Acid phosphatase associated with discharging secretory vesicles (mucocysts) of Tetrahymena thermophila

The localization of acid phosphatase in discharging mucocysts of Tetrahymena thermophila is reported. Electron dense, lead phosphate enzyme reaction product is found associated with the fibrillar meshwork of secreting mucocysts and in cytoplasmic vesicles (e.g. lysosomes). Resting mucocysts, containing highly condensed contents, exhibit no label. The specificity of the stain was controlled by reaction media without exogenous substrate and reaction media containing the inhibitor sodium fluoride. No lead phosphate deposits were found in these controls. Secreting organelle contents have no preferential affinity to lead phosphate as shown by tests with reaction medium lacking substrate and subsequent incubation in phosphate containing medium.     

Ultracytochemical study of ATP-ase activity in mucosal parietal cells and in the cells of human adenocarcinoma of the stomach]

Ultracytochemical investigation of ATP-ase activity was carried out in parietal cells of the mucosa and in cancer cells of human stomach carcinoma possessing a similar ultrastructure. In parietal cells the reaction product of ATP-ase was observed on the membranes of microvilli of intracellular canaliculi, on the membranes delineating the lateral intercellular space, on the basal plasmolemma and in the nucleoli. The reaction product was absent on the membranes of tubuvesicles and on the apical surface of the plasmalemma. In cancer cells the reaction product was found on the membranes of the microvilli of the intracellular canaliculi, basal plasmolemma and in the nucleoli. Comparative examination of ATP-ase activity in these cells implies that at least the part of the mechanism of hydrochloric acid secretion which is involved in the transfer of H+ and Cl- is retained in cancer cells. A steady decrease in hydrochlorid acid secretion observed in the stomach mucosa in cancer as well as in the tumour itself seems to be associated with other mechanisms.     

基于ITS1-5.8S rRNA-ITS2序列的猪源结肠小袋纤毛虫种群特征分析

【目的】为了揭示结肠小袋纤毛虫病在环境中的分子传播机制,研究了猪源结肠小袋纤毛虫的种群特征。【方法】用粪便涂片镜检和改进型DMEM培养基从病猪结肠内容物分离结肠小袋纤毛虫滋养体,然后用显微观察、吖啶橙荧光染色法和基于ITS1-5.8S rRNA-ITS2序列的分子标记技术分析了豫西地区猪群中结肠小袋纤毛虫的种群特征。【结果】结果显示,从来自豫西地区8个县市的病猪中共分离了15株小袋纤毛虫,5.8S rRNA序列相似性高达99.4%以上,同属于结肠小袋纤毛虫,根据ITS1/2序列分析结果,MJ-2和SX-1株属于结肠小袋纤毛虫基因型A,其余13株均属于结肠小袋纤毛虫基因型B。MJ-2和SX-1株滋养体形态特征明显区别其他13株,绝大多数呈球形,运动缓慢,粪便中和体外培养的虫体密度较低;而其他13株的滋养体均呈多形性,运动快速活跃,虫体密度较大。吖啶橙荧光染色显示,2种基因型滋养体的核结构没明显差别。【结论】首次报道中国猪源结肠小袋纤毛虫存在2个基因型,其中基因型B为优势种群,为防控人和动物结肠小袋纤毛虫病提供重要参考。     

The formation of primary and secondary lysosomes in Balantidium coli, Ciliata

Trophozoites, vegetative forms of Balantidum coli isolated from pigs affected by acute and asymptomatic balantidiasis were studied. Lysosomes and food vacuoles were revealed by cytochemical detection of lysosomal marker, acid phosphatase. The cytoplasm of all the B. coli trophozoites examined was found to contain numerous structures which differed widely in shape, size and location in the cells. One of them was located among the rough endoplasmic reticulum membranes and another one in the vicinity of endosomes. Those structures were regarded as the primary lysosomes. The two types of vesicular structures most probably represent two stages of the primary lysosome formation. Trophozoites were also found to contain secondary lysosomes which are formed by fusion of several primary lysosomes with phagosomes. The ultrathin sections of B. coli trohozoites showed the presence of two types of phagosomes. They were divided, based on their contents, into auto- and heterophagosomes.     

Discrepancies in the occurrence of Balantidium coli between wild and captive African great apes

Balantidium coli is a ciliate reported in many mammalian species, including African great apes. In the former, asymptomatic infections as well as clinical balantidiasis have been reported in captivity. We carried out a cross-sectional study of B. coli in African great apes (chimpanzees, bonobos, and both species of gorillas) and examined 1,161 fecal samples from 28 captive facilities in Europe, plus 2 sanctuaries and 11 wild sites in Africa. Samples were analyzed with the use of Sheather's flotation and merthiolate-iodine-formaldehyde (MIFC) sedimentation. MIFC sedimentation was the more sensitive technique for diagnostics of B. coli in apes. Although not detected in any wild-ape populations, B. coli was diagnosed in 52.6% of captive individuals. Surprisingly, in the apes' feces, trophozoites of B. coli were commonly detected, in contrast with other animals, e.g., Old World monkeys, pigs, etc. Most likely reservoirs for B. coli in captive apes include synantropic rats. High starch diets in captive apes are likely to exacerbate the occurrence of balantidiasis in captive apes.     

Conjugation Rescue of Exocytosis Mutants in Tetrahymena thermophila Indicates the Presence of Functional Intermediates in the Regulated Secretory Pathway

ABSTRACT. Tetrahymena thermophila possesses a regulated secretory pathway in which mucin proteins are stored in dense-core granules, called mucocysts. Exocytosis-defective mutants exist that fail to secrete mucin in response to secretagogues. Four of the mutants (SB281, SB283, SB285 and SB715) appear to be blocked at different steps of the regulated secretory pathway. SB281 and SB285 accumulate mucin proteins in heterogeneous cytoplasmic organelles which have not yet been identified; SB283 makes mucocyst-like structures but they contain no immunologically identifiable 80-kDa or 50-kDa mucin proteins; and SB715 has more than normal amounts of immature and undocked mucocysts. The organelles that accumulate in exocytosis-defective mutants could be either normal intermediates in the biosynthetic pathway or aberrant structures that form as a result of the mutations. We have used conjugation rescue to analyze steps in the biogenesis of exocytosis-competent mucocysts and to identify functional intermediates. The cytoplasmic organelles that accumulate in SB281 appear to be unidentified biosynthetic intermediates, and the defect is in a cytosolic protein essential for mucocyst maturation. The organelles which accumulate in the other mutants are likely biosynthetic, but their mutations are in proteins which are labile or not free to diffuse into the mutant conjugant.     

Makeup and properties of E. coli cells with a varying level of resistance to tetracycline]

Lipopolysaccharide composition of tetracycline sensitive and resistant strains of E. coli was studied comparatively. It was shown that that resistance of E. coli to tetracycline was probably due to the differences in the lipopolysaccharide component composition of the outer membrane. On the basis of the activity comparison of the Mg2+- and Ca2+-activated ATP-ase of the membrane fraction of the tetracycline sensitive and resistance strains of E. coli it was concluded that the resistance development in the strains tested to tetracycline was not associated with the changes in the ATP-ase activity.     

Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica.

Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa.     

Reactivity of Monoclonal Antibodies to Species-Specific Antigens of Entamoeba histolytica

Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica -like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica -like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia , and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis     

Selection for and Enrichment of Non-Discharge Mucocyst Variants of Tetrahymena thermophila 1,2

The present protocol for selection for and enrichment of potential non-discharge mucocyst variants of Tetrahymena thermophila is based on the ability of wild-type cells to discharge their mucocyst contents simultaneously when stimulated with alcian blue 8 GS. Under appropriate ionic conditions, the discharged mucocyst contents form a capsule around each cell and prevent its locomotion. Non-discharge variants unable to shed a capsule are assumed to retain their ability to swim and are simulated in this study by cells not induced to shed their capsule. For mass phenotype screening, the conditions for maximum capsule shedding were established for wild-type cells. One hour starvation in Wagner's solution rendered 100% of the cells competent to shed a capsule when triggered with a 0.4% solution of alcian blue 8 GS. Decontamination of the shedding mixture by addition of egg albumin in a final concentration of 0.1% guaranteed survival of >95% of these cells that were now encapsulated, but allowed up to 5% of the cells to escape their capsule and swim freely. Cells with intact mucocysts and cells with emptied mucocysts were separated in reconstruction experiments by density-gradient centrifugation in which 95% of the cells with intact mucocysts appeared in a discrete band. Using the same protocol, the efficiency of separation was tested with mixtures of morphologically marked (food vacuoles stained with India ink) and genetically marked (resistance to cycloheximide) cells. Using 1:1 mixtures of marked cells with intact mucocysts and cells with emptied mucocysts (or vice versa), the cells with intact mucocysts were efficiently separated from other cells; one cell with emptied mucocysts per 100 cells with intact mucocysts was found in the upper discrete gradient band.     

Dibucaine-induced synchronous mucocyst secretion in Tetrahymena

Synchronous secretion of all available mature mucocysts was induced in late log phase cultures of Tetrahymena thermophilia (B III) by the local anaesthetic dibucaine. No assembled fusion rosettes were seen within the plasma membrane after release until 2-3 hrs of regrowth, thus proving that the rosettes are not permanent sites within the plasma membrane but have to be reassembled each time for a new fusion event to occur. Concomitant with the reappearance of assembled fusion rosettes, the cell cytoplasm fills up with precursors of new mucocysts thus linking the two events together.     

The verification of cytochemical tests for ATP-ase activity in plant cells using X-ray microanalysis

Summary Electron microprobe analysis (EMMA 4) was carried out on two types of electron opaque deposit found in thin sections of barley root tips as a result of cytochemical tests for ATP-ase activity.The granular type of deposit, which mainly occurs in radial and tangential cell walls of epidermal and sub-epidermal cells, was shown to contain lead, whereas lead was absent from the opaque globular deposits, which are much more generally distributed and always associated with membrane structures. Thus the latter deposits, which contain osmium and have often been interpreted as ATP-ase reaction products, should be regarded as artifacts of the fixation and rinsing procedures. It is suggested that calcium may play a role in the formation of the osmiophilic deposits.     

ULTRASTRUCTURE OF MUCOCYSTS AND PELLICLE OF TETRAHYMENA PYRIFORMIS

Tetrahymena pyriformis GL was fixed with glutaraldehyde and/or OsO₄ for a study of cytoplasmic ultrastructure. Many small vacuoles 0.05 to 0.5 µ in diameter were found to contain each a dense particle enveloped by a limiting membrane. This membrane is continuous with the membrane of the vacuole. The particles are irregular in shape and size, but similar to the mucocysts in the appearance of the matrix. It is suggested that they are the first morphologically distinguishable stages in the development of mucocysts. In the course of this development, amorphous material becomes crystalline with a longitudinal period of 150 A and a lateral period of 100 A. The mature mucocysts are rather uniform in size and have a spheroidal shape. During discharge, the crystalline pattern disappears and the mucocysts assume a spherical configuration. The inner limiting membrane of a mucocyst seems to disintegrate during the process of discharge while the outer membrane becomes continuous with the outermost pellicular membrane; the inner pellicular membrane is continuous with the cytoplasmic membrane. Rows of few to 15 or more microtubules were found either between the cytoplasmic membrane and the ectoplasmic layer (longitudinal fibrils) or underneath the ectoplasmic layer (transverse fibrils). The outer and inner pellicular membranes are uniformly spaced and connected by "cross-bridges." Details of these structures are described.     

Analysis of Tetrahymena Mucocyst Material with Lectins and Alcian Blue1

ABSTRACT. There are numerous mucocysts in Tetrahymena; however, little is known about their composition, organization, biosynthesis, or function. Mucocysts of Tetrahymena are membrane-bounded vesicles located at the cell cortex. They are torpedo-shaped structures (0.9 μm x 0.3 μm) lined up in longitudinal rows along the surface. It is estimated here that each cell contains about 5000 mucocysts. Mucocyst contents are organized in a crystalline manner, but when that material is released by exocytosis, it swells and forms a gel. Using fluorescence microscopy, we demonstrate that mucocysts contain concanavalin A (Con A)-binding material. First, intracellular fluorescent particles in fixed cells incubated with fluorescein-derivatized Con A (F-Con A) have the same distribution, shape, and orientation as mucocysts in living cells. Also, mucocysts were induced to undergo synchronous exocytosis, and the released material formed a capsule around the cell. The capsule was fluorescent after incubation with F-Con A. In both cases fluorescence was abolished by competition with α methyl mannoside, indicating that Con A is binding specifically to a glycosidic component of the mucocyst. Mucocyst capsules also bind wheat germ agglutinin but not soybean agglutinin, pea lectin, or lentil lectin. Preparations of mucocyst material were analyzed by SDS-PAGE. Silver stain revealed a high molecular weight band that had not previously been detected by Coomassie blue staining. That band also stained with Alcian blue, indicating that it is a mucopolysaccharide. Finally, that same band was shown to be Con A binding. Thus the Con A-binding and Alcian blue-staining properties of mucocysts can be attributed to the same high molecular weight mucopolysaccharide component. This study indicates that it may be possible to purify a specific carbohydrate component of mucocysts which may be helpful in analyzing their function, biogenesis, and structural organization.     

Cytochemical localization and biochemical evaluation of a lysosomal serine protease in lung: dipeptidyl peptidase II in the normal rat

Dipeptidyl peptidase II (DPP II) in normal rat lung was evaluated by the enzymes' ability to hydrolyze Lys-Ala or Lys-Pro derivatives of 4-methoxy-2-naphthylamine (MNA). For visualization of this activity, the liberated MNA was coupled with fast blue B for light microscopy (LM) or hexazotized pararosaniline with osmication for electron microscopy (EM). Granular to diffuse reaction product was noted in many lung cells in frozen sections for LM, including alveolar and tissue macrophages, fibroblasts, chondrocytes, bronchial and bronchiolar epithelial cells and mast cells. Reaction product at the EM level was seen in the lysosomal structures of the above cells, although lysosomal heterogeneity with regard to reactivity was noted. Cellular content of reaction product by EM correlated with LM staining intensity. Additional structures, not obviously reactive by LM, such as the lamellar bodies of type II cells and lysosomes in other cell types, were seen to contain reaction product ultrastructurally. A modified biochemical assay for the quantitation of DPP II in tissue homogenates was used to determine the activity of the enzyme in rat lung. Enzyme activity in polyacrylamide isoelectric focusing gels indicate that Lys-Ala-MNA was the more specific substrate but, by virtue of its rapid hydrolysis, Lys-Pro-MNA was more sensitive.     

Carbonic anhydrase activity in primary sensory neurons

Summary Subpopulations of primary sensory neurons in mammalian dorsal root ganglion (DRG) exhibit carbonic anhydrase (CA) activity. To identify these subpopulations in DRG cells of mouse and chicken, the reliability of the cytochemical localization of the enzyme requires that several conditions be fulfilled:(1) Preservation of the enzyme activity in glutaraldehyde-containing fixative; (2) accessibility of the cytoenzymatic reaction throughout 20-m thick Vibratome sections; (3) retention of the reaction product in situ during OsO₄ post-fixation; (4) specificity of the cytoenzymatic reaction for CA activity as corroborated by the immunocytochemical detection with antibodies anti-CA II in mouse DRG; (5) strict correlation between the CA activity and the cytological characteristics in a given subclass of neurons. On the basis of these criteria, it is concluded that the CA activity may be used as a cell marker to identify cytologically defined neuronal subpopulations and their axons in mouse DRG. In chicken DRG, CA activity is not consistently expressed in a given subclass of ganglion cells and their axons. Hence, it is assumed that the expression of CA activity by DRG cells in chicken is modulated by functional or environmental conditions.     

Localization of acid phosphatase activity in Giardia lamblia and Giardia muris trophozoites

Numerous membrane-bounded vacuoles are found adjacent to the plasma membrane of the pathogenic protozoan Giardia lamblia. The function of these vacuoles has been discussed by several authors. Approximately 100-400 nm in diameter with a core of low electron density, they have been suggested to be mitochondria, mucocysts, lysosomes, and endocytotic vacuoles. Enzyme cytochemical localization for acid phosphatase activity using cerium as a capturing agent demonstrates reaction product in these vacuoles as well as in the endoplasmic reticulum and nuclear envelope cisternae. The distribution of reaction product suggests the vacuoles are lysosome-like; however, their function and development remain in question.     

Fatal ulcerative colitis in a western lowland gorilla (Gorilla gorilla gorilla)

A captive western lowland gorilla (Gorilla gorilla gorilla) presented with watery diarrhoea that progressed to become profuse and haemorrhagic. Faecal analyses revealed Balantidium (B.) coli trophozoites and salmonella-like bacteria. Despite treatment the gorilla died on the 5th day after onset of symptoms. Post-mortem examination revealed a severe erosive-ulcerative superficial and deep colitis. Histological examination of post-mortem samples of the colon showed plentiful B. coli invading into the mucosa and submucosa, whilst PCR screening of bacterial DNA could not confirm any bacteria species which could be connected to the clinical picture. As B. coli is usually a non-pathogenic gut commensal, and as this animal previously showed evidence of non-symptomatic infection of B. coli, it is possible that the switch in pathogenicity was triggered by an acute bacterial infection. Despite successful treatment of the bacterial infection the secondary deep invasion of B. coli was not reversed, possibly because of the failure of the treatment regimen, and led to the death of the gorilla.     

百合花粉母细胞腺苷三磷酸酶反应产物的X-射线微区分析

百合早前期花粉母细胞经腺苷三磷酸酶反应处理后,一部分经过锇酸后固定和铀染色,一部分不经锇酸后固定和铀染色,其余的经锇酸固定,但不经铀染色,在此三种情况下,细胞质膜和染色质中都出现有致密的,电子不通透的沉淀。进一步的X-射线微区分析表明这些沉淀物中含有一定量的铅。X-射线微区分析结果也表明核膜和胞间连丝通道内部的酶反应沉淀中也含有铅,并且质膜、染色质和胞间连丝通道中酶反应沉淀中的铅较为丰富,细胞融合期染色质酶反应沉淀物中的铅含量较高,进入粗线期后,酶反应沉淀物中铅的含量下降。本研究结果表明百合早前期花粉母细胞的质膜、染色质、核膜及胞间连丝通道内部的确具有ATP酶活性;ATP酶在细胞融合过程中可能起重要作用。     

Localization of Acid Phosphatase Activity in Giardia lamblia and Giardia muris Trophozoites1

Numerous membrane-bounded vacuoles are found adjacent to the plasma membrane of the pathogenic protozoan Giardia lamblia. The function of these vacuoles has been discussed by several authors. Approximately 100–400 nm in diameter with a core of low electron density, they have been suggested to be mitochondria, mucocysts, lysosomes, and endocytotic vacuoles. Enzyme cytochemical localization for acid phosphatase activity using cerium as a capturing agent demonstrates reaction product in these vacuoles as well as in the endoplasmic reticulum and nuclear envelope cisternae. The distribution of reaction product suggests the vacuoles are lysosome-like; however, their function and development remain in question.