A herpes-like virus in king crabs: Characterization and transmission under laboratory conditions.

A herpes-like virus was found infecting the antennal gland and bladder epithelium in the blue king crab Paralithodes platypus from the eastern area of the Sea of Okhotsk. Electron microscopic analysis of antennal gland samples from blue king crabs with histologically confirmed signs of disease revealed virus particles, which were mostly hexagonal in shape and located primarily in the nucleus; these particles were rarely observed in the cytoplasm of infected cells. Most virus particles ranged in size from 115 to 125nm. Hemocytes of the red king crab Paralithodes camtschaticus in cell culture could be experimentally infected with virus from thawed antennal gland samples of the blue king crabs with histologically confirmed signs of viral infection. Clear signs of infection were observed in hemocyte cultures at 3-4days post-inoculation as small foci of highly vacuolated formations. These formations included several nuclei and were surrounded by a halo of small cytoplasmic bubbles containing actin and tubulin. As demonstrated by electron microscopic studies, no virus-like particles were found in the cells 1day post-inoculation, but particles become abundant at 7days post-inoculation. We developed a consensus primer PCR method for amplification of a region of the herpesviral DNA-directed DNA polymerase. Primers were designed to target sequences encoding highly conserved amino acid motifs covering a region of approximately 800bp. Thus, macroscopic, histological and ultra-structural examinations of blue king crabs infected with a virus and the molecular identification of the pathogen revealed the presence of herpesviruses. The frequency of the herpes-like viral infection in natural populations of blue king crabs in the Sea of Okhotsk ranged from 0% to 3% in different years.

[Effect of exogenous factors on the induction of spicule formation in sea urchin embryonic cell cultures].

The effect of exogenous factors on the realization of the spicule formation program in two sea urchin species, Strongylocentrotus intermedius and S. nudus, has been studied in primary embryonic cell cultures derived from the blastula and gastrula stages. It has been shown that the process of spicule formation depends on the type of substrate and the composition of the medium. An original finding is that calf or horse serum necessary for spicule formation in vitro can be replaced by a complex of factors including insulin, transferrin, and lectins. Methods allowing control over the growth and differentiation of marine invertebrate embryonic cells in vitro open prospects for their application to practical problems such as the establishment of cell cultures producing certain mineral structures.

The fatty acid profile changes in marine invertebrate larval cells during cryopreservation.

The development of cryopreservation methods for embryonic cells and larvae of sea animals offers a great potential for marine biotechnology. Larval cells of bivalves and sea urchins were frozen to -196 degrees C using traditional cryoprotectants (Me(2)SO and trehalose) and the cryoprotective mixture developed by us. In addition to Me(2)SO and trehalose, this mixture contained an exogenous lipid extract from mussel tissues and antioxidants. A positive effect of antioxidants (alpha-tocopherol acetate, ascorbic acid or echinochrome, the quinoid pigment of sea urchins) on cell viability became significant only in the presence of exogenous lipids. Antioxidants added to cryoprotective mixtures did not reveal visible cryoprotective activity when used separately. To better understand the mechanism of the protective effect of exogenous lipids on cell membranes of sea animals, a comparative analysis of the fatty acid (FA) composition of total lipids in larval cells before and after freezing was carried out using a gas-liquid chromatography. The results indicate that freezing-thawing has direct effects on the FA composition of major lipid classes in marine invertebrate cells, and these effects can vary depending on the provenance of the cells. We have found that (I) both cell viability and the FA profile of cell lipids after cryopreservation depend on the cryoprotectants used; (II) an amount of saturated, monoenic and polyenic FAs changes significantly after cryopreservation. We assume that the addition of the exogenous lipid extract in form of liposomes could promote a renewal of disturbance areas and prevent from membrane damages during freezing-thawing.

[Stem cells of marine invertebrates: regulation of proliferation and differentiation processes in vitro].

Own and literary data pointing to plasticity of stem cells of marine invertebrates are viewed. Stem and embryonic cell cultures of marine invertebrates are model cell systems with a high level of all physiological and synthetic processes. The production of biological active substances in vitro may become an alternative to the chemical synthesis or marine aquaculture. The factors involved in determination and maintenance of the pluripotency of marine invertebrate stem cells are analyzed. The in vitro technology of directional differentiation of marine invertebrate stem cells in certain functionally active cell species includes the use of different growth factors, various natural and artificial substrates and also the unique biological active substances from marine invertebrate tissues. To increase expression levels of the cell growth regulatory genes and thus achieve enhanced cell growth, one method of attack was reported using the genetically engineered constructions with foreign genes. Our knowledge gained in invertebrate systems can help to determine a general mechanism of eukaryote pluripotency, and cultivated marine invertebrate cells can be used in marine biotechnology.

[Changes in the lipid composition of embryonic cells of the mussel Mytilus trossulus during cryopreservation].

A comparative analysis of the lipid composition of embryonic cells of the mussel Mytilus trossulus prior to, and after, cryopreservation in liquid nitrogen was carried out in the presence of two types of cryoprotectors: 1) dimethylsulfoxide and trehalose; and 2) dimethylsulfoxide, trehalose, total lipid extract from the mussel Crenomytilus grayanus, alpha-tocopherol and ascorbic acid. It was found that not only the cell viability but also the fatty acid composition of cell lipids after cryopreservation depend on the composition of cryoprotectors used. The content of saturated fatty acids, monoenic and polyenic fatty acids, the omega 3/omega 6 ratio, and the index of nonsaturation in the fatty acid composition of lipids was shown to change remarkably after cryopreservation. Possible reasons of these changes are discussed.

[Development of the muscle system and contractile activity in the mussel Mytilus trossulus (Mollusca, Bivalvia)].

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and florescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96 h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 microm in both mussle larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing videosystem. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult mollusks.

[Influence of the activator of transcription GAL4 on growth and development of embryos and embryonic cells in primary cultures of sand dollar]. / Vliianie aktivatora transkriptsii GAL4 na rost i razvitie émbrionov i émbrional'nykh kletok v pervichnykh kul'turakh ploskogo morskogo ezha.

In order to solve many tasks of biotechnology, constant lines of the cells of marine invertebrates with a high growth potential are required, which are absent at present. We used the universal activator of transcription gal4 to change the degree of expression of genes of growth factors in embryonic sea urchin cells and, thereby, increase their proliferative activity. The fertilized sea urchin eggs and dissociated embryonic cells at the blastula stage were treated with plasmids containing both the functional gene gal4 and the gene devoid of the regions encoding the activator domain. The transfection of embryonic sea urchin eggs with the functional gene led to cell dedifferentiation and formation of tumor-like structures in the embryos or increased number of embryonic cells in culture. In the cells obtained from the transfected embryos, the pigments were found within two months of cultivation, whose absorption spectrum coincided with that of echinochrome.

Gal4-gene-dependent alterations of embryo development and cell growth in primary culture of sea urchins.

Primary cell cultures from sea urchins have a low proliferative level that prevents the establishment of long-term cultures. To increase expression levels of the genes regulating cell growth in sea urchins, and thus enhance cell growth, we used the transcriptional activator gene Gal4 found earlier in yeast. Sea urchin embryos were treated with plasmid DNA containing the Gal4 gene. Expression of the transgene was confirmed by reverse transcriptase polymerase chain reaction. When the fully functional gene was used, embryos effectively formed teratoma-like structures after 50 to 55 hours of cultivation. In contrast, the Gal4 gene, devoid of acidic activating regions, possessed little activity as a teratogen. The Gal4-treated cells in blastula-derived culture showed higher DNA synthesis and higher proliferative activity than control cells. We suggest that formation of the teratoma-like structures in embryos, activation of DNA synthesis, and significant increase of cell number in embryo-derived cell cultures could be attributed to Gal4 gene action.

[Myogenic differentiation of Mytilus larval cells in vitro]. / Miogennaia differentsirovka lichinochnykh kletok midii in vitro.

A myogenic differentiation program can be realized during the cultivation of Mytilus trossulus cells derived from larvae in premyogenic developmental stages. About 10-15% of cells in such cultures showed that they are capable of contracting actively. The shape of such cells and the high concentration of actin microfilaments indicate a similarity with smooth muscle cells. However, the pattern of contractile activity and the protein composition of these cells differ significantly from the corresponding characteristics of differentiated smooth muscle cells. The proportion between the main proteins of the thick fiber, paramyosin, and myosin in cultivated cells is far lower than in the muscles of larvae or adult molluscs. We also found that substrates with different adhesional characteristics may determine cell development towards one or the other phenotype. Cells attached to the collagen substrate, but not spread on it, had high proliferative potential; the collagen substrate, however, inhibited myogenic differentiation.

Effect of lectin from the ascidian on the growth and the adhesion of HeLa cells.

Cell adhesion molecules, some of which are lectins, play a key role in the control of normal and pathological processes of various living organisms. We found herein that N-acetyl-D-glucosamine-specific lectin, isolated from the ascidian Didemnum ternatanum (DTL), alters the growth properties of HeLa tumor cells depending on the anchorage. DTL was shown to increase the proliferation of HeLa cells grown in soft agar greatly (in anchorage-independent fashion). In contrast, DTL inhibits the proliferative activity of HeLa cells grown on solid substrate and acts as inductor of differentiation, slowing cell growth, increasing the cell attachment and spreading. Scanning electron microscopic data have demonstrated that DTL treatment resulted in pronounced changes of the shape and surface of HeLa cells. Changes of cellular morphology correlated with essential redistribution of actin microfilaments.

Isolation and partial characterization of myogenic cells from mussel larvae in vitro.

The main finding of the present study is the discovery of the possibility of a morphofunctional myogenic differentiation of larval mussel cells in vitro. The shape and extensive cytoskeletal network of the cultured contracting cells mimic largely those of smooth muscle cells in vivo. However, the behavior and protein composition of these cells are not completely identical with those of smooth muscle cells. Contracting mussel cells in vitro, as well as differentiated smooth muscles, demonstrate both phasic and tonic contractions. The paramyosin to myosin ratio in the cultured mussel cells is far less than that in the muscles of veliger larvae and adult mussels. We have found the protein carpets with various adhesive characteristics determine different development pathways. Myogenic differentiation is only observed in spreading cells. Non-spreading adherent cells plated on collagen carpet show high synthetic activity but the commitment of contractile phenotype is inhibited. Our results confirm that the myogenic program established in early embryogenesis of molluscs can be realized during the cultivation of cells from premyogenic larval stages.

Tumor-like lesions in the mantle of the mussel Modiolus difficilis from the Sea of Japan.

Two inner growths in the mantle beneath the epithelium were found in 1 of 1000 mussels Modiolus difficilis from Amursky Bay, Sea of Japan, within the city precincts of Vladivostok. Both growths were about 2000 microns in maximal diameter in section and elevated slightly above the mantle surface. The mantle epithelium near the growths formed deep invaginations, and clusters of mucous cells were numerous beneath the epithelium. Histological and histochemical methods were employed. Two different kinds of growth were revealed. The off-white growth consisted of cells with thin granular or vesicular cytoplasm containing glucosaminoglycans, proteins and a small amount of neutral polysaccharides. Growth cells were pure white in color after treatment of preparations with 1% H2SO4 and differed markedly from the mantle cells. The yellow growth consisted of large granular cells with neutral polysaccharides and proteins. Although growths were composed of different kinds of cells, they seemed to be derived from subepithelial mucous cells. This was supported by histological and histochemical staining reactions of some tumor and mantle epithelial cells. Mitotic indices (MI) of growths and subepithelial mucous cells were zero, MI of ciliated mantle epithelium reached 0.07%. The lesions were areas of strongly altered mucous cells of mantle epithelium and were non-neoplastic.

Adhesive and growth properties of lectin from the ascidian Didemnum ternatanum on cultivated marine invertebrate cells.

The effects of N-Acetyl-D-glucosamine-specific lectin (M(r) 27 kDa) isolated from the ascidian Didemnum ternatanum on cultivated cells of molluscs and echinoderms were studied. This lectin was found to stimulate the growth or the differentiation of cultivated marine invertebrate cells depending on the stage of embryonic development at which primary cell cultures were obtained. In addition, it has been shown to increase the attachment of cells in primary cultures of these animals. The degree of attachment is considerably increased when collagen or polylysine substrates are used. Using scanning electron microscopy we have demonstrated the stage-specific effect of this lectin on embryonic sea urchin and molluscan cells. Intensive cell spreading and an alteration of cell shape were observed only at the gastrula stage, when the switching from maternal information to embryonic genes occurred. The ascidian lectin seems to have some characteristics of both an adhesive factor and a growth factor.

Hyperplastic growth of mucous cells in the mantle of the mussel Modiolus kurilensis from a heavily polluted area of Amursky Bay, Sea of Japan.

This histology of an external mantle growth in 1 of 500 Modiolus kurilensis from a heavily polluted area of Amursky Bay, Sea of Japan, was described. The growth consisted of subepithelial basophilic mucous cells containing glucosaminoglycans, eosinophilic large-granular cells with proteins and neutral polysaccharides, and mixed (acid + neutral mucopolysaccharides) cell types. Some subepithelial eosinophilic and basophilic gland cells were dividing and seemed to be the source of tumor growth. The mitotic index of growth cells reached 0.5% on some growth sections; however, many mitoses were pycnotic. The emergence of the tumor on the mussel mantle is probably related to a compensatory or regenerative hyperplasia of subepithelial mucous cells.

[The effect of trophoblast-specific beta 1-glycoprotein and oncoprecipitin A on the growth and morphology of HeLa-M tumor cells]. / Vliianie trofoblast-spetsificheskogo beta 1-glikoproteina i onkopretsipitina A na rost i morfologiiu opukholevykh kletok linii HeLa-M.

Pregnancy-specific beta 1-glycoprotein (PS-1), one of a large family in oncofetal antigens, and oncoprecipitin A, a specific glycoprotein isolated from ascidians, have been shown to reduce cell proliferative activity in HeLa-M cells but almost not to change RNA synthesis. Using scanning electron microscopy, distinct surface transductions in the studied cells were demonstrated. After the incubation of cells with oncoprecipitin A, cell shape alteration and intensive cell spreading were observed. The treatment of tumor cells with PS-1 changed cell shape and essentially reduced cell contacts, as compared to control cells. The changes in cell surface and shape correlated with essential reorganization of actin microfilaments. The use of substances influencing the growth and adhesive characteristics of tumor cells may help in understanding mechanisms of cell transformation.

Effect of inducers of lipid peroxidation on the behavior of ciliated epithelial cells.

Television microscope and original image treatment system were used for monitoring and recording the ciliary activity (beat frequency) of gill ciliated epithelia of the mussel Mytilus edulis (Bivalvia) and of the rat tracheal ciliated epithelia in response to the following prooxidants: H2O2, Fe+2, Fe+2 + ascorbic acid and NADP-H + ADP + Fe+2. Mussel ciliated cells proved to be more sensitive to the influence of the prooxidants than rat cells. The reactions of ciliated epithelial cells of mollusks and rats to the inducers of lipid peroxidation were not similar to behavioral responses of these cells under the action of low-dose ionizing radiation.

[Isolation and general characteristics of lectin from the ascidian Didemnum ternatum]. / Vydelenie i obshchaia kharakteristika lektina iz astsidin Didemnum ternatum.

DTL was isolated from the Didemnum ternatum colonial ascidian and purified by affinity chromatography on cross-linked ovalbumin followed by gel-filtration on Sephadex G-100. SDS-PAGE of the preparation showed a major intense band with a relative mol. wt. of ca. 27 kDa. In the presence of 2-mercaptoethanol, DTL gave rise to a single band of 10 kDa. The lectin isolated was found to agglutinate Ehrlich's carcinoma cells and trypsinized human A, B, O erythrocytes, the hemagglutination being inhibited by GlcNAc, chitobiose, desialylated glycoproteins. Comparison of the sugar moieties of the glycoproteins used as inhibitors led to suggestion that the specific sugar binding site contains, as a dominant sugar, a "bisecting" glucosamine attached through a beta-1,4-linkage to the beta-linked mannose residue of N-glycosidic chains of a complex or hybrid type. An absence of sialic acid residues linked with galactose residues appeared to be necessary for binding DTL with the complex-type sugar chains. DTL inhibits proliferation and reduces DNA biosynthesis ([3H]thymidine incorporation) in tumour cells (HeLa). Principal morphological differences were detected to occur between intact and DTL-treated HeLa cells.

[Growth factor from the tissues of the marine mollusk Mytilus edulis]. / Rostovoi faktor iz tkanei morskogo molliuska Mytilus edulis.

The absence of specific stimulators of cell proliferation, i.e. of growth factors, is one of the causes of failure in long-term cultivation of the marine invertebrate cells. In search for such stimulators in tissues of marine invertebrates, we succeeded in discovering in some tissues a stimulator of cell proliferation, similar to EGF, with a comparatively high content of it in tissues of Mytilus edulis. The similarity of the obtained factor with EGF was shown by the substitution of 125I-EGF, connected with EGF receptors on the surface of A431 cells, with analysed extracts, as well as by the ability of this extract to induce the internalization of EGF-receptor complexes. The fraction of acid/ethanol extract of M.edulis stimulating the cell proliferation in resting mouse fibroblasts Swiss 3T3, was obtained in the same conditions as EGF did, using reversed phase chromatography. Thus, the factor from tissues of M.edulis may belong to the family of EGF-like factors.

Primary cell culture from embryos of the Japanese scallop Mizuchopecten yessoensis (Bivalvia).

Primary cell cultures obtained from embryos of Mizuchopecten yessoensis (Bivalvia) survived for four months. Although the number of cells progressively decreased during the cultivation, mitotic cells were observed both at the first stages and at the end. A possibility of growing marine invertebrates cells in long term primary culture is discussed.