane whorls were observed in some chloragosomes, indicating the possibility that these organelles perform autophagosomal functions.

Lysosomal origin of the chloragosomes in the chloragogenous tissue of the earthwormEisenia foetida: cytochemical demonstration of acid phosphatase activity

Lysosomal origin of the chloragosomes in the chloragogenous tissue of the earthwormEisenia foetida: cytochemical demonstration of acid phosphatase activity

Histochemical demonstration of acid phosphatase activity in terminal Schwann cells associated with Ruffini endings in the periodontal ligament of rat incisors

Histochemical staining for acid phosphatase, a marker for lysosomal elements, distinguished rounded, intensely reactive cells from less reactive fibroblasts and osteoblasts in the lingual periodontal ligament. The highly reactive cells were located exclusively in the alveolar half of the ligament. Double staining for acid phosphatase and S-100 protein confirmed that these reactive cells were identical with the terminal Schwann cells associated with periodontal Ruffini endings. Electron microscopically, reaction products for acid phosphatase were observed in the lysosomes and Golgi apparatus in the paranuclear cytoplasm of the terminal Schwann cells. As the terminal Schwann cells associated with the Ruffini endings are assumed to be capable of synthesizing exportable proteins, acid phosphatase in this type of cell may be involved in the processing of macromolecules in synthetic and/or secretory pathways.

In vivo demonstration of acid phosphatase activity in the rhizosphere of soil-grown plants

For in vivo demonstration of acid phosphatase activity in the rhizosphere of soil-grown plants filter papers were treated with a mixture of 1-naphthyl phosphate as substrate and the diazonium salt Fast Red TR as an indicator. After enzymatic hydrolysis, 1-naphthol forms a red complex with Fast Red TR. This method was applied to 8-day old maize plants and 3-year old Norway spruce plants growing in rhizoboxes in soil under non-sterile conditions. The treated filter paper is placed at the surface of roots and soil and acid phosphatase activity is visualized as a red-coloured ‘root print’ on the filter paper. The method can be used as a qualitative analysis of acid phosphatase in the rhizosphere. It also allows a rough estimate of phosphatase activity in different root zones.

The Histochemical Localization of Osteoclasts in EDTA Decalcified Bone

AbstractWe describe a method for the histochemical localization of acid phosphatase (AcP) activity in sections of paraffin embedded bone, following demineralization in ethylene diamine tetra acetic acid (EDTA). AcP activity is consistently demonstrated as a bright red color in cells after incubation in naphthol phosphate/hexazotized new fuchsin substrate. Because this method has been used to demonstrate AcP activity in human and animal tissues, it has applications in both diagnostic and research areas. It is well suited to smaller laboratories because no expensive resins or specialized microtome equipment are needed. Furthermore, the time required for complete decalcification is often shorter than that required to process the same tissue into a suitable resin block. (The J Histotechnol 13:189, 1990)

Density gradient separation of two populations of lysosomes from rat parotid acinar cells.

Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.

Phagocytosis of collagen by mouse decidual cells.

Collagen fibrils were present within membrane-bound vacuoles in the cytoplasm of mouse decidual cells on the 7th day of pregnancy. The space between the vacuole membranes and the fibrils was narrow and frequently filled with a granular electron-dense material. The loss of banding of the collagen fibrils, their association with lysosomelike bodies, and the demonstration of acid phosphatase activity in the vacuoles indicate that the fibrils were internalized by the decidual cells and were being digested. It is suggested that phagocytosis of collagen is a mechanism of remodeling of the mouse decidua.

Isolation of osteoclasts from pagetic bone tissue morphometry and cytochemistry on isolated cells

Giant osteoclasts and other cells were isolated from Pagetic bone tissue using 0.5 mM ethylene diamine tetraacetic acid on bone samples from 8 patients with Paget's disease. The cell suspension contained osteoclasts and osteoblasts as well as some mononuclear cells such as monocytes. The number of nuclei in isolated osteoclasts (33.85 ± 20.92 nuclei/osteoclast) correlates fairly well ( p < 0.02) with the number of nuclei counted on histologic sections (15.88 ± 11.80 nuclei/osteoclast) for samples from each patient. Enzyme histochemistry demonstrated acid phosphatase activity in isolated osteoclasts and in mononucleated cells, such as monocytes. Alkaline phosphatase was detected only in osteoblasts while succinate dehydrogenase was observed in osteoclasts, osteoblasts and monocytes. Esterases, such as nonspecific aliesterase and specific naphthol AS-D acetate esterase, were identified in osteoclasts and in macrophages. Inhibition of specific naphthol AS-D acetate esterase in osteoclasts by addition of sodium fluoride suggests that the enzyme could be of monocytic origin.

An immunohistochemical analysis of human aortic fatty streaks

Recent studies have shown both macrophages and lymphocytes in very early intimal lesions of experimental aortic atherosclerosis. The authors obtained fresh samples of human aortic wall, which had been removed in the course of aortocoronary bypass graft surgery. Intimal fatty streaks were identified macroscopically and six were studied immunohistochemically. The fatty streaks contained foam cells that were virtually all labeled by antibodies directed against members of the mononuclear phagocyte series (RFD-2 and RFD-7). Macrophages demonstrated acid phosphatase activity and marked expression of HLA-DR, suggesting activation. Other monoclonal antibodies (UCHT-1, OKT-4, and RFT-8) identified T lymphocytes, of both helper and suppressor phenotypes, within the fatty streaks. T lymphocytes of suppressor phenotype appeared to predominate over helper cells. B lymphocytes were not detected. The presence of activated macrophages and T lymphocytes in the fatty streaks indicates that components of a cell-mediated immune response are present. Such an immune process may be important in the pathogenesis of human atherosclerosis.

Cytochemical demonstration of acid phosphatase activity in the pineal organ of the rainbow trout, Salmo gairdneri.

Activity of acid phosphatase (ACP) was investigated cytochemically in the pineal organ of the rainbow trout, Salmo gairdneri. Intense reaction product for ACP activity was observed in lysosomes varying in size and shape and in endoplasmic reticulum associated with the Golgi complex of the pineal photoreceptor and supporting cells, vascular endothelial cells, and macrophages inhabiting pineal lumen, parenchymal epithelium and perivascular spaces. This localization of ACP is discussed with particular reference to the capacity for lysosomal digestion in a pineal organ combining photoreceptive and secretory functions, and lacking a blood-brain barrier, as holds true for the pineal of the rainbow trout. Taking advantage of its capacity for endocytotic uptake and lysosomal digestion, the pineal organ of the rainbow trout may serve as a barrier between the blood circulation and the cerebrospinal-fluid compartment. Furthermore, the macrophages may be considered as an essential component in pineal function of fish.

Peroxisomes in the nervous system of Aplysia californica: a cytochemical study.

We have studied the distribution of peroxisomes in the abdominal ganglion of Aplysia californica using electron microscopic cytochemical methods. Reaction product for catalase was observed in small ovoid or dumb-bell-shaped bodies in the perikarya of many of the neurons. The abundance of these catalase-reactive peroxisomes is considerably greater than is the case in vertebrate neurons. While the non-neuronal cells of the Aplysia abdominal ganglion do contain appreciable peroxisome populations, there were few peroxisomes in glial cytoplasm directly adjacent to the perikarya, again contrasting with vertebrate ganglia in which the satellite cells are a principal site of peroxisomes. Peroxisomes are present throughout the perikaryal cytoplasm. In the regions in which lipochrome granules abound, peroxisomes are frequently seen closely associated with these granules; glycogen is abundant nearby. The association of peroxisomes, lipochrome granules and glycogen is interesting in view of the propinquities of peroxisomes to lipid droplets and lipofuscin granules reported for non-neuronal vertebrate tissues, and in view of the growing evidence indicating that some of the roles of peroxisomes are in lipid metabolism and in gluconeogenesis. Some of the lipochrome granules themselves show reaction product in ganglia incubated to demonstrate catalase activity and some react in tissue incubated to demonstrate acid phosphatase activity. Such observations suggest that the enzymatic capacities of the lipochrome granules merit further studies, and that the granules may be of complex or heterogeneous nature.

Lipid storage disease: Part I. Ultrastructure of xanthoma cells in various xanthomatous diseases.

The fundamental ultrastructure of lipid storage in the xanthoma cells of various xanthomatous diseases, including familial hyperlipoproteinemia type IIa, III, and V, cerebrotendinous xanthomatosis, Wolman's disease, Tangier disease, Hand-Schüller-Christian disease, and normolipidemic cutaneous xanthomatosis, revealed lipid vacuoles, either membrane-bound or with no single unit membrane, cholesterol crystals, multivesicular or multilocular lipid bodies, myelin-like bodies, and ceroid granules (residual bodies). According to the presence or absence of such a single unit membrane and enzyme cytochemical demonstration of acid phosphatase activity, these lipid storage inclusions were largely classified into lysosomal and non-lysosomal ones; the former included membrane-bound lipid vacuoles, cholesterol crystals, multivesicular or multilocular lipid bodies, myelin-like bodies, and ceroid granules and the latter was lipid vacuoles with no limiting membrane. The ultrastructural relationship on formation of these lysosomal and non-lysosomal lipid storage inclusions and pathogenesis of the lipid storage in the xanthoma cells of the disorders were presented. As for the origin of the xanthoma cells, the majority of them were considered to be derived from macrophages in many of the disease; however, transformation of fibroblasts into xanthoma cells was confirmed in xanthomatous diseases, such as Hand-Schüller-Christian disease.

Mechanisms of metal--salt methods in enzyme cytochemistry with special reference to acid phosphatase.

This review is concerned with theoretical and experimental aspects of the factors governing the localizing potentialities of cytochemical enzyme reactions that are based on the metal-salt principle, that is, the precipitation of the primary product of the enzymatic reaction by a heavy-metal ion at the enzymatic site. Special attention is given to the lead phosphate precipitation process in acid phosphatase cytochemistry. The various model systems developed for the study of the factors involved in precipitation are described and their advantages and disadvantages discussed. Furthermore, the various cytochemical methods so far used for the demonstration of acid phosphatase activity are critically evaluated in the light of the results obtained with the model systems.

Enzyme histochemical studies of acid phosphatase isoenzymes in induced heterotopic bone formation in guinea pigs.

A bone induction model was used to study the effect of different pretreatments and inhibitors added to the substrate media used for the histochemical demonstration of acid phosphatase activity. Two different acid phosphatases (AcP) were demonstrated. The AcP of matrix-producing cells exhibited resistance to pretreatment with EDTA while it was sensitive to tartrate added to the incubation medium. The AcP in dentin resorbing cells was EDTA sensitive but resistant to tartrate. The AcP activities in both resorbing and matrix-producing cells generally sensitive to fluoride, copper and molybdate. In some of the dentinoclasts, fluoride resistant AcP was observed. The results suggest previous investigations on normal bone formation, suggesting that at least two different isoenzymes of AcP exist. One of these seemed to be related to matrix production and mineralization of the new heterotopic bone.

Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase.

This paper deals with the progress made over the last few years in our understanding of enzyme cytochemical staining methods as studied using a fundamental approach with the aid of a model system of thin gel films. Although model films with a matrix of polyacrylamide have been mostly used, the properties and possible applications of other matrices are also reviewed. The chemical aspects of the entrapment of enzyme molecules into a matrix are summarized. Special attention has been paid in model film studies to the principles of the trapping reaction of a diffusable precursor resulting from the enzymatic conversion of a substrate. They are considered here as they concern the cytochemical demonstration of acid phosphatase activity with a lead salt. The effect of fixatives on different enzyme activities, the diffusion rate of substrates and chromogenic compounds to the enzyme site, and enzyme kinetics under cytochemical conditions are also discussed, since they are factors which influence the final results of the staining procedures. The advantage of model film studies in enabling the direct correlation of cytochemical and biochemical results is outlined with special reference to the cytochemical determination of glucose-6-phosphate dehydrogenase with Tetra Nitro BT. A method for determining enzyme activities in the soluble fraction of isolated cells after incorporation in model films is described for the first time. This method has proved to be highly appropriate for microscopical observations of glucose-6-phosphate dehydrogenase activity in single cells, because it results in a good morphology and no formazan precipitates outside the cells. On the other hand, this type of model film forms a bridge between fundamental model film studies using purified enzyme and quantitative enzyme cytochemistry performed in situ.

Exocytosis of intact lysosomes from skeletal muscle after chloroquine treatment

Following short-term chloroquine treatment (25 mg/kg daily for 7 days), intact myeloid bodies were extruded from the anterior latissimus dorsi (ALD) muscle of the chicken. These myeloid bodies demonstrated acid phosphatase activity both within the cell and after extrusion into the extracellular space. Tannic acid staining confirmed that the plasma membrane remained intact and the “extracellular lysosomes” observed had not resulted from fixation damage. Lysosome extrusion following overloading in skeletal muscle and the fate of the lysosomal contents after extrusion are discussed.

Translocation of hepatocyte lysosomes following partial hepatectomy and its inhibition by colchicine

Hepatocyte microtubules were studied during the translocation of lysosomes which follows partial hepatectomy. In hepatocytes of normal rat liver the lysosomes are seen mainly along the bile canaliculi. After partial hepatectomy, these lysosomes lose their pericanalicular arrangement and move towards large inclusions (‘protein droplets’) which result from the marked pinocytosis induced by the removal of about two-thirds of the liver mass. The lysosomes fuse with the inclusions, and by 4 h after partial hepatectomy most of the inclusions demonstrate acid phosphatase activity histochemically. Many microtubules are found in close proximity to the lysosomes. When the rats are treated with colchicine prior to the partial hepatectomy, events are markedly different. The lysosomes change their location but do not hit the cytoplasmic inclusions, and the inclusions remain negative for acid phosphatase activity even 4 h post-hepatectomy. Quantitative ultrastructural study shows that the volume density of microtubules in the hepatocytes decreases to one-third of that in control hepatocytes. This strongly suggests an involvement of microtubules in giving orientation to the translocation of hepatocyte lysosomes under these conditions.