Cytoplasmic Contraction Research Articles

Transcriptional activity and splicing factors are preserved during physiological apoptosis

Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism’s life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the progression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome elements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.

Hypoxia-inducedoxidative stress and apoptosisin gills of scaleless carp (Gymnocypris przewalskii).

Scaleless carp (Gymnocypris przewalskii) are well adaptedtolow oxygen environment, but their specific adaptation mechanism to hypoxic condition remains unclear. The gill is an important respiratory organ that plays a crucial role in regulating hypoxic stress. Here, we established fish hypoxic stress model, as well as investigated oxidative stress, apoptotic responses, and relative enzyme activities in the gills of scaleless carp after exposure to various levels of hypoxic stress. The results demonstrated that gill lamellar height and basal length increased significantly under severe hypoxic stress, and interval lengths between lamellae increased significantly under hypoxic stress. Furthermore, lamellar epithelial cells underwent apoptosis, cytoplasmic contraction, and mitochondrial expansion, and the number of apoptotic cells increased significantly after exposure to severe hypoxic stress for 24h. Subsequently, Bcl-2 and Caspase 3 mRNA levels, as well as Bcl-2/Bax expression ratio were significantly increased after exposure to severe hypoxic stress for 24h, indicating upregulation of anti-apoptotic processes. Moreover, malondialdehyde and hydrogen peroxide levels were significantly increased after exposure to hypoxic stress for 24h. Superoxide dismutase activity increased significantly after exposure to severe hypoxia for 8h and then decreased, while glutathione peroxidase activity and total antioxidant capacity increased significantly under hypoxic stress. Taken together, the results indicated that scaleless carp gills respond to acute hypoxic conditions by undergoing lamellar morphology remodeling, enhanced apoptosis, and increased antioxidant enzymatic activity. The study findings provided new insight into the adaptation mechanisms of scaleless carp in response to hypoxic challenge.

Interplay between cell height variations and planar pulsations in epithelial monolayers.

Biological tissues change their shapes through collective interactions of cells. This coordination sets length and time scales for dynamics where precision is essential, in particular during morphogenetic events. However, how these scales emerge remains unclear. Here, we address this question using the pulsatile domains observed in confluent epithelial MDCK monolayers where cells exhibit synchronous contraction and extension cycles of [Formula: see text]h duration and [Formula: see text] length scale. We report that the monolayer thickness changes gradually in space and time by more than twofold in order to counterbalance the contraction and extension of the incompressible cytoplasm. We recapitulate these pulsatile dynamics using a continuum model and show that incorporation of cell stiffness dependent height variations is critical both for generating temporal pulsations and establishing the domain size. We propose that this feedback between height and mechanics could be important in coordinating the length scales of tissue dynamics.

Anatomy and ultrastructure floral osmophores of Catasetum fimbriatum (Orchidaceae).

Catasetum fimbriatum is a dioecious species whose flowers fully adapted to an euglossinophilic mode of pollination. Euglossini male bees collect the volatile fragrances which are produced in osmophores on the flowers. In order to understand the mechanism of scent secretion and floral interaction with the pollinator, we describe the location, histochemistry, anatomy, and ultrastructure of osmophores in pistillate and staminate flowers of this species. Fresh flowers were submerged in neutral red solution to locate the position of the osmophores. Other histochemical test performed includes the NADI reaction to detect terpenoids, Sudan IV for lipids, and Lugol's iodine solution to detect starch. Anatomical and ultrastructural traits were studied with bright field and transmission electron microscopes, respectively. The location of osmophores differs between pistillate and staminate flowers. In pistillate flowers, secretory tissues were observed on the ribbed adaxial surface of the labellum, but not on its margins, whereas in staminate flowers, they were found throughout the adaxial surface of the labellum and especially in the fimbriae. Anatomy and ultrastructure of the osmophores in the labellum of both types of flowers were similar. They present characteristics of metabolically active cells, such as abundant mitochondria, rough endoplasmic reticulum, vesicles, plastids with starch grains, and lipid globules. Granulocrine secretion and cycles of cytoplasmic contraction and expansion appear to allow the release of products without involving the rupture of the cuticle. Individuals of Eufriesea auriceps and Euglossa sp. were captured in staminate and pistillate flowers but, it seems likely, that only the former pollinates this orchid species.

Antifungal activity and potential mechanism of magnoflorine against Trichophyton rubrum.

Coptis alkaloids show potent antifungal activity against Trichophyton rubrum (T. rubrum), which was a Tinea pedis fungus, but little of the literature was reported to investigate the antifungal activity of magnoflorine against it. Meanwhile, the potential mechanism of magnoflorine against T. rubrum is unknown. In the present study, we found that Coptis alkaloids, especially magnoflorine had significant antifungal activities against T. rubrum and Trichophyton mentagrophyte (T. mentagrophyte). The MIC values of magnoflorine against T. rubrum and T. mentagrophyte were both 62.5 μg ml-1, but magnoflorine exerted a better fungicidal efficiency against T. rubrum than T. mentagrophyte. Magnoflorine inhibited the conidia germination and hyphal growth, and changed the mycelial morphology such as deformation growth, surface peeling, and cytoplasmic contraction in T. rubrum. Magnoflorine had no significant effect on cell wall integrity. However, magnoflorine destroyed the fungal cell membrane of T. rubrum through increasing the nucleic acid leakage, reducing the activities of squalene epoxidase and CYP51 enzyme, and decreasing the content of ergosterol in hyphae. Our study supported the potential use of magnoflorine as an antifungal agent against T. rubrum and made contributions to the clinical application of magnoflorine against fungi.

Effects of graphene oxide on algal cellular stress response: Evaluating metabolic characters of carbon fixation and nutrient removal

The effects of different concentrations of graphene oxide (GO) on intracellular metabolism in Chlorella vulgaris (C. vulgaris) and removal of nitrogen and phosphorus nutrients by C. vulgaris from synthetic wastewater were studied. The results demonstrated that cell division of Chlorella vulgaris increased at 24 h and decreased at 96 h after exposure to different concentrations of GO. The removal rates of total nitrogen (TN), ammoniacal nitrogen (NH3–N), phosphate (PO43--P), and chemical oxygen demand (COD) were 24.1%, 70.0%, 37.0%, and 39.6%, respectively, when the concentration of GO was 0.01 mg/L 10 mg/L GO induced severe plasmolysis and cytoplasmic contraction. Furthermore, the protein-like exopolysaccharide (EPS) content of algal cells exposed to 10 mg/L GO decrease to 10.8% of the control group. Simultaneously, the reactive oxygen species (ROS) level was 175.4% of control group. The biological responses to 10 mg/L GO included increase in ROS level, inhibition of saccharide metabolism, and degradation of amino acids. In addition, high concentrations of 10 mg/L GO weakened the carbon fixation process in algal cells. These stress-response behaviors increased cell permeability and oxidative stress. Overall, these findings provide new insights regarding the effects of GO on algal cellular stress responses.

Developmental programmed cell death during asymmetric microsporogenesis in holocentric species ofRhynchospora(Cyperaceae)

Members of the Cyperaceae family exhibit an asymmetric microsporogenesis that results in the degeneration of three out of four meiotic products. Efforts have been made previously to describe the resulting structure, named the pseudomonad, but mechanisms concerning the establishment of cell domains, nuclear development, and programmed cell death are largely unknown. Using the Rhynchospora genus as a model, evidence for cell asymmetry, cytoplasmic isolation, and programmed cell death was obtained by a combination of electron microscopic, cytochemical, immunocytochemical, in situ hybridization, and flow cytometric methods. Degenerative cells were identified at the abaxial region, with the cytoskeleton marking their delimitation from the functional domain after meiosis. After attempting to initiate cell division with an unreplicated genome and abnormal spindle assembly, these cells exhibited a gradual process of cytoplasmic contraction associated with hypermethylation of cytosines and differential loss of DNA. These results indicate that the asymmetric tetrad establishes a functional cell, where one nucleus is preferentially selected to survive. Degenerative haploid cells are then eliminated in a multistep process associated with mitotic disorder, non-random elimination of repetitive DNA, vacuolar cell death, and DNA fragmentation.

Excessive EDTA induces morphologic changes in bone marrow smears that mimic specific features of dysplasia

The WHO 2008 guidelines recommend that bone marrow (BM) slides for the morphological assessment of dysplasia should be made from freshly obtained BM specimens, and that specimens exposed to anticoagulants for more than two hours are unsatisfactory. However, BM aspirates may be exposed to excessive concentrations of anticoagulant, due to underfilling of the tube or inadequate mixing of the specimen. Here, we document the morphologic changes in BM smears resulting from exposure to excessive concentrations of ethylenediaminetetraacetic acid (EDTA). Subjects with normal morphology in BM smears without anticoagulant were studied. Smears without anticoagulant and smears made from BM stored in excessive EDTA for 2 h at room temperature were stained with May-Grünwald Giemsa, and cell morphology was evaluated microscopically. Neutrophil and megakaryocyte size were measured using a calibrated microscope eyepiece graticule. Excessive EDTA concentrations induced progressive nuclear and cytoplasmic contraction with respective membrane damage, cell smudging and pyknotic nuclei. In the granulocytic and megakaryocytic series, hypolobated neutrophils, small neutrophils and micromegakaryocytes were increased in number. In erythroblasts, nuclear contour irregularities were induced, but in general, artifactual changes did not mimic dyserythropoiesis. With excessive EDTA concentrations, morphologic features of erythroblasts and megakaryocytes were obscured by nuclear and cytoplasmic contraction. Excessive EDTA induces morphologic changes in BM smears that mimic the specific dysplastic features of hypolobated neutrophils, small neutrophils, and micromegakaryocytes. BM aspirates should be collected into an appropriate concentration of EDTA to minimize such artifacts.

Mammalian erythroblast enucleation requires PI3K-dependent cell polarization

Enucleation, the final step in terminal differentiation of mammalian red blood cells, is an essential process in which the nucleus surrounded by the plasma membrane is budded off from the erythroblast to form a reticulocyte. Most molecular events in enucleation remain unclear. Here we show that enucleation requires establishment of cell polarization that is regulated by the microtubule-dependent local activation of phosphoinositide 3-kinase (PI3K). When the nucleus becomes displaced to one side of the cell, actin becomes restricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ are highly localized at the cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation. We propose that enucleation is regulated by microtubules and PI3K signaling in a manner mechanistically similar to directed cell locomotion.

Bacterial surface appendages strongly impact nanomechanical and electrokinetic properties of Escherichia coli cells subjected to osmotic stress.

The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM) and electrokinetics (electrophoresis). Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus). From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO3, cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (∼700–900 kPa and ∼100–300 kPa respectively). Under similar ionic strength condition, a dramatic ∼50% to ∼70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the intimate relationship between structure/flexibility and charge of bacterial envelope and propensity of bacterium and surface appendages to contract under hypertonic conditions.

Living-cell imaging of transgenic rat anterior pituitary cells in primary culture reveals novel characteristics of folliculo-stellate cells

Folliculo-stellate (FS) cells in the anterior pituitary gland appear to possess multifunctional properties. Recently, the development of transgenic rats (S100b-green fluorescent protein (GFP) rats) that express GFP specifically in FS cells in the anterior pituitary gland has allowed us to distinguish and observe living FS cells in other kinds of pituitary cells. We used S100b-GFP rats to investigate the topographic affinity of FS cells for other pituitary cells. We observed living FS cells in enzymatically dispersed anterior pituitary cells of S100b-GFP rats under a fluorescent microscope, and noted that FS cells markedly extended and contracted cytoplasmic processes and formed interconnections with neighboring FS cells. In addition, FS cells adhered to small clusters of GFP-negative cells, which were primarily hormone-producing cells, and these clusters further aggregated during the course of cytoplasmic contraction. In the presence of laminin, fibronectin, and varying types of collagen, FS cells showed marked changes in shape and specific proliferative activity; however, GFP-negative cells did not. On reverse transcription-PCR analysis and immunohistochemistry, FS cells were shown to express integrin subunits, which are the cell surface receptors for extracellular matrix (ECM). In the anterior pituitary gland, FS cells and the various types of hormone-producing cells generate a unique topography in the presence of basement membrane components and interstitial collagens. The novel characteristics of FS cells observed in the present study suggest that in the anterior pituitary gland, FS cells play important roles in determining and/or maintaining local cellular arrangement in the presence of ECM components.

Role of TWIST in the apoptosis of Hep-2 cells induced by paclitaxel

To explore the relationship between the expression of transcription factor TWIST and apoptosis of Hep-2 cells induced by paclitaxel. Morphological changes of Hep-2 cells were observed by reserved microscopy and acridine orange cytochemistry staining. Viability of Hep-2 cells treated with various concentrations of paclitaxel was detected by MTT assay. Apoptosis was examined by flow cytometry. The expressions of transcription factor TWIST at both mRNA and protein level in response to paclitaxel at 24 h, 48 h and 72 h were then examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. Typical morphological changes of apoptotic cells, i.e., cellular rounding-up, cytoplasmic contraction, chromatin condensation and, particularly, apoptotic body, the main morphological characteristic of apoptosis, were observed by reserved microscopy and acridine orange cytochemistry staining. The cell surviving rates significantly decreased in a concentration- and time-dependent manner as evidenced by MTT assay (P < 0.05). Percent of apoptosis after 24 h, 48 h or 72 h paclitaxel-treatment was (22.6 +/- 5.3)%, (38.7 +/- 7.9)% and (52.4 +/- 14.3)%, respectively, whereas the percent of control was (9.85 +/- 5.83)%. There existed a statistically significant difference between treatment and control (F = 12.621, P < 0.05). The expression of TWIST at both mRNA and protein levels for 24 h, 48 h or 72 h in the paclitaxel-induced apoptosis of Hep-2 cells were decreased by 16.7%, 46.8%, 76.9% (F = 10.407, P < 0.05) and 16.4%, 33.6%, 69.6% (F = 18.013, P < 0.05) respectively. TWIST, which is significantly decreased in expression in response to paclitaxel in Hep-2 cells, may play a pivotal role in paclitaxel-induced apoptosis of Hep-2 cells.

Lethal effect of chitosan-Ag (I) films onStaphylococcus aureusas evaluated by electron microscopy

This article investigated the lethal effect and morphological changes on Staphylococcus aureus strains ATCC 25923 and ATCC 6538P produced by chitosan-Ag (I) films as observed by electron microscopy. The antimicrobial activity of films against staphylococci was determined using the broth dilution method and agar diffusion test. Killing curves, transmission and scanning electron microscopy (TEM and SEM) techniques were employed to evaluate the bacterial death and morphological changes in bacterial cells after exposure to chitosan-Ag (I) films. Films affected the cell structure of Staph. aureus, causing elongation of cells, disaggregation of grape-like cluster, contraction of bacterial cytoplasm, thickening of cell wall, increase in cell wall roughness, cell disruption with loss of intracellular material, filamentation and bacteriolysis, as seen in the micrographs following 3, 6, 12 and 16 h of incubation. Obtained images clearly show that chitosan-Ag (I) films have a notable antistaphylococcal activity. Information from this study can be employed in guiding future strategies to improve the design of materials for the food industry packaging.

Role of Antibiosis in Suppression of Charcoal Rot Disease by Soybean Endophyte Paenibacillus sp. HKA-15

Four defective (AFM(-)) mutants of Paenibacillus sp. HKA-15 that no longer produced the peptide antifungal metabolites were developed through ethyl methane sulfonate (EMS) mutagenesis and used for in vivo experimentation. Reduced percentage of seed germination by mutants DM1 and DM2 (22.5% and 25%, respectively) and a high percent of disease incidence (69.3% and 67%, respectively) compared to wild-strain HKA-15 (80% seed germination and 27% disease incidence) indirectly indicated the role of peptide metabolite on disease suppression. Plants treated with AFM(-) clones showed stunted growth and the presence of pepperlike microsclerotia in the stem tissues. Light and scanning electron microscopic studies clearly showed the effect of peptide antibiosis on hyphal morphology. Exposure to crude extracts of antibiotics produced abnormal contraction of fungal cytoplasm, granulation, and fragmentation of hyphal mycelia and cell lysis. The presence of bacterial cells in the lumen of degrading fungal mycelium suggested a direct involvement of Paenibacillus sp. HKA-15 in the lysis of Rhizoctonia bataticola.

Cytoplasmic contractions in growing fungal hyphae and their morphogenetic consequences

Video-enhanced light microscopy of the apical and subapical regions of growing hyphae of several fungal species revealed the existence of momentary synchronized motions of subcellular organelles. First discovered in a temperature-sensitive morphological mutant (ramosa-1) of Aspergillus niger, these seemingly spontaneous cytoplasmic contractions were also detected in wild-type hyphae of A. niger, Neurospora crassa, and Trichoderma atroviride. Cytoplasmic contractions in all fungi lasted about 1 s. Although the cytoplasm recovered its motility and appearance, the contraction usually led to drastic changes in Spitzenkörper (apical body) behavior and hyphal morphology, often both. Within 10 s after the contraction, the Spitzenkörper commonly became dislodged from its polar position; sometimes it disassembled into phase-dark and phase-light components; more commonly, it disappeared completely. Whether partial or complete, the dislocation of the Spitzenkörper was always accompanied by a sharp reduction or cessation of growth, and was usually followed by marked morphological changes that included bulbous hyphal tips, bulges in the hyphal profile, and formation of subapical and apical branches. The cytoplasmic contractions are vivid evidence that the most conspicuous cell organelles (membrane-bound) in living hyphae are interconnected via a contractile cytoskeletal network.

Ionic currents and cytoplasmic streaming in Amoeba proteus

Amoeba proteus may drive a current through themselves, and this current may determine the direction of cytoplasmic streaming. Cytoplasm streams from the more contracted tail or uroid region to the extending pseudopods. The positions of the uroid and pseudopods change as the amoeba changes direction. The membrane potential of the amoeba may change with location in the cell. This voltage gradient may regulate the magnitude and direction of cytoplasmic streaming. Bingley and Thompson (1962), recorded from amoebae using intracellular microelectrodes and reported that the membrane potential of the uroid region was more negative than that of the pseudopods. In contrast, Nuccitelli et al. (1977), using a vibrating probe, observed that current entered the uroid region and exited through the pseudopods. They concluded that the uroid region was more depolarized than the pseudopods. We revisited this issue by recording membrane potential simultaneously from two regions of the amoeba and by recording ionic currents using the two‐electrode voltage clamp. Our data support the findings of Nuccitelli et al. Cytoplasm may stream from more depolarized to more hyperpolarized regions. Moreover, overshooting depolarizations of the cell may produce global cytoplasmic contractions and rounding of the amoeba.

Mechanical Properties of Xenopus Egg Cytoplasmic Extracts

Cytoplasmic extracts prepared from Xenopus laevis eggs are used for the reconstitution of a wide range of processes in cell biology, and offer a unique environment in which to investigate the role of cytoplasmic mechanics without the complication of preorganized cellular structures. As a step toward understanding the mechanical properties of this system, we have characterized the rheology of crude interphase extracts. At macroscopic length scales, the extract forms a soft viscoelastic solid. Using a conventional mechanical rheometer, we measure the elastic modulus to be in the range of 2–10 Pa, and loss modulus in the range of 0.5–5 Pa. Using pharmacological and immunological disruption methods, we establish that actin filaments and microtubules cooperate to give mechanical strength, whereas the intermediate filament cytokeratin does not contribute to viscoelasticity. At microscopic length scales smaller than the average network mesh size, the response is predominantly viscous. We use multiple particle tracking methods to measure the thermal fluctuations of 1 μm embedded tracer particles, and measure the viscosity to be ∼20 mPa-s. We explore the impact of rheology on actin-dependent cytoplasmic contraction, and find that although microtubules modulate contractile forces in vitro, their interactions are not purely mechanical.

Key differences between lateral and apical branching in hyphae of Neurospora crassa

We examined in fine detail growth kinetics and intracellular events during lateral and apical branching in hyphae of Neurospora crassa. By high-resolution video-enhanced light microscopy, we found remarkable differences in the events preceding lateral vs apical branching. While apical branching involved a significant disturbance in the apical growth of the parental hypha, lateral branching occurred without any detectable alterations in the growth of the parental hypha. Prior to the emergence of a lateral branch, an incipient Spitzenkörper was formed about 12–29 μm behind the apex. Lateral branch formation did not interfere with the elongation rate of the primary hypha, the shape of its apex or the behavior of its Spitzenkörper. In sharp contrast, apical branching was preceded by marked changes in physiology and morphology of the parental hypha and by a sharp drop in elongation rate. The sequence involved a cytoplasmic contraction, followed by a retraction, dislocation, and disappearance of the Spitzenkörper; hyphal elongation decreased sharply and a transient phase of isotropic growth caused the hyphal apex to round up. Growth resumed with the formation of two or more apical branches, each one with a Spitzenkörper formed by gradual condensation of phase-dark material (vesicles) around an invisible nucleation site. The observed dissimilarities between lateral and apical branching suggest that these morphogenetic pathways are triggered differently. Whereas apical branching may be traced to a sudden discrete disruption in cytoplasmic organization (cytoplasmic contraction), the trigger of lateral branching probably stems from the subapical accumulation of wall precursors (presumably vesicles) reaching a critical concentration. Index Descriptors: Apical branching; Lateral branching; Hyphae; Neurospora crassa; Spitzenkörper; Video microscopy

Reactivation of Ca2+-dependent cytoplasmic contraction in permeabilized cell models of the heliozoon Echinosphaerium akamae.

Permeabilized cell models of the large heliozoon Echinosphaerium akamae were prepared by treatment with 100 mM EGTA or 1% Triton X-100. When > 10(-6) M Ca(2+) was added to the EGTA-permeabilized cells, axopodial cytoplasm became contracted and several swellings were formed along the axopodial length. Axonemal microtubules remained intact, while higher concentration of Ca(2+) (> 10(-4) M) induced microtubule disassembly and complete breakdown of the axopodia. In Triton-permeabilized cells, cytoplasmic contraction and relaxation of the cell body were induced repeatedly by successive addition and removal of Ca(2+). The contraction did not require ATP, and was not inhibited by cytochalasin B. Electron microscopy showed, in EGTA-permeabilized axopodia, contractile tubules became granulated by the addition of Ca(2+). From these observations, it is strongly suggested that Ca(2+)-dependent granulation of the contractile tubules is responsible for the axopodial contraction.

The status of Haemogregarina mansoni Sambon and Seligmann from Zamenis flagelliformis Laurenti.

Haemogregarina mansoni Sambon and Seligmann 1907, a parasite of the coachwhip snake (Masticophis flagellum) is redescribed on the basis of sporogonic stages obtained in the experimental vector Aedes aegypti and designated as Hepatozoon mansoni from hosts collected in north Florida. Gamonts average 14.7 x 4.7 (13-16 x 4-6) and are not recurved, with nuclei situated in the second quarter of the gamont. Erythrocyte cytoplasm is often thin, appearing partially dehemoglobinized, or contracted into a central mass with infected erythrocytes always distorted, longer, and more slender than uninfected cells. Sporogony occurred within the head and thorax of A. aegypti. Oocysts were spherical to ovoid, 144.2 x 126.1 (79-198 x 69-178), containing on average 29.2 (7-64) sporocysts. Sporocysts were spherical to ovoid, 33.1 x 29.8 (19-48 x 18-44), with 20.2 (12-32) sporozoites contained within. Experimental infection in Tantilla relicta produced gamonts that did not differ from those in M. flagellum, but dehemoglobinization and cytoplasmic contraction of the host erythrocyte did not occur, and persistent merogonic stages were not present in the tissues.