Acid Phosphatase Reaction Research Articles

Menstrual-cycle-dependent and -independent features of the human Fallopian tube fimbrial epithelium: an ultrastructural and cytochemical study.

Ultrastructural and cytochemical features of 100 specimens of human Fallopian tube fimbrial epithelium were studied to determine whether they were dependent on or independent of the ovarian hormones. Characteristics which were independent of hormonal control included the presence of alkaline phosphatase reaction product along the apical cell membrane of the secretory cell and its absence in the ciliary cell. Acid phosphatase reaction product was found in ciliary and secretory cell lysosomes in similar numbers and calcium deposits were observed in ciliary and secretory cell nuclei, nucleoli and cell membranes. On the other hand, characteristics dependent on stages of the cycle included the presence of mitochondrial-bound calcium during the preovulatory phase and its depletion during the periovulatory phase. The same sequence was noticed in the content of the large ciliary cell perinuclear globules, which displayed accumulations of glycogen and lipid in preovulatory phase and their depletion during the periovulatory phase. It seems that during the periovulatory phase the increased metabolic activity of the cells is accompanied by utilization of some of the material stored within the cells.

Biochemical and fine structure evidence for cellular specialization in a large spumellarian radiolarian Thalassicolla nucleata

Thalassicolla nucleata is a skeletonless, large spumellarian radiolarian (ca 3 mm diameter) occurring abundantly in the surface water of the Sargasso Sea. It has a complex cellular organization consisting of (1) a well-defined central capsule surrounded by a non-living capsular wall perforated by numerous cytoplasmic strands and (2) an extracapsulum containing a frothy layer of alveolate cytoplasm penetrated by numerous rhizopodia. Evidence is presented for physiological specialization complementing this structural compartmentalization. Acid acryl phosphatase activity (a digestive vacuole marker enzyme) is approximately 3 times greater in the extracapsulum compared to the intracapsulum; whereas, cytochrome oxidase (a respiratory marker enzyme) is approximately 1.5 times greater in the intracapsulum compared to the extracapsulum. Furthermore, cytochemical and electron microscopic evidence indicates that the extracapsulum, containing large vacuoles and copious amounts of acid phosphatase reaction product, is a major site of catabolism; while the intracapsulum, containing the nucleus, numerous Golgi bodies, and a rich supply of mitochondria, is a substantial site of anabolism, lysosomal secretory activity, and food product storage. This marked level of cellular specialization in T. nucleata may enhance its biological efficiency and thus partially account for its abundance in oligotrophic environments.

Acute megakaryoblastic Leukemia.

Acute megakaryoblastic leukemia is a rare and rapidly fetal disorder characterized by extensive proliferation of megakaryoblasts and atypical megakaryocytes in bone marrow and extramedullary sites, thrombocytopenia and only a few blasts in peripheral blood. Three cases of this leukemia were studied morphologically, cytochemically, and electron microscopically. The leukemia blasts varied from 10-20 mu in diameter, had coarsely reticular nuclear chromatin, and numerous cytoplasmic projections and vacuoles. Except for intense granular PAS positivity and diffuse acid phosphatase reactivity, all of the usual cytochemical stains were negative. The blasts had no specific differentiating features identifying them as megakaryoblasts even at the ultrastructural level. In such instances demonstration of platelet peroxidase will confirm the megakaryocytic origin. All three patients in this series were men and all died within 90 days. Two patients also had other malignancies.

Redistribution of material labelled with [3H]mannose in amoebae induced to undergo pinocytosis.

The synthesis, transport, and disposition of material labelled with [3H]mannose were studied by electron microscopic radioautography in normal amoebae and in cells that had internalized cell surface as a result of being induced to undergo pinocytosis. Control amoebae were injected with the precursor and placed in normal medium. The Golgi apparatus and rough endoplasmic reticulum were heavily labelled at the earliest intervals, while radioactivity of the cell surface peaked 12 h after injection of precursor. The experimental cells were injected, placed in bovine serum albumin solution from 15 to 60 min after injection, and then removed to normal medium until fixation. Incorporation of the precursor into the rough endoplasmic reticulum was near normal, but the proportions of grains associated with the Golgi apparatus and the cell surface were greatly reduced. The percentage of grains overlying vacuoles increased 12 h after injection, notably in the case of polymorphous vacuoles and dense vacuoles, both of which were identified as lysosomes with the acid phosphatase reaction. The results suggest that addition to the surface of components labelled with [3H]mannose was diminished following induction of pinocytosis. Incorporation of the precursor appeared to be shifted from cell surface material to lysosomal contents, possibly lysosomal enzymes. It is thought that this shift occurred in response to the need for the cell to digest unusually large amounts of endocytosed protein. Recycling of cell surface under these conditions is considered possible.

Quantitative x-ray microanalysis of spleen lysosomes after acid phosphatase reaction.

A quantitative X-ray microanalytical study of 80 spleen lysosomes after histochemical reaction for acid phosphatase was carried out. Lysosomal Fe concentrations showed a skewed distribution, with most lysosomes having relatively low concentrations. The concentration of Pb in the lysosomes (indicative of acid phosphatase activity) showed a close to normal distribution. Although the plot of lysosomal Pb concentrations against Fe concentrations showed a marked scattering of the points, there is a statistically highly significant correlation between accumulated metal and acid phosphatase activity as determined from the Pb concentrations. The relation between concentrations of P and those of Pb in the lysosomes points to the possibility that Pb(H2PO4)2 is the main reaction product of the histochemical acid phosphatase reaction.

Acid phosphatase and alpha-naphthyl acetate esterase in neoplastic and non-neoplastic lymphocytes. A statistical analysis.

Acid phosphatase and alpha-naphthyl acetate esterase reaction patterns were evaluated in lymphocytes from patients with a variety of neoplastic and non-neoplastic conditions: leukemia, 59; NHL, 53; and reactive follicular hyperplasia, 23. Fifteen individuals with normal peripheral blood were also studied. For both enzymes, statistical analysis showed a strong correlation between a globular reaction pattern and T lymphocytic origin in both non-neoplastic lymph nodes and normal peripheral blood specimens (P less than 0.0001). A similarly strong correlation was found between a granular acid phosphatase pattern and T lymphocytic origin in cell isolated from non-neoplastic lymph nodes (P less than 0.0001) but not in those obtained from normal peripheral blood where this pattern was observed with equal frequency in B, T, and "null" lymphocytes (P = 0.415). A granular alpha-naphthyl acetate esterase pattern was correlated with non-T lymphocytes from normal peripheral blood (P less than 0.0001), but was observed with equal frequency in B, T, and "null" lymphocytes fron non-neoplastic lymph nodes (P = 0.76). In the eight T cell neoplasias studied, a globular pattern was evident in the majority of cells for both enzymes. In the majority of the B cell neoplasias, however, a granular pattern was observed for both enzymes.

Amebocytic Accumulations in Biomphalaria Glabrata: Fine Structure

Internal pathologic conditions in four stocks of Biomphalaria glabrata involve amebocytes. Most snails exhibiting these conditions are also nonsusceptible to infection with Schistosoma mansoni. Fine structure studies reveal that perisortic and atrial amebocytic accumulations show two responses that involve heart tissue: a generalized response in which amebocytes have infiltrated the myocardium and focal areas of nodules in which heart tissue is encapsulated and destroyed.In pericardial accumulations amebocytes are subspherical at the periphery with their numerous filopodia intertwining; centrally they elongate and flatten to form a nodule. Hemocoelic accumulations are similar to pericardial ones but more compact.Hyalinocytes and granulocytes were both found in the four types of amebocytic accumulations; each contained phagocytized material. Acid phosphatase reaction product was detected in the Golgi bodies, primary phagosomes, multivesicular bodies and myelin bodies of granulocytes.

Ultrastructural localization of alkaline and acid phosphatases in the human fallopian tube epithelium during the menstrual cycle.

Ultrastructural and cytochemical methods were utilized to study the human Fallopian tube fimbrial epithelium during the different stages of the menstrual cycle. Alkaline phosphatase reaction product was located along the apical and lateral plasma membranes of the secretory cells only, regardless of the stage of the cycle. The ciliated cells were almost devoid of any reaction product at all stages of the cycle. Acid phosphatase reaction product depicted the lysosomes. These appeared as electron-dense bodies, of almost equal numbers in the ciliated and the secretory cells at all stages of the cycle. Thus the number of lysosomes did not vary appreciable during the different stages of the menstrual cycle. Many lipid droplets were found in both cells; these were rimmed by acid phosphatase reaction product, and some were partially enveloped by electron-dense bodies containing acid phosphatase deposits. Acid phosphatase deposits were also found on the inner face of Golgi vesicles.

The fine localization of acid phosphatase activity in the unvacuolated notochordal cells of the early chick embryo.

The electron microscopical localization of acid phosphatase activity was investigated in ultra-thin and semi-thin sections of unvacuolated notochordal cells of chick embryos from stages 9 to 14 (as defined by Hamburger & Hamilton). At stage 9, many notochordal cells show a lightly positive reaction for acid phosphatase activity. Thereafter, the acid phosphatase-positive cells of the notochord increase in number and, at stage 14, the reaction products for the enzymes are distributed throughout almost all the cisternae of the nuclear envelope and a well-differentiated endoplasmic reticulum, the parallel cisternal and reticular parts of the Golgi complex, and various lysosomes in nearly all notochordal cells. In the cisternae of the nuclear envelope and endoplasmic reticulum, the acid phosphatase reaction products are in a fine granular form. In the outermost layer of the cisternal parts of the Golgi complex, faint lead deposits similar to those in the endoplasmic reticulum are found, but in other cisternal and reticular regions which may correspond to the GERL, considerable amounts of reaction products are present. Knob-like projections are also seen protruding from the reticular parts of the Golgi complex. These results suggest that, at least up to stage 14, the notochordal cells are actively synthesizing acid phosphatase which is directly transported from the endoplasmic reticulum to the Golgi complex. The enzyme may be accumulated by the Golgi complex from which primary lysosomes are formed. Furthermore, the pattern of the ultrastructural localization of acid phosphatase activity in embryonic notochordal cells of the chick differs from that of adult cells of other animals.

Reaction of rat liver acid phosphatase with p-chloromercuribenzoate

Reaction of rat liver acid phosphatase with p-chloromercuribenzoate

Presence of matrix vesicles in the trabecular meshwork of glaucomatous eyes.

In the last few years matrix vesicles (M.V.) have been found in the vessel wall in various diseases and are considered to be related with pathological changes of the extracellular fibers of the tissue (atypical collagen etc.). Since the trabecular meshwork, at least the cribriform region, may be looked upon as a part of a vessel wall, it was an obvious conclusion to look for M.V. even in this area. Lysosomal and non-lysosomal M.V. were found in 138 trabeculectomy-specimens of various kinds of glaucoma, located for the most part within the cribriform or juxta-canalicular region of the meshwork. In ten cases small, electron-dense M.V. were found which ultrahistochemically showed a positive reaction for acid phosphatase. This proves that these vesicles are indeed extracellular lysosomes.

Isolation of autofluorescent ‘aged’ human fibroblasts by flow sorting: Morphology, enzyme activity and proliferative capacity

In cultures of human fibroblasts the percentage of bright autofluorescent (AF) cells increases with increasing passage number. These autofluorescent cells were isolated using a FACS II cell sorter and compared with sorted non-fluorescent (NF) cells. The AF cells showed an increase in population doubling time (2.3-fold), cell protein (1.9-fold), and in specific activities of the lysosomal enzymes: β-hexosaminidase (4.2-fold), β-galactosidase (3.8-fold) and acid phosphatase (2.5-fold). The specific activities of two non-lysosomal enzymes glucose-6-phosphate dehydrogenase and lactate dehydrogenase had increased only slightly (1.1-fold) respectively (1.5-fold). The autofluorescence in the AF cells was restricted to small round organelles. The distribution and size of these autofluorescence granules were similar to the acid phosphatase-containing granules in the cytochemically stained cells. Electronmicroscopical examination showed that these AF cells contained a large amount of small electron-dense granules containing amorphosmophilic material. These granules which were positive for the acid phosphatase reaction, were classified as secondary lysosomes. The low percentage of the sorted AF cells which incorporate [ 3H]thymidine during a 24 h test period (19%) as compared with the labelling percentage of sorted NF cells (73%) from the same culture, indicate that the autofluorescent cells in a ‘young’ culture have a very limited remaining proliferative capacity. The results imply, that by flow sorting it is possible to isolate ‘aged’ cells with characteristics of ‘phase III’ cells out of non-aged fibroblast cultures.

Cytochemical demonstration of DNAse in pollen.

Pollen DNAse of Chlorophytum comosum was demonstrated cytochemically by coupling the breakdown of DNA (due to the endogenous DNAse) with acid phosphatase supplied to the reaction mixture. Precipitation of the free phosphate groups (from acid phosphatase reaction on the freed nucleotides) as lead phosphate results in a electron dense deposit, visible with the electron microscope. Enzyme activity using this technique was localized in vesicles along the intine (principally along the aperture region) and around the generative cell.

ULTRASTRUCTURAL EVALUATION OF BIOCHEMICAL EVENTS OF BONE RESORPTION IN HUMAN CHRONIC OTITIS MEDIA

Bone resorption is a significant component of chronic inflammatory ear disease. Bone is a unique tissue requiring both demineralization and collagen degradation for breakdown. Mineral removal probably occurs prior to collagen destruction. A localized change in pH may be one of the mechanisms that induces demineralization. Localized accumulations of lysosomal acid hydrolases could provide the acidic environment necessary for mineral removal. The present study utilizes the electron microscope to localize the lysosomal enzyme acid phosphatase in specimens removed from patients with chronic otitis media. Areas of localized bone resorption exhibited mononuclear inflammatory cells attached to the resorption margin. These cells contained abundant acid phosphatase, as did isolated fibroblasts within the zone of inflammation at the resorption margin. Extra cellular acid phosphatase was seen in these areas. Within the bone, osteocytes adjacent to resorption displayed increased cytoplasmic organelle patterns and contained the acid phosphatase reaction. The localization of destructive enzymes in mononuclear inflammatory cells and osteocytes explains the bony destructive changes observed in human chronic otitis media in the absence of multinucleated osteoclasts.

Proliferation and transformation of lymphocytes in the lung capillaries of the rat: ultrastructure, acid phosphatase and peroxidase cytochemistry.

After fixation by vascular perfusion numerous leukocytes, among which 91% lymphocytes - not only small but particularly medium- and large-sized categories - were observed in the lung capillaries of healthy Wistar rats. We are struck by the presence of lymphocytes belonging to different intermediate stages between small lymphocytes and plasma cells. Lymphocyte mitosis can be found in the lung capillaries, resembling 'blast cell mitosis'. The different kinds of lymphocytes can be observed with a variable amount of uranylophilic membrane-bound granules with a diameter ranging from 200 to 400 nm. Acid phosphatase (AP) and peroxidase (PO) reaction product was found in several granules. The distribution of azurophil granules corresponds to that of the AP and PO positive granules. Since staining with uranyl acetate increases the density of the azurophil granules and thereby allows a direct electron mircoscopical identification of azurophils, AP reaction azurophil granules in lymphocytes correspond to a from of lysosome containing AP and PO. Thus, it would appear that movement of blood cells to the lung capillaries is not random but preferentially favors cells of the lymphocytic series. The morphology and cytochemical reactions for AP and PO of the medium- and large-sized lymphocytes is characteristic of cells subjected to antigenic stimulation.

Lysosomal proliferation in rachitic avian intestinal absorptive cells following 1,25-Dihydroxycholecalciferol

Lysosomes in chick intestinal absorptive cells from rachitic (vitamin D-deficient) and vitamin D-replete animals were studied utilizing transmission electron microscopic histochemistry and ultrastructural morphometry. Absorptive cells from rachitic animals, serum calcium = 7.3±0.3 mg%, contained an average of 4.0±0.3 supranuclear lysosomes. In rachitic chicks sacrificed 9 hr post-injection of 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D, the values for both serum calcium, 9.8 ± 0.2 mg%, and the number of apical absorptive cell lysosomes, 12.9±0.6, were increased over non-injected or vehicle-only injected animals. Lysosomes in vitamin D-replete absorptive cells were characterized by their intense staining with pyroantimonate, indicative of their high calcium content. The same organelles also produced a positive reaction for acid phosphatase. Rachitic lysosomes, also acid phosphatase positive, were only lightly stained with pyroantimonate. The lysosomal proliferation apparently induced by 1,25-dihydroxycholecalciferol may be a further indication that these organelles play a role in intestinal calcium transport and/or intracellular calcium homeostasis within the absorptive cell.

Lead staining in the Golgi complex

Lead ions at similar concentrations to those used for Gomori type phosphatase localization stain some parts of the vacuolar system, particularly compartments of the Golgi complex (GC) and isolation envelopes (im) in a characteristic way in both vertebrates and invertebrates. After fixation in 2.5% glutaraldehyde, lead citrate in acetate or aspartate buffer (pH 5.5–7.2) leaves the contents of GC cisternal compartments with a fine particulate stippling. In the fat body of Calpodes ethlius and in mouse pancreas the staining is falnt but definite without further enhancement of contrast, although it is easily overlooked after section staining. The distribution of lead staln differs from that of the lead phosphate precipitated after Gomori type acid phosphatase reactions. Whereas lead stain may be in all GC and im compartments, acid phosphatase is restricted to the innermost saccules and nearby vacuoles. The compartment specific staining by lead also differs from the generalized staining in all compartments given by uranyl. Thus the contents or luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC cisternae are devoid of uranyl stainable material. However, lead staining and acid phosphatase activity in the GC continue. We presume that they mark the environment within these cisternae rather than the proteins passing through them. This environment is itself not static. Several observations suggest that the function of cisternae that is detectable by lead staining is temporally discontinuous and related to a stage of maturation or development. Only early stage ims stain: the staining ceases by the beginning of autophagy after hydrolytic enzymes are presumed to have been added. Condensing vacuoles cease to stain as the central core crystallizes out. Stain may be absent from one or two GC saccules at any position in the stack as though the phase of lead staining (or lack of it) can move progressively through the system. We conclude that in studies characterizing components of the vacuolar system it is necessary to separate those that mark transient occupants of a compartment from those that mark the compartment itself. Both may vary temporally independently from one another.

Intranuclear crystalloids associated with abnormal granules in eosinophilic leukocytes

Ultrastructural evaluation of eosinophilic leukocytes from a 2-yr-old asymptomatic girl with chronic benign neutropenia (CBN) revealed a variety of morphological abnormalities. All eosinophils obtained from blood and marrow specimens contained multipole microcrystalloids in most of the mature cytoplasmic granules. An increase in crystalloid-free, immature granules in late (bilobed nuclei) eosinophils suggested a delay in granule maturation. The eosinophil granules appeared to be of normal size and demonstrated normal acid phosphatase reactivity. Eosinophilic myelocytes contained abnormal cisternae of rough endoplasmic reticulum (RER) and lacked abundant elongated RER cisternae seen in normal cells. A few eosinophilic myelocytes in specimens of bone marrow from the child contained large intranuclear crystalloids measuring up to 3 mu in length. The intranuclear crystalloid contained as cubic lattice of dense material with a periodicity similar to that described for cytoplasmic crystalloids. The ultrastructural morphology of marrow neutrophils was normal, as described in other cases of CBN. Ultrastructural examination of blood eosinophils from the father demonstrated microcrystalloids in cytoplasmic granules identical to those seen in the child. The father was asymptomatic and had normal leukocyte counts. Thus, anomalous crystalloid granule genesis occurred in the father and daughter and was not necessarily associated with neutropenia or clinical symptomatology. This anomaly is associated with the accumulation of intranuclear crystalloid material in eosinophilic myelocytes, which do not appear to be released from the marrow compartment.

Intranuclear Crystalloids Associated With Abnormal Granules in Eosinophilic Leukocytes

Abstract Ultrastructural evaluation of eosinophilic leukocytes from a 2-yr-old asymptomatic girl with chronic benign neutropenia (CBN) revealed a variety of morphological abnormalities. All eosinophils obtained from blood and marrow specimens contained multipole microcrystalloids in most of the mature cytoplasmic granules. An increase in crystalloid- free, immature granules in late (bilobed nuclei) eosinophils suggested a delay in granule maturation. The eosinophil granules appeared to be of normal size and demonstrated normal acid phosphatase reactivity. Eosinophilic myelocytes contained abnormal cisternae of rough endoplasmic reticulum (RER) and lacked abundant elongated RER cisternae seen in normal cells. A few eosinophilic myelocytes in specimens of bone marrow from the child contained large intranuclear crystalloids measuring up to 3 mu in length. The intranuclear crystalloid contained as cubic lattice of dense material with a periodicity similar to that described for cytoplasmic crystalloids. The ultrastructural morphology of marrow neutrophils was normal, as described in other cases of CBN. Ultrastructural examination of blood eosinophils from the father demonstrated microcrystalloids in cytoplasmic granules identical to those seen in the child. The father was asymptomatic and had normal leukocyte counts. Thus, anomalous crystalloid granule genesis occurred in the father and daughter and was not necessarily associated with neutropenia or clinical symptomatology. This anomaly is associated with the accumulation of intranuclear crystalloid material in eosinophilic myelocytes, which do not appear to be released from the marrow compartment.

Carbohydrate-specified endocytosis: localization of ligand in the lysosomal compartment.

Carbohydrate-directed endocytosis is mediated by a receptor, the hepatic binding protein; it is responsible for the clearance of galactose-terminated glycoproteins from the circulation. This process was investigated by using lactosaminated ferritin which is recognized by this receptor. Ferritin was seen in elements of an extensive "lysosomal compartment" that includes secretory vacuoles, coated vesicles, and GERL. The compartment marked in hepatocytes by the distribution of ligand is similar to that previously described in Kupffer cells (marked by acid phosphatase reaction product and horseradish peroxidase reaction product).