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Vladimir Parpura

Publications and source records attributed to Vladimir Parpura.

16 recordsLinked to original sources
PubMed2006

Vesicular transmitter release from astrocytes.

Astrocytes can release a variety of transmitters, including glutamate and ATP, in response to stimuli that induce increases in intracellular Ca(2+) levels. This release occurs via a regulated, exocytotic pathway. As evidence of this, astrocytes express protein components of the vesicular secretory apparatus, including synaptobrevin 2, syntaxin, and SNAP-23. Additionally, astrocytes possess vesicular organelles, the essential morphological elements required for regulated Ca(2+)-dependent transmitter release. The location of specific exocytotic sites on these cells, however, remains to be unequivocally determined.

Animals↗
PubMed2006

Single molecule mechanical probing of the SNARE protein interactions.

Exocytotic release of neurotransmitters is mediated by the ternary soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) complex, comprised of syntaxin (Sx), synaptosome-associated protein of 25 kDa (SNAP25), and synaptobrevin 2 (Sb2). Since exocytosis involves the nonequilibrium process of association and dissociation of bonds between molecules of the SNARE complex, dynamic measurements at the single molecule level are necessary for a detailed understanding of these interactions. To address this issue, we used the atomic force microscope in force spectroscopy mode to show from single molecule investigations of the SNARE complex, that Sx1A and Sb2 are zippered throughout their entire SNARE domains without the involvement of SNAP25. When SNAP25B is present in the complex, it creates a local interaction at the 0 (ionic) layer by cuffing Sx1A and Sb2. Force loading rate studies indicate that the ternary complex interaction is more stable than the Sx1A-Sb2 interaction.

Animals↗
PubMed2005

Nanofabricated carbon-based detector.

The procedure for the fabrication of a detector termed NACAD, nanofabricated carbon-based detector, is described. I characterized the mechanical properties of this detector using atomic force and scanning electron microscopy techniques. This detector exhibits a smooth surface (rms 2-4 nm) and easy access for atomic force microscopy tips, necessary features for biological studies of secretory granules and vesicles, which store transmitters and release them via exocytosis. The NACAD does not impair the elastic properties of granular matrixes deposited onto a detector, as they show their typical Young's moduli and ion exchanger properties; divalent histamine shrinks them, while monovalent sodium causes their swelling. Additionally, the NACADs' electrochemical properties allowed amperometric measurements of serotonin released from intact secretory granules isolated from mast cells, after removal of their granular membranes using a mild detergent treatment. Thus, this detector will aid future studies of single secretory granules and vesicles and their insoluble matrixes.

Animals↗
PubMed2005

Chemically functionalized water soluble single-walled carbon nanotubes modulate neurite outgrowth.

We report the use of chemically-functionalized water soluble single-walled carbon nanotube (SWNT) graft copolymers for modulation of outgrowth of neuronal processes. The graft copolymers were prepared by the functionalization of SWNTs with poly-m-aminobenzene sulphonic acid and polyethylene glycol. When added to the culturing medium, these functionalized water soluble SWNTs were able to increase the length of various neuronal processes.

Animals↗
PubMed2005

Detection of botulinum toxins: micromechanical and fluorescence-based sensors.

Botulinum neurotoxins (BoNTs) are the most lethal of known human toxins, exerting their actions by cleaving the soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) required for neurotransmitter release. Early detection of these toxins is important for appropriate medical treatment. To detect BoNT activity, traditional assays monitor the effects of the toxins on a mammalian organism (observing signs of botulism in mice), or identify cleaved substrate molecules (electrophoresis and immunoblot). Similarly, enzyme-linked assays were used for screening potential toxin inhibitors in vitro in attempt to select antitoxins that could be used for therapeutic purposes. Here we review two recently developed sensor systems for detection of toxin activity in vitro and in living cells. In vitro detection was carried out using a micromechanosensor that relies on the attachment of a bead to the micromachined cantilever through the interactions between SNARE proteins, with synaptobrevin 2 deposited onto beads and syntaxin 1A deposited onto cantilevers. The presence of toxin is indicated by the detachment of the bead, resulting from cleavage of synaptobrevin 2. Additional in vitro detection is possible using fluorescent sensors constructed by inserting linkers, containing fragments of SNARE proteins acting as toxin substrates, between cyan and yellow fluorescent proteins (CFP and YFP). Toxins cause the cleavage of these linkers and thereby abolish fluorescence resonance energy transfer (FRET) between CFP and YFP. This approach, combined with an additional sensor based on subcellular redistribution of YFP fluorescence in cells, was used for cell-based screening of toxin activity.

Botulinum Toxins↗
PubMed2004

cCXCR1 is a receptor for cIL-8 (9E3/cCAF) and its N- and C-terminal peptides and is also activated by hIL-8 (CXCL8).

Chemokines are chemotactic cytokines that play important roles in immune responses and wound healing, as well as in pathological conditions such as chronic inflammation and tumorigenesis. The chemokines and their receptors are highly conserved and maintain similar functions in different species. One noteworthy exception is the chemokine interleukin (IL)8/CXC ligand 8 and its specific receptor CXCR1, which are found in humans but are not found in the traditional model organisms, mice and rats. As a consequence, we are using model organisms other than mice to study the functions of IL-8 and CXCR1, as well as the mechanisms involved in receptor activation by IL-8. Toward this goal, we have isolated and characterized a new receptor that is highly homologous to human (h)CXCR1, which we named chicken (c)CXCR1. To determine whether this receptor is activated by cIL-8 and its N- and C-terminal peptides and whether it responds to hIL-8, we expressed cCXCR1 in NIH3T3 cells, which naturally lack this receptor, and used single-cell Ca(2)(+) imaging to detect increases in intracellular Ca(2)(+) and immunoblot analysis to detect extracellular signal-regulated kinase 1/2 phosphorylation. We show that cIL-8, its N and C peptides, and hIL-8 activate cCXCR1. We further show that cIL-8 and hIL-8 stimulate chemotaxis of chicken embryonic fibroblasts, cells that express cCXCR1, and that this effect is specific for each chemokine and this receptor. These results strongly suggest that cCXCR1 is the ortholog for hCXCR1 and that chickens can be used as an effective model system to study the functions of IL-8, its terminal peptides, and its specific receptor CXCR1.

3T3 Cells↗
PubMed2004

Separation of individual neurons using dielectrophoretic alternative current fields.

Experimental investigations into the dynamics of neuronal networks are a fundamental step towards understanding how the nervous system works. Memory formation and development are associated with changes in the electrical activity of the neurons. To understand the changes in the electrical activity, it is essential to conduct in vitro studies on individual neurons. Hence, there is an enormous need to develop novel ways for isolating and localizing individual neurons. To this end, we designed and fabricated a 4x4 multiple microelectrode array system to spatially arrange neurons by generating dielectrophoretic traps using gradient alternating current (AC) fields. We characterized the electric field distribution inside our test platform by using three-dimensional finite element modeling (FEM) and estimated the location of neurons over the electrode array. As the first stage in forming a neuronal network, dielectrophoretic AC fields were employed to separate the neurons from the glial cells and to position individual neurons over single electrodes. The extracellular electrical activity from a single neuron was recorded. The frequency spectrum of the electrical activity was generated using fast Fourier transformation analysis (FFT) to determine the characteristic burst rates of individual neurons.

Action Potentials↗
PubMed2004

C(a2+)-dependent glutamate release involves two classes of endoplasmic reticulum Ca(2+) stores in astrocytes.

Astrocytes can modulate synaptic transmission by releasing glutamate in a Ca(2+)-dependent manner. Although the internal Ca(2+) stores have been implicated as the predominant source of Ca(2+) necessary for this glutamate release, the contribution of different classes of these stores is still not well defined. To address this issue, we cultured purified solitary cortical astrocytes and monitored changes in their internal Ca(2+) levels and glutamate release into the extracellular space. Ca(2+) levels were monitored by using the Ca(2+) indicator fluo-3 and quantitative fluorescence microscopy. Glutamate release was monitored by an L-glutamate dehydrogenase-linked detection system. Astrocytes were mechanically stimulated with a glass pipette, which reliably caused an increase in internal Ca(2+) levels and glutamate release into the extracellular space. Although we find that the presence of extracellular Cd(2+), a Ca(2+) channel blocker, significantly reduces mechanically induced glutamate release from astrocytes, we confirm that internal Ca(2+) stores are the predominant source of Ca(2+) necessary for this glutamate release. To test the involvement of different classes of internal Ca(2+) stores, we used a pharmacological approach. We found that diphenylboric acid 2-aminoethyl ester, a cell-permeable inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist, greatly reduced mechanically induced glutamate release. Additionally, the preincubation of astrocytes with caffeine or ryanodine also reduced glutamate release. Taken together, our data are consistent with dual IP(3)- and caffeine/ryanodine-sensitive Ca(2+) stores functioning in the control of glutamate release from astrocytes.

Aniline Compounds↗
PubMed2004

Vesicular glutamate transporter-dependent glutamate release from astrocytes.

Astrocytes exhibit excitability based on variations of their intracellular Ca2+ concentrations, which leads to glutamate release, that in turn can signal to adjacent neurons. This glutamate-mediated astrocyte-neuron signaling occurs at physiological intracellular Ca2+ levels in astrocytes and includes modulation of synaptic transmission. The mechanism underlying Ca2+-dependent glutamate release from astrocytes is most likely exocytosis, because astrocytes express the protein components of the soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors complex, including synaptobrevin 2, syntaxin, and synaptosome-associated protein of 23 kDa. Although these proteins mediate Ca2+-dependent glutamate release from astrocytes, it is not well understood whether astrocytes express functional vesicular glutamate transporters (VGLUTs) that are critical for vesicle refilling. Here, we find in cultured and freshly isolated astrocytes the presence of brain-specific Na+-dependent inorganic phosphate cotransporter and differentiation-associated Na+-dependent inorganic phosphate cotransporter that have recently been identified as VGLUTs 1 and 2. Indirect immunocytochemistry showed a punctate pattern of VGLUT immunoreactivity throughout the entire cell body and processes, whereas pharmacological inhibition of VGLUTs abolished mechanically and agonist-evoked Ca2+-dependent glutamate release from astrocytes. Taken together, these data indicate that VGLUTs play a functional role in exocytotic glutamate release from astrocytes.

Amino Acid Transport Systems, Acidic↗
PubMed2004

The N- and C-terminal peptides of hIL8/CXCL8 are ligands for hCXCR1 and hCXCR2.

Chemokines are small cytokines that function in immune responses, wound healing, and pathological conditions such as chronic inflammation and tumorigenesis. This multifunctionality has been attributed primarily to ligand interaction with multiple or dimerized receptors. However, multifunctionality could also result from interactions of the receptors with small peptides produced by processing of the chemokines. Chemokine peptides are functional in vivo, but it is not yet known whether they can interact with and activate their receptors. The work presented here examines the interactions between the two forms of human interleukin 8 (hIL-8), and its N- and C-peptides, with the chemokine receptors hCXCR1 and hCXCR2. We used a Tet-on retroviral system to introduce CXCR1 into mouse NIH 3T3 cells (that lack endogenous CXCR1) and monitored activation of this receptor by the ligands by using quantitative Ca2+ imaging and mitogen-activated protein kinase (MAPK) activation. We found that the N and C termini of the chemokine can stimulate the respective CXCR1 to induce intracellular Ca2+ release and MAPK activation independent of the other regions of the molecules. Furthermore, we showed that these peptides can also stimulate chemotaxis of several cell types, including primary human microvascular endothelial cells, and that this function is specific and mediated by hCXCR1 and/or hCXCR2. These findings advance understanding of the multifunctionality exhibited by chemokines, reveal a new mode of functional regulation, and may serve as the basis for therapeutic targeting.

Animals↗
PubMed2003

Botulinum toxin type B micromechanosensor.

Botulinum neurotoxin (BoNT) types A, B, E, and F are toxic to humans; early and rapid detection is essential for adequate medical treatment. Presently available tests for detection of BoNTs, although sensitive, require hours to days. We report a BoNT-B sensor whose properties allow detection of BoNT-B within minutes. The technique relies on the detection of an agarose bead detachment from the tip of a micromachined cantilever resulting from BoNT-B action on its substratum, the synaptic protein synaptobrevin 2, attached to the beads. The mechanical resonance frequency of the cantilever is monitored for the detection. To suspend the bead off the cantilever we use synaptobrevin's molecular interaction with another synaptic protein, syntaxin 1A, that was deposited onto the cantilever tip. Additionally, this bead detachment technique is general and can be used in any displacement reaction, such as in receptor-ligand pairs, where the introduction of one chemical leads to the displacement of another. The technique is of broad interest and will find uses outside toxicology.

Antigens, Surface↗
PubMed2003

Dopamine-induced oxidative stress in neurons with glutathione deficit: implication for schizophrenia.

Glutathione (GSH) is the main non-protein antioxidant and plays a critical role in protecting cells from damage by reactive oxygen species (ROS) generated by dopamine (DA) metabolism. We reported a decrease of GSH levels ([GSH]) in CSF and in prefrontal cortex in vivo in schizophrenics [Eur. J. Neurosci. 12 (2000) 3721]. A GSH deficit may lead to membrane peroxidation and microlesions around dopaminergic terminals, resulting in loss of connectivity. To test this hypothesis, we studied the effect of DA in cultured cortical neurons with low [GSH]. DA alone decreased [GSH] by 40%. This effect appears to result from direct conjugation of DA semiquinone/quinone with GSH. Ethacrynic acid (EA) decreased [GSH] in a concentration-dependent manner. When added to EA, DA further lowers [GSH]. As this additional decrease is blocked by superoxide dismutase (SOD) or D(1)/D(2) receptor antagonists, it likely involves the generation of superoxide via activation of DA receptors. It also reduces the mitochondrial membrane potential. Most interestingly, a significant decrease in number of neuronal processes (spines analogous) was induced by 24-h application of DA only in low [GSH]. These data, compatible with our hypothesis, is consistent with the dendritic spines reduction reported in schizophrenia and could be related to abnormalities in synaptic connectivity.

Animals↗
PubMed2002

Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes.

Low-intensity ultrasound accelerates fracture healing in humans. In rat femur fracture models, ultrasound advanced healing is associated with increased proteoglycan expression. Here we report that ultrasound stimulation of primary rat chondrocytes elevated the intracellular concentration of calcium [Ca2+]i. The [Ca2+]i increase was rapid and transient at lower pressures (175-320 kPa), but rapid and sustained at higher ultrasound exposures (350-500 kPa). Chelating internal [Ca2+]i with 1,2-bis(2-aminophenoxy) ethane-N-N-N',N'-tetraacetic acid (BAPTA-AM), stopping the Ca2+/ATP-ase induced mitochondrial release of [Ca2+]i with Thapsigargin, or removing [Ca2+]i from the medium with EGTA inhibited the stimulatory effects of ultrasound on proteoglycan synthesis. These results imply that ultrasound-stimulated synthesis of cell matrix proteoglycan, associated with accelerated fracture healing, is mediated by intracellular calcium signaling.

Aggrecans↗
PubMedDate not supplied

Mechanisms of glutamate release from astrocytes: gap junction "hemichannels", purinergic receptors and exocytotic release.

Neuronal exocytotic release of glutamate at synapses involves a highly specialized vesicular apparatus, consisting of a variety of proteins connected to the vesicles or required for vesicular fusion to the presynaptic membrane. Astrocytes also release glutamate, and recent evidence indicates that this release can modify neuronal function. Several mechanisms have been proposed for astrocytic release of glutamate under pathological conditions, such as reversal of glutamate transporters and opening of volume sensitive ion channels. In this review we limit our discussion to findings supporting the exocytotic release of glutamate, as well as two new pathways implicated in this release, the ionotropic (P2X) purinergic receptors and gap junction hemichannels.

Animals↗