Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 x 10(-1) and 1.14 x 10(4) cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 +/- 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (> or =98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.     

Specificity of bacteriolytic enzyme II from a soil amoeba, Hartmannella glebae.

Acetylene reduction activity of intact rice plants was measured in closed assay chambers with plants grown in water culture. Acetylene was added to the liquid medium, and the ethylene formed was measured from both gas and liquid phases. After cutoff of mineral nitrogen supply and inoculation of fresh soil, rice plants grown from the seedling stage in water culture exhibited acetylene reduction activity after a lag period. However, rice plants grown in a paddy field and transferred to water culture were more suitable for N₂ fixation studies because of their higher, less variable acetylene reduction activity. The time course of acetylene reduction was monitored by continuous circulation of gas between the gas phase and the liquid phase, and the result showed an initial 2- or 3-h period of lower activity, followed by increased and almost constant activity up to 24 h. The effects on acetylene reduction activity of aeration, ammonium, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea addition are reported. Ammonium was inhibitive at 0.33 mM, and its depressive effect was alleviated by ammonium uptake by the plants.     

Inhibition of cytokinesis by a lipid metabolite, psychosine

Although a number of cellular components of cytokinesis have been identified, little is known about the detailed mechanisms underlying this process. Here, we report that the lipid metabolite psychosine (galactosylsphingosine), derived from galactosylceramide, induced formation of multinuclear cells from a variety of nonadherent and adherent cells due to inhibition of cytokinesis. When psychosine was added to the human myelomonocyte cell line U937, which was the most sensitive among the cell lines tested, cleavage furrow formed either incompletely or almost completely. However, abnormal contractile movement was detected in which the cellular contents of one of the hemispheres of the contracting cell were transferred into its counterpart. Finally, the cleavage furrow disappeared and cytokinesis was reversed. Psychosine treatment also induced giant clots of actin filaments in the cells that probably consisted of small vacuoles with filamentous structures, suggesting that psychosine affected actin reorganization. These observations could account for the formation of multinuclear globoid cells in the brains of patients with globoid cell leukodystrophy, a neurological disorder characterized by the accumulation of psychosine due to galactosylceramidase deficiency.     

Inhibition of plant cytokinesis by deoxyguanosine and caffeine

The efficacy of 2′-deoxyguanosine (GdR) on the induction of binucleate cells in onion root tips was studied. GdR inhibits plant cytokinesis during the last part of the mitotic period. Apparently, this deoxynucleoside causes a considerable slowing down in the mitotic rate (50%) and also has a slight effect as an inhibitor of cytokinesis (30%). The effect on the mitotic rate was established with 1 mM colchicine (metaphase rate) and with 5 mM caffeine (telophase rate). Since 1 mM GdR has a remarkable potentiating action on inhibition of cytokinesis by caffeine, we postulate that GdR, or a phosphorylated derivative from it, inhibits cytokinesis by a similar mechanism as caffeine. According to our postulation, GdR interferes with a certain ATPase activity required for membrane fusion of the Golgi vesicles during plant cytokinesis.     

Associations of the soil amoeba Hartmannella rhysodes with the bacterial causative agents of plague and pseudotuberculosis in an experiment]

Some aspects of relationships between soil ameba and the causative agents of plague and pseudotuberculosis, capable of forming natural associations, are considered. Ameba can phagocytose bacteria causing plague and pseudotuberculosis. Cases of the preservation of individual bacterial cells at the stationary phase and in precysts of amebae have been registered.     

The movement of fluids and substances in the testis

Three aspects of the control of movements of fluids and substances into, out of and inside the testis are discussed: the tubular barrier, the interstitial extracellular fluid and the testicular blood vessels. The functional basis for the tubular barrier is twofold; there are significant differences in the concentration of many substances inside and outside the tubules and marker substances enter or leave the tubular fluid at widely different rates, depending on lipid solubility and the presence of specific carrier systems. The anatomical basis for this barrier appears to be the specialized junctions between adjacent pairs of Sertoli cells. The barrier develops only at puberty, as the first cells undergo meiosis, but the development may not be as sudden as previously believed. The barrier breaks down after efferent duct ligation when spermatogenesis is disrupted. Techniques for measuring the volume, the turnover rate, the composition and fate of the interstitial extracellular fluid are described, and the unsatisfactory features of the presently available techniques for collecting this fluid for analysis are emphasized. There is a relationship between the fluid in the testis and lymph from vessels in the spermatic cord and lymph may be important for the transport of hormones to the general circulation in some circumstances and to other organs close to the testis. The testicular blood vessels display certain unusual features, a very high susceptibility to the toxic effects of cadmium salts, a high level of alkaline phosphatase activity in all endothelial cells but only after puberty and a high level of gamma-glutamyl transpeptidase in the endothelial cells of the arterioles and the testicular artery. These same cells are the site for a specific transport system for leucine and phenylalanine, with kinetic characteristics similar to the system in brain. Flow of blood may limit hormone secretion by the aspermatogenic testis, but diffusion limitation may also be important under some circumstances. A fuller understanding of the ways in which substances move around in the testis, particularly how they cross the endothelial cell layer or penetrate into the tubules, will be important for a better appreciation of testicular function.     

Inhibition of in vitro attachment of Giardia trophozoites by mucin

An enzyme-linked immunoabsorbent assay was developed to quantify the number of Giardia sp. trophozoites attached to a substratum. Trophozoites were allowed to attach for 5 or 10 min to untreated or modified substratum, or allowed to attach in the presence of cytoskeletal inhibitors. Microtubule inhibitors, cochicine and nocodazole, were ineffective in blocking attachment, although the actin-disrupting agent cytochalasin B produced significant inhibition of attachment. Three different mucins were associated with significant inhibition of Giardia trophozoite attachment, which was reversed by treating the mucin-coated wells with 0.1% poly-L-arginine. Examination of mucin-treated substrata by high-resolution field emission scanning electron microscopy showed the presence of thin linear fibrils, whereas treatment of mucin-coated wells with poly-L-arginine revealed similar fibrils but of thicker dimensions. These data support the concept that mucin may inhibit Giardia sp. trophozoite attachment in vitro by electrostatic repulsion and that this can be eliminated by treatment of mucin-coated surfaces with polycationic agents, such as poly-L-arginine or poly-L-lysine.     

Inhibition of cytokinesis by lead (Pb2+) in a centric diatom.

Effect of inorganic and organo lead has been studied on the mitosis of a centric diatom Cyclotella meneghiniana f. unipunctata. Binucleate cells were formed in the presence of different concentrations of Pb2+ (1.0, 2.0, 3.0 and 5.0 mM) due to inhibition of cell plate formation. Lead at 5.0 mM concentration was more inhibitory than the other concentrations. Organo lead was a powerful depressant of cytokinesis than inorganic lead. Failure of cytokinesis might be due to disruption of microtubules. Formation of distinct nuclei delayed post incubation cell divisions suggest partial damage of mitotic spindles.     

The role of centromere-binding factor 3 (CBF3) in spindle stability, cytokinesis, and kinetochore attachment.

The spindle midzone is critical for spindle stability and cytokinesis. Chromosomal passenger proteins relocalize from chromosomes to the spindle midzone after anaphase onset. The recent localization of the inner-kinetochore, centromere-binding factor 3 (CBF3) complex to the spindle midzone in budding yeast has led to the discovery of novel functions for this complex in addition to its essential role at kinetochores. In G1/S cells, CBF3 components are detected along dynamic microtubules, where they can "search-and-capture" newly replicated centromeres. During anaphase, CBF3 is transported to the microtubule plus-ends of the spindle midzone. Consistent with this localization, cells containing a mutation in the CBF3 subunit Ndc10p show defects in spindle stability during anaphase. In addition, ndc10-1 cells show defects during cytokinesis, resulting in a defect in cell abscission. These results highlight the importance of midzone-targeted proteins in coordinating mitosis with cell division. Here we discuss these findings and explore the significance of CBF3 transport to microtubule plus-ends at the spindle midzone.     

Inhibition of ADH-enhanced transepithelial urea and water movement by prostaglandins

The effects of PGF_2α and PGE₂ on transepithelial urea flux and osmotic water flow were evaluated in toad bladders. Mucosal to serosal urea flux and osmotic water flow were not changed from basal values by the addition of either prostaglandin to the serosal bath. However, treatment with either PGF_2α or PGE₂ inhibited both urea flux and osmotic water flow in response to ADH stimulation in a concentration-dependent manner. The hydrosmotic response to ADH was more sensitive to prostaglandin inhibition than was urea flux. The inhibitory effect of the prostaglandins on ADH-enhanced urea flux was not dependent upon inhibition of the hydrosmotic response, since both PGF_2α and PGE₂ decreased urea flux in the absence of a trans-epithelial osmotic gradient. Prostaglandin E₂ was a more potent inhibitor than PGE_2α of both ADH-enhanced urea flux and osmotic water flow. The PGF_2α antagonism of osmotic water flow was apparently competitive, while antagonism of urea flux was apparently non-competitive. The results are consistent with the hypothesis of the existence of a “spare” population of prostaglandin receptors that modulate water flow, but the absence of a “spare” prostaglandin receptor population with respect to the modulation of urea flux.     

Inhibition of cytokinesis and altered contractile ring morphology induced by cytochalasins in synchronized PtK2 cells

PtK2 cells and antigen affinity-purified antibodies to actin and tubulin were used to study the effects on mitosis of cytochalasin B (CB) and dihydrocytochalasin B (H₂CB). PtK2 cells were synchronized in S phase by a double thymidine block and CB or H₂CB was added at various concentrations at the time of release from the block. CB- and H₂CB-treated populations, and control populations not treated with either drug, progressed synchronously through G2 and into mitosis with similar time courses. By both phase contrast and immunofluorescence microscopy, CB- and H₂CB-treated cells appeared normal in terms of chromosome condensation, spindle formation and spindle dynamics throughout prophase, metaphase and early anaphase. At late anaphase, contractile ring staining with actin antibody was not normal. High actin antigenicity remained localized in the region of the contractile ring; however, it appeared atypically as a punctate line of fluorescence across the midzone. Although some degree of furrowing was often seen to occur, at suitable concentrations of CB or H₂CB only binucleate G1 cells formed. Scanning electron microscopy (SEM) of normal and CB- and H₂CB-treated cells verified that cleavage furrowing did not proceed normally in treated cells. Large numbers of microvilli and surface blebs occurred in the normally smooth furrow region in these treated populations. These results suggest that intact microfilament function is not necessary for progression from S phase into mitosis, for spindle formation or for chromosome movement. They indicate that CB and H₂CB lead to formation of binucleated cells by causing aberrant cleavage furrowing and inhibition of contractile ring microfilaments.