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A V Belyavsky

Publications and source records attributed to A V Belyavsky.

At least 19 recordsLinked to original sources
PubMed2003

Helix peptide immunoreactivity pattern in the nervous system of juvenile aplysia.

Distribution of neurons immunopositive to antibody against the small peptides encoded by the Helix Command-Specific 2 (HCS2) gene in the central nervous system of juvenile Aplysia californica was investigated. The HCS2 gene is specifically expressed in the withdrawal behavior neurons of the terrestrial snail Helix lucorum. In Aplysia, 20-25 immunopositive neuronal somata were observed on dorsal surface of each pleural ganglion (including a giant pleural neuron). The HCS2-encoded peptide immunopositive fibers were observed in neuropiles of all ganglia and in many nerves. Functional significance of Aplysia immunopositive cells is discussed.

Animals↗
PubMed2001

Overexpression of camello, a member of a novel protein family, reduces blastomere adhesion and inhibits gastrulation in Xenopus laevis.

Vertebrate gastrulation involves complex coordinated movements of cells and cell layers to establish the axial structures and the general body plan. Adhesion molecules and the components of extracellular matrix were shown to be involved in this process. However, other participating molecules and detailed mechanisms of the control of gastrulation movements remain largely unknown. Here, we describe a novel Xenopus gene camello (Xcml) which is expressed in the suprablastoporal zone of gastrulating embryos. Injection of Xcml RNA into dorsovegetal blastomeres retards or inhibits gastrulation movements. Database searches revealed a family of mammalian mRNAs encoding polypeptides highly similar to Xcml protein. Characteristic features of the camello family include the presence of the central hydrophobic domain and the N-acetyltransferase consensus motifs in the C-terminal part, as well as functional similarity to Xcml revealed by overexpression studies in Xenopus embryos. Xcml expression results in the decrease of cell adhesion as demonstrated by the microscopic analysis and the blastomere aggregation assay. Cell fractionation and confocal microscopy data suggest that Xcml protein is localized in the secretory pathway. We propose that Xcml may fine tune the gastrulation movements by modifying the cell surface and possibly extracellular matrix proteins passing through the secretory pathway.

Acetyltransferases↗
PubMed2001

Two-dimensional gene expression fingerprinting.

In the present study we have developed the two-dimensional Gene Expression Fingerprinting (2-D GEF) procedure suitable for gene expression analysis and new gene discovery. The procedure is based on the two-dimensional gel display of 3'-terminal cDNA restriction fragments produced by one primary (first dimension) and several sequential secondary restriction digestions. Many thousands of individual sequences per cDNA sample can be visualized using this approach, which is also characterized by a high reproducibility, predictable spatial location of cDNA fragments on 2-D gels, and the potential for identifying cDNA fragments solely on the basis of their two-dimensional coordinates. Using this 2-D GEF method, we analyzed and compared the gene expression patterns of two related primitive hematopoietic cell lines, Kg-1 and Kg-1a. A total of 25 candidate differentially expressed sequences were identified, and for 75% of them the presumed expression pattern was confirmed by Northern blotting or reverse transcription-polymerase chain reaction. We also demonstrated that for 70% of bands, correct prediction of their identity could be made on the basis of two-dimensional coordinates, whereas the major part of incorrect predictions was caused by insufficient database quality.

Blotting, Northern↗
PubMed2001

Up- and down-regulation of Helix command-specific 2 (HCS2) gene expression in the nervous system of terrestrial snail Helix lucorum.

A novel gene named Helix command-specific 2 (HCS2) was shown to be expressed predominantly in four giant parietal interneurons involved in withdrawal behavior of the terrestrial snail Helix lucorum L. and several single neurons in other ganglia. Decrease in spontaneous electrophysiological activity of neurons in the isolated CNS by 24h incubation in saline with elevated Mg(2+) concentration significantly decreased the number of HCS2-expressing neurons. Five short-term serotonin applications (each of 10microM), during a 24h incubation of the nervous system in saline induced expression of the HCS2 gene in many cells in cerebral, parietal, pleural and pedal ganglia. Dopamine applications under similar conditions were not effective. Application of anisomycin or cycloheximide, known to block protein synthesis, did not prevent the induction of HCS2 expression under serotonin influence. Skin injury elicited a significant increase in the number of HCS2-expressing cells 24h later in pleural and cerebral ganglia. Incubation of the isolated nervous system preparations for three days in culture medium elicited close to a maximum increase in number of HCS2-expressing cells. Elevation of the normal Mg(2+) concentration in the culture medium significantly decreased the number of cells demonstrating HCS2 expression. Application of the cAMP activator forskolin (10microM) increased the expression under Mg(2+), indicating that cAMP was involved in the up-regulation of HCS2. Application of thapsigargin (10microM), known to release Ca(2+) from intracellular stores, was also effective in increasing expression, suggesting participation of Ca(2+) in regulation of HCS2 expression. Cellular groups expressing the HCS2 gene under different conditions seem to be functionally related since it was demonstrated earlier that some neurons constituting these clusters are involved in the withdrawal behavior and the response of the organism to stress stimuli. From these results we suggest that the HCS2 pattern of expression can be down-regulated by a decrease in synaptic activity in the nervous system, and up-regulated by external noxious inputs, as well as the application of neurotransmitters and second messengers known to be involved in the withdrawal behavior and maintenance of isolated ganglia in culture medium. When up-regulated, the HCS2 expression appears, at least in part in neurons, to be involved in the withdrawal behavior.

Animals↗
PubMed2000

Analysis of gene expression in subpopulations of murine hematopoietic stem and progenitor cells.

OBJECTIVE: The aim of the present work was to study how functional differences between subsets of the murine hematopoietic stem/progenitor cell compartment are manifested on the level of different patterns of gene expression in these subsets. MATERIALS AND METHODS: Amplified 3' terminal total cDNA fragment populations from four stem and progenitor cell fractions sorted using differential staining with Rhodamine 123 were prepared, and gene expression patterns were analyzed by Southern hybridization with a panel of gene markers. RESULTS: For the vast majority of lineage-specific markers, no expression was detected in the long-term repopulating stem cell fraction. Expression of a number of key genes positively regulating entry and progression through the cell cycle was down-regulated in long-term repopulating cells, in accordance with the quiescent state of the latter. In contrast, certain but not all cell division kinase inhibitors were significantly up-regulated in long- and short-term repopulating stem cell fractions. Expression of several genes important for entry into the apoptotic pathway was moderately reduced in long-term repopulating cells. Messenger RNA levels of the transcription factors GATA-1, GATA-2, c-Myb and SCL were down-regulated in long-term repopulating cells, as compared to more mature stem/progenitor cells. Finally, expression of the MDR1a gene encoding the Pgp efflux pump was highest in long-term repopulating cells, and progressively decreased with maturation. CONCLUSION: The patterns of gene expression in the stem/progenitor cell fractions are in good correlation with the known properties of adult hematopoietic stem/progenitor cells and may provide insight into molecular mechanisms underlying stem cell physiology.

Animals↗
PubMed1998

Putative neuropeptides and an EF-hand motif region are encoded by a novel gene expressed in the four giant interneurons of the terrestrial snail.

Nine giant interneurons located in the pleural and parietal ganglia of the terrestrial snail Helix lucorum L. were reported to be a key element in the network controlling withdrawal behaviour of the animal. Using a combination of complementary DNA subtraction cloning and differential screening approaches we have isolated a novel gene named HCS2 which is expressed predominantly in a subset of these interneurons. The predicted amino acid sequence of the HCS2 protein contains at the N-terminus a hydrophobic leader sequence and four putative neuropeptides, and at the C-terminus a perfect match to the consensus motif of the EF-hand family of the Ca2+-binding proteins. All four predicted neuropeptides bear a C-terminal signature sequence Tyr-Pro-Arg-X (where X is Ile, Leu, Val or Pro), and three of them are likely to be amidated. Physiological action of three synthetic peptides corresponding to the predicted mature HCS2 peptides mimics fairly well the described action of parietal interneurons on follower motoneurons controlling pneumostome closure. In situ hybridization experiments demonstrated that the HCS2 gene is selectively expressed in the four parietal giant interneurons, as well as in several small unidentified neurons. The onset of the HCS2 transcription during embryogenesis coincides temporally with the time-point when the first withdrawal responses of the embryo to tactile stimulation appear. We propose that the HCS2 gene encodes a hybrid precursor protein whose processed products act as neuromodulators or neurotransmitters mediating the withdrawal reactions of the snail, and in addition may participate in the calcium regulatory pathways or calcium homeostasis in command neurons.

Amino Acid Sequence↗
PubMed1998

A novel neuropeptide precursor gene is expressed in the terrestrial snail central nervous system by a group of neurons that control mating behavior.

We report the isolation of a cDNA clone encoding a neuropeptide precursor named preproGFAD from the central nervous system (CNS) of the snail Helix lucorum. Analysis of the expression of this gene shows that it is neurospecific and expressed in several groups of CNS neurons. Most notable is the expression of preproGFAD gene in the right mesocerebrum, where the neurons controlling mating behavior are located. The expression in this particular region is observed in adult animals but not in juvenile ones. The preprohormone is 108 amino acids long and contains a hydrophobic leader peptide and eight Lys-Arg recognition sites for endoproteolysis. The post-translational processing of the prohormone may lead to the generation of seven tetrapeptides, Gly-Phe-Ala-Asp-COOH (GFAD). This peptide has the same sequence as two previously isolated peptides from a related snail, Achatina fulica. The first of them (achatin-I) contains D-Phe; the second (achatin-II) is its L-Phe-containing stereoisomer. Injection of synthetic D-GFAD in nanomolar concentrations into intact animals caused an increase of the heartbeat rate and opening of the genital atrium. In preparations containing CNS with intact innervation of reproductive organs, bath application of D-GFAD caused extensive movements of the penis but not of other reproductive organs. Intracellular activation of individual neurons expressing the preproGFAD gene also elicited penis movements. D-GFAD also suppressed activity of neurons modulating feeding behavior. Our data therefore indicate that the preproGFAD gene encodes the precursor of a neuropeptide that participates in the regulation of male mating behavior.

Amino Acid Sequence↗
PubMed1997

Characterization of a cDNA clone encoding pedal peptide in the terrestrial snail.

We report the isolation of a Helix lucorum cDNA clone encoding a precursor of neuropeptides that are closely related to Aplysia and Tritonia pedal peptides (Pep). The predicted propeptide contains 20 copies of the two variants of Helix Pep interspersed with Lys-Arg endopeptidase cleavage sites. Northern blot hybridization revealed multiple Pep-hybridizing species in the Helix CNS RNA. The Pep gene was expressed by several identified serotonergic neurones in pedal and cerebral ganglia, groups of sensory neurones in procerebrum, peripheral neurones in olfactory bulb, mantle and foot, and group of neurones in pedal ganglia presumably involved in locomotion control. Pep mRNA was detected in several neurones at the early stages of nervous system development.

Amino Acid Sequence↗
PubMed1997

A novel marker of early epidermal differentiation: cDNA subtractive cloning starting on a single explant of Xenopus laevis gastrula epidermis.

To understand the molecular mechanism underlying in the earliest steps of the embryonic ectoderm subdivision into epidermis and neuroectoderm, it would be important to isolate differentially expressed genes in presumptive neuroectoderm and epidermis at the gastrula stage, the period of the divergence of the two adjacent ectodermal compartments. Meanwhile, the most direct approach for such a task, i.e. subtractive enrichment of cDNA from neuroectodermal and epidermal explants with differentially expressed gene sequences, was difficult to realize because of the high number of explants needed for this technique. In the present paper we report a novel effective and quite simple method of cDNA subtractive enrichment, based on amplification of cDNA in vitro by polymerase chain reaction (PCR) and allowing to use a very small amount of initial cDNA samples. With this method we have cloned cDNA of a novel gene of Xenopus laevis, which was named XEP-1 for its specific expression in the presumptive epidermis starting from the midgastrula stage.

Amino Acid Sequence↗
PubMed1996

A novel murine cathelin-like protein expressed in bone marrow.

A novel cDNA encoding a putative secreted protein was isolated from murine bone marrow. The encoded protein named MCLP (murine cathelin-like protein) was found to be highly homologous to the pig cathelin, and to four neutrophil antimicrobial polypeptides: CAP 18, indolicidin, Bac 5 and FALL-39. Secondary structure prediction studies identified a highly cationic region in the C-terminal part of prepro-MCLP with a tendency to adopt an amphipathic alpha-helical conformation, as observed in many antimicrobial peptides. However, no antibacterial activity was observed with the synthetic peptide corresponding to this region of MCLP.

Amino Acid Sequence↗
PubMed1996

Identification of two novel genes specifically expressed in the D-group neurons of the terrestrial snail CNS.

A search for genes specifically expressed in the giant interneurons of parietal ganglia of the snail Helix lucorum yielded, among others, two genes named HDS1 and HDS2. According to data obtained by Northern hybridization and whole-mount in situ hybridization, both genes are neurospecific and expressed almost exclusively in the peptidergic D-group neurons (Sakharov, 1974) located in the right parietal ganglion. In situ hybridization of the HDS1 and HDS2 probes with CNS of several related species of the Helicoidea superfamily identified in all cases similarly located homologous groups of neurons. Sequencing of the near full-length cDNA copies of the HDS1 and HDS2 genes revealed open reading frames 107 and 102 amino acids long for HDS1 and HDS2, respectively. Both putative proteins contain a hydrophobic leader peptide and putative recognition sites for furin-like and PC-like endopeptidases. Predicted amino acid sequences of the HDS1 and HDS2 proteins were found to be moderately homologous to each other, as well as to the LYCP preprohormone expressed by the light yellow cells of the freshwater snail Lymnaea stagnalis. These results confirm an earlier hypothesis that the D-group of the Helix family and the light yellow cells of Lymnaea stagnalis represent homologous neuronal groups. Our data suggest that the HDS1 and HDS2 genes encode precursors of secreted molecules, most likely neuropeptides or neurohormones.

Amino Acid Sequence↗
PubMed1995

Genomic structure and alternative splicing of the murine bhk/ctk/ntk gene.

Recently, we and others have cloned cDNAs encoding a second member of the Csk family of inhibitory protein kinases, which we termed Bhk [M.A. Ershler et al. (1994) Dokl. Akad. Nauk. 339, 679-683]. In the present study, two new distinct types of bhk mRNA were found in addition to the third form described previously. Analysis of the bhk genomic structure established that three exons participate in the alternative splicing of bhk mRNA.

Alternative Splicing↗
PubMed1995

Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting.

A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3' terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3'-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially in murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes.

Animals↗
PubMed1994

Sequence of the cDNA encoding murine CRK4 protein kinase.

A cDNA clone encoding murine CRK4 protein kinase [Ershler et al., Gene 124 (1993) 305-306] has been isolated and sequenced. The deduced amino acid (aa) sequence shows 85% overall identity with the Xenopus laevis MO15 PK (the catalytic subunit of CAK). The central parts of the CRK4 and MO15 proteins are characterized by an unusually high (97.5%) degree of aa sequence conservation.

Amino Acid Sequence↗
PubMed1994

Novel gene HCS1 is specifically expressed in the giant interneurones of the terrestrial snail.

The terrestrial snail Helix lucorum is a promising model for molecular neurobiology since its central nervous system (CNS) is simple and contains many morphologically and functionally identified large neurones. Among these, the giant interneurones located in pleural and parietal ganglia are especially interesting because they trigger the withdrawal behaviour of the snail and participate in aversive conditioning. Here we describe the identification and characterization of a gene named HCS1 which is preferentially expressed in these interneurones. It encodes a putative protein 100 amino acids long containing an N-terminal hydrophobic leader peptide. No sequences with significant homology to HCS1 were found in the protein (Swiss-Prot) and nucleotide (EMBLbank) data libraries. We suppose that the product of this gene is a secreted protein, presumably a neuropeptide or a growth factor.

Amino Acid Sequence↗
PubMed1992

A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo.

To obtain gene sequences controlling the early steps of amphibian neurogenesis, we have performed differential screening of a subtractive cDNA library prepared by a novel PCR-based method from a single presumptive neural plate of a Xenopus laevis late-gastrula embryo. As a result we have isolated a fragment of a novel homeobox gene (named XANF-1, for Xenopus anterior neural folds). This gene is expressed predominantly in the anterior part of the developing nervous system. Such preferential localization of XANF-1 mRNA is established from its initially homogenous distribution in ectoderm of early gastrula. This change in the expression pattern is conditioned by a differential influence of various mesoderm regions on ectoderm: anterior mesoderm activates XANF-1 expression in the overlying ectoderm, whereas posterior axial and ventral mesoderm areas inhibit it. The data obtained demonstrate for the first time that selection of genes for specific expression in the CNS of the early vertebrate embryo is affected not only by chordamesoderm (a neural inductor) but also by ventral mesoderm.

Amino Acid Sequence↗
PubMed1991

Distribution of high mobility group proteins 1/2, E and 14/17 and linker histones H1 and H5 on transcribed and non-transcribed regions of chicken erythrocyte chromatin.

Quantitative analysis of distribution of chromosomal proteins on single copy DNA sequences has been further developed. Our approach consists of DNA-protein crosslinking within whole cells or isolated nuclei, specific immunoaffinity isolation of crosslinked complexes via protein and identification of crosslinked DNA by hybridisation with single-stranded DNA probes. The present study shows that transcribed chromatin of chicken embryonic erythrocyte beta globin gene is characterized by about 1.5-2.5-fold higher density of HMG 14/17 and 2-fold lower density of H1 and H5 as compared with non-transcribed chromatin of ovalbumin and lysozyme genes, whereas HMG 1/2, E proteins were equally distributed between DNA of both transcribed and non-transcribed genes. The depletion of H1/H5 in beta globin sequences was verified by the 'protein image' hybridisation technique (1). The DNase I hypersensitive site located 5' upstream from beta globin gene is deficient in all the proteins assayed, what implies a drastic disruption in the nucleosomal array. Minor quantitative changes of protein pattern suggest transient local perturbation of the chromatin on transcription.

Animals↗