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G Enikolopov

Publications and source records attributed to G Enikolopov.

16 recordsLinked to original sources
PubMed2005

Transgenic mice overexpressing nNOS in the adult nervous system.

Nitric oxide (NO) has a profound role in the generation, differentiation, survival, and physiology of neurons. We have created a novel transgenic model to study the action of NO in the adult brain, in which the neuronal isoform of NO synthase (nNOS) is expressed under control of the promoter of the calcium-calmodulin multifunctional kinase IIalpha (CaMKIIalpha) gene. We show that the transgenic nNOS RNA and protein are expressed in the cortex, the hippocampus and the striatum of the transgenic mice. We also show that expression of several genes involved in the protection of neurons from oxidative stress and cell death is not affected in neurons of the transgenic mice. Furthermore, generation of new cells is depressed in the neurogenic brain areas in transgenics. In addition, we analyze gene expression in the hippocampus of the transgenic animals using microarray RNA profiling and Q-PCR. Our experiments describe specific changes in cell division and gene activity in the CaMKII-nNOS transgenic model and demonstrate its utility for studying the action of NO in the adult brain.

Animals↗
PubMed2001

Nitric oxide is an essential negative regulator of cell proliferation in Xenopus brain.

Mechanisms controlling the transition of a neural precursor cell from proliferation to differentiation during brain development determine the distinct anatomical features of the brain. Nitric oxide (NO) may mediate such a transition, because it can suppress DNA synthesis and cell proliferation. We cloned the gene encoding the neuronal isoform of Xenopus NO synthase (XNOS) and found that in the developing brain of Xenopus tadpoles, a zone of XNOS-expressing cells lies adjacent to the zone of dividing neuronal precursors. Exogenous NO, supplied to the tadpole brain in vivo, decreased the number of proliferating cells and the total number of cells in the optic tectum. Conversely, inhibition of NOS activity in vivo increased the number of proliferating cells and the total number of cells in the optic tectum. NOS inhibition yielded larger brains with grossly perturbed organization. Our results indicate that NO is an essential negative regulator of neuronal precursor proliferation during vertebrate brain development.

Animals↗
PubMed2001

The Drosophila nitric-oxide synthase gene (dNOS) encodes a family of proteins that can modulate NOS activity by acting as dominant negative regulators.

Nitric oxide (NO) is involved in organ development, synaptogenesis, and response to hypoxia in Drosophila. We cloned and analyzed the only gene in the fly genome that encodes Drosophila nitric-oxide synthase (dNOS). It consists of 19 exons and is dispersed over 34 kilobases of genomic DNA. Alternative transcription start sites and alternative splice sites are used to generate a remarkable variety of mRNAs from the dNOS gene. We identified eight new transcripts that are widely expressed throughout Drosophila development and encode a family of DNOS-related proteins. Alternative splicing affects both the 5'-untranslated region and the coding region of the dNOS primary transcript. Most of the splicing alterations in the coding region of the gene lead to premature termination of the open reading frame. As a result, none of the alternative transcripts encode an enzymatically active protein. However, some of these shorter DNOS protein products can effectively inhibit enzymatic activity of the full-length DNOS1 protein when co-expressed in mammalian cells, thus acting as dominant negative regulators of NO synthesis. Using immunoprecipitation, we demonstrate that these short DNOS protein isoforms can form heterodimers with DNOS1, pointing to a physical basis for the dominant negative effect. Our results suggest a novel regulatory function for the family of proteins encoded by the Drosophila NOS gene.

Alternative Splicing↗
PubMed2000

Specific pattern of p53 phosphorylation during nitric oxide-induced cell cycle arrest.

Nitric oxide (NO) is an efficient inhibitor of cell proliferation. Here we show that part of the antiproliferative activity of NO in fibroblasts is mediated through p53 signaling pathway. Cells from p53-/- knockout mice are compromised in their ability to stop dividing in the presence of NO. NO strongly induces expression of genes which are transcriptional targets of p53, and p53 is necessary for some, but not all, of the transcription activation effects of NO. Furthermore, NO strongly increases the cellular level of p53 protein. Since phosphorylation of particular residues of the p53 molecule has been correlated with its functional activity, we determined the phosphorylation pattern of p53 molecule after exposure to NO and compared it with the phosphorylation patterns that develop upon treatment with gamma-irradiation, UV light, and adriamycin. We found that NO induces a specific signature pattern of p53 phosphorylation, distinct from the patterns evoked by other inducers. This study suggests that NO activates specific signaling pathways that may partially overlap, but that do not coincide, with signaling pathways activated by other known inducers of p53 activity.

3T3 Cells↗
PubMed2000

The anti-dementia drug nefiracetam facilitates hippocampal synaptic transmission by functionally targeting presynaptic nicotinic ACh receptors.

Nefiracetam, a pyrrolidone derivative developed as an anti-dementia drug, persistently potentiated currents through neuronal nicotinic acetylcholine (ACh) receptors (alpha7, alpha4beta2) expressed in Xenopus oocytes, and the potentiation was blocked by either the selective protein kinase C (PKC) inhibitors, GF109203X and staurosporine, or co-expressed active PKC inhibitor peptide. In primary cultures of rat hippocampal neurons, nefiracetam increased the rate of nicotine-sensitive miniature excitatory postsynaptic currents, without affecting the amplitude, and the increase was inhibited by GF109203X. In addition, the drug caused a marked increase in the glutamate release from electrically stimulated guinea pig hippocampal slices, and the effect was abolished by the nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. Nefiracetam induced a long-lasting facilitation of synaptic transmission in both the CA1 area and the dentate gyrus of rat hippocampal slices, and the facilitation was inhibited by alpha-bungarotoxin and mecamylamine. Such facilitatory action was still found in the hippocampus with selective cholinergic denervation. The results of the present study, thus, suggest that nefiracetam enhances activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to a sustained facilitation of hippocampal neurotransmission. This may represent a cellular mechanism underlying the cognition-enhancing action of nefiracetam. The results also provide the possibility that nefiracetam could be developed as a promising therapeutic drug for senile dementia or Alzheimer's disease.

Animals↗
PubMed2000

Nitric oxide interacts with the retinoblastoma pathway to control eye development in Drosophila.

Animal organ development requires that tissue patterning and differentiation is tightly coordinated with cell multiplication and cell cycle progression. Several variations of the cell cycle program are used by Drosophila cells at different stages during development [1] [2]. In imaginal discs of developing larvae, cell cycle progression is controlled by a modified version of the well-characterized mammalian retinoblastoma (Rb) pathway [3] [4], which integrates signals from multiple effectors ranging from growth factors and receptors to small signaling molecules. Nitric oxide (NO), a multifunctional second messenger [5], can reversibly suppress DNA synthesis and cell division [6] [7]. In developing flies, the antiproliferative action of NO is essential for regulating the balance between cell proliferation and differentiation and, ultimately, the shape and size of adult structures in the fly [8] [9] [10]. The mechanisms of the antiproliferative activity of NO in developing organisms are not known, however. We used transgenic flies expressing the Drosophila nitric oxide synthase gene (dNOS1) and/or genes encoding components of the cell cycle regulatory pathways (the Rb-like protein RBF and the E2F transcription factor complex components dE2F and dDP) combined with NOS inhibitors to address this issue. We found that manipulations of endogenous or transgenic NOS activity during imaginal disc development can enhance or suppress the effects of RBF and E2F on development of the eye. Our data suggest a role for NO in the developing imaginal eye disc via interaction with the Rb pathway.

Animals↗
PubMed2000

Targeting of synaptotagmin to neurite terminals in neuronally differentiated PC12 cells.

We have investigated structural elements that determine the accumulation of synaptotagmin, a major synaptic vesicle protein, in neurite terminals of neuronally differentiated neuroendocrine pheochromocytoma PC12 cells. We performed extensive deletion and point mutagenesis of rat synaptotagmin II, expressed mutant proteins in PC12 cells differentiated by nerve growth factor (NGF) and monitored their intracellular distribution by immunofluorescence. We found a structural element located at the carboxy-terminal domain of the synaptotagmin molecule, which is necessary for its accumulation at the terminal. Using alanine-scanning mutagenesis, we have identified two amino acids in this element, tryptophan W405 and leucine L408, that are critical for correct targeting of synaptotagmin II to neurite terminals. Changing either one of them to alanine prevents the accumulation of the protein at the terminals. These amino acids are evolutionarily conserved throughout the entire synaptotagmin family and also among synaptotagmin-related proteins, suggesting that different synaptotagmins may have similar mechanisms of targeting to neuronal cell terminals.

Amino Acid Sequence↗
PubMed1999

Arachidonic acid induces a long-lasting facilitation of hippocampal synaptic transmission by modulating PKC activity and nicotinic ACh receptors.

The present study was conducted to understand the effect of arachidonic acid on nicotinic acetylcholine (ACh) receptor-mediated synaptic plasticity. Arachidonic acid persistently (>/=1 h) potentiated currents through neuronal nicotinic ACh receptors (alpha7 and alpha4beta2) expressed in Xenopus oocytes, and the effect was blocked by the selective protein kinase C (PKC) inhibitors, such as GF109203X, PKCI, and co-expressed active PKC inhibitor peptide. This free fatty acid markedly increased nicotine-sensitive glutamate release from hippocampal slices and enhanced the rate of nicotine-sensitive miniature excitatory postsynaptic currents without affecting the amplitude in cultured hippocampal CA1 neurons under the influence of PKC. Furthermore, arachidonic acid induced a long-lasting (>/=3 h) facilitation of hippocampal CA1 synaptic transmission in slices, and the effect was blocked by nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. The facilitation, whereas independent of N-methyl-D-aspartate (NMDA) receptors, shares a common mechanism with long-term potentiation (LTP) induced by tetanic stimulation. The results of the present study thus suggest that arachidonic acid sustains enhanced activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to an 'LTP-like' facilitation of hippocampal synaptic transmission.

Animals↗
PubMed1999

Arachidonic acid potentiates currents through Ca2+-permeable AMPA receptors by interacting with a CaMKII pathway.

The present study investigated the effect of arachidonic acid on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, presumably heteromeric receptors formed of GluR1, GluR2, and GluR3, expressed in Xenopus oocytes. Arachidonic acid (10 microM) potentiated currents through receptors expressing GluR1 and 3 (GluR1,3) to 170% of basal level during initial 20 min following application, being still evident at 60-min washing-out of the drug, while it never or little enhanced currents through receptors expressing GluR1 and 2 (GluR1,2) or GluR1, 2, and 3 (GluR1,2,3) (110% 30 min after treatment). The effect of arachidonic acid on GluR1,3 currents was not observed in Ca2+-free extracellular solution, and the potentiation was blocked by either KN-93, a selective Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, or NP217, an active CaMKII inhibitor peptide, when co-expressed with the receptors. In contrast, the protein synthesis inhibitor, cycloheximide, the selective inhibitor of cAMP-dependent protein kinase (PKA), H-89, the selective inhibitors of protein kinase C (PKC), PKCI and GF109203X, the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059, or the inactive CaMKII inhibitors, KN-92 and NP218, had no effect on the currents. In the assay of intracellular calcium mobilizations, Ca2+ influx in response to receptor activation was greatest with receptors formed in oocytes expressing GluR1,3. The results of the present study indicate that arachidonic acid induces a long-lasting potentiation of GluR1,3 currents, possibly as a result of the interaction with a CaMKII pathway.

Animals↗
PubMed1999

Nitric oxide and Drosophila development.

Mechanisms controlling the transition of precursor cells from proliferation to differentiation during organism development determine the distinct anatomical features of tissues and organs. NO may mediate such a transition since it can suppress DNA synthesis and cell proliferation. Inhibition of NOS activity in the imaginal discs of Drosophila larvae results in hypertrophy of tissues and organs of the adult fly, whereas ectopic overexpression of NOS has the reciprocal, hypotrophic, effect. Furthermore, NO production is crucial for the establishment of ordered neuronal connections in the visual system of the fly, indicating that NO affects the acquisition of the differentiated phenotype by the neural tissue. Increasing evidence points to a broad role that NO may play in animal development by acting as an essential negative regulator of precursor cell proliferation during tissue and organ morphogenesis.

Animals↗
PubMed1999

Mutual regulation between the intercellular messengers nitric oxide and brain-derived neurotrophic factor in rodent neocortical neurons.

The diffusible factors, nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) are both suggested to be intercellular messengers that have similar synaptic activities and developmental influences in the brain. In the present study, we have analysed their mutual regulation with respect to their production in rodent neocortical neurons. Some of the cultured rat neocortical neurons exhibited immunoreactivity for both neuronal NO synthase (NOS) and the BDNF receptor trkB. Neuronal NOS appeared to be activated autonomously and produced NO in culture as monitored by nitrite accumulation. Inhibition of the endogenous NO production in culture by a NOS inhibitor, NG-monomethyl-L-arginine (NMMA), enhanced basal expression of BDNF mRNA and protein. Similarly, cerebroventricular administration of another NOS inhibitor, N-omega-nitro-L-arginine methylester (L-NAME), but not D-NAME or saline, increased BDNF content in the neocortex. In the opposite direction, however, BDNF appeared to function as a positive regulator for NO synthesis. Addition of BDNF upregulated the neuronal NOS expression as well as NO production in neocortical culture. In agreement, BDNF knock-out mice exhibited significant impairment of neuronal NOS expression in the neocortex. Taken together, these observations suggest that the trans-synaptic signalling molecules, NO and BDNF, influence the production of each other and mutually regulate the strength of their intercellular communications.

Animals↗
PubMed1996

Nitric oxide regulates cell proliferation during Drosophila development.

Cell division and subsequent programmed cell death in imaginal discs of Drosophila larvae determine the final size of organs and structures of the adult fly. We show here that nitric oxide (NO) is involved in controlling the size of body structures during Drosophila development. We have found that NO synthase (NOS) is expressed at high levels in developing imaginal discs. Inhibition of NOS in larvae causes hypertrophy of organs and their segments in adult flies, whereas ectopic expression of NOS in larvae has the opposite effect. Blocking apoptosis in eye imaginal discs unmasks surplus cell proliferation and results in an increase in the number of ommatidia and component cells of individual ommatidia. These results argue that NO acts as an antiproliferative agent during Drosophila development, controlling the balance between cell proliferation and cell differentiation.

Animals↗
PubMed1996

A critical role of nitric oxide in human immunodeficiency virus type 1-induced hyperresponsiveness of cultured monocytes.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infection leads to a general exhaustion of the immune system. Prior to this widespread decline of immune functions, however, there is an evident hyperactivation of the monocyte/macrophage arm. Increased levels of cytokines and other biologically active molecules produced by activated monocytes may contribute to the pathogenesis of HIV disease both by activating expression of HIV-1 provirus and by direct effects on cytokine-sensitive tissues, such as lung or brain. In this article, we investigate mechanisms of hyperresponsiveness of HIV-infected monocytes. MATERIALS AND METHODS: The study was performed on monocyte cultures infected in vitro with a monocytetropic strain HIV-1ADA. Cytokine production was induced by stimulation of cultures with lipopolysaccharides (LPS) and measured by ELISA. To study involvement of nitric oxide (NO) in the regulation of cytokine expression, inhibitors of nitric oxide synthase (NOS) or chemical donors of NO were used. RESULTS: We demonstrate that infection with HIV-1 in vitro primes human monocytes for subsequent activation with LPS, resulting in increased production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin 6 (IL-6). This priming effect can be blocked by Ca(2+)-chelating agents and by the NOS inhibitor L-NMMA, but not by hemoglobin. It could be reproduced on uninfected monocyte cultures by using donors of NO, but not cGMP, together with LPS. CONCLUSIONS: NO, which is expressed in HIV-1-infected monocyte cultures, induces hyperresponsiveness of monocytes by synergizing with calcium signals activated in response to LPS stimulation. This activation is cGMP independent. Our findings demonstrate the critical role of NO in HIV-1-specific hyperactivation of monocytes.

Calcium↗
PubMed1995

Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells.

Arrest of cell division is a prerequisite for cells to enter a program of terminal differentiation. Mitogenesis and cytostasis of neuronal cell precursors can be induced by the same or by different growth or trophic factors. Response of PC12 cells to nerve growth factor (NGF) involves a proliferative phase that is followed by growth arrest and differentiation. Here we present evidence that the cytostatic effect of NGF is mediated by nitric oxide (NO), a second messenger molecule with both para- and autocrine properties that can diffuse freely and act within a restricted volume. We show that NGF induces different forms of nitric oxide synthase (NOS) in neuronal cells, that nitric oxide (NO) acts as a cytostatic agent in these cells, that inhibition of NOS leads to reversal of NGF-induced cytostasis and thereby prevents full differentiation, and that capacity of a mutant cell line to differentiate can be rescued by exogenous NO. We suggest that induction of NOS is an important step in the commitment of neuronal precursors and that NOS serves as a growth arrest gene, initiating the switch to cytostasis during differentiation.

Amino Acid Oxidoreductases↗
PubMed1993

Amplification of calcium-induced gene transcription by nitric oxide in neuronal cells.

Nitric oxide (NO) is a short-lived, highly reactive gas, which has been identified as a mediator in vasodilation, an active agent in macrophage cytotoxicity and neurotoxicity, and a neuro-transmitter in the central and peripheral nervous systems. Production of NO by neurons is critical for facilitated synaptic transmission in models of synaptic plasticity such as long-term potentiation and long-term depression, suggesting a role for NO as a retrograde messenger that could complete a hypothetical feedback loop by strengthening the connection between postsynaptic and presynaptic cells. We report here that although alone NO has no evident effect on transcription, it can act as an amplifier of calcium signals in neuronal cells. NO and Ca2+ action have to coincide in time for amplification to occur. Experiments with a series of simplified reporter genes in combination with specific recombinant protein kinase inhibitors suggest that induction of gene activity following NO-amplified calcium action involves protein kinase A-dependent activation of the transcription factor CREB.

Animals↗