Phylogenetic analysis of phagotrophic, photomorphic and osmotrophic euglenoids by using the nuclear 18S rDNA sequence.

Phylogenetic analyses of 35 strains including 25 previously published sequences and 10 which have been newly sequenced, representing two species of Euglena, five species of Phacus and three species of Astasia, were carried out using the SSU rDNA. Parsimony, distance and maximum-likelihood inferred phylogenies support (1) monophyly of the euglenoids, (2) kinetoplastids as the sister group, (3) the phagotrophic Petalomonas cantuscygni Cann et Pennick anchoring the base of the euglenoid lineage, (4) evolution of phototrophy within the euglenoids from a single event, (5) multiple origins of osmotrophic euglenoids and (6) polyphyly of the genera Euglena Ehrenberg and Phacus Dujardin. Analyses also indicate that Lepocinclis Perty, Trachelomonas Ehrenberg and Astasia Dujardin are polyphyletic. In addition, the results suggest that neither the Euglenales nor the Eutreptiales form a monophyletic lineage, thus questioning currently available classifications. Concerning the phagotrophic mode of nutrition, the data suggest that the feeding apparatus arose multiple times.

Reconstruction of the flagellar apparatus in Ploeotia costata (Euglenozoa) and its relationship to other euglenoid flagellar apparatuses.

The flagellar apparatus of Ploeotia costata Farmer and Triemer was reconstructed using serial sectioning and TEM. The flagellar apparatus is similar to other euglenoids having two flagella arising from basal bodies connected by a striated fiber, and three asymmetrically arranged roots. The flagella emerge subapically from between the two ventral pellicle strips. The dorsal flagellum is 1/2 the body length and actively pulls the cell, while the ventral flagellum is twice the body length and drags along the substrate surface. The ventral and dorsal roots are on the opposite sides of their respective basal bodies, while the intermediate root is associated with the ventral flagellum on the side closest to the dorsal basal body. The dorsal root lines the dorsal side of the reservoir and after giving rise to the dorsal band lines the right side of the reservoir/canal. The ventral and intermediate roots join at the reservoir forming the intermediate-ventral root, which lines the left and ventral sides of the reservoir/canal. There was no evidence of a microtubule-reinforced pocket in P. costata. Comparisons with Ploeotia vilrea, Lentomonas applanatum, and related flagellar apparatuses led to the conclusion that the basic euglenoid flagellar structure is symplesiomorphic but with enough variation to be taxonomically diagnostic.

A molecular study of euglenoid phylogeny using small subunit rDNA.

The euglenoids are an ancient and extremely diverse lineage of eukaryotic flagellates with unclear relationships among taxa. Synapomorphies for the euglenoids include a surface pellicle and a closed mitosis with a series of separate sub-spindles. The taxonomy currently in use is inconsistent with the available data and needs revision. Most euglenoid phylogenies are largely intuitive reconstructions based on a limited number of morphological characters. Therefore, we have added molecular characters from the Small Subunit (SSU) rDNA to generate an overall phylogenetic framework for the euglenoids. SSU rDNA sequences from photosynthetic, osmotrophic, and phagotrophic euglenoids were aligned based on secondary structure. Phylogenetic analysis using the conserved areas of the sequence was performed using parsimony, maximum likelihood, and distance methods. Trees derived using different criteria are in agreement. The euglenoids form a distinct monophyletic clade with phagotrophic members diverging prior to the phototrophic and osmotrophic members. Among photosynthetic members, the biflagellate form diverged prior to the uniflagellate form. Additionally, the genus Euglena appears to be paraphyletic, with osmotrophic taxa, such as Astasia and Khawkinea, diverging independently within the clade containing the photosynthetic genus Euglena.

Activation and enhanced contact of human T-lymphocytes with autologous red blood cells are required for their stable adherence at 37 degrees.

The adherence of human red blood cells (RBC) to autologous T-cells does not occur in the body, and in vitro is elicited at 4 degrees. Autologous E-rosetting at 37 degrees has not previously been described. In this work, lymphocyte-RBC adherence has been studied in mixed leukocyte-RBC cultures and in whole blood from healthy donors. Vital, cytochemical and electron microscopic studies have shown that T-cells may form stable E-rosettes with autologous RBC at 37 degrees. As in the previously reported cold-dependent reversible rosetting, stable rosetting is mediated by the erythrocyte LFA3 and lymphocyte CD2 molecules. Uniquely, this phenomenon requires both T-cell activation and an enhanced contact between the T-cell and RBC membranes. These requirements were met by exposure of cell cultures to: (1) PHAE, the erythroagglutinating component of PHAP, or (2) to either non-erythroagglutinating mitogens, PHAL, Con A, OKT3 or SEA, or to antigens of typhus group rickettsiae or salmonellae, provided that the RBC membrane was desialyted. Cultures derived from individuals seropositive to rickettsiae or vaccinated with salmonellae demonstrated the adherence phenomenon after antigen exposure when neuraminidase was present in the culture medium. The system 2 described here can be used as a diagnostic tool for defining activated T-cells and T-cell clones with the memory to antigens capable of inducing cell-mediated immunity.

Ultrastructure of mitosis in Diplonema ambulator larsen and patterson (euglenozoa).

The fine structural features of mitosis are described in a small biflagellated phagotrophic protist, Diplonema ambulator. Flagella replicate early in mitosis and move to opposite sides of the nucleus where they remain lateral to the spindle poles. An array of microtubules diverge from two foci located on opposite sides of the nucleus adjacent to the nuclear envelope. At metaphase, the chromosomes compact and align to form a distinct ring around the elongating nucleolus. The chromosomal plates then separate and move towards the poles followed by elongation of the nucleus. By telophase, the nucleus and nucleolus assume a dumbbell-shape. Constriction of the nuclear envelope around the chromosomal masses cleaves the nucleolus and produces the daughter nuclei. The nuclear envelope and nucleolus persist through mitosis. A comparison of the ultrastructural features of mitosis in Diplonema with that found in the euglenoids, the group with which they are believed to be most closely related, reveals marked differences.

Effect of the anti-microtubule drug oryzalin on growth and differentiation of the parasitic protozoan Leishmania mexicana.

The parasitic protozoan Leishmania mexicana differentiates from a non-motile intracellular amastigote in the mammalian macrophage phagolysosome into a motile, extracellular promastigote in the insect vector gut. This developmental program has been accomplished in vitro, thus providing a useful model for studying changes in the cytoskeleton during cell differentiation. The role of microtubules in leishmania differentiation was demonstrated by using the dinitroaniline herbicide oryzalin, which inhibited both leishmania proliferation and differentiation; 25 microM oryzalin reduced promastigote division by over 95%. Interestingly, at a sublethal dose (5 microM), promastigotes became round and multiflagellated but remained motile. At 50 microM oryzalin, the number of intracellular amastigotes decreased by 50%. However, leishmania differentiation seemed to be the most drug-sensitive stage: there was a 60% reduction in amastigote-to-promastigote differentiation at 0.5 microM oryzalin. The specific action of oryzalin on leishmania microtubules was verified by its inhibition of in vitro polymerization of leishmania microtubules, but not control mammalian microtubules (from rat brain). These findings indicate that microtubules play a major role in leishmania proliferation, maintenance of cell shape, and cytodifferentiation.

Ultrastructure of Diplonema ambulator larsen & patterson (euglenozoa) and its relationship to Isonema.

The fine structure of a small phagotrophic protist, Diplonema ambulator, associated with deteriorating leaves of some species of the common fresh-water aquarium plant, Cryptocoryne is described. The organisms bear two short flagella which arise subapically and have a flexible cell surface often exhibiting pronounced changes in shape. Under the plasma membrane is a single layer of interconnected microtubules. Adjacent to the microtubules is a peripheral reticulate mitochondrion with plate-like cristae and numerous dense beaded inclusions. A large complex microtubular-based feeding apparatus extending to the cell surface is surrounded by several food vacuoles. Because the organism fits the light microscopic description of the genus Diplonema and has ultrastructural features attributable to Isonema we recommend that these taxa be regarded as synonymous and that existing species of Isonema be transferred to the genus Diplonema.

Flagellar systems in the euglenoid flagellates.

The flagellar apparatus of euglenoids consists of two functional basal bodies, three unequal microtubular roots subtending the reservoir, and a fourth band of microtubules nucleated from one of the flagellar roots and subtending the reservoir membrane. The flagellar apparatus of some euglenoids may contain additional basal bodies, striated roots ("rhizoplasts"), fibrous roots, striated connecting fibers between basal bodies, layered structures, or various electron-dense connective substances. With the possible exception of Petalomonas cantuscygni, nearly all euglenoids are biflagellate although the length of one flagellum may be highly reduced. The flagellar transition zone and number of basal bodies are highly variable among species. In recent years a cytoplasmic pocket that branches off from the reservoir has been discovered. The microtubules of the ventral flagellar root are continuous with the microtubules which line this pocket. Based on positional and structural similarities, this structure is believed to be homologous with the MTR/cytostome of bodonids. Coupled with other ultrastructural and biochemical data, the fine structure of the flagellar apparatus supports the belief that the euglenoid flagellates are descendant from bodonid ancestors.

Phosphatase localization in the endomembrane system of the dinoflagellate Crypthecodinium cohnii.

The distribution of four enzymes within the endomembrane system of the protist Crypthecodinium cohnii has been determined using cytochemical localizations with lead as a capture agent. Nucleoside diphosphatase (NDPase) activity, using inosine diphosphate (IDP) and thiamine pyrophosphate (TPP) as substrates, was observed in the Golgi apparatus, with a gradient of increasing reaction product noted in some cells from the cis to trans cisternae. Tubules and vesicles associated with the trans cisternae also contained reaction product. The endoplasmic reticulum exhibited a high activity of glucose-6-phosphatase [with glucose-6-phosphate (G-6-P) as substrate]. Traces of reaction product were also observed in the cis-most and trans-most cisternae of the dictyosomes. Activity of acid phosphatase (AcPase) was observed in Golgi cisternae as well as in associated cytoplasmic vesicles. Heaviest deposition was localized in medial and trans dictyosome cisternae. The cytoplasmic system of flattened vesicles subtending the surface membranes in these cells did not exhibit reactivity with any of the substrates used. The distribution of these enzymes in this algal cell appears similar to that observed in animal cells and suggests that these enzymes may represent markers for algal cell endomembrane compartments.

Freeze-fracture analysis of thylakoid membranes and photosystem I and II enriched fractions from Phormidium laminosum.

Thylakoid membranes of the thermophilic cyanobacterium Phormidium laminosum have been fractionated into photosystem II and photosystem I particles. These fractions have been characterized by their partial electron transport activities, and biochemical and spectral properties. Exoplasmic fracture face and protoplasmic fracture face particles in the unfractionated thylakoid membranes were shown to correspond in size to particles in freeze-fractured photosystem II and photosystem I fractions, respectively. Differences between the histograms of the thylakoid membrane protoplasmic fracture face particles and the isolated photosystem I particles suggest that in addition to photosystem I complexes some of the particles on the thylakoid protoplasmic fracture face may be related to cytochrome b/f complexes, the hydrophobic component of the coupling factor, or respiratory complexes.

Freeze-fracture architecture and polypeptide composition of thylakoid membranes from euploid Ricinus cells.

The freeze-fracture architecture and polypeptide composition of thylakoid membranes of euploid cells of Ricinus communis L. were examined. Electron microscopic examination of the chloroplasts of 1N 2N and 4N cells revealed little variation in the size of chloroplasts, lamellar structure and internal organization of plastids, despite increases in plastid numbers per cell observed to accompany the increase in nuclear ploidy. Thylakoid membranes from euploid cells were also similar in their freeze-fracture morphology. Two basic types of intramembranous particles were observed on the fracture faces of thylakoid membranes of euploid cells. The endoplasmic fracture (EF) face of experimentally unstacked thylakoid membranes of 1N, 2N and 4N cells contain 2 size categories of particles (115-121 A and 164-166 A), whereas the protoplasmic fracture (PF) face of these membranes contain a single size category of particles (85-88 A). The distribution and size of the EF- and PF-face particles were found to be similar among membranes from cells of the 3 ploidy levels. Analysis of the polypeptide composition of thylakoid membranes from 1N, 2N and 4N cells revealed no difference in the relative proportion of the constituent polypeptides of these membranes. The possible factors involved in the regulation of the development of thylakoid structure and composition in the presence of altered nuclear genome size are discussed.

Chromosome structure and mitosis in the dinoflagellates: an ultrastructural approach to an evolutionary problem.

Chromosome structure and mitosis have been examined in three evolutionarily diverse members of the Pyrrophyta. Chromosome uncoiling, revealing the chromonema, has been correlated with the uptake of [3H]thymidine. In addition, chromosome uncoiling has been observed during gamete formation, gamete fusion, and in the nucleolar organizing region of the chromosomes suggesting that dinoflagellate chromosomes undergoing duplication, transcription or pairing have a morphology different from the characteristic tightly banded structure generally observed during most of interphase and mitosis. The dinoflagellate chromonema is composed of 2.5-nm fibers and 9.0-nm granules coiled into a helix around a central core of 9.0-nm fibers. Chromosome attachment to nuclear channels and kinetochore division and separation have been examined in several dinoflagellates. After evaluating many nuclear and cytoplasmic characteristics of the dinoflagellates it appears that this group of organisms are true eukaryotes which may be on the main line to the evolution of the mitotic spindle typical of higher plant and animals cells.U

Proteinases in cardiac and skeletal muscle.

Although changes in proteolysis in muscle tissue are now well documented for a variety of physiological and pathological conditions, the mechanism of degradation of cellular protein during normal protein turnover remains to be elucidated. Data from several laboratories have suggested the involvement of alkaline serine proteinases. Recent studies have questioned these results, and demonstrated that the serine proteinases are of mast cell origin and are not present in muscle cells. The only proteinases to date that have been shown to be present in muscle cells and capable of degrading myofibrillar proteins are Ca2+-activated proteinase, cathepsin B, and cathepsin D. Recent interest and developing awareness of endogenous enzyme inhibitors in cells may unmask many new enzymes.

The ultrastructure of cell division in Euglena gracilis.

The ultrastructure of mitosis in Euglena gracilis was investigated. At preprophase the nucleus migrates anteriorly and associates with the basal bodies. Flagella and basal bodies replicate at preprophase. Cells retain motility throughout division. The reservoir and the prophase nucleus elongate perpendicular to the incipient cleavage furrow. One basal body pair surrounded by a ribosome-free zone is found at each of the nuclear poles. The spindle forms within the intact nuclear envelope- Polar fenestrae are absent. At metaphase, the endosome is elongated from pole to pole, and chromosomes are loosely arranged in the equatorial region. Distinct, trilayered kinetochores are present. Spindle elongates as chromosomes migrate to the poles forming a dumb-bell shaped nucleus by telophase. Daughter nuclei are formed by constriction of the nuclear envelope. Cytokinesis is accomplished by furrowing. Cell division in Euglena is compared with that of certain other algae.