Development of cyst-like cells of the flagellate Leptomonas oncopelti in the midgut of the hemipteran Oncopeltus fasciatus

The structure of cyst-like cells of Leptomonas oncopelti (Trypanosomatidae) found in the midgut of the bug Oncopeltus fasciatus (Lygaeidae) was examined with light and electron microscopy. The formation of "cysts" begins with an unequal division of active flagellates with promastigote configuration. Cytokinesis starts on the lateral side of the flagellate, and then the cleavage furrow moves toward the apical end of the cell. The anterior part of a smaller daughter cell, referred to as cell C1, remains associated with the flagellum of maternal promastigote. C1 divides twice to give rise first to two equivalent cells (C2), and then to four morphologically similar cells (C3). C2 join with each other, and afterwards C3 attach between themselves as well via short cytoplasmic outgrowths, which appear instead flagella. In the point of outgrowth attachment of only one C2 and then of only one C3 to maternal flagellum zonal desmosomes occur. C1--C3 of L. oncopelti are similar to so-called straphangers (cyst-like parasites attached to the flagellum of maternal promastigote) known in some species of the genera Leptomonas and Blastocrithidia. Basal bodies are present in C1 and C2 but not in C3. DNA fibrils in the kinetoplast lack their common circular configuration, they progressively condense to form a disordered mass. C3 chromatin becomes denser to acquire eventually a characteristic "labyrinthine structure" looking like a huge bundle of whorled filaments 3-5 nm width. Inside this bundle there are channels of 10-12 nm in diameter filled with karyoplasm. On becoming ovoid, C3 are separated from the maternal promastigote flagellum and differentiate into mature "cysts". Straphangers C1--C3 and mature "cysts" lack any visible outer extracellular protective envelope (cyst wall). Instead, these cells have a cortical complex made of a reinforced plasmatic membrane underlined by a layer of a dense granular cytoplasm free of subpellicular microtubules. The mature "cyst" endoplasm shows a high electron density, and because of this identification of the majority of cellular organelles is next to impossible. Nevertheless, in both C3 and mature "cysts" some unusual membranes are seen composed of two electron lucent layers, with a single electron dense layer in between.     

Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote- containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.     

Ultrastructural visualization of lipids in trypanosomatids

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis. T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.     

Unique features of the motility and structures in the flagellate polar region of Campylobacter jejuni and other species: an electron microscopic study

Similarly to Helicobacter pylori but unlike Vibrio cholerae O1/O139, Campylobacter jejuni is non‐motile at 20°C but highly motile at ≥37°C. The bacterium C. jejuni has one of the highest swimming speeds reported (>100 μm/s), especially at 42°C. Straight and spiral bacterial shapes share the same motility. C. jejuni has a unique structure in the flagellate polar region, which is characterized by a cup‐like structure (beneath the inner membrane), a funnel shape (opening onto the polar surface) and less dense space (cytoplasm). Other Campylobacter species (coli, fetus, and lari) have similar motility and flagellate polar structures, albeit with slight differences. This is especially true for Campylobacter fetus, which has a flagellum only at one pole and a cup‐like structure composed of two membranes.     

Presence of genes of ribosomal and transfer RNAs in kinetoplast DNA from two Crithidia species

The coding properties of kinetoplast DNA from two respresentatives of the order Kinetoplastidae--Crithidia oncopelti and C. fasciculata--were studied. Experiments on hybridization of the whole network and fraction of minicircles with labelled 23S and 16S rRNA and with tRNA isolated from kinetoplasts of C. oncopelti clearly demonstrated the presence of the genes for these RNAs in the whole network and their absence in the minicircles. It may be thus concluded that the genes of ribosomal and transfer RNAs are localized in the maxicircular molecules. Similar efficiency of hybridization of rRNAs from C. oncopelti with kDNA from C. fasciculata and C. oncopelti revealed significant conservativity of ribosomal genes in the protozoa under study.     

Effect of temperature on the fluidity of boar sperm membranes

Fluidity was used to assess changes in molecular organization of boar spermatozoa plasma membranes from (1) the head and (2) the rest of the sperm body and acrosome as a consequence of temperature. The initial fluidity of the head membranes at 25 degrees C was less than that of the sperm body membranes (P less than 0.05). When held at 25 degrees C, the fluidity of the head membranes decreased for 105 +/- 8 min and then stabilized for the remainder of the 160-min incubation. Calcium (10 mM) caused a significantly greater decrease in fluidity. The fluidity of the sperm body membranes increased slightly over time in the absence of Ca2+, but decreased significantly with Ca2+. Cooling from 25 to 5 degrees C and subsequent heating to 40 degrees C (0.4 degrees C/min) caused marked alterations in the fluidity of each membrane. Cooling the head membranes prevented the fluidity increase seen at 25 degrees C, while reheating caused a dramatic decrease in fluidity. Fluidity of the head membranes was now unaffected by Ca2+. Lipid phase transitions, indicated by sharp break points in data curves, were detected at the onset of reheating (7 +/- 3 C) and at 23 +/- 4 degrees C during reheating. Fluidity of the sperm body membranes decreased slightly and in a linear fashion with Ca2+. Without Ca2+, the sperm body membranes showed an additional lipid phase shift at 31 +/- 5 degrees C, which led to a rapid fall in fluidity. These results suggest that the fluidity, and therefore the molecular structure, of sperm head and body membranes differ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the rumen flagellate Neocallimastix frontalis.

The vast increase in the population density of the rumen flagellate Neocallimastix frontalis shortly after the host animal has commenced eating is caused by stimulation of a reproductive body on a vegetative phase of the organism to differentiate and liberate the flagellates. The stimulant is a component of the host's diet. The vegetative stage of N. frontalis bears a strong morphological resemblance to that of certain species of aquatic phycomycete fungi, and consists of a reproductive body borne on a single, much branched rhizoid. The flagellates liberated in vivo within 15 to 45 min of feeding lose their motility within I h and develop into the vegetative phase, thus producing a rapid decrease in population density of the flagellates. Conditions for maximum flagellate production are similar to those occurring in the rumen: pH 6-5, 39 degrees C, absence of O2, presence of CO2. Differentiation of the reproductive body is inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The organism was cultured in vitro in an undefined medium in the absence of bacteria or other flagellates.     

Feeding spectra and pseudoconoid structure in predatory alveolate flagellates

It is revealed experimentally that predatory alveolate flagellates Colpodella angusta Simpson et Patterson, C. edax Simpson et Patterson, and Voromonas pontica (Mylnikov, 2000) Cavalier-Smith, 2004 eat its prey by sucking out the insides through the front of its own body (rostrum). Feeding spectra of these predators include bodonids, percolomonads and colorless chrysomonads, while colorless euglenoids, cryptomonads, ciliates and naked amoebas are unsuitable food for them. Predators’ rostrum contains a microtubular structure (pseudoconoid) complemented by microtubular bands, micronemas or rhoptries. It is shown that the pseudoconoid begins near the kinetosomes of flagella and passes along the flagellate pocket into the rostrum. The pseudoconoid forms an unlocked cone in Colpodella angusta and Voromonas pontica, while, in Colpodella edax, it coils significantly without forming a cone. The similarity between the pseudoconoid and associated structures of predatory flagellates with analogous structures in other colpodellids, percinzoids, dinoflagellates and sporozoans is discussed.     

DNA-dependent RNA polymerase activity of isolated zooflagellates (Crithidia oncopelti) nucleoli

A fraction of nucleoli is isolated from zooflagellates (Crithidia oncopelti) nuclei, its DNA-dependent RNA polymerase activity is studied at different temperature, ionic strength and Mg2+, Mn2+ and antibiotic concentrations. The effect of some factors and alpha-amantine on RNA polymerase activity of exonucleolar chromatin was studied as a control. A comparison of heat denaturation of nucleoli and chromatin RNA polymerase activities within the temperature range 30--55 degrees C has revealed a higher thermosensitivity of nucleoli RNA polymerase. Substitution of Mg2+ with equivalent amount of Mn2+ results in a considerable decrease of rRNA synthesis in nucleoli. Nucleoli RNA polymerase activity in the presence of Mg2+ is sensitive to the elevation of ionic strength from 0.12 to 1.30 u; chromatin RNA polymerase activity in the presence of Mn2+ is maximal at high ionic strength (1.30 mu). alpha-Amantine and cycloheximide at high concentrations (10 and 200 mkg/ml) practically do not affect RNA polymerase activity of nucleoli. Nucleoli RNA polymerase of zooflagellates (Crithidia oncopelti) is similar to the A-form of the enzyme in higher eukaryotes.     

Membrane-Sensitive Conformational States of Helix 8 in the Metabotropic Glu2 Receptor, a Class C GPCR

The recent elucidation of the X-ray structure of several class A GPCRs clearly indicates that the amphipathic helix 8 (H8) is a conserved structural domain in most crystallized GPCRs. Very little is known about the presence and the possible role of an analogous H8 domain in the distantly related class C GPCRs. In this study, we investigated the structural properties for the H8 domain of the mGluR2 receptor, a class C GPCR, by applying extended molecular dynamics simulations. Our study indicates that the amphipathic H8 adopts membrane-sensitive conformational states, which depend on the membrane composition. Cholesterol-rich membranes stabilize the helical structure of H8 whereas cholesterol-depleted membranes induce a disruption of H8. The observed link between membrane cholesterol levels and H8 conformational states suggests that H8 behaves as a sensor of cholesterol concentration.     

Cytoplasmic Membranes and the Nuclear Membrane in the Flagellate Trichonympha

The structure of the nuclear and cytoplasmic membranes of Trichonympha, a complex flagellate, has been studied in the electron microscope. The nuclear membrane consists of two 70 A membranes, penetrated by numerous pores. Small (100 A) granules occur on the outer surface, around the rims of the pores. Granule-bearing membranes, only 30 to 40 A thick, form long, ribbon-shaped sacs, with 100 A granules on their outer surface. They apparently form close to the nucleus, from which they probably derive their granules. Smooth membranes occur in the parabasal bodies, which consist of stacks of 70 A membranes, joined at their edges in pairs to form flattened sacs. These can inflate and form cytoplasmic vesicles. A protein fibre is applied laterally to the pile of sacs. New sacs, replacing those lost by inflation, appear to form by a process involving the granular membranes, and there may be a transformation of one into the other. Starving eliminates granular membranes and results in a failure in the formation of new parabasal sacs. Refeeding reverses these effects. A parabasal body is a steady-state system, in which the rates of loss and gain of sacs are normally approximately equal. Parabasal bodies resemble the Golgi apparatus.     

长毛对虾精子发生的研究：Ⅰ.精子的形态结构

利用电子显微镜技术结合细胞化学染色方法,研究长毛对虾精子形态结构,结果显示:长毛对虾精子由圆球状球体部和钉子状棘突部组成;精子全长约7μm;棘突及与之相联的球体表面平滑无突起,而与棘突相对的球体底面有指状突起。透射和冷冻蚀刻电镜显示:长毛对虾精子棘突包括顶体锥和顶体帽两部分;顶体锥向外突出形成精子的棘突,顶体帽覆盖在球体部的胞质及核区上方。成熟精子胞质极度退化,仅靠顶体帽边缘可见1—2个线粒体。球体的中央区域为近球形,呈Feul-gen阳性反应,为精子的核区。其结构松散,电子密度低,属非浓缩型,内布许多絮状物质,外由许多长短不一的膜性结构不规则排列成不连续的核膜结构。研究结果认为:长毛对虾精子属不动无鞭毛精子类型,其棘突部并非鞭毛结构而是顶体的位置。     

Kinetoplast DNA of Crithidia oncopelti. Recognition of the principles of structural organization of minicircular DNA molecules

The heterogeneity of the kinetoplast minicircular DNAs from Crithidia oncopelti was studied by electron microscopy and restriction analysis. The associate of kinetoplast DNA comprises five main classes of minicircles sizing 0.42, 0.51, 0.62, 0.78 and 0.83 microns. Examination of cleavage patterns revealed that each class of the minicircles is heterogeneous in base sequence, but there are restriction fragments of the same size common for all these classes. A model of structural arrangement of minicircles of Crithidia oncopelti was proposed. This model is based on the block structure of minicircular molecules. Each minicircle is composed of one variable block (V-block), 810 base pairs, and from 1 to 4 more conservative blocks (C-blocks) each containing 445 base pairs.     

Ubiquinone-9 of the flagellates Crithidia oncopelti and Astasia longa

The content of ubiquinones (Co Q) of the Astasia longa and Crithidia oncopelti protozoa was studied. The protozoa were grown on an artificial nutrient broth. The cells were separated, washed, freeze-dried, and refluxed with KOH and pyrogallol in ethanol media. The hydrolyzate was concentrated. The residue was stored at -20 degrees and filtered. An ubiquinone fraction was isolated from the filtrate by TLC on silica gel. Identification of the ubiquinone homologues was carried out by reverse phase TLC and mass spectrometry. Ubiquinones were quantitated with respect to the difference in the density between the oxidized and reduced forms of Co Q at 275 nm. The A. longa and C. oncopelti flagellates were shown to contain ubiquinone-9 (Co Q9) at a concentration of 0.48 and 1.14 mumole/g dry cells, respectively. The higher Co Q level in zooflagellates as compared to that in phytoflagellates is discussed.     

Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

Unusual pressure dependence of the lateral motion of pyrene-labeled phosphatidylcholine in bipolar lipid vesicles.

The lateral mobility of a pyrene-labeled phosphatidylcholine probe in liposomes containing archaebacterial bipolar lipids has been studied isothermally as a function of pressure. The pressure-dependence of the probe mobility, R, is found to be slightly positive or zero in the temperature range of 17 - 48 degrees C. At temperatures > 48 degrees C, R becomes negative and decreases with temperature. The data indicate that lateral mobility only becomes appreciable at high temperatures. In addition, the R values obtained with other lipid membranes are much lower than that obtained with bipolar liposomes, implying that the membranes of archaebacterial liposomes are laterally immobile, as compared to other lipid membranes.     

A new class of the stramenopiles,Placididea Classis nova: description of Placidia cafeteriopsis gen. et sp. nov

A marine flagellate resembling Cafeteria roenbergensis (bicosoecids, stramenopiles) in cell shape and behavior of the cell while attached to substratum was collected from the coast of Japan. The flagellate was examined by light and electron microscopy, and the 18S rDNA was sequenced to elucidate its taxonomic and phylogenetic position. Ultrastructural features suggested that the flagellate is not a bicosoecid, but a relative of the recently described stramenopile, Wobblia lunata. 18S rDNA phylogenetic trees also revealed that the flagellate forms a monophyletic clade with W. lunata and that it is distantly related to Cafeteria and other bicosoecids. The flagellate differs from W. lunata due to its lack of wobbling motion as well as intracellular features such as the number of mitochondria, flagellar apparatus architecture, the presence of a paranuclear body and cytoplasmic microtubules. The similarity of 18S rDNA sequences was 81% between the flagellate and W. lunata. This new flagellate was described as Placidia cafeteriopsis gen. et sp. nov. Because the phylogenetic lineage comprised of W. lunata and P. cafeteriopsis was one of the major, deep-branching clades of the stramenopiles, the class Placididea (= Placidiophyceae) classis nova was proposed.     

Reversible expression of flagella in Campylobacter spp.

The in vitro phase variation of flagella and the transition rates between flagellate and aflagellate phenotypes in Campylobacter species including C. jejuni, C. coli, C. lari (thermophilic campylobacters), C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis were investigated. The change from the flagellate to aflagellate phenotype was detected in all of the 12 Campylobacter strains studied. When measured in a motility medium, flagellate to aflagellate transition in thermophilic campylobacters, C. fetus and C. hyointestinalis strains occurred at a rate of 1.8 x 10(-3) to 7.5 x 10(-3), 3.0 x 10(-4) to 7.8 x 10(-4) and 1.8 x 10(-5) to 7.7 x 10(-6) per cell per generation, respectively. Transition from aflagellate to flagellate phenotype occurred at a rate of 5.8 x 10(-6) to 9.3 x 10(-6) per cell per generation in thermophilic campylobacters and 1.0 x 10(-6) to 1.5 x 10(-6) in C. fetus strains. No reversion from aflagellate to flagellate phenotype could be detected in C. hyointestinalis strains. It was concluded that the ability to reversibly express flagella was inherent in the wild-type strains and the transition rates for both directions were consistent for each strain.     

An analysis of the supramolecular organization of the olfactory neuroepithelium in the rat by freeze etching with rotary platinum-carbon shadow-casting

The rat olfactory epithelium was analysed by freeze-deep etching and Pt/C rotary replication. Ultrathin sections and freeze-etching findings of proximal (dendrite) and distal (axon) parts of bipolar olfactory neurons are examined. The supramolecular organization of neuron membranes and intracellular cytoskeleton structure is studied. The role of the Schwann cells in formation of isolated axon bundles is discussed. Methods of the whole neuroepithelium preparation for freeze-etching and different easy approaches of obtaining the Pt/C rotary shadowing replicas with high resolution (15-20A) are presented.