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Vladimir Berezin

Publications and source records attributed to Vladimir Berezin.

At least 19 recordsLinked to original sources
PubMed2006

GAP-43 regulates NCAM-180-mediated neurite outgrowth.

The neural cell adhesion molecule (NCAM), and the growth-associated protein (GAP-43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP-43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP-43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP-43. We demonstrate that phosphorylation of GAP-43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM-induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP-43, NCAM-induced neurite outgrowth requires functional association of NCAM-180/spectrin/GAP-43, whereas in the absence of GAP-43, the NCAM-140/non-receptor tyrosine kinase (Fyn)-associated signaling pathway is pivotal. Thus, expression of GAP-43 presumably acts as a functional switch for NCAM-180-induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP-43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM-180/spectrin-mediated modulation of the actin cytoskeleton, NCAM-140-mediated activation of Fyn, or both.

Animals↗
PubMed2006

Multiple effects of pentyl-4-yn-VPA enantiomers: from toxicity to short-term memory enhancement.

2-n-Pentyl-4-pentynoic acid (PE-4-yn-VPA) is a derivative of the antiepileptic and mood-stabilizing drug valproic acid (VPA). PE-4-yn-VPA exists as R- and S-enantiomers, the latter being more teratogenic. PE-4-yn-VPA also possesses antiepileptic, antiproliferative, and cell-differentiating properties. Moreover, the less teratogenic enantiomer, R-PE-4-yn-VPA, was recently shown to improve learning and memory. We here present a detailed investigation of the enantioselective properties of PE-4-yn-VPA using a range of in vitro and in vivo assays including measurements of cellular growth and migration, neuronal differentiation and survival, intracellular signal transduction, synaptic plasticity and maturation, and short-term memory as determined by the social recognition test. The results show that the enantiomers of PE-4-yn-VPA largely had similar effects in vitro. However, in all in vitro experiments the more teratogenic enantiomer, S-PE-4-yn-VPA, exhibited a stronger potency than R-PE-4-yn-VPA, and only S-PE-4-yn-VPA had a detrimental effect on cell survival. Interestingly, both the R- and S-enantiomer improved learning and memory. In contrast, the beneficial effect of S-PE-4-yn-VPA on memory was lost by time, whereas the effect of R-PE-4-yn-VPA administration was longer lasting, suggesting that the beneficial effect of the S-enantiomer on memory formation may be counteracted by its detrimental effect on neuronal cell survival.

Analysis of Variance↗
PubMed2006

An automatic procedure for evaluation of single cell motility.

BACKGROUND: Cell motility is vital in many physiological and pathological processes, such as embryogenesis, inflammation, wound healing, and metastasis. However, the time-consuming step in the evaluation of individual cell motility is the analysis of hundreds of recorded images of cell cultures in general consisting of retrieving images, one at a time, and marking the positions of individual cells by a computer mouse. Therefore, the aim of the present study was to develop a novel automatic procedure for the evaluation of cell motility. MATERIALS AND METHODS: The procedure was tested on fibroblasts and glioma and adenocarcinoma cells engineered to express the green fluorescent protein by either transient transfection or adenovirus transduction, allowing automatic recognition of cell coordinates on retrieved images. RESULTS: The effects of serum growth factors, teratogenic compounds, and overexpression of transcription factors on the motile behavior of cultured cells were determined. Cell motility was estimated by both manual and automatic marking of cell position and subsequently motility parameters were computed. The results obtained by the two procedures were found to correlate significantly. CONCLUSIONS: We developed a procedure allowing automatic video recording of sparsely seeded cells transfected with a plasmid or tranduced with a recombinant virus expressing the enhanced green fluorescent protein (EGFP).

Adenocarcinoma↗
PubMed2006

The neural cell adhesion molecule binds to fibroblast growth factor receptor 2.

The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface plasmon resonance analysis that NCAM can bind to FGFR2 with an affinity similar to that for the NCAM-FGFR1 interaction. However, the kinetic parameters for the NCAM-FGFR2 binding are different from those of the NCAM-FGFR1 binding. Both receptors were shown to cycle relatively fast between the NCAM bound and unbound states, although FGFR2 cycling was clearly faster (13 times) than the FGFR1 cycling. Moreover, ATP was more effective in inhibiting the binding of NCAM to FGFR1 than to FGFR2, indicating that the binding sites in NCAM for the two receptors are similar, but not identical.

Adenosine Triphosphate↗
PubMed2006

Neural cell adhesion molecule induces intracellular signaling via multiple mechanisms of Ca2+ homeostasis.

The neural cell adhesion molecule (NCAM) plays a pivotal role in the development of the nervous system, promoting neuronal differentiation via homophilic (NCAM-NCAM) as well as heterophilic (NCAM-fibroblast growth factor receptor [FGFR]) interactions. NCAM-induced intracellular signaling has been shown to affect and be dependent on the cytoplasmic Ca2+ concentration ([Ca2+]i). However, the molecular basis of this remains unclear. In this study, we determined [Ca2+]i regulating mechanisms involved in intracellular signaling induced by NCAM. To mimic the effect of homophilic NCAM interaction on [Ca2+]i in vitro, we used a peptide derived from a homophilic binding site of NCAM, termed P2, which triggers signaling cascades similar to those activated by NCAM-NCAM interaction. We found that P2 increased [Ca2+]i in primary hippocampal neurons. This effect depended on two signaling pathways. The first pathway was associated with activation of FGFR, phospholipase Cgamma, and production of diacylglycerol, and the second pathway involved Src-family kinases. Moreover, NCAM-mediated Ca2+ entry required activation of nonselective cation and T-type voltage-gated Ca2+ channels. These channels, together with the Src-family kinases, were also involved in neuritogenesis induced by physiological, homophilic NCAM interactions. Thus, unanticipated mechanisms of Ca2+ homeostasis are shown to be activated by NCAM and to contribute to neuronal differentiation.

Animals↗
PubMed2006

Elucidation of the mechanism of the regulatory function of the Ig1 module of the fibroblast growth factor receptor 1.

The extracellular part of the fibroblast growth factor (FGF) receptor (FGFR) consists of up to three Ig modules (Ig1-Ig3), in which the Ig2 and Ig3 modules determine affinity and specificity for FGF and heparin. The FGFR isoforms lacking the Ig1 module have higher affinity for FGF and heparin than the triple Ig-module isoforms, suggesting that the Ig1 module is involved in the regulation of the FGFR-ligand interaction. We show here by surface plasmon resonance and NMR analyses that the Ig1 module binds to the Ig2 module, and identify by NMR the binding sites involved in the Ig1-Ig2 interaction. The identified binding site in the Ig2 module was found to be in the area of the FGF-Ig2 and Ig2-heparin contact sites, thus providing direct structural evidence that the Ig1 module functions as a competitive autoinhibitor of the FGFR-ligand interaction. Furthermore, the Ig1 binding site of the Ig2 module overlaps the Ig2-Ig2 contact site. This suggests that the function of the Ig1 module is not only regulation of the FGFR-ligand binding affinity but also prevention of spontaneous FGFR dimerization (through a direct Ig2-Ig2 interaction) in the absence of FGF.

Animals↗
PubMed2006

NMR structure of the first Ig module of mouse FGFR1.

Fibroblast growth factor (FGF) receptors (FGFRs) regulate a multitude of cellular processes during embryogenesis and in the adult. The extracellular part of the prototypical FGFR consists of three Ig modules (Ig1 - Ig3), in which Ig2 and Ig3 determine affinity and specificity for FGF and heparin, while the Ig1 module is thought to have a regulatory function. The crystal structures of the Ig2 and Ig3 modules alone and in complex with FGF have previously been reported. The structure of the Ig1 module is unknown, and very little is known about the structural determinants for the regulatory function of this module. We describe here the NMR structure of the Ig1 module of mouse FGFR1. The three-dimensional fold of the module belongs to the intermediate Ig subgroup and can be described as a beta-barrel consisting of two beta-sheets. One sheet is formed by A', G, F, C, and C', and the other by A, B, B', E, and D beta-strands. The overall strand topology of the Ig1 module is similar to that of the Ig2 and Ig3 modules. However, the A/A' loop of the Ig1 module is much longer than that of the Ig2 and Ig3 modules. It contains eight extra residues compared to the Ig3 module, and five extra residues compared to Ig2.

Animals↗
PubMed2006

Hippocampal up-regulation of NCAM expression and polysialylation plays a key role on spatial memory.

Memory formation has been associated with structural and functional modifications of synapses. Cell adhesion molecules are prominent modulators of synaptic plasticity. Here, we investigated the involvement of the cell adhesion molecules, NCAM, its polysialylated state (PSA-NCAM) and L1 in spatial learning-induced synaptic remodeling and memory storage. A differential regulation of these adhesion molecules was found in the hippocampus of rats submitted to one training session in the spatial, but not cued, version of the Morris water maze. Twenty-four hours after training, synaptic expression of NCAM and PSA-NCAM was increased, whereas L1 appeared markedly decreased. The regulation of these molecules was spatial learning-specific, except for L1 reduction, which could be attributed to swimming under stressful conditions rather than to learning. Subsequent psychopharmacological experiments were performed to address the functional role of NCAM and PSA-NCAM in the formation of spatial memories. Rats received an intracerebroventricular injection of either a synthetic peptide (C3d) aimed to interfere with NCAM function, or endoneuraminidase, an enzyme that cleaves polysialic acid from NCAM. Both treatments affected acquisition of spatial information and lead to impaired spatial memory abilities, supporting a critical role of the observed learning-induced up-regulation of synaptic NCAM expression and polysialylation on spatial learning and memory. Therefore, our findings highlight NCAM as a learning-modulated molecule critically involved in the hippocampal remodeling processes underlying spatial memory formation.

Animals↗
PubMed2006

Molecular mechanisms of Ca(2+) signaling in neurons induced by the S100A4 protein.

The S100A4 protein belongs to the S100 family of vertebrate-specific proteins possessing both intra- and extracellular functions. In the nervous system, high levels of S100A4 expression are observed at sites of neurogenesis and lesions, suggesting a role of the protein in neuronal plasticity. Extracellular oligomeric S100A4 is a potent promoter of neurite outgrowth and survival from cultured primary neurons; however, the molecular mechanism of this effect has not been established. Here we demonstrate that oligomeric S100A4 increases the intracellular calcium concentration in primary neurons. We present evidence that both S100A4-induced Ca(2+) signaling and neurite extension require activation of a cascade including a heterotrimeric G protein(s), phosphoinositide-specific phospholipase C, and diacylglycerol-lipase, resulting in Ca(2+) entry via nonselective cation channels and via T- and L-type voltage-gated Ca(2+) channels. We demonstrate that S100A4-induced neurite outgrowth is not mediated by the receptor for advanced glycation end products, a known target for other extracellular S100 proteins. However, S100A4-induced signaling depends on interactions with heparan sulfate proteoglycans at the cell surface. Thus, glycosaminoglycans may act as coreceptors of S100 proteins in neurons. This may provide a mechanism by which S100 proteins could locally regulate neuronal plasticity in connection with brain lesions and neurological disorders.

Animals↗
PubMed2005

Expression, crystallization and preliminary X-ray analysis of the extracellular Ig modules I-IV and F3 modules I-III of the neural cell-adhesion molecule L1.

Four amino-terminal immunoglobulin (Ig) modules and three fibronectin type III (F3) modules of the mouse neural cell-adhesion molecule L1 have been expressed in Drosophila S2 cells. The Ig modules I-IV of L1 crystallized in a trigonal space group, with unit-cell parameters a = b = 239.6, c = 99.3 A, and the crystals diffracted X-rays to a resolution of about 3.5 A. The F3 modules I-III of L1 crystallized in a tetragonal space group, with unit-cell parameters a = b = 80.1, c = 131 A, and the crystals diffracted X-rays to 2.8 A resolution. This is a step towards the structure determination of the multimodular constructs of the neural cell-adhesion molecule L1 in order to understand the function of L1 on a structural basis.

Animals↗
PubMed2005

Structural biology of NCAM homophilic binding and activation of FGFR.

In this review, we analyse the structural basis of the homophilic interactions of the neural cell adhesion molecule (NCAM) and the NCAM-mediated activation of the fibroblast growth factor receptor (FGFR). Recent structural evidence suggests that NCAM molecules form cis-dimers in the cell membrane through a high affinity interaction. These cis-dimers, in turn, mediate low affinity trans-interactions between cells via formation of either one- or two-dimensional 'zippers'. We provide evidence that FGFR is probably activated by NCAM very differently from the way by which it is activated by FGFs, reflecting the different conditions for NCAM-FGFR and FGF-FGFR interactions. The affinity of FGF for FGFR is approximately 10(6) times higher than that of NCAM for FGFR. Moreover, in the brain NCAM is constantly present on the cell surface in a concentration of about 50 microm, whereas FGFs only appear transiently in the extracellular environment and in concentrations in the nanomolar range. We discuss the structural basis for the regulation of NCAM-FGFR interactions by two molecular 'switches', polysialic acid (PSA) and adenosine triphosphate (ATP), which determine whether NCAM acts as a signalling or an adhesion molecule.

Animals↗
PubMed2005

Neural cell adhesion molecule function is regulated by metalloproteinase-mediated ectodomain release.

The neural cell adhesion molecule (NCAM) is involved in development of the nervous system, in brain plasticity associated with learning and memory, and in neuronal regeneration. NCAM regulates these processes by influencing cell adhesion, cell migration, and neurite outgrowth. NCAM activates intracellular signaling upon homophilic NCAM binding, and this is a prerequisite for NCAM-stimulated neurite outgrowth. NCAM is synthesized in three main membrane-bound isoforms, NCAM-120, NCAM-140, and NCAM-180. Soluble forms of NCAM in blood and cerebrospinal fluid have also been found, although the functional significance of these forms remains unclear. In this report, we demonstrate that NCAM can be released from primary hippocampal neurons in culture. The release was enhanced by application of ATP and inhibited by the metalloproteinase inhibitor BB-3103. ATP also induced metalloproteinase-dependent release of all three major NCAM isoforms from NCAM-transfected fibroblastoid L-cells. In this model system, the extracellular ATP-binding site of NCAM was shown not to be necessary for ATP-induced NCAM release. Furthermore, inhibition of serine, cysteine, and aspartic proteinases could not prevent ATP-induced down-regulation of NCAM in L-cells, suggesting that NCAM is cleaved directly by a metalloproteinase. Aggregation of hippocampal neurons in culture was increased in the presence of the metalloproteinase inhibitor GM 6001, consistent with a metalloproteinase-dependent shedding of NCAM occurring in these cells. Moreover, NCAM-dependent neurite outgrowth was significantly reduced by application of GM 6001. Taken together, these results suggest that membrane-bound NCAM can be cleaved extracellularly by a metalloproteinase and that metalloproteinase-dependent shedding of NCAM regulates NCAM-mediated neurite outgrowth.

Adenosine Triphosphate↗
PubMed2005

Triple effect of mimetic peptides interfering with neural cell adhesion molecule homophilic cis interactions.

The neural cell adhesion molecule (NCAM) is pivotal in neural development, regeneration, and learning. Here we characterize two peptides, termed P1-B and P2, derived from the homophilic binding sites in the first two N-terminal immunoglobulin (Ig) modules of NCAM, with regard to their effects on neurite extension and adhesion. To evaluate how interference of these mimetic peptides with NCAM homophilic interactions in cis influences NCAM binding in trans, we employed a coculture system in which PC12-E2 cells were grown on monolayers of fibroblasts with or without NCAM expression and the rate of neurite outgrowth subsequently was analyzed. P2, but not P1-B, induced neurite outgrowth in the absence of NCAM binding in trans. When PC12-E2 cells were grown on monolayers of NCAM-expressing fibroblasts, the effect of both P1-B and P2 on neurite outgrowth was dependent on peptide concentrations. P1-B and P2 acted as conventional antagonists, agonists, and reverse agonists of NCAM at low, intermediate, and high peptide concentrations, respectively. The demonstrated in vitro triple pharmacological effect of mimetic peptides interfering with the NCAM homophilic cis binding will be valuable for the understanding of the actions of these mimetics in vivo.

Amino Acid Sequence↗
PubMed2005

The role of RhoA in the regulation of cell morphology and motility.

Members of the Rho family of small GTPases are key regulators of the actin cytoskeleton, particularly in relation to the cell shape changes and the adhesion dynamic that drive cell migration. Here, we report the effect of activation or inhibition of the function of RhoA on cell motility and morphology. Both in the presence and the absence of serum, expression of constitutively active RhoA dramatically inhibited L929 fibroblasts' cell motility, and induced a rounding of the cells and a decrease in the number of processes per cell. In contrast, expression of a dominant negative mutant of RhoA had no effect on cell motility or morphology in steady-state conditions with or without serum in the medium. Inhibition of p160ROCK, a kinase effector of RhoA, only partially inhibited cell migration. Conversely, when cells were submitted to a period of serum deprivation followed by addition of serum, inhibition of endogenous RhoA by expression of the dominant negative mutant of RhoA impeded cell motility after serum stimulation. Thus, RhoA activity is required for stimulation of cell locomotion by serum factors. It was also observed that the addition of serum factors to quiescent L929 and NR6wtEGFR fibroblasts resulted in a delayed motility response of several hours compared to the immediately induced morphological changes, indicating the absence of a previously assumed direct correlation between changes in cell motility and cell morphology in response to serum addition. The motility response of L929 and NR6wtEGFR fibroblasts to serum stimulation required protein synthesis.

3T3 Cells↗
PubMed2005

Molecular characterization of virulent Newcastle disease virus isolates from chickens during the 1998 NDV outbreak in Kazakhstan.

Newcastle disease virus (NDV) infects domesticated and wild birds throughout the world and has the possibility to cause outbreaks in chicken flocks in future. To assess the evolutionary characteristics of 10 NDV strains isolated from chickens in Kazakhstan during 1998 we investigated the phylogenetic relationships among these viruses and viruses described previously. For genotyping, fusion (F) gene phylogenetic analysis (nucleotide number 47-421) was performed using sequences of Kazakhstanian isolates as compared to sequences of selected NDV strains from GenBank. Phylogenetic analysis showed that all newly characterized strains belonged to the genetic group designated as VIIb. All strains possessed a virulent fusion cleavage site (RRQRR/F) belonging to velogenic or mesogenic pathotypes with intracerebral pathogenicity indexes (ICPI) varying from 1.05 to 1.87.

Amino Acid Sequence↗
PubMed2005

Genetic analysis of an overlapping functional requirement for L1- and NCAM-type proteins during sensory axon guidance in Drosophila.

L1- and NCAM-type cell adhesion molecules represent distinct protein families that function as specific receptors for different axon guidance cues. However, both L1 and NCAM proteins promote axonal growth by inducing neuronal tyrosine kinase activity and are coexpressed in subsets of axon tracts in arthropods and vertebrates. We have studied the functional requirements for the Drosophila L1- and NCAM-type proteins, Neuroglian (Nrg) and Fasciclin II (FasII), during postembryonic sensory axon guidance. The rescue of the Neuroglian loss-of-function (LOF) phenotype by transgenically expressed L1- and NCAM-type proteins demonstrates a functional interchangeability between these proteins in Drosophila photoreceptor pioneer axons, where both proteins are normally coexpressed. In contrast, the ectopic expression of Fasciclin II in mechanosensory neurons causes a strong enhancement of the axonal misguidance phenotype. Moreover, our findings demonstrate that this functionally redundant specificity to mediate axon guidance has been conserved in their vertebrate homologs, L1-CAM and NCAM.

Animals↗
PubMed2005

Distinct roles of PKC isoforms in NCAM-mediated neurite outgrowth.

The role of protein kinase C (PKC) isoforms in the neural cell adhesion molecule (NCAM)-mediated neurite outgrowth was tested using a co-culture system consisting of fibroblasts with or without NCAM expression upon which either primary cerebellar granular neurones (CGN) or pheochromocytoma (PC12-E2) cells were grown. The latter transiently expressed various PKC isoforms and domains derived from selected PKCs. PKC inhibitors of various specificity inhibited NCAM-stimulated neuritogenesis from CGN, indicating that PKC is involved in this process. Moreover, stimulation by the NCAM-mimetic peptide, C3d, elicited phosphorylation of PKC in CGN. Expression of kinase-deficient forms of PKCalpha, betaI and betaII blocked NCAM-mediated neurite extension, but had no effect on nerve growth factor (NGF)-mediated neurite outgrowth. Expression of two PKCepsilon constructs: (i) a fragment from PKCepsilon encompassing the pseudosubstrate, the C1a domain (including the actin-binding site, ABS), and parts of the V3 region, or (ii) the PKCepsilon-specific ABS blocked NCAM-mediated neurite extension in both cases. These two constructs also partially inhibited NGF-stimulated neuritogenesis indicating that PKCepsilon is a positive regulator of both NCAM- and NGF-mediated differentiation. We suggest that PKCepsilon is a common downstream mediator for several neuritogenic factors, whereas one or more conventional PKCs are specifically involved in NCAM-stimulated neurite outgrowth.

Animals↗