TAXONOMIC REVISIONS OF MORPHOLOGICALLY SIMILAR SPECIES FROM TWO EUGLENOID GENERA: EUGLENA (E. GRANULATA AND E. VELATA) AND EUGLENARIA (EU. ANABAENA,EU. CAUDATA,AND EU. CLAVATA)1

The establishment of epitypes (together with the emended diagnoses) for three species of Euglenaria Karnkowska, E. W. Linton et Kwiatowski [Eu. anabaena (Mainx) Karnkowska et E. W. Linton; Eu. caudata (Hübner) Karnkowska et E. W. Linton; and Eu. clavata (Skuja) Karnkowska et E. W. Linton] and two species of Euglena Ehrenberg [E. granulata (Klebs) Schmitz and E. velata Klebs] was achieved due to literature studies, verification of morphological diagnostic features (cell size, cell shape, number of chloroplasts, the presence of mucocysts), as well as molecular characters (SSU rDNA). Now all these species are easy to identify and distinguish, despite their high morphological similarity, that is, spindle‐shaped (or cylindrically spindle‐shaped) cells and parietal, lobed chloroplasts with a single pyrenoid, accompanied by bilateral paramylon caps located on both sides of the chloroplast. E. granulata is the only species in this group that has spherical mucocysts. E. velata is distinguished by the largest cells (90–150 μm) and has the highest number of chloroplasts (>30). Eu. anabaena has the fewest chloroplasts (usually 3–6), and its cells are always (whether the organism is swimming or not) spindle‐shaped or cylindrically spindle‐shaped, in contrast to the cells of Eu. clavata, which are club‐shaped (clavate) while swimming and only after stopping change to resemble the shape of a spindle or a cylindrical spindle; Eu. clavata has numerous chloroplasts (15–20). Eu. caudata is characterized by asymmetrical spindle‐shaped (fusiform) cells, that is, with an elongated rear section and a shorter front section; the number of chloroplasts normally ranges from 7 to 15.     

DEVELOPMENT OF MUCILAGINOUS SURFACES IN EUGLENOIDS. II. FLAGELLATED,CREEPING AND PALMELLOID CELLS OF EUGLENA1

Critical-point dried (CPD) cells from clonal cultures of Euglena gracilis Klebs (Z strain), E. deses Ehrb., E. tripteris (Duj.) Klebs and E. myxocylindracea Bold & MacEntee were examined by scanning electron microscopy. Flagellated motile cells of E. gracilis are naked except for a few strands of mucilage on the posterior tip. Flagellated cells of E. tripteris have a permanent mucilage coating often of uneven distribution and usually not as well developed as that of nonflagellated creeping cells which have a distinctive mucilage. In E. deses the coating appears rough due to the aggregation of isolated groups of strands above the cell surface. In E. tripteris the coating appears smooth except for breaks near the articulation of the pellicular strips where the mucilage may rise above the surface to form waves. At high magnification this mucilage consists of a network of strands generally lying parallel to the cell surface; the strands become obscure in some specimens. In E. myxocylindracea elongated, mucilage-coated cells contract to form spheres which undergo further mucilage deposition producing the mucilage covering of palmellae. As palmellae mature, the mucilage surface becomes less porous and the individuality of most mucilage strands is lost.     

SEXUAL REPRODUCTION IN THE DINOFLAGELLATE NOCTILUCA MILIARIS SURIRAY1,2

The existence of sexuality in dinoflagellates has long been controversial. Events described as the differentiation and release of isogametes and sexual fusion in cultures of Noctiluca miliaris were studied with the light microscope. In cultures fed. twice weekly with Dunaliella sp., kept under a 12-hr day, 12-hr night light regime at 25–30 ft-c and held at 18 C, certain vegetative cells, termed gametocyte mother cells, were observed to undergo what is interpreted as meiosis. This was followed by several synchronous mitotic divisions occurring at 45-min to 1-hr intervals and. resulting in the production of up to 1024 mature uniflagellated isogametes attached on the surface of the gametocyte mother cell body. After several hours gametes now seen swimming freely in the media were observed to fuse and form zygotes. One zygote subsequently became vacuolated and eventually differentiated into a small but reasonably typical vegetative cell. The life cycle of N. miliaris appears thus to be of the diplontic type. The taxonomic relationships between Noctiluca and other mesocaryotic dinoflagellates are discussed.     

PHOSPHATE UPTAKE KINETICS IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) GROWN ON LIGHT/DARK CYCLES.' I. SYNCHRONIZED BATCH CULTURES1

The characteristics of phosphate uptake in synchronized populations of Euglena gracilis Klebs (Z) were studied. The cells were grown autotrophically in batch culture and synchronized with a cycle of 14:10 LD. Incorporation of P was nonlinear with time for the first 2 h of incubation over a wide range of P concentrations and completely inhibited by darkness. The kinetics of P uptake as a function of P concentration were triphasic between 0 and 100 μM PO₄, obeying Michaelis-Menten kinetics over the 0–3 μM PO₄ range-only. Uptake velocity increased linearly with, concentration above 3 μM PO₄. The kinetics of P uptake varied with stage in the cell cycle. The half-saturation constant for uptake at the lower concentrations oscillated between 0.7 and 2.8 μM PO₄, reaching a peak immediately before the onset of cell division (beginning of the dark period). V_max was largest in the middle of the light period, as was the slope of the linear portion of the kinetic pattern. Further analysis of the kinetics suggests that changes in this slope are responsible for the oscillation in K_s values calculated for the lower concentrations. This analysis assumes 2 uptake mechanisms, one which saturates at low concentrations of phosphate, and one which is nonsaturable over the entire concentration range examined.     

Unique GH18 chitinase from Euglena gracilis: full-length cDNA cloning and characterization of its catalytic domain

A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [(GlcNAc)_4–6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)₂ and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)₂ or GlcNAc, which are easily taken up by the cells.     

LIGHT/DARK-PHASED CELL DIVISION IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) IN PO4-LIMITED CONTINUOUS CULTURE1

Eugene gracilis Klebs (Z) was grown in a cyclostat (continuous culture on a light/dark cycle) at growth limiting levels of phosphate. Cell division was restricted to the dark period regardless of the proportion of the cells dividing during each 24 h period. Growth rate, as reflected by the amplitude of the cell density oscillation, was correlated with dilution rate. The width of the division gate was analyzed using a phasing index and found to be narrowest at dilution rates where the mean generation time of the cell population was an even multiple of 24 h. The effect was attributed to enhanced phasing of the cell division process by the biological clock of Euglena. Residual phosphate levels in the cyclostat were less than 0.3 μM PO₄ at all submaximal growth rates. Cellular phosphorus concentration increased with dilution rate as described by a hyperbola saturating at D_max= 0.74 day⁻¹ with 8 × 10⁻⁸μM P/cell as the minimum intracellular phosphorus concentration for growth. The results are discussed, in terms of the inherent similarities and differences between a cyclostat and a steady state chemostat, and the advantages of the cyclostat for studies in phytoplankton ecology.     

CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN-STARVED EUGLENA GRACILIS1

Growth of Euglena gracilis Z Pringsheim under photoheterotrophic conditions in a nitrogen-deprived medium resulted in progressive loss of chloroplastic material until total bleaching of the cells occurred. Biochemical analysis and ultrastructural observation of the first stages of the starvation process demonstrated an early lag phase (from 0 to 9 h) in which cells increased in size, followed by a period of cell division, apparently supported by the mobilization of some chloroplastic proteins such as the photosynthetic CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. The degradation of the enzyme started after 9 h of starvation and was preceded by a transient concentration of this protein in pyrenoidal structures. Protein nitrogen and photosynthetic pigments as well as number of chloroplasts per cell decreased during proliferation through mere distribution among daughter cells. However, after 24 h, when cell division had almost ceased, there was a slow but steady decline of photosynthetic pigments. This was paralleled by observable ultrastructural changes including progressive loss of chloroplast structure and accumulation of paramylon granules and lipid globules in the cytoplasm. These findings reinforce the role of chloroplastic materials as a nitrogen source during starvation of E. gracilis in a carbon-rich medium. The excess of ribulose-1,5-bisphosphate carboxylase/oxygenase acts as a first reservoir that, once exhausted, is superseded by the generalized disassembly of the photosynthetic structures, if the adverse environment persists more than 24 h.     

Induction of fumarase in resting Euglena

Exposure of dark-grown resting (carbon deficient) Euglena to light, ethanol or malate produced a transient increase in the specific activity of fumarase. Fumarase levels decreased 8–12 h after the start of induction and this decrease could not be prevented by additional inducer. During the period of fumarase accumulation, cycloheximide prevented further fumarase synthesis and enzyme levels decreased at a rate comparable to the rate of decline normally observed 8–12 h after the start of induction. Although the addition of ethanol to ethanol-induced cultures or malate to malate-induced cultures 12 or 24 h after the initial induction failed to maintain or induce additional fumarase synthesis, the addition of organic carbon to photoinduced cells 8 or 24 h after light exposure induced additional enzyme synthesis. Additional enzyme synthesis was not induced when ethanol- or malate-induced cells were exposed to light 12 or 24 h after organic carbon addition. Light exposure or ethanol addition failed to induce fumarase synthesis during balanced growth indicating that fumarse inducibility is a property of resting cells.     

SYNCHRONOUS GROWTH IN THE COLONIAL EUDORINA ELEGANS (CHLOROPHYCEAE)1,2

Eudorina elegans Ehrenberg has been synchronized by growing the organisms at 32 C in an enriched medium on a 16:8 LD cycle. Nuclear division occurred during the light period and 2–4 h was required, for the population to complete the sequence of 4 or 5 divisions each cell normally undergoes. Each cell division required 2–3 min with ca. 15 min as the intermitotic time. The sequence was repealed, every 24 h as long as synchrony was maintained.     

KINETICS OF CELL PROLIFERATION IN THE PRE-IMPLANTED MOUSE EMBRYO IN VIVO AND IN VITRO

The cell proliferation of pre-implanted mouse embryos was investigated after development in vivo and in vitro. The studies were started at the pronuclear stage, 2 h post conception (p.c.) and continued until the hatching of blastocysts, 120–144 h p.c. The number of cell nuclei, the DNA content of each nucleus, the mitotic index and the labelling index were determined. From these data it was possible to calculate the length of the cell generation cycle and its various phases. With the exception of the first cell cycle the S-phase was constant. The G₁- as well as the G₂-phase varied in length during the different cell cycles. From 31–72 h p.c. the increase in cell number was exponential. After cultivation in vitro this increase was smaller than in vivo. At later periods the proliferation rate decreased with proceeding development. In late blastocysts most of the cells were in the G₁-phase. The development of the embryos was somewhat faster in vivo than in vitro. But in principle conditions were comparable.     

COENOBIAL CELL NUMBER IN SCENEDESMUS QUADRICAUDA (CHLOROPHYCEAE) AS A FUNCTION OF GROWTH RATE IN NITRATE-LIMITED CHEMOSTATS1

The relative numbers of cells growing as coenobia of different cell number are functions of growth rate when Scenedesmus quadricauda (Turp.) Bréb. is grown axenically in nitrate-limited, steady-state chemostats in continuous light. Unicells decreased from a maximum fraction of 0.04–0.05 of the total number of cells at 0.1 day^?1 to 0.01 or less as growth rate increased. The fraction of cells that grew as two-celled coenobia decreased from about 0.2 to 0.01–0.02. The fraction that grew as 4-celled coenobia increased from about 0.7–0.8 at 0.1 day^?1 to near unity at 0.5–0.6 day^?1, and then decreased sharply. The fraction of 8-celled coenobial cells increased from very small values below 0.6 day^?1, to ca. 0.9 at 1.0 day^?1.     

CHOLINE ACETYLTRANSFERASE, ACETYLCHOLINESTERASE AND AROMATIC l-AMINO ACID DECARBOXYLASE IN SINGLE IDENTIFIED NERVE CELL BODIES FROM SNAIL HELIX ASPERSA

—The distribution of choline acetyltransferase, aromatic l -amino acid decarboxylase and acetylcholinesterase in the nervous system of Helix aspersa has been studied using homogenates of whole ganglia, microdissection from freeze-dried sections and dissection of single neurons from fresh tissue. Choline acetyltransferase was found in both the cell body and neuropil layers of all the Helix ganglia. The enzyme was not specifically localized to any ganglion or region of ganglion. Between 10 and 30 per cent of the isolated single cell bodies contained the enzyme. The enzymic activity corresponded to 50–200 mmol ACh/1 cell bodies/h. Choline acetyltransferase is probably a specific marker for cholinergic cells in this species. Aromatic l -amino acid decarboxylase was more selectivity localized and its distribution corresponded well with that of monoamine containing cells as visualized by the fluorescence histochemical technique. A large proportion of cell bodies were localized in the boundary between the visceral and right parietal ganglia and in the pedal ganglion. The other ganglia contained few such cells. The activity of aromatic l -amino acid decarboxylase corresponded 10–50 mmol dopamine/1 cell bodies/h. A method was developed to measure the enzyme activity towards 5-hydroxytryptophan and DOPA in single cells simultaneously. The ratio between the activity towards both substrates did not vary significantly for the different cells. The enzyme is probably a specific marker for monoamine cells, but cannot be used to differentiate between the different monoamine cells. Acetylcholinesterase was uniformly distributed in the ganglia and was probably present in all nerve cells.     

Altering textural properties of fermented milk by using surface‐engineered Lactococcus lactis

Lactic acid bacteria are widely used for the fermentation of dairy products. While bacterial acidification rates, proteolytic activity and the production of exopolysaccharides are known to influence textural properties of fermented milk products, little is known about the role of the microbial surface on microbe–matrix interactions in dairy products. To investigate how alterations of the bacterial cell surface affect fermented milk properties, 25 isogenic Lactococcus lactis strains that differed with respect to surface charge, hydrophobicity, cell chaining, cell‐clumping, attachment to milk proteins, pili expression and EPS production were used to produce fermented milk. We show that overexpression of pili increases surface hydrophobicity of various strains from 3–19% to 94–99%. A profound effect of different cell surface properties was an altered spatial distribution of the cells in the fermented product. Aggregated cells tightly fill the cavities of the protein matrix, while chaining cells seem to be localized randomly. A positive correlation was found between pili overexpression and viscosity and gel hardness of fermented milk. Gel hardness also positively correlated with clumping of cells in the fermented milk. Viscosity of fermented milk was also higher when it was produced with cells with a chaining phenotype or with cells that overexpress exopolysaccharides. Our results show that alteration of cell surface morphology affects textural parameters of fermented milk and cell localization in the product. This is indicative of a cell surface‐dependent potential of bacterial cells as structure elements in fermented foods.     

Stimulation of Plastidogenesis Induced by 5-Azacytidine in Euglena gracilis Klebs

When etiolated Euglena gracilis was treated with 10 mM 5-azacytidine (5-azaC), an inhibitor of DNA methylation, stimulation of plastidogenesis in both dark and light conditions was observed. The phenomenon occurred in 10–15% of the cells possibly due to the asynchronicity of the cultures. The main features of this sub-population, as evaluated by electron and fluorescence microscopy, were the following: 1. the presence in darkness of differentiating proplastids that were red fluorescent under UV, positive to TCNBT cytochemical reaction (specific for PSII) and negative to DAB (specific for PSI); 2. the acceleration of proplastid differentiation during the first 20–30 h of illumination; 3. the occurrence in both culture conditions of concentric lamellar bodies (LB_S). These structures were considered to be proplastids blocked in the first step of evolution, since they emitted a red fluorescence, were contained within compartments limited by a triple-layered envelope, were reactive to TCNBT in darkness and to both TCNBT and DAB in light conditions. Even if the action mechanism of 5-azaC on plastidogenesis in Euglena remains to be defined, the induced stimulatory effect on plastid differentiation pointed to a relationship between DNA methylation and plastid development. Furthermore, the presence of LB_S opens the possibility of studying early aspects of plastid development in Euglena.     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

PHOSPHATE UPTAKE KINETICS IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) GROWN IN LIGHT/DARK CYCLES. II. PHASED PO4-LIMITED CULTURES1

The characteristics of phosphate uptake and photosynthetic capacity were studied in P-limited populations of Euglena gracilis Klebs (Z), using both P-limited batch cultures in stationary phase and cyclostat cultures grown on 14:10 LD. P uptake obeyed Michaelis-Menten kinetics between 0 and 150 μM PO₄ under both growth conditions. The value of V_max was 35% lower in the dark than in the light in the stationary phase cells. The value of K₈ was not affected by light conditions, and uptake was completely inhibited in the presence of 1 mm KCN. P uptake (at 2.0 μM PO₄) and photosynthetic capacity showed diel periodicity with peak rates occurring just before the beginning of the dark period for P uptake, and 8 h into the light period for photosynthetic capacity. V_max for P uptake increased by a factor of 1.5 over the light period, whereas K₈ remained constant at 1.4 μM PO₄. These patterns were displayed by both nondividing stationary phase cells and populations in which less than a third of the cells divided each day, indicating that the rhythmicity is not coupled to cell division.     

MORPHOLOGICAL CHANGES IN TOXIC AND NON-TOXIC MICROCYSTIS ISOLATES AT DIFFERENT IRRADIANCE LEVELS1

Light intensity (4.5–40.0 μEin m^?2 s^?1) and culture age had a pronounced effect on cell size and size range of a non-toxic axenic Microcystis isolate. The rate of cell volume increase (μm³ d^?1) was 1.03 × light intensity (μEin m^?2 s^?1) – 6.49. Average cell volume varied from 33 to 119 μm³, cells at higher light intensities being larger and having a larger size range. The effects on a toxic axenic Microcystis isolate were similar but less pronounced. Average cell volume ranged from 21–74 μm³. In general, cell size and especially size variability appear to be sensitive indicators of physiological state, with cells under stress conditions being larger and associated with a larger size range. The wide range of cell diameters observed at different irradiance levels (3.4–7.2 μm for the non-toxic and 1.8–6.4 μm for the toxic isolate), makes questionable the use of cell size as a taxonomic character without careful consideration of environmental conditions.     

CELL DIVISION PATTERNS AND DIURNAL CYCLE OF PORPHYRA ABBOTTAE (RHODOPHYTA,BANGIALES) CARPOGONIAL AND CARPOSPORE DEVELOPMENT1

Post-fertilization development of carpospores in Porphyra is a well-documented phenomenon. Development of the pre-fertilization carpogonial cells from vegetative cells, however, has not been previously described. In Porphyra abbottae Krishn., a rock? intertidal monostromatic species occurring from British Columbia to central California, large cells, designated hue CIS “procarpogonial mother cells” (PMCs), initiated the formation of the carpogonial cells. The PMCs formed during late night mitoses beginning at 0200 h with cytokinesis from 0300-0500 h during short day periods of 10:14 h LD in northern California (38°20′N, 123°03′W and 36°37′N, 121°55′W). The PMC cut off numerous smaller cells which in turn divided equal. Approximately 12 h Inter, at 1500 h (day 1) the Smaller cells were recognizable as carpogonial cells by the presence of trichogynes growing from the cytoplasm out through the cell wall to the thallus surface. In another 24 h (day 2), the fertilized carpogonia had divided into carpospore packets. Spores were released at 1500 h the following day (day 3), their projection creating escape channels through the cell walls.     

FLAGELLAR DEVELOPMENT AND REGENERATION IN VOLVOX CARTERI (CHLOROPHYTA)1

The biflagellate somatic cells of Volvox carteri f. nagariensis lyengar exhibit an asymmetric pattern of flagellar development. Initiallt each somatic cell has two short (4 μm) flagella but after several hours one flagellum on each cell elongates unitl it reaches a length of 12 μm. Due to the regular arrangement of somatic cells in the Volvox spheroid it is apparent that the same flagellum on each somatic is the first to elongale. The asymmetric flagellar length is maintained for about 8 h after which the second flagellum on each somatic cell elongates. When the second flagellum attains the same length (12 μm) as the first flagellum, both flagella elongale at the same rate until reaching a final length of 22 μm. Experimental removal of somatic cell flagella results in their regeneration. Somatis cells regenerate both flagella simultaneously and full length flagella are produced in about 2 h. The intial rate of flagellar regeneration is about ten times faster than the intial rate of flagllar growth in development. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial rate of flagellar regeneration but the flagella produced in the presence of the drug are half the length of flagella produced in its absence. Somatic cells are able to regenerate flagella up to the time of α and β tubulin, the major structural proteins of the flagellar axoneme, and other cellular proteins.     

Expression and purification of the recombinant porcine NK-lysin in Pichia pastoris and observation of anticancer activity in vitro

Natural killer (NK)-lysin, a broad-spectrum antimicrobial peptide, has antitumor and antibactericidal activities against both gram-positive and gram-negative bacteria. In this study the recombinant porcine NK-lysin was expressed and purified in the Pichia pastoris system, and then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to assess its anticancer activity in vitro. The results showed that the recombinant porcine NK-lysin possesses potent antitumor activity against the human hepatocellular carcinoma cell line SMMC-7721 in a time- and dose-dependent manner, but has negligible hemolysis activity against human erythrocytes. Scanning electronic microscopy was used to directly observe the ultrastructure of SMMC-7721 cells treated with NK-lysin; untreated cells showed lamellipodia and filopodia scattered with the cell surface, with good cell–cell contacts among neighboring cells. In contrast, treated tumor cells exhibited marked alterations in cell morphology, and cell–cell contacts disappeared among neighboring cells. Compared with the untreated tumor cells, the tumor cells treated with NK-lysin for 12 and 24 hr were suppressed for the expression of fascin 1. Thus, the recombinant porcine NK-lysin potentially could be developed as a therapeutic agent for inhibiting tumor growth.