Nonspecific esterase activity expressed in Weibel-Palade bodies of cloned guinea pig aortic endothelial cells

We studied the localization of nonspecific esterase activities in cloned guinea pig aortic endothelial cells using ultrastructural cytochemistry. Weibel-Palade bodies (WPB), which are known to contain von Willebrand protein, were positive for esterase, defining a heretofore unrecognized activity of these organelles. Esterase activity was also found localized to the external surface of the plasma membrane, to cytoplasmic lipid bodies, and to the outer (cytoplasm-facing) surface of certain membrane-bound cytoplasmic vacuoles. Localization of esterase activity to these four discrete sites probably reflects the presence of a number of endothelial cell enzymes capable of hydrolyzing alpha-naphthyl acetate or butyrate. The physiological substrate and biological function of these enzyme activities are not presently understood.     

Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation

We used a morphometric and autoradiographic approach to analyze changes in specific cytoplasmic granules and cytoplasmic lipid bodies associated with human lung mast cell degranulation. Mast cells were dissociated from lung tissue by enzymatic digestion and were then enriched to purities of up to 99% by countercurrent centrifugation elutriation and recovery from columns containing specific antigen bound to Sepharose 6 MB. Degranulation was induced by goat anti-IgE. At various intervals after stimulation, parallel aliquots of cells were recovered for determination of histamine release or were fixed for transmission electron microscopy. We found that lipid bodies, electron- dense structures that lack unit membranes, were present in both control and stimulated mast cells. Autoradiographic analysis showed that lipid bodies represented the major repository of 3H-label derived from [3H]arachidonic acid taken up from the external milieu. By contrast, the specific cytoplasmic granules contained no detectable 3H-label. In addition, lipid bodies occurred in intimate association with degranulation channels during mast cell activation, but the total volume of lipid bodies did not change during the 20 min after stimulation with anti-IgE. This result stands in striking contrast to the behavior of specific cytoplasmic granules, the great majority of which (77% according to aggregate volume) exhibited ultrastructural alterations during the first 20 min of mast cell activation. These observations establish that mast cell cytoplasmic granules and cytoplasmic lipid bodies are distinct organelles that differ in ultrastructure, biochemistry, and behavior during mast cell activation.     

Morphological, cytochemical and ultrastructural studies of a case of systemic mastocytosis

Morphological, cytochemical and ultrastructural studies of mast cells were carried out in a patient affected with systemic mastocytosis. Neoplastic mast cells showed morphological features between classic tissue mast cells and circulating basophils. They showed strong granule metachromasia after toluidine blue, faint positivity to Hotchkiss reaction, strong positivity to chloroacetate esterase, while they were negative to alkaline phosphatase. Ultrastructural observations showed heterogeneity of granules, most of which had homogenous fine dotted contents. The origin and linkage between mast cells and circulating basophils are discussed.     

A macrophage-monocyte cell line from a dog with malignant histiocytosis

Summary The DH82 cell line was established from the neoplastic progenitor cells of canine MH and was characterized as histiocytic in origin based on light microcopic and ultrastructural morphology, positive staining reactions for alpha naphthyl acetate esterase and acid phosphatase, presence of Fc receptors, phagocytosis of latex beads, and plastic adherence in culture.     

Preliminary characterisation of esterase and platelet-activating factor (PAF)-acetylhydrolase activities from cat flea (Ctenocephalides felis) salivary glands

Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters alpha-naphthyl acetate (approximately 210pmol/min/gland pair; 10.0micromol/min/mg specific activity; K(m) approximately 59microM) and beta-naphthyl acetate (approximately 110pmol/min/gland pair; 5.2micromol/min/mg specific activity; K(m) approximately 132microM). Salivary gland extracts have PAF-acetylhydrolase activity (approximately 5pmol/min/gland pair; 0.24micromol/min/mg specific activity) but do not have detectable acetylcholinesterase activity. Native-PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native-PAGE. Renaturation of esterase activity after SDS-PAGE gave approximately 56kDa, approximately 57kDa and approximately 58kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of approximately 59kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain alpha- and beta-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.     

A role for vesicles in human basophil secretion

The evidence for vesicular transport as a mechanism for secretion by human basophils is reviewed. Initially, direct electron-microscopic inspection of experimentally produced and sequentially biopsied contact allergy skin lesions revealed a unique form of secretion termed piecemeal degranulation, characterized by the slow emptying of secretory granule contents (with retention of empty containers) in the absence of extrusion of entire granules. Budding of small vesicles to/from secretory granules was observed, and cytoplasmic vesicles were abundant. A generalized degranulation model was proposed to unify classical regulated secretion and this new form of secretion. Investigation of the mechanism(s) of secretion from human basophils required the development of numerous tools and resources. Chief among these were: (a) isolation and purification of circulating basophils; (b) identification of specific growth factors to increase the supply of this rare granulocyte; (c) understanding of secretogogue mechanisms and reliable analyses of secreted basophil products; and (d) development of ultrastructural preparations allowing imaging of small vesicles and quantifiable small electron-dense tags for granule materials in small vesicles. Applications of these tools to well-defined models of basophil secretion have established a role for vesicles as a mechanism for effecting secretion of histamine and the Charcot-Leyden crystal protein from activated human basophils.     

A method for the ultrastructural demonstration of non-specific esterase in human blood and lymphoid tissue

Summary Using the substrate 2-naphthylthiol acetate (NTA), we developed a reproducible method of demonstrating a non-specific esterase while retaining nuclear and cytoplasmic details at the ultrastructural level. The NTA esterase had a distribution and pattern of staining similar to those of esterases demonstrable at the light microscopic level by the -naphthyl acetate or naphthol AS-D acetate esterase reaction.The NTA esterase appeared as intensely electron-dense granules of varying size and shape in the cytoplasm. The granules were most abundant in the cells of the histiomonocytic series. The large number of diffusely scattered granules in the cytoplasm of the histiocytes and monocytes made it possible to separate these cells from other haematopoietic elements. There was usually no direct relationship between the NTA esterase positivity and the amount or the location of lysosomes or mitochondria, although in some histoocytes the granules appeared to the associated with lysosomes. The NTA esterase-positive granules were usually more numerous than lysosomes and were located outside the lysosomal granules. Some of the lymphocytes outside the germinal centres and most of the lymphocytes in the blood showed a punctate positivity in the form of 1–4 electron-dense dots. Plasma cells were usually negative but, in rare cases, contained an occasional single dot-like reaction product similar to that in some of the lymphocytes. Granulocytes were always negative.The method described in this paper can be used effectively for identification and study of human haematopoietic cell lines at the ultrastructural level.     

Ultrastructural localization of tyrosine hydroxylase in human peripheral blood mononuclear cells: effect of stimulation with phytohaemagglutinin

Using immunocytochemistry coupled to fluorescence and electron microscopy, we investigated the expression and ultrastructural localization of tyrosine hydroxylase (TH, EC 1.14.16.2), the rate-limiting enzyme in the biosynthesis of catecholamines, in human peripheral blood mononuclear cells (PBMCs), with PC12 cells as positive controls. In unstimulated PBMCs, TH-specific immunoreactivity was localized to the plasma membrane. However, after stimulation with the polyclonal mitogen phytohaemagglutinin (PHA), TH immunoreactivity was almost completely localized to electron-dense cytoplasmic granules, which resembled those found in PC12. TH-positive granules, however, were larger (300-500 nm) than in PC12 cells (100-200 nm). Flow cytometry analysis of TH expression showed about 46-50% positive cells in unstimulated PBMCs and in PHA-stimulated PBMCs in the G0/G1 phase of the cell cycle, but more than 80% positive cells in PHA-stimulated PBMCs in the S+G2/M phase. In agreement with previous observations, PHA stimulation also induced de novo expression of TH mRNA as well as increased intracellular catecholamine content, suggesting the occurrence of TH upregulation at the level of both gene expression and enzyme activity. The ultrastructural localization of TH in human PBMCs seems therefore regulated by cell stimulation and related to the functional activity of the enzyme.     

Cytochemical localization of mercury in Saccharomyces cerevisiae treated with mercuric chloride

We describe a cytochemical method for localizing mercury at the electron microscopic level in the yeast Saccharomyces cerevisiae. After addition of a lethal concentration of mercuric chloride to growing yeast cells, mercury was associated with the cell wall and cytoplasmic membrane. Little or no mercury was present in the cytoplasm. Electron probe X-ray microanalysis (EPMA) confirmed that the cytochemical reaction, visualized as mercury-silver complexes, was localized in dense bodies consisting of a core of mercury sulfide polymers surrounded by a shell of silver atoms.     

Enzyme Histochemistry on Paraffin Embedded Tissue Sections

To obtain diagnostic enzyme reactions in paraffin embedded tissue sections, we compared four fixatives (buffered formol sucrose, Baker's formol calcium, periodate lysin paraformaldehyde, and buffered formalin acetone) and subsequent acetone dehydration with or without graded concentrations of Triton X-100. Four spleens and 14 lymph nodes were tested for peroxidase, naphthol ASD chloroacetate esterase, acid phosphatase, alpha naphthyl acetate esterase, and alpha naphthyl butyrate. Best results were obtained by a processing method using buffered formalin acetone, Holt's gum sucrose, dehydration in acetone with 0.03% Triton X-100, and paraffin for embedding.     

Pyridoxal 5'-phosphate: a possible physiological substrate for alkaline phosphatase in human neutrophils

The intracellular localization of pyridoxal phosphatase activity was demonstrated in human neutrophils by electron microscope cytochemistry. Under alkaline conditions, an enzyme active against pyridoxal phosphate was localized to a cytoplasmic granule population, the phosphasome. These granules have previously been shown by electron microscope cytochemical techniques and by subcellular fractionation to be rich in alkaline phosphatase. Under acidic conditions, a phosphatase activity against pyridoxal phosphate was localized to intracellular multilamellar bodies resembling secondary lysosomes. These were quite distinct from the primary, secondary and phosphasome granules and this unique localization corresponds to that previously demonstrated (tertiary granules) by subcellular fractionation studies of these cells. The similarity in the enzyme reaction requirements of alkaline pyridoxal phosphatase and alkaline phosphatase, and their localization to the same subcellular organelle, suggests that pyridoxal phosphate may be a physiological substrate for human neutrophil alkaline phosphatase.     

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10^–8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10^–8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.     

Ultrastructural cytochemistry of chronic T-cell leukaemias. A study with four acid hydrolases

The ultrastructural localization of four acid hydrolases (acid phosphatase, beta-glucuronidase, beta-glucosaminidase and alpha-naphthylacetate esterase) has been studied in lymphocytes from 16 patients with three types of chronic T-cell leukaemia, namely, T-prolymphocytic leukaemia (T-PLL), T-chronic lymphocytic leukaemia (T-CLL) and adult T-cell lymphoma leukaemia (ATLL). Different patterns of enzyme distribution were observed in the leukaemic T-cells from these disorders. In T-PLL, reactivity for the four acid hydrolases was confined to single or a few large granules. Gall bodies were reactive for beta-glucuronidase, b-glucosaminidase and alpha-naphthylacetate esterase but apparently unreactive for acid phosphatase. In T-CLL, scattered small- to medium-size cytoplasmic granules and many parallel tubular arrays were strongly reactive for acid phosphatase, beta-glucuronidase and beta-glucosidase but showed no reactivity for alpha-naphthylacetate esterase. Intermediate features were observed in ATLL. The observed differences in enzyme reactivity reflect a different content of lysosomal granules in the various types of leukaemic T-cells. They also suggest that similar differences may be found in normal T-lymphocyte subsets.     

ON THE ASSOCIATION OF NON-SPECIFIC ESTERASE ACTIVITY WITH CENTRAL NERVE MYELIN PREPARATIONS

Abstract— Isolated bovine central nerve myelin sheath preparations showed non-specific esterase activity towards naphthyl ester substrates of increasing chain length from acetate to palmitate. Short chain esters were hydrolysed much faster than long chain substrates by myelin, the specific activity for the hydrolysis of β-naphthyl acetate being the highest. Micro-somal fractions from brain white matter were much higher in esterase activity to all naphthyl ester substrates. NADPH-cytochrome c reductase activity was absent from isolated myelin samples. Distilled water and salt and buffer solutions of different ionic strengths and pH were ineffective in releasing non-specific esterase activity from myelin although tri-potassium citrate caused marked inhibition of the membrane-bound esterase activity. The detergent Triton X-100 released esterase activity from the myelin preparations but at a concentration of 0.1 per cent was also inhibitory.     

Ultrastructural localization of acid phosphatase in nonhuman primate vaginal epithelium

Summary The vagina of the rhesus monkey is lined by a stratified squamous epithelium. However, little is known regarding the cytochemical composition of its cell organelles and the substances found in the intercellular spaces. In this study we have examined the ultrastructural distribution of acid phosphatase in the vaginal epithelium. In basal and parabasal cells reaction product was found in some Golgi cisternae and vesicles and in a variety of cytoplasmic granules. Reaction product was also found in some, but not all, membrane-coating granules. In the upper layers of the epithelium, the membrane-coating granules extruded their contents and acid phosphatase was localized in the intercellular spaces. The possible roles of acid phosphatase in keratinization, desquamation, or modification of substances in the intercellular compartment are discussed.     

Ultrastructural localization of peroxidase activity in normal human bone marrow cells

Summary The ultrastructural localization of peroxidase activity has been studied in the cells of normal human bone marrow using the diaminobenzidine peroxidase technique. Peroxidase activity has been localized within the primary (azurophil) granules of the neutrophilic series as well as in the cytoplasmic granules of eosinophils, basophils and monocytes. Peroxidase activity appears within the cisternal system (nuclear envelope, Golgi complex and rough endoplasmic reticulum) of these cells during the period of peroxidase-containing lysosome production. With the cessation of granulogenesis, peroxidase activity disappears from the cisternal system and does not reappear in subsequent developmental stages. In cells incubated in peroxide-free media, staining of granular components, but not of cisternae, is reduced. The inclusion of catalase in peroxide-free media eliminates all staining. This indicates that an endogenous peroxide is present within the cisternae and granules of these cell types.Supported by Grant No. AM-HE-12084-12 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Anita Topson and Barbara Jordan for their technical assistance and to Dr. Arthur Sagone who performed the marrow aspirations.     

Hydrolysis of organophosphorus compounds by an esterase isozyme from insecticide resistant pest Helicoverpa armigera

Esterase activity of resistant and susceptible H. armigera were compared in gels with different substrate such as naphthyl acetate, naphthyl phosphate, paraoxon and monocrotophos. Whole body extract of resistant H. armigera hydrolyzed paraoxon, monocrotophos and naphthyl phosphate in gels. Resistant H. armigera showed high esterase, phosphatase and paraoxon hydrolase activity compared to susceptible ones.     

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae. International Journal for Parasitology16: 491–510. Naive guinea pigs and guinea pigs vaccinated 4 weeks previously with highly irradiated cercariae were challenged percutaneously with normal cercariae. Skin samples from the challenge site were then harvested at varying times to provide histological, quantitative and ultrastructural data on the respective cellular responses to cercarial invasion. The primary cutaneous reaction was characterised by neutrophils; these cells reached peak numbers (16% of total cells) by 18 h. Eosinophils and basophils made only a small contribution to the infiltrate (2.9 and 5.7% respectively). Some basophils showed evidence of anaphylactic degranulation, others seemed to be damaged, but most appeared normal. Mononuclear cells of varied morphology were present at all times, but activated fibroblasts were prominent, and collagen deposition increased with time. Degranulating mast cells were recognised at 24 and 48 h. Dead schistosomula were never seen in naive-challenged skin, although one or two of the observed larvae showed minor tegumental abnormalities. In vaccinated guinea pigs, the cutaneous cellular response to challenge was significantly enhanced, with basophils dominating the reaction (33% of total cells at 24 h). Many basophils were already degranulating by the anaphylactic mechanism at 3 h post challenge, and free basophil granules were seen frequently. Both intact cells and free granules congregated in close proximity to invading larvae. Eosinophils were also present in greater numbers at secondary reaction sites, but they never exceeded 6% of the total infiltrate. Mononuclear cells believed to be immature eosinophils were prominent from 3 h. For the first time, the mechanism of eosinophil attachment and degranulation onto a multicellular target, described previously only from in vitro investigations, was recognized in vivo. Neutrophil numbers matched those recorded in naive-challenged skin at 3 and 6 h, but declined thereafter, while mast cells were seen degranulating at these early times. Mononuclear cells again presented a variety of morphological appearances; of particular note were large cells that had phagocytosed debris and were presumed to be macrophages, and small rounded cells with scant cytoplasm and few organelles, that may have been lymphocytes. By 12 h, large areas of the dermis had become severely disorganised and numerous, free basophil granules were distributed amongst the other cellular constituents. Dead schistosomula, denuded of tegument, were clearly recognised at 6 h, and such individuals invariably had neutrophils attached to their exposed muscle layers. Since dead schistosomula were not identified in naive-challenged guinea pig skin, it is concluded that a percentage of the challenge larvae, however small, is preferentially killed in the skin of the vaccinated animals.     

Cytochemical observations of the coiled bodies in neurons of rat sympathetic ganglia

Coiled bodies were investigated by means of ultrastructural cytochemistry. Preferential staining methods for localization of various proteins (ribonucleoproteins, basic proteins, phosphoproteins and glycoproteins) and DNA were applied. The results of cytochemical tests revealed that coiled bodies have a proteinaceous nature. They are composed of ribonucleoproteins, probably of nucleolar origin. They also contain phosphoproteins and glycoproteins but lack cytochemically detectable DNA. Coiled bodies present ultrastructural and cytochemical characteristics similar to the fibrillar part of the nucleous and to the interchromatin granules. The origin and possible functional role of coiled bodies are briefly discussed.     

Cytochemical observations of the coiled bodies in neurons of rat sympathetic ganglia

Summary Coiled bodies were investigated by means of ultrastructural cytochemistry. Preferential staining methods for localization of various proteins (ribonucleoproteins, basic proteins, phosphoproteins and glycoproteins) and DNA were applied. The results of cytochemical tests revealed that coiled bodies have a proteinaceous nature. They are composed of ribonucleoproteins, probably of nucleolar origin. They also contain phosphoproteins and glycoproteins but lack cytochemically detectable DNA. Coiled bodies present ultrastructural and cytochemical characteristics similar to the fibrillar part of the nucleous and to the interchromatin granules. The origin and possible functional role of coiled bodies are briefly discussed.