Electron Microscopic Cytochemistry Research Articles

Metabolic Glycoengineering Enables the Ultrastructural Visualization of Sialic Acids in the Glycocalyx of the Alveolar Epithelial Cell Line hAELVi.

The glycocalyx—a plethora of sugars forming a dense layer that covers the cell membrane—is commonly found on the epithelial surface of lumen forming tissue. New glycocalyx specific properties have been defined for various organs in the last decade. However, in the lung alveolar epithelium, its structure and functions remain almost completely unexplored. This is partly due to the lack of physiologically relevant, cost effective in vitro models. As the glycocalyx is an essential but neglected part of the alveolar epithelial barrier, understanding its properties holds the promise to enhance the pulmonary administration of drugs and delivery of nanoparticles. Here, using air-liquid-interface (ALI) cell culture, we focus on combining metabolic glycoengineering with glycan specific electron and confocal microscopy to visualize the glycocalyx of a recently immortalized human alveolar epithelial cell line (hAELVi). For this purpose, we applied different bioorthogonal labeling approaches to visualize sialic acid—an amino sugar that provides negative charge to the lung epithelial glycocalyx—using both fluorescence and gold-nanoparticle labeling. Further, we compared mild chemical fixing/freeze substitution and standard cytochemical electron microscopy embedding protocols for their capacity of contrasting the glycocalyx. In our study, we established hAELVi cells as a convenient model for investigating human alveolar epithelial glycocalyx. Transmission electron microscopy revealed hAELVi cells to develop ultrastructural features reminiscent of alveolar epithelial type II cells (ATII). Further, we visualized extracellular uni- and multilamellar membranous structures in direct proximity to the glycocalyx at ultrastructural level, indicating putative interactions. The lamellar membranes were able to form structures of higher organization, and we report sialic acid to be present within those. In conclusion, combining metabolite specific glycoengineering with ultrastructural localization presents an innovative method with high potential to depict the molecular distribution of individual components of the alveolar epithelial glycocalyx and its interaction partners.

Electron microscopy cytochemistry and three-dimensional reconstruction of labeled structures in Trypanosoma cruzi.

Significant advances have occurred in the area of high-resolution scanning electron microscopy (SEM), especially related to methodologies that allow the observation of intracellular structures that are exposed either by successive abrasion with a gallium ion beam or by sectioning in epoxy-embedded cells. Images of series of successively exposed surfaces can then be rendered into 3D models. Here, we report our observations by combining this approach with classical cytochemical methods to facilitate the 3D reconstruction of labeled structures and organelles. We used epimastigotes of Trypanosoma cruzi whose endocytic pathway was labeled with horseradish peroxidase, followed by fixation and detection of the peroxidase activity using the classical diaminobenzidine-osmium method followed by incubation with thiocarbohydrazide, which increases the concentration of osmium at the sites where the enzyme is located as well as the contrast of lipid-containing structures. This procedure allows not only a better visualization of membranous structures and lipid inclusions but can also easily identify the endocytic tracer (HRP) inside the cell. All structures involved in the endocytic activity could be traced and reconstructed.

Three-dimensional morphology of the Golgi apparatus in osteoclasts: NADPase and arylsulfatase cytochemistry, and scanning electron microscopy using osmium maceration.

This study was designed to observe osteoclasts in the rat femora by light and electron microscopic cytochemistry for nicotinamide adenine dinucleotide phosphatase (NADPase) and arylsulfatase, and scanning electron microscopy using osmium maceration to assess the three-dimensional morphology of the Golgi apparatus in osteoclasts. The Golgi apparatus showed strong NADPase activity and surrounded each nucleus with the cis-side facing the nucleus. The Golgi apparatus could be often traced for a length of 20 μm or longer. Observations of serial semi-thin sections confirmed that a single line of reaction products (=lead precipitates) intervened somewhere between any two neighboring nuclei. The nuclear membrane showed strong arylsulfatase activity as well as rough endoplasmic reticulum and lysosomes. Scanning electron microscopy showed that the Golgi apparatus covered the nucleus in a porous sheet-like configuration. Under magnification, the cis-most saccule showed a sieve-like configuration with fine fenestrations. The saccules decreased fenestration numbers toward the trans-side and displayed a more plate-like appearance. The above findings indicate the following. (1) The Golgi saccules of osteoclasts have a three-dimensional structure comparable with that generally seen in other cell types. (2) The Golgi apparatus forms a porous multi-spherical structure around nuclei. Within the structure, in most cases a Golgi stack partitions the room into several compartments in each of which a nucleus fits. (3) The nuclear membrane synthesizes some kinds of proteins more stably and sufficiently than the rough endoplasmic reticulum. Consequently, the Golgi apparatus accumulates around nuclei with the cis-side facing the nucleus.

Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas (Thunberg)

In this study, a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase, and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules (YGI, YGII, and YGIII) that differed in electron density and acid phosphatase reaction were identified in early cleavage, morula, blastula, gastrula, trochophore, and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation, indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI (free of acid phosphatase reaction) with YGII (rich in acid phosphatase reaction) could be the way by which yolk degradation is triggered.

Electron microscopic identification of hydrogen peroxide detected in fixed human polymorphonuclear leukocytes during phagocytosis.

Polymorphonuclear leukocytes (PMNs) engaged in phagocytosis produce reactive oxygen species (ROS), such as those that occur in an activated NADPH oxidase reaction, to eliminate ingested microorganisms. The translocation of NADPH oxidase components to produce antimicrobial free radicals from the vesicles to the phagosomes may be important. Hydrogen peroxide (H2O2) derived from O2- has been observed by electron microscopy using a cerium method. However, 2'-7'-dichlorofluorescin diacetate can also detect H2O2 through fluorescence. The main objective of the present study was to measure the H2O2-dependent fluorescence of PMNs after opsonized zymosan A (OPZ) phagocytosis using a microplate reader under different fixation conditions, including 0.5, 1, and 10% glutaraldehyde (GA) individually for 1, 5, 10, or 30 min. An additional objective was to visualize, through the use of electron microscopic cytochemistry, the process of H2O2 generation in OPZ phagocytic fixed PMNs. The fixed PMNs showed that the largest fluorescent value was produced by a concentration of 0.5% GA for all fixation times. This suggested that the fixation of PMNs with a high concentration of GA inhibited phagocytosis and produced ROS. In the fixed PMNs, electron microscopic results showed that after 1 min of mixing, some PMNs attached to particles and exhibited mild deposits in their secretory vesicles. When PMNs engulfed particles, free radical-producing vesicles had enhanced reaction deposits 10 min later and fused to the phagosomal membrane, releasing numerous free radicals into the lumen. Time-dependent H2O2 production was enhanced in the secretory vesicles, some of which were fused exactly to the phagosome membranes.

Unchanged Distribution Density of Anionic Sites on the Glomerular Wall in Rats with Active Heymann Nephritis

In various kinds of glomerulonephritis, alteration of anionic charge on the glomerular basement membrane (GBM) and podocytes has been controversial for more than decade. To elucidate the relation between glomerular protein leakage and anionic sites on the glomerular wall, we examined the distribution of anionic sites on the GBM and podocytes of rats with active Heymann nephritis (AHN). Urinalysis for protein levels was conducted, and the kidneys were examined using electron microscopic cytochemistry for the assessment of anionic charge with two cationic probes. The anionic sites on podocytes were decreased in number in the AHN rats; however, the distributions of anionic sites on the GBM were similar in density to those seen in the control animals. From these results, we consider that the decrease in anionic charge density on podocytes might be attributable to protein leakage and that the charge barrier of the GBM is irrelevant to the protein leakage in AHN rats.

Unchanged Distribution Density of Anionic Sites on the Glomerular Wall in Rats with Streptozotocin-Induced Diabetic Nephropathy

As a cause of proteinuria in diabetic nephropathy, a decrease in anionic charge on the glomerular basement membrane (GBM) is considered to be related to protein leakage. However, the constancy of the anionic charge has been reported in several types of nephropathy. To elucidate the relation between glomerular protein leakage and anionic charge, we examined the distribution of anionic sites on the GBM and podocytes in diabetic rats induced by a single intravenous injection of 60 mg/kg of streptozotocin (STZ). Five months after the treatment with STZ, urinalysis for glucose and protein levels was conducted, and the kidneys were examined using electron microscopic cytochemistry for the assessment of anionic charge with two cationic probes. The distributions of anionic sites on the GBM demonstrated by two kinds of cationic markers in the diabetic rats were similar in density to those seen in the control animals. The distributions of anionic sites on the foot processes and cell membrane of podocytes were regular and also similar in density to that of the control group. From these results, we consider that the charge barrier of the GBM and podocytes is irrelevant to the protein leakage in diabetic rats.

Characterization of Adherent Umbilical Cord Blood Stromal Cells Regarding Passage, Cell Number, and Nano-biomarking Utilization

Adherent umbilical cord blood stromal cells (AUCBSCs) are multipotent cells with differentiation capacities. Therefore, these cells have been investigated for their potential in cell-based therapies. Quantum Dots (QDs) are an alternative to organic dyes and fluorescent proteins because of their long-term photostability. In this study we determined the effects of the cell passage on AUCBSCs morphology, phenotype, and differentiation potential. QDs labeled AUCBSCs in the fourth cell passage were differentiated in the three mesodermal lineages and were evaluated using cytochemical methods and transmission electron microscopy (TEM). Gene and protein expression of the AUCBSCs immunophenotypic markers were also evaluated in the labeled cells by real-time quantitative PCR and flow cytometry. In this study we were able to define the best cellular passage to work with AUCBSCs and we also demonstrated that the use of fluorescent QDs can be an efficient nano-biotechnological tool in differentiation studies because labeled cells do not have their characteristics compromised.

Ultrastructure and cytochemical localization of acid phosphatase of laticifers in Euphorbia kansui Liou

Acid phosphatase (AcPase) activities are involved in the degeneration process of cytoplasm in plants. In this study, acid phosphatase was detected by the method of lead nitrate and cytochemical electron microscopy during the development of nonarticulated laticifers in Euphorbia kansui Liou. The most important feature in the differentiation of the laticifers in E. kansui is that the development of small vacuoles arises from endoplasmic reticulum (ER). The mature laticifers possess a thin layer of electron-dense peripheral cytoplasm in which the organelle cannot be distinguished and a large central vacuole filled with latex particles. AcPase cytochemistry studies show AcPase reaction products congregated into heaps are distributed along the tonoplast of central vacuole and around the mitochondria and plastids. Some small vacuoles which develop at later developmental stages of laticifers contain AcPase reaction products. As a result, the central vacuole is formed by cellular autophagy and fusion of small vacuoles which apparently arises from ER.

Subcellular location of horseradish peroxidase in horseradish leaves treated with La(III), Ce(III) and Tb(III)

The agricultural application of rare-earth elements (REEs) would promote REEs inevitably to enter in the environment and then to threaten the environmental safety and human health. Therefore, the distribution of the REEs ion, 141Ce(III) and effects of La(III), Ce(III) and Tb(III) on the distribution of horseradish peroxidase (HRP) in horseradish mesophyll cells were investigated with electron microscopic radioautography and transmission electron microscopic cytochemistry. It was found for the first time that REEs ions can enter into the mesophyll cells, deposit in both extra and intra-cellular. Compared to the normal condition, after the horseradish leaves treated with La(III) or Tb(III), HRP located on the tonoplast is decreased and HRP is mainly located on the cell wall, while HRP is mainly located on the plasma membrane after the horseradish leaves were treated with Ce(III). This also indicated that REEs ions may regulate the plant growth through changing the distribution of enzymes.

Cytosolic Phospholipase A2 Activity in Neutrophilic Oxidative Stress of Platelet-activating Factor-induced Acute Lung Injury

Background: The present investigation was performed in rats and isolated human neutrophils in order to confirm the presumptive role of the positive feedback loop of cytosolic phospholipase () activation by plateletactivating factor (PAF). Methods: The possible formation of the positive feedback loop of the activation and neutrophilic respiratory burst was investigated in vivo and in vitro by measurement of the parameters denoting acute lung injury. In addition, morphological examinations and electron microscopic cytochemistry were performed for the detection of free radicals in the lung. Results: Five hours after intratracheal instillation of PAF (), the lung leak index, lung myeloperoxidase (MPO) activity, the number of neutrophils and the concentration of cytokine-induced neutrophil chemoattractant (CINC) in bronchoalveolar lavage fluid were increased by PAF as compared with those of control rats. The NBT assay and cytochrome-c reduction assay revealed an increased neutrophilic respiratory burst in isolated human neutrophils following exposure to PAF. Lung and neutrophilic activity were increased following PAF exposure and exposure to hydrogen peroxide increased activity in the lung. Histologically, inflammatory findings of the lung were observed after PAF treatment. Remarkably, as determined by cytochemical electron microscopy, increased production of hydrogen peroxide was identified in the lung after PAF treatment. Conclusion: PAF mediates acute oxidative lung injury by the activation of , which may provoke the generation of free radicals in neutrophils.

OP24 Myelodysplastic syndrome management in Slovakia

Two main forms of acute leukemia have been recognized by the French-American-British (FAB) group: myeloid (AML) and lymphoblastic (ALL). Some types of AML can be diagnosed on well prepared bone marrow films stained with May-Grünwald-Giemsa. Poorly differentiated types, myeloblastic (M1) and monoblastic (M5PD), need confirmation by positive cytochemical reactions (Sudan Black B, myeloperoxidase and non-specific esterase). There are 2 sub-types of promyelocyte leukemia: M3 typical, hypergranular and M3 variant, microgranular. The M3 variant has a more acute course, higher WBC and may require cytochemistry to demonstrate promyelocytic differentiation. Electron microscopic cytochemistry can also help in the classification of difficult AML cases; the ‘platelet-peroxidase’ reaction, for example, is essential for the diagnosis of megakaryoblastic leukemia, a disorder often presenting as ‘acute’ myelosclerosis.Three morphological types are seen in ALL: L1, predominantly in children, L2, more frequently in adults, and the relatively rare L3 or Burkitt type. Immunological and enzyme markers (ALL and la antigens, terminal transferase, etc.) help define the cell phenotype: (1) non-B, non-T ALL with 3 forms (common, null and pre-B), (2) T-ALL, related to but distinct from T-lymphoblastic lymphoma, and (3) B-ALL, usually with L3 morphology. There is growing evidence that the FAB morphological types correlate with prognosis in ALL independently of other factors. The immunologically defined types also correlate with prognosis but not as an independent variable.

Structural asymmetry and discrete nucleic acid subdomains in the Trypanosoma brucei kinetoplast

The mitochondrial genome of Trypanosoma brucei is contained in a specialized structure termed the kinetoplast. Kinetoplast DNA (kDNA) is organized into a concatenated network of mini and maxicircles, positioned at the base of the flagellum, to which it is physically attached. Here we have used electron microscope cytochemistry to determine structural and functional domains involved in replication and segregation of the kinetoplast. We identified two distinct subdomains within the kinetoflagellar zone (KFZ) and show that the unilateral filaments are composed of distinct inner and outer filaments. Ethanolic phosphotungstic acid (E-PTA) and EDTA regressive staining indicate that basic proteins and DNA are major constituents of the inner unilateral filaments adjoining the kDNA disc. This evidence for an intimate connection of the unilateral filaments in the KFZ with DNA provides support for models of minicircle replication involving vectorial export of free minicircles into the KFZ. Unexpectedly however, detection of DNA in the KFZ throughout the cell cycle suggests that other processes involving kDNA occur in this domain. We also describe a hitherto unrecognized, intramitochondrial, filamentous structure rich in basic proteins that links the kDNA discs during their segregation and is maintained between them for an extended period of the cell cycle.

Pretreatment of Diltiazem Ameliorates Endotoxin-Induced Acute Lung Injury by Suppression of Neutrophilic Oxidative Stress

Background : Acute respiratory distress syndrome (ARDS) is characterized by severe inflammatory pulmonary edema of unknown pathogenesis. To investigate the pathogenesis of ARDS associated with neutrophilic oxidative stress, the role of phospholipase () was evaluated by the inhibition of calcium channel. Methods : In Sprague-Dawley rats, acute lung injury (ALI) was induced by the instillation of E.coli endotoxin (ETX) into the trachea. At the same time, diltiazem was given 60 min prior to tracheal instillation of ETX. Parameters of ALI such as lung and neutrophil , lung myeloperoxidase (MPO), BAL neutrophils, protein, surfactant were measured. Production of free radicals from neutrophils was measured also. Morphological studies with light microscope and electron microscope were carried out and electron microscopic cytochemistry for detection of free radicals was performed also. Results : Diltiazem had decreased the ALI parameters effectively in ETX given rats and decreased the production of free radicals from neutrophils and lung tissues. Morphological studies denoted the protective effects of diltiazem. Conclusion : Diltiazem, a calcium channel blocker, was effective in amelioration of ALI by the suppression of neutrophilic oxidative stress mediated by activation.

Sources of Activator Calcium Ions in the Contraction of Smooth Muscles in Aplysia kurodai

The physiological and pharmacological properties of contraction and the ultrastructure of buccal mass retractor muscle (I4) and gill-pinnule closure muscle (GPCM) in Aplysia kurodai were studied to learn more about the sources of activator Ca2+ in molluscan smooth muscle. Acetylcholine (ACh) and high K+-induced contractions were reduced by lowering the external Ca2+ concentration, and eliminated by the removal of extracellular Ca2+. Nifedipine appreciably reduced ACh- and high K+-induced contractions, while amiloride decreased only ACh-induced contractions and had no significant effect on high K+-induced contractions. When nifedipine and amiloride were applied together, either type of contraction was still appreciable. Serotonin (5-HT) could potentiate subsequent ACh- and high K+-induced contractions in I4; potentiated tension was significantly reduced by nifedipine and amiloride, whereas 5-HT inhibited ACh-and high K+-induced contractions in GPCM. The potentiating effects of 5-HT may be mediated by the activation of the Ca2+-channel to increase the influx from extracellular Ca2+. Caffeine caused contractions in Ca2+-free solution in both muscles. Electron microscopy revealed sarcolemmal vesicles underneath the plasma membrane in both muscle fibers. Electron microscopical cytochemistry demonstrated that pyroantimonate precipitates were localized in the sarcolemmal vesicles and in the inner surface of plasma membranes in the resting fibers. Present results indicate that the contractions of I4 and GPCM fibers are caused not only by Ca2+-influx but also by Ca2+ release from the intracellular storage sites, such as the sarcolemmal vesicles and the inner surface of plasma membranes.

Protective Effect of the Inhibition of PAF Remodeling and Adhesion Molecule on the Oxidative Stress of the Lungs of Rats Given Endotoxin Intratracheally

Backgraound : There have been many reports on the pathogenesis of sepsis-induced acute respiratory distress syndrome(ARDS) but, the precise mechanism has not been elucidated. This study examined the protective effect of an inhibition of platelet activating factor(PAF) remodeling and the adhesion molecule on the oxidative stress of the lungs in rats with an endotoxin induced acute lung injury(ALI). Methods : ALI was induced in Sprague-Dawley rats by instilling an E-coli endotoxin into the trachea. Ketotifen and fucoidan were used respectively to inhibit PAF remodeling and adhesion molecule. The lung leak index, lung myeloperoxidase(MPO) activity, bronchoalveolar lavage(BAL) fluid neutrophil count and lyso PAF acetyltransferase activity(AT), were measured and an ultrastructural study and cytochemical electron microscopy were performed. Results : The lung leak index, lung MPO activity, BAL fluid neutrophil count and lyso PAF AT activity was higher in the endotoxin-treated rats. In addition, severe destruction of the pulmonary architecture and increased hydrogen peroxide production were identified. These changes were reversed by ketotifen. However, fucoidan did not appear to have any protective effects. Conclusion : The inhibition of PAF remodeling appeared to be effective in decreasing the endotoxin-induced ALI. In addition, this effect might be derived from the inhibition of neutrophilic oxidative stress. However, the inhibition of the adhesion molecules by fucoidan appeared to be ineffective in decreasing the endotoxin-induced ALI.

Placentation in the African Elephant, Loxodonta africanus: III. Ultrastructural and Functional Features of the Placenta

Successful transfer of nutrients to the elephant fetus during pregnancy relies on a variety of placental modifications. Our light and electron microscopical investigations show that the structure is endotheliochorial from implantation to term, with unicellular, never syncytial trophoblast. Light and electron microscope immunocytochemistry shows the restriction of the glucose transporter 1 isoform to the basolateral surfaces of the trophoblast, with the glucose transporter 3 restricted to the apical plasmalemma of the trophoblast. Glucose transport to the fetus therefore requires a sequential use of both isoforms. Light and electron microscope cytochemistry indicate the presence of iron deposits only in the haemophagous zones confirming their iron transport function. No trophoblast areas with high concentrations of Calcium binding protein, specialised for Calcium transport were found. In situ hybridisation demonstrated the presence of IGF-II mRNA in the trophoblast from the earliest stage, with TGFbeta1 and HGF-SF mRNA expressed subsequently but only IGF-II and HGF mRNA present in the second half of pregnancy. The results are briefly discussed in terms of placental growth and function and indicate that the elephant placenta is another example of a unique solution to the variety of problems posed by a resident fetus.

Electron Microscopic Cytochemistry of Adenylyl Cyclase Activity in Mouse Spermatozoa

We investigated adenylyl cyclase activity of mouse spermatozoa by electron microscopic cytochemistry. Subcellular localization of enzyme activity was determined in the presence and absence of bicarbonate ions. Results confirm the existence in sperm of a bicarbonate-regulated adenylyl cyclase, which suggests microdomain signaling.

Calcium in the Control of Nitrogenase Activity in Vicia faba L. Root Nodules: Calcium Release from Symbiosomes and the Accompanying Decrease in Their Nitrogenase Activity Is Blocked by Verapamil

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron microscopy demonstrates a considerable calcium depletion in symbiosomes of the infected nodule cells by EGTA and A23187. Ca2+ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca2+ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached the baseline in symbiosomes. The experiments on isolated symbiosomes and arsenazo III demonstrated that verapamil inhibited Ca2+ transport induced by valinomycin at the background of K+ ions. These data suggest the presence of a verapamil-sensitive transporter on the peribacteroid membrane responsible for Ca2+ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of the root nodules is discussed.

Quinone Oxidoreductases of the Plasma Membrane

Quinone oxidoreductases of the plasma membrane relate functionally to the operation of a cell surface redox chain, where cytosolic NAD(P)H is oxidized. Plasma membrane quinones serve as lipid-soluble transmembrane shuttles to transfer the 2H + + 2e - from NAD(P)H to 1/2 O 2 to form water. The reduction of 1/2 O 2 is at the expense of hydroquinone catalyzed by cell surface hydroquinone oxidases. The first demonstration of a redox-related plasma membrane enzyme was that of an NADH-ferricyanide reductase observed with purified fractions of plasma membranes isolated from rat liver. A plasma membrane location was subsequently confirmed by electron microscope cytochemistry. Involvement in plasma membrane electron transport was inferred from observations in which ferricyanide and other impermeant oxidants were reduced by intact cells. Quinone oxidoreductases, of the plasma membrane, have physiological significance as components in support of plasma membrane electron transport. Among these are the ECTO-NOX proteins that comprise a family of NAD(P)H oxidases of plants and animals that exhibit both oxidative and protein disulfide isomerase-like activities that alternate and serve as terminal oxidases for plasma membrane electron transport. A unique feature of the ECTO-NOX proteins is that the two enzymatic activities they catalyze, hydroquinone (NADH) oxidation and disulfide–thiol interchange, alternate within a 24-min period.