Ultrastructural localization of Trypanosoma cruzi lysosomes by aryl sulphatase cytochemistry

Lysosomes of trypanosomatid protozoa are poorly known. In this work we have cytochemically detected the lysosomal enzyme aryl sulphatase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata, by using p-nitrocatecholsulphate as substrate. Positive reaction was located exclusively inside membrane-bound cytoplasmic vesicles distributed throughout the cell body. Electron-dense reaction was either dispersed homogeneously through the vesicular matrix or located at the vesicle periphery, apposed to the membrane, with fine granular deposits occasionally found at the vesicular matrix. Trypomastigote and epimastigote forms of T. cruzi lacked electron-dense deposits at the plasma membrane, thus indicating that aryl sulphatase was not secreted to the environment. Furthermore, no positive reaction was detected in epimastigote reservosomes, which are organelles considered as pre-lysosomal compartments. Thus, our data show that reservosomes and lysosomes are organelles that can be distinguished by the cytochemical localization of aryl sulphatase in T. cruzi epimastigotes and trypomastigotes. Positive reaction in cytoplasmic vesicles of C. fasciculata choanomastigotes confirmed the specificity of the reaction for lysosomes in other trypanosomatid species.     

Effect of usnic acid from the lichen Cladonia substellata on Trypanosoma cruzi in vitro: an ultrastructural study

Chemotherapy for Chagas' disease is still unsatisfactory due to toxicity and limited effectiveness of the available drugs. In this work we have investigated the effect of usnic acid, isolated from lichen Cladonia substellata, against Trypanosoma cruzi, in vitro. Incubation of culture epimastigotes with 5-30microg/ml of this compound resulted in growth inhibition in a dosis-dependent manner. Ultrastructural analysis of treated epimastigotes showed damage to mitochondria, with a marked increase in kinetoplast volume and vacuolation of the mitochondrial matrix. Intense lysis of bloodstream trypomastigotes was observed with all drug concentrations tested. Besides mitochondrial and kinetoplast damage, trypomastigotes also presented enlargement of the flagellar pocket, as well as intense cytoplasm vacuolation. Treatment of infected macrophages with 40 or 80microg/ml usnic acid induced marked cytoplasm vacuolation in intracellular amastigote forms, with disorganization of parasite kinetoplast and mitochondria, but with no significant ultrastructural damage to the host cells.     

Radiation response of cell organelles

The cellular responses to various form of radiation, including ionizing- and UV-irradiation or exposure to electromagnetic fields is manifested as irreversible and reversible structural and functional changes to cells and cell organelles. Moreover, beside the morphological signs related to cell death, there are several reversible alterations in the structure of different cell organelles. The radiation-induced changes in the supramolecular organization of the membranes, including plasma membrane, and different cell organelle membranes, play a significant role in the development of acute radiation injury. These signs of radiation-induced reversible perturbation biological membranes reflect changes in the organization and/or composition of the glycocalix, modified activity and/or distribution of different membrane domains, including enzymes and binding sites. The observed changes of the cell surface micromorphology and the alteration of intercellular connections are closely related to the reorganization of the cytoskeletal elements in the irradiated cells. The mitochondria, endoplasmic reticulum, Golgi-complex, the lysosomal system have long been considered to be direct intracellular targets of irradiation. The listed morphological alterations of nuclear chromatin (e.g. changes of fine structure, altered number of nucleolar organizing regions and micronuclei, development of chromosome aberrations) may originate from the radiation-induced damage to the supramolecular organization of DNA and/or nucleus specific proteins. These endpoints of radiation effects resulted as direct consequence(s) of absorbed radiation energy, and indirectly altered intra-, intercellular communication or modified signal transduction. Some complementary data suggest that all these effects are not strictly specific to radiation and may be best considered as general stress responses, similar to those observed after application of various injurious agents and treatments to cells. Moreover, they may be equally responsible for direct degradation of supramolecular component of cells, altered signal transduction, or changes in the amount or ratio of any extracellular mediators upon irradiation. Nevertheless, qualitative and/or quantitative evaluation of any changes of chromosomes by different techniques (morphological analysis of metaphase chromosomes, fluorescent in situ hybridization, development of micronuclei etc.) are useful biological indicators as well as "biological dosimeters" of radiation injury. It is suggested, that some modern methods such as immunohistochemical detection of different proteins, specific markers of cell organelles and cytoskeleton, inspection of distribution of cell surface charged sites and different membrane domains and application of tracer substances may all be included into protocols for evaluation of cell alterations induced by different types and intensities of radiation.     

Ultrastructural and biophysical comparison of the membrane effects induced by different stressing agents on K562 cells

The morphological (using scanning electron microscopy) and biophysical (determination of membrane order by electron paramagnetic resonance spectroscopy) changes occurring in the erythroleukemic cell line K562 were determined after heat-shock (physical) and gliotoxin treatment (chemical) stress. The data seem to indicate that although similarities in the response of these cells to different types of stress agents exist, there are profound differences in the mechanisms of cell death. Specifically, gliotoxin seems to induce free-radial cytoskeletal cross-linking of actin microfilaments and subsequent surface blebbing and increases in membrane order while heat-shock appears to bring about an increase in membrane order due to changes in membrane proteins.     

Damage effects of protoporphyrin IX - sonodynamic therapy on the cytoskeletal F-actin of Ehrlich ascites carcinoma cells

In this study, we report evidence of the damage effects of sonodynamic therapy (SDT) on a novel intracellular target, cytoskeletal F-actin, that has great importance for cancer treatment. Ehrlich ascites carcinoma (EAC) cells suspended in PBS were exposed to ultrasound at 1.34MHz for up to 60s in the presence and absence of protoporphyrin IX (PPIX). To evaluate the polymeric state and distribution of actin filaments (AF) we employed FITC-Phalloidin staining. The percentage of cells with intact AF was decreased with 10-80muMu PPIX after ultrasonic exposure, while only few cells with disturbed F-actin were observed with 80muMu PPIX alone. The fluorescence intensity of FITC-Phalloidin labeled cells was detected by flow cytometry. The morphological changes of EAC cells were observed by scanning electron microscope (SEM). The nuclei were stained with Hoechst 33258 to determine apoptosis. Cytoskeletal F-actin and cell morphological changes were dependent on the time after SDT. Some cells suffered deformations of plasma membrane as blebs that reacted positively to FITC-Phalloidin at 2h after SDT treatment. Many of the cells showed the typically apoptotic chromatin fragmentation. The alterations were more significant 4h later. Our results showed that cytoskeletal F-actin might represent an important target for the SDT treatment and the observed effect on F-actin and the subsequent bleb formation mainly due to apoptosis formation due to the treatment.     

Phagocytosis, endosomal/lysosomal system and other cellularaspects of macrophage activation by Canova medication

Canova is a homeopathic medication with immunomodulatory properties, recommended for diseases where the immune system is depressed. Our research aims to study the activation of mice peritoneal macrophages when submitted to in vivo and in vitro Canova treatment. Morphological parameters and acid phosphatase activity were analyzed using light and transmission electron microscopy. Differential interference contrast microscopy, including serial time acquisition in living cells, was also performed. The results demonstrated a greater spreading ability in Canova treated macrophages, a higher phagocytic activity of non-infective microorganisms (Saccharomyces cerevisiae and Tripanosoma cruzi epimastigotes) and a tendency to lower the phagocytic activity of the infective microorganisms T. cruzi trypomastigotes and Leishmania amazonensis, when compared with control cells. Acid phosphatase activity was analyzed and showed that Canova treatment stimulates an increase of the endosomal/lysosomal system. Treated macrophages that do or do not interact with yeast present a higher number of acid phosphatase marked vesicles compared to control cells. In contrast, the activity of tartrate resistant acid phosphatase (TRAP), is lower in Canova treated macrophages. The net results demonstrate that Canova medication is an effective stimulator of macrophage activity.     

The ultrastructure of infective larvae (L3) of Wuchereria bancrofti after treatment with diethylcarbamazine

Although the large use of diethylcarbamazine (DEC), as the major anti-filaricide drug, its mechanism of action remains a matter of controversy. Several authors defend the hypothesis that DEC has no direct effect on nematodes. This study demonstrated that infective larvae (L3) of Wuchereria bancrofti treated in vitro with DEC presented several behaviour and morphological changes. The first alteration produced by treatment for 2 h with 3, 5, 10 microg/ml of DEC was the reduction of motility. Larvae treated with 5, 10 microg/ml DEC showed severely affected organelles, formation of several vacuoles, mainly in neurocytes and in the muscle cells, and dissolution of cytoplasm. Some larvae showed extreme cellular disorganization with abundance of large and dense mitochondria and numerous large vacuoles containing residual organelles. Lamellar bodies, probably related to an assembly of hipodermal membranes, were also observed in some damaged larvae. Thus, undoubtedly in vitro treatment with concentrations of DEC similar to therapeutic conditions, which are 1-5 microg/ml (Hawking, 1979), had a direct effect on infective larvae of W. bancrofti by causing, primarily neuromuscular alterations with subsequent damage to organelles.     

Effect of AEE788 and/or Celecoxib on colon cancer cell morphology using advanced microscopic techniques

Analysis of changes in cancer cell morphology and cytoskeletal element induced by external stimuli is focus of current cancer chemotherapeutic studies. Cancer cell cytoskeleton is complex network of interwoven protein fibers composed of microtubules, microfilaments and intermediate filaments. These interwoven protein fibers are responsible for maintaining cell morphology, movement, adhesion and transmembrane signal transmission. In this study, morphological and cytoskeletal changes induced by AEE788 and/or Celecoxib on colon cancer cell HCT 15 were analyzed using advanced microscopic techniques. Cell proliferation assay was used for determining IC₅₀ of AEE788 and/or Celecoxib on HCT 15. Confocal microscopic analysis of AEE788 and/or Celecoxib treated HCT 15 was performed using Rhodamine-Phalloidin (actin stain) and Hoechst 33342 (nuclear stain). Atomic force (AFM) and scanning electron microscopic (SEM) studies were also performed to analyze cell morphology and cell wall extension (filopodia and lamellipodia). In addition, quantitative analysis of morphological parameters was studied using cellular image processing technique. This is the first report that combination of AEE788 and Celecoxib additively increase growth inhibition and cell death on human colon cancer cell HCT 15 as estimated by cell proliferation assay. Morphological analysis of AEE788 or Celecoxib treated HCT 15 cell for 24 h have not revealed significant change in morphology under phase contrast microscopy. But, slight morphological changes were observed in combination (AEE788 + Celecoxib) treated HCT 15 for 24 h. In contrast, high resolution confocal laser fluorescence and atomic force microscopic studies have revealed cell shrinkage, disorganized actin filament and, loss of filopodia and lamellipodia. These changes were more prominent in combination of AEE788 and Celecoxib treated HCT 15 than either drug alone. These results may suggest antiproliferative and antimetastatic activity of AEE788 and/or Celecoxib. Quantitative analysis of morphological parameters using cellular image processing technique have shown decrease in mean area, perimeter, compactness and eccentricity of combination drug treated cells than either drug alone. These results further support the confocal and AFM study. Scanning electron microscopic study of AEE788 and/or Celecoxib treated HCT 15 has also shown morphological changes and loss of filopodia and lamellipodia. In conclusion, this investigation of morphological and cytoskeletal changes using advanced microscopic techniques present a significant foundation for evaluating anticancer activity of a drug and form a new strategy for evaluating effect of AEE788 and/or Celecoxib on colon cancer.     

Spin labeling studies of the interactions of potentially useful therapeutic agents for the treatment of Alzheimer’s disease with cytoskeletal proteins in erythrocyte ghosts and brain synaptosomal membranes

Alzheimer’s disease (AD) is the major dementing disorder of the elderly with four million victims of this disease in the United States. The molecular basis of AD remains unknown, but the biochemical and biophysical state of cytoskeletal proteins is reportedly altered in AD cortical neurons. No approved effective therapy for AD is available. We have used electron spin resonance (ESR) and a protein-specific spin label to investigate the interactions of three potential therapeutic agents in AD: tacrine, velnacrine, and acetylcarnitine, with cytoskeletal proteins in erythrocyte membranes. Further, we report for the first time the effects of tacrine on the physical state of membrane proteins in brain neocortex synaptosomal membranes. All three agents lead to decreased segmental motion and increased cytoskeletal protein-protein interactions in erythrocyte membranes in order of effectiveness: tacrine > velnacrine > acetylcarnitine. In addition, we have synthesized N-methylacridinium methosulfate which has a positive charge on the opposite side of the molecule relative to tacrine. This former agent gave a less pronounced diminution of the relevant ESR parameter than that caused by tacrine, implying that the orientation of the molecule with its interaction site is important in the increased cytoskeletal protein-protein interactions induced by tacrine. With synaptosomal membranes tacrine also significantly decreased segmental motion of membrane proteins. Our ESR results on erythrocyte and brain membranes suggest that in addition to their biochemical effects, these potential AD therapeutic agents function to strengthen cytoskeletal protein-protein interactions. These results are discussed with reference to possible molecular mechanisms involving cytoskeletal proteins in AD.     

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.     

Ultrastructural analysis of midgut cells from Culex quinquefasciatus (Diptera: Culicidae) larvae resistant to Bacillus sphaericus

The larvicidal action of the entomopathogen Bacillus sphaericus towards Culex quinquefasciatus is due to the binary (Bin) toxin present in crystals, which are produced during bacterial sporulation. The Bin toxin needs to recognize and bind specifically to a single class of receptors, named Cqm1, which are 60-kDa α-glucosidases attached to the apical membrane of midgut cells by a glycosylphosphatidylinositol anchor. C. quinquefasciatus resistance to B. sphaericus has been often associated with the absence of the α-glucosidase Cqm1 in larvae midgut microvilli. In this work, we aimed to investigate, at the ultrastructural level, the midgut cells from C. quinquefasciatus larvae whose resistance relies on the lack of the Cqm1 receptor. The morphological analysis showed that midgut columnar cells from the resistant larvae are characterized by a pronounced production of lipid inclusions, throughout the 4th instar. At the end of this stage, resistant larvae had an increased size and number of these inclusions in the midgut cells, while only a small number were observed in the cells from susceptible larvae. The morphological differences in the midgut cells of resistant larvae found in this work suggested that the lack of the Cqm1 receptor, which also has a physiological role as being an α-glucosidase, can be related to changes in the cell metabolism. The ultrastructural effects of Bin toxin on midgut epithelial cells from susceptible and resistant larvae were also investigated. The cytopathological alterations observed in susceptible larvae treated with a lethal concentration of toxin included breakdown of the endoplasmic reticulum, mitochondrial swelling, microvillar disruption and vacuolization. Some effects were observed in cells from resistant larvae, although those alterations did not lead to larval death, indicating that the receptor Cqm1 is essential to mediate the larvicidal action of the toxin.This is the first ultrastructural study to show differences in the cell morphology of resistant larvae and further investigation is needed to understand the impact of the lack of expression of midgut enzymes on the physiology of resistant insects.     

Modifications of nuclear envelope during differentiation and depolyploidization of rat trophoblast cells

An increased activity of membranes of the nuclear envelope (NE) was observed electron microscopically in the trophoblast cells of the rat placenta. The activity of the membranes was manifested as formation of various NE derivatives, such as the annulate lamellae (AL), the intranuclear tubules, and the concentric membranous structures. At the period of terminal differentiation of the secondary giant trophoblast cells (SGTC) the NE derivatives play active role in subdivision of the initial highly polyploid nuclei into the numerous low-ploidy fragments. (3)H-thymidine labeling showed that attenuation of the DNA replication precedes the nuclear fragmentation. In the course of the nuclear fragmentation the narrow deep NE invaginations subdivide the nucleus into the separate lobes that subsequently are detached from the initial nucleus. By the beginning of the fragmentation, the accumulated membranous structures, i.e. the intranuclear AL, tubules, clusters of pore complexes, etc., seem to be the source of the reserve material that is necessary for formation of the great amount of the NE membranes of the newly formed nuclear fragments. Thus, the intranuclear membranous structures that seem to increase the active surface of the growing endopolyploid nucleus at the earlier stage of differentiation then take part in genome isolation that results in formation of a multinucleate cell with diploid and low-polyploid nuclei. The outer NE membrane of the initial nucleus plays an active role in compartmentalization of cytoplasmic areas around the nuclear fragments within the giant polykaryocyte. Apart from the membranous structures the bundles of intermediate filaments (IF) located in the cytoplasm perinuclear zones seem to participate in the nuclear fragmentation. These processes are likely to provide formation of the giant polykaryocytes incapable for further proliferation.     

Live morphological analysis of taxol-induced cytoplasmic vacuolization [corrected] in human lung adenocarcinoma cells

Taxol (paclitaxel), one of the most active cancer chemotherapeutic agents, can cause programmed cell death (PCD) and cytoplasmic vacuolization. The objective of this study was to analyze the morphological characteristics induced by taxol. Human lung adenocarcinoma (ASTC-a-1) cells were exposed to various concentration of taxol. CCK-8 was used to assay the cell viability. Atomic force microscopy (AFM), plasmid transfection and confocal fluorescence microscopy were performed to image the cells morphological change induced by taxol. Fluorescence resonance energy transfer (FRET) was used to monitor the caspase-3 activation in living cells during taxol-induced cell death. Cells treated with taxol exhibited significant swelling and cytoplasmic vacuolization which may be due to endoplasmic reticulum (ER) vacuolization. Caspase-3 was not activated during taxol-induced cytoplasmic vacuolization and cell death. These findings suggest that taxol induces caspase-3-independent cytoplasmic vacuolization, cell swelling and cell death through ER vacuolization.     

Immunolocalization of an osteopontin-like protein in dense granules of Toxoplasma gondii tachyzoites and its association with the parasitophorous vacuole

Toxoplasma gondii is an apicomplexan parasite infecting a broad host range, including humans. The parasite invades host cell by active penetration with the participation of its secretory organelles proteins during this process. Until now, only a limited number of secretory proteins have been discovered, and the effectors molecules involved in parasite invasion and survival are not well understood. Osteopontin (OPN) is a multifunctional glycophosphoprotein, secreted by different cell types, which is involved in various physiological and pathological events including cell signaling and survival. For the first time we demonstrated in this work by immunofluorescence and immunoelectron microscopy approaches the localization of an OPN-like protein in dense granules of extracellular T. gondii tachyzoites. Western blotting and RT-PCR confirmed this protein expression by the parasites. Our results also showed, after macrophage invasion, an intense positive labeling for OPN-like protein at the sub-apical portion of tachyzoites, the site of dense granules secretion, and the localization of this protein at the parasitophorous vacuole membrane. These data suggest that dense granules secrete an OPN-like protein, and we speculate that this protein participates during the parasite interaction process with host cells and parasitophorous vacuole formation.     

Nuclear alterations associated to programmed cell death in larval salivary glands of Apis mellifera (Hymenoptera: Apidae)

The silk glands of bees are a good model for the study of cell death in insects. With the objective to detect the nuclear features during glandular regression stage, larvae at the last instar and pre-pupae were collected and their silk glands were dissected and processed for ultrastructural analysis and histologically for cytochemical and imunocytochemical analysis. The results showed that the cellular nuclei exhibited characteristics of death by atypical apoptosis as well as autophagic cell death. Among the apoptosis characteristic were: nuclear strangulation with bleb formation in some nuclei, DNA fragmentation in most of the nuclei and nucleolar fragmentation. Centripetal chromatin compaction was observed in many nuclei, forming a perichromatin halo differing from typical apoptotic nuclei. With regards to the characteristics of autophagic-programmed cell death, most relevant was the delay in the collapse of many nuclei.     

Ultrastructural changes to rat hippocampus in pentylenetetrazol- and kainic acid-induced status epilepticus: A study using electron microscopy

A pentylenetetrazol (PTZ)-induced status epilepticus model in rats was used in the study. The brains were studied one month after treatment. Ultrastructural observations using electron microscopy performed on the neurons, glial cells, and synapses, in the hippocampal CA1 region of epileptic brains, demonstrated the following major changes over normal control brain tissue. (i) There is ultrastructural alterations in some neurons, glial cells and synapses in the hippocampal CA1 region. (ii) The destruction of cellular organelles and peripheral, partial or even total chromatolysis in some pyramidal cells and in interneurons are observed. Several astrocytes are proliferated or activated. Presynaptic terminals with granular vesicles and degenerated presynaptic profiles are rarely observed. (iii) The alterations observed are found to be dependent on the frequency of seizure activities following the PTZ treatment. It was observed that if seizure episodes are frequent and severe, the ultrastructure of hippocampal area is significantly changed. Interestingly, the ultrastructure of CA1 area is found to be only moderately altered if seizure episodes following the status epilepticus are rare and more superficial; (iv) alterations in mitochondria and dendrites are among the most common ultrastructural changes seen, suggesting cell stress and changes to cellular metabolism. These morphological changes, observed in brain neurons in status epilepticus, are a reflection of epileptic pathophysiology. Further studies at the chemical and molecular level of neurotransmitter release, such as at the level of porosomes (secretory portals) at the presynaptic membrane, will further reveal molecular details of these changes.     

Brownian dynamics in Fourier space: membrane simulations over long length and time scales

A simulation algorithm for elastic membrane sheets is presented. Overdamped stochastic dynamics including hydrodynamic coupling to surrounding solvent and arbitrary external forces are generated by employing Fourier modes of the sheet as the primary dynamic variables. Simulations over the micron length scale and second time scale are easily achieved. The dynamics of a lipid bilayer attached to an underlying network of cytoskeletal filaments is used to estimate the diffusion constant of membrane-bound proteins on the surface of the red blood cell.     

Thermotropic phase transitions in model membranes of the outer skin layer based on ceramide 6

The lipid intercellular matrix stratum corneum of the outer skin layer is a multilayer membrane consisting of a complex mixture of different lipids: ceramides, fatty acids, cholesterol, and its derivatives. The basis of the multilayer membrane is the lipid bilayer, i.e., a two-dimensional liquid crystal. Currently, it is known that the main way of substance penetration through the skin is the lipid matrix. The complexity of the actual biological system does not allow reliable direct study of its properties; therefore, system modeling is often used. Phase transitions in the lipid system whose composition simulates the native lipid matrix are studied by the X-ray synchrotron radiation diffraction method.