[Hormone-Induced In Vitro Maturation and Ovulation of Danio rerio Oocytes and Production of Eggs Capable of Fertilization and Futher Development].

It is common knowledge that zebrafish, Danio rerio, oocytes in their follicular envelope that have reached definitive size undergo in vitro maturation in 90% Leibovitz's medium, pH 9.0, when treated with 17α,20ß-dihydroxyprogesterone and acquire developmental competence but do not ovulate (Seki et al., 2008). We have demonstrated that zebrafish oocytes that have undergone maturation under the indicated conditions ovulate when treated with prostaglandin F2α (5 µg/mL) and/or 20% carp ovarial fluid and are capable of development towards the actively feeding larvae upon fertilization (the maximum follow-up period).

[ag1 Is Required for the Fin Regeneration in Danio rerio].

In the current research, we have demonstrated that Ag1 protein is necessary for the fin regeneration in the fish Danio rerio. Robust activation of gene ag1 expression in cells of the wound epithelium is observed after caudal fin amputation. Besides, inhibition of translation of ag1 mRNA leads to retardation of the caudal tail fin regeneration. Results of our research are important because only lower vertebrates (fish and amphibians) with good regenerative capacity have ag1, whereas this gene is missing in higher vertebrates, which are not capable to effectively regenerate limbs. Our data confirm that reduction of the regenerative abilities in higher vertebrates, including human, could be explained by extinction of some genes essential for the regeneration, in particular, of ag1.

[Hormonal Induction of In Vitro Maturation and Ovulation of Loach Oocytes and Obtaining Egg Cells Capable of Fertilization and Development].

Loach oocytes that have reached a definitive size and are surrounded by follicular envelopes are capable of maturation and ovulating under the effect of 1 µg/mL progesterone in 75% Leibovitz medium with 1 g/L sodium bicarbonate or with pH adjustment to 9.0 by 1 N sodium hydroxide. Inseminated eggs are developed until the stage of when adding 20% bovine serum to the incubation medium. Substitution of the bovine serum with 10-20% loach ovarian fluid or 20% carp ovarian fluid provides more complete development of inseminated eggs until the stage of prolarvae that pass to active nutrition.

[Fish ovarian fluid contains protease inhibitors].

Studies of the conditions under which fish egg is activated spontaneously without the sperm showed that the egg retains the ability for fertilization in the ovarian (coelomic) fluid, which surrounds it in the gonad cavity after ovulation. Earlier, we showed that, in artificial media, the spontaneous activation is suppressed by protease inhibitors. In this study, we investigated the presence of natural protease inhibitors in the ovarian fluid and showed that the ovarian fluid of zebrafish and loach contains protease inhibitors, in particular, type I serpin a, a protein inhibitor of trypsin proteases.

[Modern opportunities and prospects for studying pathogenesis, diagnosing and treating hereditary optic neuropathies].

OBJECTIVE: To evaluate modern opportunities and prospects for studying pathogenesis and improving diagnostics and treatment of hereditary optic neuropathies (HON). MATERIAL AND METHODS: The article presents summarized data on the pathogenesis, diagnostics, and treatment of HON based on modern methods of assessment. RESULTS: The results of long-term worldwide studies and those performed in the Research Institute of Eye Diseases in collaboration with several other institutions are presented. Genetic testing for mitochondrial and nucleus DNA mutations that have a known association with Leber's hereditary optic neuropathy (LHON) and autosomal dominant optic neuropathy (ADON) allow verification only in half of the cases. Particular features of hereditary diseases, such as incomplete penentrance, variable expression, clinical polymorphism, difficulties in detection of hereditary sings, and genetic heterogeneity, are shown to complicate the diagnosis of HON. Spectral retinal tomography revealed characteristic morphometric changes in the macular region and peripapillary nerve fiber layer in the acute stage of LHON. Hereditary optic neuropathies result from a genetically determined decrease in mitochondrial respiratory chain complexes activity, which is associated with a decrease in ATP production. From that standpoint, studying of mitochondrial oxidative phosphorylation biochemical defects in LHON and ADON is an option for detection of mitochondrial dysfunction. Results of a newly proposed method of mitochondrial membrane potential assessment in skin fibroblasts, which can be used for differential diagnosis of mitochondrial optic nerve diseases, are presented. Possible therapeutic measures for HON are discussed. CONCLUSION: In the prevailing number of cases the described clinical, molecular genetic, and cytological methods ensure proper diagnosis of hereditary optic neuropathies. Prospects of HON treatment, rather ambiguous, are associated with further studying of pathogenesis, development of drugs and gene therapy.

[Homeostatic responses of plants to modern climate change: spatial and phenological aspects].

A series of dates of unfolding of the first leaves and duration of the season of vegetation in the silver birch (Betulapendula Roth. (B. verrucosa Ehrh.)), as well as the duration of flowering of the bird cherry (Padus avium), mountain ash (Sórbus aucupária), and small-leaved lime (Tilia cordata Mill.) for the period 1970-2010 in the central part of European Russia were studied in order to assess the trends. Differences in phenological responses to homogeneous climate changes in the trees of the same species from the northern and southern parts of the range were revealed. If spring events occur 3-7 days earlier in the northern part, no such effect is observed in the south. This fact can be interpreted as a manifestation of the different mechanisms of homeostasis in different populations determined by their biological characteristics (in particular, by the need to pass successfully the periods of organic rest and vegetation).

[Role of vimentin in cell migration].

Cell migration plays a crucial role in embryonic development, wound healing, regeneration, inflammation, and immune response, as well as in dissemination of malignant tumors. Vimentin is the marker of migrating cells, but its role in cell migration is still unclear. However, recent studies have revealed novel functions for vimentin related to the migration, such as determination of cellular polarity, regulation of cell contact formation, and arrangement and transport of signal proteins involved in cell motility. The review sums up the latest data on vimentin functions and its involvement in molecular mechanisms underlying cell migration. Early studies demonstrated that vimentin expression during embryonic development is associated with cell migration. However, having obtained vimentin knockout mice without apparent impairments in development and ability to reproduce, doubts have appeared ifvimentin is required for cell migration during embryonic development. In the present review, we also discuss involvement of vimentin in migration processes at different stages of development and try to resolve current contradictions concerning the role of vimentin in various events of cell migration.

[Influence of primary adhesive interactions with fibronectin on clonal growth and osteogenic potential of rat mesenchymal stromal cells].

Interactions with extracellular matrix including fibronectin (Fn) play an important role in regulation of cell growth and differentiation. Influence of Fn and its individual domains on adhesion and osteogenic potencies of rat mesenchymal stromal cells (MSCs) was estimated. Investigation of bone marrow or fetal liver MSCs adhesion dynamics showed that after 7 days of cultivation on Fn the number of adhered clonogenic cells derived from both sources was comparable to their number observed on plastic but their content in suspension was commonly decreased. Population of fetal liver MSCs differed from bone marrow-derived population by greater fraction of cells that adhered for the first 7 days. Bone marrow MSC cultures on Fn were characterized by reduced activity of alkaline phosphatase as compared with cultivation on plastic; furthermore, they deposed significantly smaller amount of calcium salts under cultivation in osteogenic medium. Cultivation of MSCs on Fn fragments demonstrated the primary role of its cell-binding domain in the inhibition of osteogenesis.

[Spontaneous activation of fish eggs is abolished by protease inhibitors].

During spawning, eggs of most fish species entering the aquatic environment remain fertilizable for a relatively short period of time. This is due to the "spontaneous egg activation" giving rise to the fertilization membrane, which prevents the penetration of excessive and foreign sperm into the egg during normal fertilization. This work demonstrates that the fertilization membrane formation and the loss of fertilizability in aqueous solutions of different composition are inhibited by protease inhibitors, in particular, leupeptin and aprotinin. The presence of natural protease inhibitors in the ovarian fluid that prevent spontaneous egg activation is proposed. The decrease in the concentration of these inhibitors as the ovarian fluid is diluted in aquatic medium during spawning can explain egg activation in the absence of sperm.

[A study of proteins of annexin group in early fish development. IV. Identification of calcium-binding proteins in zebrafish egg by mass spectrometry].

Calcium-binding proteins were isolated from zebrafish (Brachidanio rerio) eggs by the method of precipitation with acidic phospholipids in the presence of calcium ions. The revealed proteins separated by SDS-PAGE were identified by mass spectrometric tryptic peptide fingerprinting. The proteins included annexins A2a, A1a, A13.1, and A5. In addition, copine III, a member of the recently described copine family of C2 domain-containing proteins, was identified. Total RNA was analyzed in mature oocytes by RT-PCR and transcripts of two different annexin A13 forms (A13.1 and A13.2) as well as of annexin A3 were detected. Thus, the presence of both proteins and mRNAs of annexins has been shown in the zebrafish egg.

Intermediate vimentin filaments and their role in intracellular organelle distribution.

Intermediate filaments (IF) represent one of three main cytoskeletal structures in most animal cells. The human IF protein family includes about 70 members divided into five main groups. The characteristic feature of IF is that in various cells and tissues they are formed by proteins of different groups. Structures of all IF proteins follow a unique scheme: a central alpha-helical part is flanked at the N and C ends by positively charged polypeptide chains devoid of a clear secondary structure. The central part is highly conserved for all proteins in all animals, whereas the N and C termini strongly differ both in size and amino acid composition. This review covers the broad spectrum of recent investigations of IF structure and diverse functions. Special attention is paid to the regulatory mechanisms of IF functions, mainly to phosphorylation by different protein kinases whose role is well studied. The review gives examples of hereditary diseases associated with mutations of some IF proteins, which point to an important physiological role of these cytoskeletal structures.

[Study of the mitochondria movement using videomicroscopy]. / Videomikroskopicheskoe issledovanie dvizheniia mitokhondrii.

A stable cell line CV-1 was obtained for vital observation of the transport of mitochondria in animals cells, which express a fragment of the resident protein of mitochondria marked by yellow fluorescent protein. The parameters and conditions of movement of the mitochondria in living cells were established using fluorescence videomicroscopy. Under the normal conditions, only a small part of mitochondria (ca. 7%) was transported over significant distances, while others were in the state of relative rest. The effective transport of mitochondria strictly depended on the dynamic properties of microtubules. Incubation of cell in a serum-free medium suppressed active transport of mitochondria, thus suggesting its dependence on certain, not yet determined environmental factors.

[A study of annexin family protein in early development of fish. II. Analysis of complete amino acid sequence]. / Izuchenie belka gruppy anneksinov v rannem razvitii ryb. II. Opredelenie polnoi aminokislotnoi posledovatel'nosti.

We studied mRNA structure of 31 kDa annexin of zebra fish Brachydanio rerio using previously obtained 3'-terminal incomplete cDNA. The size of this protein mRNA was determined by Northern hybridization. PCR screening of cDNA library of zebra fish gastrula allowed us to obtain cDNA of the 5'-terminal regions of the mRNA. The primary structure of the protein deduced from the mRNA sequence allowed us to identify it as an annexin IV with threonine in position 6--a phosphorylation target for protein kinase C.

An isoform of kinesin light chain specific for the Golgi complex.

Conventional kinesin is a motor protein implicated in the transport of a variety of cytoplasmic organelles along microtubules. The kinesin molecule consists of two heavy chains with motor domains at their amino termini and two light chains, which, together with the carboxyl termini of the heavy chains, are proposed to mediate binding to cargoes. Since the light chains are represented by multiple isoforms diverging at their carboxyl termini they are presumed to specify kinesin targeting to organelles. Previously, we isolated five cDNAs, encoding hamster kinesin light chain isoforms, and found that one of them (B or C) preferentially associated with mitochondria. To obtain additional evidence proving the specific location of various kinesin light chain isoforms on organelles, we made an antibody against a 56 amino-acid sequence found at the carboxyl-terminal regions of the hamster D and E isoforms. By indirect immunofluorescence, this antibody specifically labeled the Golgi complex in cultured cells. In western blots of total cell homogenates, it recognized two close polypeptides, one of which co-purified with the Golgi membranes. Thus, the results of this and previous studies demonstrate that different kinesin light chains are associated with different organelles in cells.

Regulation of melanosome movement in the cell cycle by reversible association with myosin V.

Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161-164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis.

Protein kinase C and casein kinase 2 phosphorylate in vitro proteins of the annexin family from eggs of loach Misgurnus fossilis.

A mixture of proteins of the annexin family was obtained from the cytoplasm of mature eggs of loach Misgurnus fossilis (by reprecipitation with acid phospholipids in the presence of Ca2+). This mixture comprised five proteins with molecular weights of 58, 38, 36, 35, and 31 kD. Polyclonal rabbit antibodies against the major 31-kD protein were obtained. Western blot analysis showed that the obtained antibodies exhibit a high specificity towards the 31-kD protein from eggs and other tissues of loach and zebrafish (Brachydanio rerio). The analysis of cDNA corresponding to the 31-kD protein by screening the zebrafish cDNA library confirmed that this protein belongs to the annexin family. Phosphorylation of the obtained annexins in vitro was studied. It is shown that the 58-kD protein is phosphorylated by casein kinase 2 (CK2), whereas the 38-, 36-, 35-, and 31-kD proteins are phosphorylated by protein kinase C (PKC).

[A protein of the annexin group in early fish development. I. Identification and biosynthesis of the protein in embryogenesis]. / Izuchenie belka gruppy anneksinov v rannem razvitii ryb. I. Identifikatsiia belka i ego biosintez v émbriogeneze.

Ca-binding proteins were isolated from the loach (Misgurnus fossilis) eggs and embryos, using their capacity for binding to acidic phospholipids in the presence of Ca ions. It was shown that the major protein present in a mixture of these proteins was synthesized at the early developmental stages. Polyclonal rabbit antibodies were raised against this protein. These antibodies proved to be monospecific both to the protein of the loach eggs and embryos and that of the Brachydanio rerio embryos. The antibodies were used for screening cDNA library from the B. rerio embryos at 6 to 9 h of development. As a result of screening, a cDNA clone was obtained, which, when converted into a peptide sequence, shows a high degree of homology with proteins of the annexin group.

A specific light chain of kinesin associates with mitochondria in cultured cells.

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1-3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.