The enzyme cytochemistry and composition of elasmobranch granulocytes

Peroxidase, alkaline phosphatase, acid phosphatase, β-glucuronidase, α-naphthyl acetate esterase (ANAE), α-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, acetyl-L-tyrosine-α-naphthyl esterase (ATNE), tosyl-L-lysine-α-naphthyl esterase (TLNE) and periodic acid-Schiff (PAS) were studied in 17 species of elasmobranchs in which granulocytes had previously been identified at the ultrastructural level.
Eosinophils, eosinophilic and neutrophilic granulocytes contained variable acid phosphatase, esterases and PAS, but they were strongest in neutrophilic granulocytes; particularly ANAE. Esterases were released into surrounding plasma and therefore probably function as ectoenzymes. In eosinophils and some neutrophilic granulocytes there were indications of weak peroxidase, but this could not be conclusively demonstrated. Alkaline phosphatase was diffuse between granules in some eosinophils of Pavoraja , and (β-glucuronidase was diffuse in neutrophilic granulocytes of Etmopterus baxteri , otherwise granulocytes lacked these enzymes. Neutrophilic granulocytes stained moderately to strongly for ATNE and weakly and inconsistently for TLNE in Squalus acanthias and Dalatias licha . with a similar reaction in granular lymphocytoid and thrombocytoid cells of Galeorhinus ausiralis and Raja nasuta . The enzyme composition of these granulocytes is discussed.     

Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) II: Monocytes

Granulation staining and cytochemistry of peripheral blood monocytes in healthy carp ( Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. For representation of different granulations triazide-staining for eosinophil and neutrophil granules and aqueous methylere-blue staining for basophil granules were used. Lipids were shown by sudan-black-reaction. Monocytes showed only basophil granulation and weak lipid reaction. All tested enzymes were detected, with the exception of peroxidase. The PAS reaction for glycogen proof was negative.     

Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) I. Granulocytes

The cytochemistry and staining of granula in peripheral blood granulocytes in healthy carp ( Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl-acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. Different granula types were shown by triazid-staining (eosinophil and neutrophil granula) and methylenblue-staining for basophil granulation. Toluidinblue-staining was used for basophil granulocytes. Lipids were shown by the Sudan-black-reaction. Four granulocyte subpopulations are described: neutrophil, heterophil, basophil and eosinophil granulocytes. Neutrophils possess all tested granula types, whereas heterophil and basophil granulocytes show only basophil granula. Neutrophils and heterophils show no activity of the tested esterases with the exception of AS-D. Only neutrophils were peroxidase-positive. Alkaline phosphatase and β-glucuronidase were not detected in granulocytes. Basophils and especially eosinophils were rarely found in peripheral blood.     

团头鲂外周血细胞的显微结构及细胞化学特征

采用常规瑞氏染色和细胞化学染色方法对团头鲂(Megalobrama amblycephala)外周血细胞的显微结构及细胞化学特征进行了观察。在团头鲂外周血细胞中可区分出六类细胞: 红细胞、淋巴细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞和血栓细胞。其中淋巴细胞是除红细胞外含量最多的细胞, 其次分别为血栓细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞。成熟红细胞多为卵圆形, 表面光滑, 胞核呈椭圆形或圆形, 染色质较为致密; 淋巴细胞多呈圆形, 胞质较少, 胞核常偏位; 单核细胞多为圆形, 胞核呈圆形或椭圆形, 胞质内可见空泡状结构; 嗜中性粒细胞近似圆形, 胞核常偏于细胞一侧, 呈分叶状、肾形或椭圆形, 核质界限清晰; 嗜酸性粒细胞一般为圆形, 胞核为肾形或椭圆形, 胞质中充满紫红色颗粒; 血栓细胞形态多样, 主要有椭圆形、纺锤形、长杆状和泪滴形, 核质比较大。淋巴细胞呈α-醋酸萘酚酯酶(ANAE)阳性, 呈过碘酸-雪夫(PAS)、氯乙酸AS-D萘酚酯酶(AS-DCE)弱阳性, 呈苏丹黑B(SBB)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)及过氧化物酶(POX)阴性; 单核细胞呈POX、ACP强阳性, PAS、SBB、AS-DCE和ANAE为阳性, 呈AKP阴性; 嗜中性粒细胞除PAS和ANAE为弱阳性外, 其他染色结果和单核细胞相同; 嗜酸性粒细胞呈POX、ANAE强阳性, SBB、ACP阳性, PAS及AS-DCE则为弱阳性, 呈AKP阴性; 血栓细胞呈PAS、AS-DCE及ANAE弱阳性, 呈SBB、ACP、AKP及POX阴性。团头鲂外周血细胞的显微结构及细胞化学特征与其他鱼类具有相似之处, 但亦有其明显的物种特异性。该研究结果可作为监测团头鲂健康状态的依据, 为其养殖及病理诊断提供基础资料。     

Light and electron microscopic studies on the enzyme cytochemistry of leucocytes of eels, Anguilla species

The leucocytes of three anguillid eels were studied using enzyme cytochemistry. Leucocytes were stained for peroxidase, alkaline phosphatase, acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, lysozyme, a variety of non-specific esterases, chloroacetate esterase and two proteases. All cells were negative for aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and β-galactosidase. Very few neutrophils, thought to be mature, and all eosinophils contained peroxidase-positive granules, and some monocytes showed very weak peroxidase staining. All leucocytes lacked alkaline phosphatase, but all cells except lymphocytes and thrombocytes of A. dieffenbachii contained acid phosphatase. Neutrophil acid phosphatase released into phagosomes was associated with Escherischia coli bacteriolysis. Neutrophils also secrete lysozyme and, with monocytes, produce and secrete a variety of esterases. The possible interaction of lysozyme, acid phosphatase and esterases in bacteriolysis is discussed.     

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.     

Investigation of α-naphthyl acetate esterase and acid phosphatase in the peripheral blood leukocytes of greyhounds

The purpose of our study was to determine the percentages of α-naphthyl acetate esterase (ANAE)- and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL), the presence of the ANAE and ACP enzymes in leukocytes, and the proportion of PBL in greyhounds. Peripheral blood samples were collected from the cephalic antebrachial vein of 14 (7 animals of each sex) healthy 1-2-year-old greyhounds. Mean percentages of ANAE-positive PBL were found to be 73.29 ± 0.95% in female and 74.29 ± 2.21% in male dogs. The difference between mean values of the genders was not statistically significant. The ACP values were 36.00 ± 2.94% for females and 33.57 ± 2.15% for males. No significant differences were found with regard to gender. For both enzymes, although monocytes and eosinophilic granulocytes displayed a positive reaction, neutrophils gave negative reactions. The proportion of PBL was 36.29 ± 5.31% and 33.00 ± 2.38 % in female and male dogs, respectively. The differences were not significant.     

The leucocyte enzyme cytochemistry of the Indian eel, Anguilla bicolor bicolor McClelland

Activity of peroxidase, alkaline phosphatase, acid phosphatase, α-naphthyl acetate esterase and naphthol-AS-D chloroacetate esterase in the leucocytes of the Indian eel, Anguilla bicolor bicolor McClelland, was studied. But for some minor differences, the enzyme pattern of the leucocytes, especially neutrophil peroxidase, of the Indian eel closely resembled that of the New Zealand eels, suggesting that the Indian eel is phylogenetically more closely allied to the New Zealand eels than to the European eel.     

Cytochemical identification of the leukocytes of torpedoes (Torpedo marmorata Risso and Torpedo ocellata Rafinisque)

The authors subjected peripheral blood smears of Torpedoes to cytochemical analysis of lipids, protein, neutral and acid polysaccahrides and of some enzymatic activities, i.e. adenosine triphosphatase (ATP-ase), acid and alkaline phosphatase, aliesterase and peroxidase. It was found that neutrophilic granulocytes are intensely PAS and aliesterase positive and weakly ATP-ase positive. Eosinophilic granulocytes show the presence of neutral polysaccharides in the matrix (which is PAS positive) and strong ATP-ase and acid phosphatase activities in the granules. Lymphocytes sometimes contain weakly PAS and aliesterase positive granules. Monocytes show some small PAS positive granules and weak acid phosphatase and aliesterase activities. Thrombocytes contain some peripheral granules which are PAS positive and slightly ATP-ase positive. There are no transitional forms between the various cellular types. The results confirm the classification of leukocytes of Torpedoes into neutrophilic granulocytes, eosinophilic granulocytes, lymphocytes, monocytes and thrombocytes and contribute some informations about the histoenzymatic content of Elasmobranch leukocytes.     

Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half‐smooth tongue sole Cynoglossus semilaevis

The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non‐specific esterase (NSE), peroxidase (POD) and mucous‐cell types was evaluated in the gastrointestinal tract of the half‐smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH 2·5) periodic acid Schiff (AB‐PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB‐) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS‐AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.     

American paddlefish leukocytes demonstrate mammalian‐like cytochemical staining characteristics in lymphoid tissues

American paddlefish Polyodon spathula leukocytes demonstrated cytoplasmic staining patterns very similar to mammalian leukocytes when stained with acid phosphatase, α‐naphthyl butyrate esterase and β‐glucuronidase. American paddlefish monocytes, lymphocytes and granulocytes stained positive for acid phosphatase. Monocytes stained positive for α‐naphthyl butyrate esterase. Lymphocytes that stained positive for α‐naphthyl butyrate esterase were designated type A. Lymphocytes that stained positive with antibodies to the L chain of white sturgeon Acipenser transmontanus immunoglobulin (Ig) were designated type B. Type A and type B lymphocytes stained positive for β‐glucuronidase. All leukocytes observed were negative for Sudan Black B. Monocytes, lymphocytes and granulocytes were present in the renal haematopoietic tissue, spleen, thymus, pericardial myeloid tissue, lamina propria of the spiral valve, and in meningeal myeloid tissue located dorsal to the brain, at the base of the brain and around the notochord. Peyer's patches were present in the gut. Morphological characteristics of leukocytes stained with Wright's and haematoxylin and eosin and appeared very similar to those of other fish species.     

Cytochemical studies of normal feline blood and bone marrow cells

Blood and bone marrow cells of ten clinically healthy cats were stained for alkaline phosphatase (ALP), peroxidase (PO), chloroacetate esterase (CAE), alpha-naphthyl butyrate esterase (NBE), sudanophilia, and periodic acid-Schiff (PAS) reaction. Mature neutrophils in blood and bone marrow were devoid of ALP and NBE, but exhibited modest to strong PO, CAE, sudanophilia, and PAS reaction. In bone marrow, sudanophilia, PO, and CAE were prominent at the promyelocyte stage and diminished with cellular differentiation and maturation, while PAS reactivity increased with cell maturation usually from the myelocyte stage onwards. Myeloblasts were negative for all cytochemical reactions, but some large unidentifiable cells reacted strongly for ALP. Eosinophils were slightly reactive for ALP, CAE, and PAS, but not for PO, sudanophilia, and NBE. Basophil granules stained strongly for CAE, revealed PAS positivity, and stained negatively for PO, NBE, ALP, and sudanophilia. Slight ALP activity was detected in the intergranular cytoplasm of basophils. Lymphocytes and monocytes, with few exceptions, stained negatively. An occasional lymphocyte revealed slight globular NBE activity (NaF-resistant) and diffuse PAS reaction, while an occasional monocyte contained a few PO-positive and sudanophilic granules. Monocytes reacted modestly, whereas bone marrow macrophages reacted strongly for NBE (NaF-sensitive). Cells of the erythroid series stained negatively for all cytochemical reactions, megakaryocytes were PAS-positive, and platelets gave positive reactions for PAS and CAE.     

Investigation of α-naphthyl acetate esterase and acid phosphatase in the peripheral blood leukocytes of greyhounds

The purpose of our study was to determine the percentages of α-naphthyl acetate esterase (ANAE)- and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL), the presence of the ANAE and ACP enzymes in leukocytes, and the proportion of PBL in greyhounds. Peripheral blood samples were collected from the cephalic antebrachial vein of 14 (7 animals of each sex) healthy 1-2-year-old greyhounds. Mean percentages of ANAE-positive PBL were found to be 73.29 ± 0.95% in female and 74.29 ± 2.21% in male dogs. The difference between mean values of the genders was not statistically significant. The ACP values were 36.00 ± 2.94% for females and 33.57 ± 2.15% for males. No significant differences were found with regard to gender. For both enzymes, although monocytes and eosinophilic granulocytes displayed a positive reaction, neutrophils gave negative reactions. The proportion of PBL was 36.29 ± 5.31% and 33.00 ± 2.38 % in female and male dogs, respectively. The differences were not significant.     

牛蛙消化道黏膜5种酶的分布

目的研究牛蛙(Rana catesbeinana)消化道黏膜碱性磷酸酶(ALP)、酸性磷酸酶(ACP)、ATP酶、酯酶和脂酶的分布。方法在消化道的8个部位取材,采用冰冻切片技术和酶的组织化学方法。结果 ALP主要分布于十二指肠、空肠和回肠,胃中酶反应呈弱阳性。ACP主要分布于胃中,食道和肠道酶反应呈弱阳性。ATP酶在消化道各部位均有较多分布,十二指肠、空肠和回肠显著较多,胃各部位其次。酯酶和脂酶均主要分布于肠道,胃各部位其次。结论牛蛙消化道黏膜酶的分布同其它动物有相似之处,也有其自身特点。十二指肠、空肠和回肠是牛蛙的主要消化吸收部位。     

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.     

Blood cells of Fundulus heteroclitus (L.)

Much of the parenchyma of the normal pronephros of the adult Fundulus heterodilus , a euryhaline teleost, is haematopoietic tissue which was examined in cytocentrifuge preparations and plastic embedded thick sections. As we have not characterized many of the blood cells functionally, the terminology used is based on their morphological resemblance to similarly named cells in higher vertebrates. Approximately 80% of the non-erythroid elements observed in the pronephros are mature eosinophilic granulocytes (48%), immature eosinophilic granulocytes (25%), or cells likely to be their precursors [e.g. small blast cells (6%) and large blast cells (2%)]. Although there are macrophages in the pronephros that are capable of endocytotic activity, the mature and immature granulocytes are not. The granulocytes are non-specific esterase positive, PAS positive, acid phosphatase negative, and are capable of being mobilized by a RES activating agent, Ecteinascidia turbinata .     

Morphological,cytochemical, and biochemical observations on the blood of the tuatara,Sphenodon punctatus

Abstract

Morphological features of the circulating blood cells of an adult female tuatara are described and illustrated. Mitosis in circulating erythrocytes and the occurrence of an intraerythrocytic haemogregarine parasite are noted. The packed cell volume (haematocrit) was 35%. There were about 310 000 erythrocytes and 7200 leucocytes per mm³ of blood. The lymphocyte was the predominant leucocyte, followed by the eosinophil, monocyte, neutrophil, and basophil. The eosinophils and neutrophils reacted positively to acid phosphatase and peroxidase; the neutrophils were also esterase positive, and all three granulocytes were PAS positive. Haemoglobin, plasma protein, glucose, sodium, potassium, cholesterol, and triglyceride levels were determined. The primitive nature of the tuatara is reflected in the morphological. mensural, and biochemical data presented.     

Biochemical characterisation of esterases active in hydrolysing xenobiotics in wheat and competing weeds

Esterase activities toward model xenobiotic substrates ( p -nitrophenyl acetate, naphthyl acetate) and pesticide esters (diclofop methyl, bromoxynil octanoate, binapacryl) have been compared in crude extracts from wheat (Triticum aestivum L.) and Triticum progenitors of wheat. Esterase activities were also determined in the weeds, wild oat ( Avena fatua ) and two populations of black-grass ( Alopecurus myosuroides ), one of which (Rothamsted) is susceptible to herbicides, while the other (Peldon) shows cross-resistance to multiple classes of herbicides. Esterase activity toward the model substrates was highest in wheat, while the weeds were more active in hydrolysing the pesticides. Using isoelectric focussing (pH 4–8), 13 proteins with esterase activity toward α -naphthyl acetate could be resolved in hexaploid wheat (genome AABBDD). The pattern of these activities was most similar to that of the diploid progenitor T. tauschii (DD), excepting a major acidic esterase (pI 4.6), which originated from T. urartu (AA). Resolved esterase activities in the weeds were distinct from those observed in the Tritcum species. However, unlike the case with other classes of xenobiotic-metabolising enzymes, the complement of esterases in the Peldon and Rothamsted populations of black-grass appeared to be identical. In all species, the more basic esterases (>pI 5.0) were sensitive to inhibition by organophosphate and carbamate insecticides, suggesting that they were B-class esterases. In contrast, the acidic wheat esterase (pI 4.6) with the greatest activity toward α -naphthyl acetate was insensitive to insecticides. This wheat-specific esterase was purified 7000-fold by a combination of hydrophobic interaction chromatography, gel filtration and anion-exchange chromatography. The purified esterase behaved as a monomeric 45-kDa protein showing high activity toward p -nitrophenyl acetate and α -naphthyl acetate, but limited activity toward the pesticides.     

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.