Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.     

The Fine Structure of Acanthamoeba astronyxis,with Special Emphasis on Encystment1

ABSTRACT. The fine structure of the trophozoite, encysting cells, and the cyst of Acanthamoeba astronyxis has been examined. In the trophic form a microtubule organizing center was associated with a well developed Golgi complex. During encystment the organelles of the amoeba changed considerably. The profiles of rough endoplasmic reticulum elongated and were often arranged in circles of multilayered concentric systems, enclosing mitochondria, the nucleus, or other inclusions. The mitochondria showed a tendency toward elongation and constriction. One or two nucleolus-like bodies appeared in the nucleus. Lipid droplets increased considerably in amount and were distributed individually or as aggregates. The mature cyst was star-shaped and surrounded by an almost circular exocyst and an endocyst that was closely apposed to the cell membrane. Both walls differed in their thickness and granulation. The exocyst was continuous over the entire cyst, while the endocyst was interrupted by gaps, ostioles. in the region of the rays. Within the ostioles was a bell-shaped structure, the operculum. The latter was composed of a granular material comparable in electron density to that of the endocyst.     

A specific primer pair for the diagnosis and identification of Acanthamoeba astronyxis by random amplified polymorphic DNA-polymerase chain reaction

Random amplified polymorphic DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that is useful for species identification. In this study, a distinctive 485-bp band in Acanthamoeba astronyxis band patterns was found, using the OPC20 primer (ACTTCGCCAC). The band specificity was confirmed by hybridization, using it as a probe, against all OPC20 amplifications from different Acanthamoeba species. Once the fragment was sequenced, we used it to design a specific primer pair that was useful for the identification of different isolates as A. astronyxis species.     

Mitotic Inhibition and Nucleic Acid Synthesis in Amoeba Nuclei

SEVERAL strains of large mononucleate amoebae contain specific proteins which inhibit mitosis and eventually kill amoebae of strains other than their own^1,2. We now describe experiments to show that this lethal antimitotic factor (AF) inhibits RNA synthesis when injected into susceptible cells.     

Association of Human SCFSubunit p19with Interphase Centrosomes and Mitotic Spindle Poles

InSaccharomyces cerevisiae,the initiation of DNA replication and mitotic progression requires SKP1p function. SKP1p is an essential subunit of a newly identified class of E3 ubiquitin protein ligases, the SCF complexes, that catalyze ubiquitin-mediated proteolysis of key cell-cycle-regulatory proteins at distinct times in the cell cycle. SKP1p is also required for proper kinetochore assembly. Little is known about the corresponding human homolog, p19^SKP1, except that it is expressed throughout the cell cycle and that it too is a component of an S-phase-regulating SCF–E3 ligase complex. Here we show by immunofluorescence microscopy that p19^SKP1localizes to the centrosomes. Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis. These findings suggest that p19^SKP1is a novel component of the centrosome and the mitotic spindle, which, in turn, implies a physiological role of this protein in the regulation of one or more aspects of the centrosome cycle.     

Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.     

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis Survival within Amoebal Cysts

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.     

Comparative Fine Structural Investigations of Interphase and Mitotic Nuclei of Vampyrellid Filose Amoebae

The nuclei of trophozoites and digestive cysts as well as mitotic nuclei of several species of the vampyrellids Vampyrella, Gobiella, Hyalodiscus, Arachnula , and Leptophrys were investigated by electron microscopy. Except for some species of the genus Hyalodiscus , the vampyrellids are generally multinucleate. The nuclei of the trophozoite stage are in interphase. These nuclei are spherical, except for the genus Arachnula , which reveals elongated nuclei. In digestive cysts of all vampyrellids the nuclei enlarge and the pars granulosa of the nucleoli becomes prominent. Karyokineses take place synchronously in older digestive cysts, which transform into reproductive cysts. The nuclei divide by closed intranuclear orthomitosis. In telophase the old nuclear envelope disintegrates and a new one is rearranged. Only in the genus Leptophrys the nuclear envelope decomposes before telophase. Neither centrioles nor MTOC-plaques have been found in any stage of mitosis. After karyokinesis the cell divides inside the cyst or when leaving the cyst.     

Evaluation of taxonomic validity of four species of Acanthamoeba: A. divionensis, A. paradivionensis, A. mauritaniensis, and A. rhysodes, inferred from molecular analyses

The taxonomy of Acanthamoeba spp., an amphizoic amoeba which causes granulomatous amoebic encephalitis and chronic amoebic keratitis, has been revised many times. The taxonomic validity of some species has yet to be assessed. In this paper, we analyzed the morphological characteristics, nuclear 18s rDNA and mitochondrial 16s rDNA sequences and the Mt DNA RFLP of the type strains of four Acanthamoeba species, which had been previously designated as A. divionensis, A. parasidionensis, A. mauritaniensis, and A. rhysodes. The four isolates revealed characteristic group II morphology. They exhibited 18S rDNA sequence differences of 0.2-1.1% with each other, but more than 2% difference from the other compared reference strains. Four isolates formed a different clade from that of A. castellanii Castellani and the other strains in morphological group II on the phylogenetic tree. In light of these results, A. paradivionensis, A. divionensis, and A. mauritaniensis should be regarded as synonyms for A. rhysodes.     

Francisella tularensis Type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks Postinfection

Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown and to the organism''s potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype is caused by factor(s) secreted by amoebae and/or F. tularensis into the coculture medium. Further, our results indicate that in contrast to the live vaccine strain LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks postinfection and that the induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that interactions between F. tularensis strains and amoebae may play a role in the environmental persistence of F. tularensis.Francisella tularensis is the etiological agent of the zoonotic disease tularemia, also known as rabbit fever (35, 53). Strains belonging to F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, which are both prevalent in the Northern Hemisphere, cause the majority of reported cases of tularemia (36). Subspecies tularensis is highly contagious, with an infectious dose of 1 to 10 bacteria, and is associated with more severe disease (21). Though described more than a century ago as a disease common among hunters and trappers, tularemia has recently been reported in areas with no previous known risk (20, 25, 31, 42). F. tularensis infects a broad range of wildlife species (36), and a number of arthropods, such as ticks and flies, are known to be vectors (36, 49). Humans are usually infected either through an insect bite or by inhalation of aerosolized bacteria (49). Tularemia can be fatal in up to 30% of untreated cases (36, 49), with the mortality rate reaching 90% in pneumonic infections, as described in early studies conducted with vaccinated human volunteers (44-46, 49). Due to its highly infectious nature and its potential for use as a bioterrorism agent, F. tularensis has been classified as a class A biothreat pathogen by the Centers for Disease Control and Prevention (CDC), which has mandated that human tularemia be a reportable disease since 2000 (15, 37). In addition, the absence of a licensed vaccine for prophylaxis (36) makes understanding the virulence mechanisms used by this pathogen imperative for the development of efficacious measures to prevent or treat human disease.Though F. tularensis has been isolated from more than 250 wildlife species (21), the acute nature of the infections and the resultant high mortality rates in these hosts indicate that the bacterial reservoir(s) in nature have yet to be identified. Tularemia outbreaks involving F. tularensis subsp. holarctica have often been linked to water sources (6, 40), and a positive PCR field test was reported for Francisella during such an outbreak in Norway (5). Abd et al. reported that the F. tularensis live vaccine strain LVS is able to survive and replicate in the amoeba Acanthamoeba castellanii (1), suggesting a potential link between amoeba-Francisella interactions and environmental persistence. A. castellanii, a free-living environmental amoeba, is known to serve as a reservoir for a number of pathogenic microorganisms (24). However, to date, interactions of virulent F. tularensis subspecies tularensis strains with amoebae have not been documented. The ability of several human intracellular pathogens, including Legionella pneumophila and Mycobacterium avium, to infect and survive within amoebae has been well characterized (10, 12). In addition to playing a role in environmental survival and dissemination, growth in A. castellanii has been shown to enhance the ability of L. pneumophila and M. avium to survive and replicate in host macrophages (10, 12) and to enhance the virulence of both species in mice (7, 12). Since F. tularensis species are facultative intracellular pathogens that primarily survive in macrophages, probing the Francisella-amoeba interaction may provide insights into Francisella pathogenesis, as well as environmental survival. In this study, we investigated the ability of virulent type A strains of F. tularensis to survive in A. castellanii with a focus on understanding the role of Francisella-amoeba interactions in environmental persistence.     

Nuclei, septation, branching and growth of Geotrichum candidum.

A study was made of growth, septation and branching in Geotrichum candidum, a mould which forms physiologically complete septa. A correlation was observed between septation and branch initiation; branches were almost invariably formed just behind septa. Primary branches and their parent intercalary compartments initially increased in length at an exponential rate before eventually attaining a constant rate of extension. The whole branching system (which eventually contained seven tips) produced by an intercalary compartment increased in length exponentially until it attained a total length of at least 1-5 mm. The total length and the number of nuclei of undifferentiated mycelia increased exponentially at the same specific growth rate. The results suggest that nuclei divide just before or just after arthrospore formation.     

Taxonomic structure of phytoplankton assemblages in Crater Lake, Oregon, U.S.A.

SUMMARY. 1. A taxonomic analysis of 171 phytoplankton samples obtained from Crater Luke, Oregon, between 1985 and 1987 revealed 132 taxa in the upper 250 m of the water column. The greatest temporal variation in taxonomic structure occurred between 40 and 80 m below the water surface, a depth range which corresponded to the zone of maximum primary production.
2. Phytoplankton cell biovolume in the upper 20 m of the water column was relatively high during the summer months, a period when Nitzschia gracilis was dominant in the epilimnion. However, 72% or more of the cell biovolume between 0 and 200 m was distributed below 20 m and, during the winter and spring months, 61% was found below 80 m.
3. Cluster analysis identified a sparse, temporally ubiquitous flora which was modified to various degrees when environmental conditions became favourable for the growth of a few dominant taxa. These surges ot dominance by individual taxa accounted for 2 to 5-fold increases in cell biovolume and generated a pronounced taxonomic discontinuity between the floras in the epilimnion and hypolimnion.
4. While the taxonomic structure of the phytoplankton in the epilimnion corresponded closely with the structure found in a 1978–80 study, the flora below the metalimnion was more diverse and less predictable in species composition than the pattern reported in the earlier study.     

Permeability and Structural Characteristics of Isolated Nuclei from Chaetopterus Eggs

The germinal vesicle of the mature Chaetopterus egg is invested by an envelope which can be seen in electron micrographs to contain "pores" in its bilaminar structure. While under continuous microscopic observation, individual germinal vesicles were isolated in various test solutions by an extremely gentle method. Repeated measurements of nuclear diameter and of optical path differences with an interference microscope provided data on changes in mass after isolation. It was found that bovine serum albumin can readily penetrate the nuclear envelope of the isolated nucleus and that there are soluble elements which rapidly diffuse out. A relatively non-diffusible mass is lost at a much slower rate, the proportion of soluble to non-diffusible mass being dependent on the ionic environment. Calcium and manganese increase the proportion of the non-diffusible mass at the expense of the soluble components, while potassium decreases it. The shape and size of the isolated nucleus is at least partially dependent on the non-diffusible mass of its interior. Digestion with trypsin causes a complete structural collapse and loss of the non-diffusible elements, along with disappearance of the nucleolus. The nucleus shrinks and becomes wrinkled. A small residual mass is left which is probably associated with the nuclear envelope. Digestion with RNase or DNase causes no detectable effect on the isolated nucleus. Micromanipulation of the isolated nucleus consistently indicates that there are strands emanating from the nucleus. They may be up to several hundred microns long, are structurally strong, and are not destroyed by trypsin, RNase, or DNase. Electron micrographs of thin sections of intact cells show that the germinal vesicle is highly irregular in outline with complex evaginations extending into the cytoplasm. With the light microscope the isolated nucleus looks spherical and smooth and no emanating strands can be seen. The nature of the strands is not known.     

Mitotic Entry: A Matter of Oscillating Destruction

Entry into mitosis is essentially driven by cyclin B1 and its associated catalytically active partner Cdk1. While cyclin B1 is kept cytoplasmic throughout interphase, nuclear accumulation occurs just prior to mitosis. This restriction is thought to be part of an oscillating mechanism to properly time mitotic entry. A novel nuclear SCF-type mammalian E3 ligase defined by the F-box containing protein NIPA (nuclear interaction partner of ALK), SCFNIPA, targets nuclear cyclin B1 in interphase while it allows for accumulation at G2/M. Thus, oscillating ubiquitination of nuclear cyclin B1 driven by the SCFNIPA complex contributes to the timing of mitotic entry in the mammalian cell cycle.