Dur3 and nrt2 genes in the bloom-forming dinoflagellate Prorocentrum minimum: Transcriptional responses to available nitrogen sources.

The increasing inflow of nitrogen (N) substrates into marine nearshore ecosystems induces proliferation of harmful algal blooms (HABs) of dinoflagellates, such as potentially toxic invasive species Prorocentrum minimum. In this study, we estimated the influence of NO3-, NH4+ and urea on transcription levels and urea transporter dur3 and nitrate transporter nrt2 genes expression in these dinoflagellates. We identified dur3 and nrt2 genes sequences in unannotated transcriptomes of P. minimum and other dinoflagellates presented in MMETSP database. Phylogenetic analysis showed that these genes of dinoflagellates clustered to the distinct clade demonstrating evolutionary relationship with the other known dur3 and nrt2 genes of microalgae. The evaluation of expression levels of dur3 and nrt2 genes by RT-qPCR revealed their sensitivity to input of the studied N sources. Dur3 expression levels were downregulated after the supplementation of additional N sources and were 1.7-2.6-fold lower than in the nitrate-grown culture. Nrt2 expression levels decreased 1.9-fold in the presence of NH4+. We estimated total RNA and DNA synthesis rates by the analysis of incorporation of 3H-thymidine and 3H-uridine in batch and continuous cultures. Addition of N compounds did not affect the DNA synthesis rates. Transcription levels increased up to 12.5-fold after the N supplementation in urea-limited treatments. Investigation of various nitrogen sources as biomarkers of dinoflagellate proliferation due to their differentiated impact on expression of dur3 and nrt2 genes and transcription rates in P. minimum cells allowed concluding about high potential of the studied parameters for future modeling of HABs under global N pollution.

Natural killer cell activity irreversibly decreases after Cryptosporidium gastroenteritis in neonatal mice.

Cryptosporidiosis causes persistent diarrhoea in infants, immunocompromised patients and elderly persons. Long-term consequences of the disease include increased risk of malignancy, cardiomyopathy and gastrointestinal inflammation. This study aimed to investigate prolonged effects of cryptosporidiosis on innate immunity and growth in neonatal C3HA mice. The disease was challenged by Cryptosporidium parvum oocyst inoculation into 7-day-old animals. The mice whose intestine smears contained 3-5 or 6 and more oocysts per microscopic field at the day 5 after infection were considered as mildly or severely infected, correspondingly. To determine natural killer cell (NK) activity, we applied 3 H-uridine cytotoxic assay to the animals at 5-68 days after infection using K562 cells as targets. At severe infection, there was a statistically significant 1.5-2.0 fold decline of body mass, spleen mass and spleen cellularity that persisted in animals of all ages. Accordingly, NK cytotoxicity showed even more drastic drop reaching 2.7-3.0 folds that was statistically significant in all animals. At mild infection, the discovered effects were less pronounced and reached significance only in some age groups. Thus, our study provides evidence that NK cells show long-term cytotoxic activity decrease following Cryptosporidium infection in neonatal mice, particularly in severe disease.

MECHANICAL IMPACT ON THE CELL COVERING FINE STRUCTURE OF DINOFLAGELLATES PROROCENTRUM MINIMUM.

Complex cell coverings (amphiesma) of potentially toxic dinoflagellates Prorocentrum minimum include plasma membrane and flattened amphiesmal vesicles with thecal cellulose plates. Two largest thecal plates surround the major portion of dinoflagellate cell as shell valves. We have revealed that P. minimum cells appear to be extremely sensitive to the physical stress: even low speed centrifugation (1200 and 2000 g) leads to a dropping of old coverings shedding (ecdysis) and the formation of viable spheroplasts. Spheroplasts are surrounded only by the plasma membrane beneath which the new amphiesma is formed. These spheroplasts can be a convenient model system for investigation of numerous aspects of cell and molecular biology of the dinoflagellates.

[ANALYSIS OF THE DINOFLAGELLATE PROROCENTRUM MINIMUM TRANSCRIPTOME: IDENTIFYING THE MEMBERS OF THE VOLTAGE-GATED CATION CHANNELS SUPERFAMILY].

Dinoflagellates are an ecologically important group of aquatic single-cell eukaryotes. At the present time relatively little is known about physiological features that determine the role of these protists in natural ecosystems. Lack of knowledge on the diversity, structure, and functioning of dinoflagellate ion channels significantly hampers the interpretation of physiological reactions and adaptations in these microorganisms. We performed the analysis of the translated transcriptome databases that belong to two strains of the dinoflagellate Prorocentrum minimum in order to identify the members of the voltage-gated cation channels superfamily. We found out that transcriptomes of these potentially toxic microorganisms contained the homologues of: 1) inwardly rectifying potassium channels (K(ir)), 2) voltage-gated potassium channels (K(v)), 3) calcium-activated potassium channels (K(Ca)), 4) cyclic nucleotide-gated channels (EAG and HCN/CNG), 5) TRPV and TRPP channels, 6) two-pore calcium channels TPC, 7) voltage-gated sodium (Na(v)) and calcium (Ca(v)) channels, 8) voltage-gated proton channels (H(v)).

[Comparative morphology of the subphilum Conosa Cavalier-Smith 1998].

Comparative analysis of archamoebae and slime molds morphology revealed that this organisms have a marked similarity in organization of locomotive forms, structure of glycocalix and also in organization of nuclear and flagellar apparatus. A possible scheme of formation the modern diversity of Conosa group was proposed.

[Mixotrophy in microorganisms: ecological and cytophysiological aspects].

Mixotrophy is the ability to combine autotrophic and heterotrophic modes of nutrition. It is widely spread in various microorganisms, particularly in such important plankton groups as dinoflagellates and cyanobacteria. Mixotrophy has a significant impact on our comprehension of the matter and energy flows in marine ecosystems, and therefore, it is an object of much attention for several recent decades. Nevertheless, the precise data on the balance of auto- and heterotrophy during the mixotrophic growth have been absent so far, which is due, first of all, to insufficient understanding of physiological and molecular ground of this phenomenon. In this review we discuss some ecological and cytophysiological aspects of investigation of mixotrophy in microorganisms as well as possible reasons for relatively slow progress in this area.

[Correlations between salinity-persistence of ciliate species and their constitutive heat shock protein of 70 kDa contents].

Our own studies of alterations in the level of constitutive heat shock protein of 70 kDa family (Hsp70) in freshwater (Paramecium jenningsi), meta-freshwater (Tetrahymena pyriformis) and curyhaline (P. nephridiatum) ciliates acclimated to salt-water and fresh-water medium were reviewed. It has been shown that the level of constitutive Hsp70 content correlates with the salinity-resistance of ciliate species: in P. jenning i it was lower in freshwater, than in marine water, in euryhaline P. nephridiatum it was higher in freshwater, than in marine water, and was more or less stable in T. pyriformis, the ciliate that occupies intermediate position between two mentioned above species according its salinity-resistance. The ecological importance of constitutive heat shock protein level is discussed in the context of salinity adaptations studies.

[Transcriptional regulation macronuclear protein-coding genes in stichotrichous ciliates].

The concept of transcriptional regulation and base promoter structure of genes in eukaryotic organisms rests on two approaches--the experimental (using different methods of directional mutagenesis) and the comparative molecular (comparison of nucleotide sequences in promoter regions of different genes). Investigation of ciliates has led researchers to the conclusion that the protein-coding genes of these organisms lack the classical eukaryotic regulatory elements in the promoter region. This conclusion is based mainly on the usage of the comparative approach, while experimental investigations of such genes are practically absent so far. In the present paper, the comparative molecular analysis of the promoter regions of genes and analysis of the functional role of tubulin, cathepsin and Hsp 70 5'-uncoding areas was performed using new experimental data obtained during investigation of Stylonychia lemnae alpha-tubulin gene. We suggest a new classification of mechanism of transcriptional regulation of protein-coding genes in stichotrichous ciliates.

[Three-dimensional organization of nucleoli of Didinium nasutum (Ciliata)].

A comparative study of nucleolar organization in the somatic nuclei of the ciliate Didinium nasutum was carried out using 3D reconstruction on the basis of serial ultrathin sections. Recently fed interphase ciliates, starved interphase ciliates and cysts were studied. The nucleoli at the interphase stage were shown to have a complex architecture: the fibrillar component forms a complicated network, the granular component is located inside of it. It was shown that nucleoli, which look like individual structures in single sections, are in fact parts of branched nucleolar networks. A 30-h starvation doesn't lead to disintegration of these networks. However in the starved cells the granular component becomes more dense and vacuolized. In the fed ciliates there are many holes in the fibrillar component, whereas in starved ones the fibrillar component is virtually devoid of them. These holes can be proposed to ensure the transport of newly synthesized rRNP. The nucleolar networks didn't occur in D. nasutum cysts. Nucleoli in the cysts look like small individual structures, mainly consisting of fibrogranular component.

[Stylonychia lemnae as a model organism for studies of macronuclear minichromosomes of the ciliates].

The unique structural and functional organization of macronuclear (somatic nucleus) genome of the spirotrichous ciliates, exemplified by Stylonychia lemnae, has been reviewed. Data on the architecture of S. lemnae nuclear apparatus at interphase and during vegetative cell division, conjugation or autogamy are summarized. Special attention being paid to the structural and functional peculiarities of short macronuclear minichromosomes known to contain protein-coding regions, 5'- and 3'-flanking nontranslated regions, and telomeres. A hypothesis, previously put forward, according to which in the spirotrichous ciliates the telomeres themselves may serve as starting points of replication in minichromosomes, has now received its further substantiation. The recent experimental data, which confirm that 5'-nontranscribed DNA leader sequence of alpha1- and alpha2-tubulin-encoding minichromosomes display at least several regulatory elements typical for eukaryote promoter (TATA-box, CAAT-box, transcriptional initiator), are discussed. Up to now, there is no confirmation with regard to a possible existence in the spirotrichous minichromosomes of specific regulatory sequences capable of controlling both replication and transcription processes.

[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.

[The fine structure of chromatin in Paranosema grylli (Microsporida)].

Nucleosomes were found for the first time in the nuclear chromatin of Microsporida--organisms known among the smallest eukaryotes on Earth. Chromatin of Paranosema grylli sporoplasm was studied by Miller's technique. On low ionic-strength cell spreads, this chromatin was represented by 10 nm nucleosome filaments, 20 nm filaments, and "smooth" (nucleosome-free) filaments of 3-4 nm in diameter. Nucleosome filaments display structural heterogeneity seen as irregular arrangement of nucleosome particles along the filament length. Different nucleosome filaments show 13-30 nucleosomes per 1 microm with the length of linker DNA ranging from 10 to 45 nm. The present results suggest that microsporidian chromatin is weakly condensed. Only lower-order chromatin packaging levels displayed some structural peculiarities.

[Study of DNA-containing structures in the nucleus and cytoplasm of the Crithidia flagellates using the Miller method]. / Izuchenie DNK-soderzhashchikh struktur iadra i tsitoplazmy zhgutikonostsev Crithidia metodom Millera.

Weakly condensed interphase chromosomes made of chromatin, and the tightly packed kinetoplast DNA (kpDNA) of a single mitochondrion of Crithidia (Kinetoplastidea, Trypanosomatida) flagellates were studied on the Miller-type spread preparations by electron microscopy. Chromatin of organisms lysed at low ionic strength conditions for 5-10 min unfolds up to 10-nm nucleosomal filaments and 20-nm chromatin fibers. The initial indications of kpDNA decompaction become visible after a 10-13 min dispersion of lysed flagellates. However, at least a 15 min long procedure is required for well-defined identification of intrakinetoplast structures. In this case, the kinetoplast looks like a heterogeneous network disposed close to the kinetosome of a single flagellum. Cells with diameters of 162.5 nm and contour wall length of 510 nm dominated within the network. With the prolongation of the dispersion time up to 20 min both these parameters increased up to 218 and 686 nm, respectively. Further prolongation of the treatment up to 60 min results in wall disruption in many cells. Within these cells, some isolated circular kpDNA molecules appear with the contour length of 588-792 nm. The circles of this size correspond to individual minicircles of the Crithidia kpDNA. Partly unfolded maxicircles of the kpDNA can be found only at early stages of dispersion (10 min). Special features of compaction of DNA-containing structures in both the nucleus and cytoplasm of Crithidia are discussed.

[Chromosome-like bodies of dysentery ameba Entamoeba histolytica]. / Khromosomopodobnye tela dizenteriinoi ameby Entamoeba histolytica.

Intact and surface stretched amembraneous nuclei of Entamoeba histolytica (Rhizopoda, Lobosea, Entamoebidae) trophozoites were studied by light and electron microscopy. A moderately dense karyosome about 1.5 microns in diameter, localized in the central part of the interphase nucleus, contains the bulk of nuclear DNA. Within the karyosome, beaded and ribbon-like chromatin bodies surrounding a loose fibrillar core are commonly recognized. The peripheral domain of both interphase and several mitotic nuclei is filled with a heterogeneous material similar in its ultrastructure to the nucleolar substance. A wide fibrogranular domain lies between this unusual nucleolus and the karyosome. Rosette-like intranuclear inclusions 0.2-0.4 micron in diameter are often seen in both the fibrogranular and nucleolar domains. At the prophase-metaphase, nearly 50 linear chromosome-like bodies are detected as being in close association with several large beaded and ribbon-like chromatin bodies. At the anaphase-telophase, the chromatin bodies per surface-stretched daughter nucleus of live entamoebae, and in each amembraneous daughter nuclear preparation number nearly 14 and 6, respectively. Besides, in each amembraneous DAPI-stained nucleus a set of 50 or so linear chromosome-like bodies are clearly identified. We infer that the nucleus of E. histolytica contains more than 50 linear chromosomes which at different stages of the cell cycle can unite into several beaded and ribbon-like associations. These form a single moderately dense chromatin karyosome in the central part of the interphase nucleus.

[Ultrafine organization of Leptomonas peterhoffi flagellates cultured in broth and solid nutrient media]. / Issledovanie ul'tratonkoi organizatsii zhgutikonostsev Leptomonas peterhoffi, kul'tiviruemykh na zhidkoi i tverdoi pitatel'nykh sredakh.

The morphology of in vitro grown lower trypanosomatids L. peterhoffi was studied by means of electron microscopy. The flagellates from both liquid and solid culture media are represented by uninucleate cells of two structural types. Type I flagellates are characterized by dense cytoplasm enriched with numerous ribosomes. Type II flagellates are most abundant in the cultures; they display a less dense cytoplasm and fewer ribosomes. The flagella of L. peterhoffi of both types form enlargements, which are most expressed at the outlet of the flagellar pocket. The nuclei of some cells contain twisted threads about 10 nm in diameter. L. peterhoffi from the liquid media usually possess long, narrow and curved flagellar pockets. On the solid medium, amoeboid and hemispherical colonies composed of both uninucleate and giant multinucleate cells are formed. In these cells the flagellar pockets are usually short and straight. Outside the flagellar pocket, the axoneme often becomes looped in the flagellar enlargements of the colonial uninucleate cells.