Biomedical subjects

J D Andreadis

Publications and source records attributed to J D Andreadis.

9 recordsLinked to original sources

PubMed2002

Gene modulation in total brain induced by exposure to the bicyclic phosphorus ester trimethylolpropane phosphate (TMPP).

The effect of a single subconvulsive dose of the GABAergic convulsant trimethylolpropane phosphate (TMPP) on gene expression in total rat brain was examined using cDNA array analysis. Using threshold criteria that reduce the number of false positives to <1 gene per 3551 actively transcribed genes on the cDNA array, 41 genes/EST sequences were reproducibly modulated in response to 0.25 mg/kg TMPP. Several genes that were consistent with epileptogenesis and/or neuronal damage and repair mechanisms, such as trkB, alphaB-crystallin, and decorin, were modulated by TMPP exposure in the absence of clinical convulsions. Previous research indicates that rats exposed to subconvulsive doses of TMPP exhibit both "absence-like" EEG paroxysms and persisting central nervous system (CNS) sensitization, as evidenced by increased susceptibility to audiogenic seizures (AGS). Results of this study suggest that cDNA arrays can be used to identify gene modulation events induced by low-level exposure to a chemical convulsant in a reproducible manner.

Animals↗

PubMed2001

Detection of physiologically active compounds using cell-based biosensors.

Cell-based biosensors are portable devices that contain living biological cells that monitor physiological changes induced by exposure to environmental perturbations such as toxicants, pathogens or other agents. Methods of detecting physiological changes include extracellular electrical recordings, optical measurements, and, in the future, functional genomics and proteomics. Several technical developments are occurring that will increase the feasibility of cell-based biosensors for field applications; these developments include stem cell and 3D culture technologies. Possible scenarios for the use of cell-based biosensors include broad-range detectors of unknown threat agents and functional assessment of identified agents.

Biosensing Techniques↗

PubMed2001

Ethanol blocks cytosolic Ca2+ responses triggered by activation of GABA(A) receptor/Cl- channels in cultured proliferating rat neuroepithelial cells.

GABA(A) receptor/Cl- channels and voltage-gated Ca2+ channels are believed to be important sites of ethanol action in the CNS. Acute exposure of ethanol potentiates GABA(A) receptor/Cl- channel activity and inhibits voltage-gated Ca2+ channels in a number of preparations, mostly post-mitotic neurons. The effects of ethanol on these channels in primary cultures of undifferentiated neural precursor cells remain unknown. To address this issue, we examined the effects of ethanol on GABA(A) agonist-activated elevation of cytosolic Ca2+ in an in vitro model of the cortical neuroepithelium derived from rat basic fibroblast growth factor-expanded neural precursor cells. We found a potent inhibition of GABA(A)-activated elevation of cytosolic Ca2+ by ethanol in actively proliferating cells. Since we had recently demonstrated that GABA(A) receptor activation depolarizes these cells and elevates their cytosolic Ca2+, we tested whether the effects of ethanol involved both GABA(A) receptors and voltage-gated Ca2+ channels. Both extracellular K+- and muscimol-induced cytosolic Ca2+ elevations were abolished by nitrendipine, indicating that both depolarizing stimuli triggered Ca2+ influx through L-type voltage-gated Ca2+ channels. Exposure of proliferating cells to different concentrations of ethanol revealed that the drug was more potent in blocking muscimol-induced compared to K+-evoked cytosolic Ca2+ elevations. These results raise the possibility that ethanol blocks GABAergic stimulation of cytosolic Ca2+ levels in proliferating precursors primarily by interacting with GABA(A) receptor/Cl- channels and secondarily with voltage-gated Ca2+ channels.

Animals↗

PubMed2001

Identification of differential gene expression profiles in rat cortical cells exposed to the neuroactive agents trimethylolpropane phosphate and bicuculline.

Recent technological advancements in microfabrication combined with the rapid acquisition of full genome sequence data have led to the development of DNA arrays that have the capacity to monitor the expression levels of thousands of genes simultaneously. The development of this technology enables the use of functional genomics approaches to identify molecular markers associated with cellular responsiveness to cytotoxic exposures. Databases containing unique cell-response profiles associated with specific toxicants or classes of toxicants can then be used in conjunction with cell-based biosensor platforms for environmental surveillance and toxicological assessment. An important issue that must be addressed, however, is whether DNA arrays can be used to identify transient gene modulation events in a reproducible manner. To address this issue, we utilized a primary embryonic rat (day 18) cortical cell model system and examined the RNA of both chemically treated and untreated cells using radioisotope-labeled cDNA probes and commercially available nylon membrane arrays. Using this approach, we examined experimental variability, basal gene expression variability, the occurrence of false positives, and the reproducibility of gene expression profiles obtained after chemical exposure. Minimal differences in gene modulation were observed between RNA samples from independently cultured cortical cells when array experiments were conducted in parallel (Pearson correlation coefficient for gene intensities =0.98). In contrast, significant differences in gene expression were observed between array experiments conducted at different times with an identical RNA source (Pearson correlation coefficient for gene intensities=0.91). Our results suggest the effect of basal gene activity differences in independently isolated cell cultures is negligible and that experimental variability possibly associated with the handling of RNA samples, differences in reverse transcription efficiency, hybridization, and/or signal acquisition are the primary contributors to variability in measurements. Using cDNA array analysis of unexposed cells from three independent cell culture preparations, we calculated false positive gene modulation events as a function of the threshold absolute value of log(2) >1.0. The number of false positives using this criteria was 1-10 gene/ESTs/5109 actively transcribed gene/ESTs represented on the array. Using three independent replicate experiments of untreated cortical cell cultures, we determined that a threshold criterion of absolute value of log(2) >0.63 for triplicate experiments would reduce the expected number of false positives in our experiments to less than one. Using this criterion, reproducible gene expression profiles were identified in cortical cells exposed to the neuroactive agents trimethylolpropane phosphate and bicuculline.

Animals↗

PubMed2000

Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells.

Ionotropic glutamate receptors mediate fast forms of excitatory synaptic transmission in mature neurons and may play critical roles in neuronal development. However, the developmental stage at which neuronal cells begin to express functional receptors and their roles in lineage progression remain unclear. In the present study, neural precursor cells were isolated from the cortical neuroepithelium of embryonic day 13 rats, and rapidly expanded in serum-free medium in response to basic fibroblast growth factor. RT-PCR revealed the presence of mRNAs encoding AMPA(A), AMPA(C), KA(1), KA(2), NMDA(1), and NMDA(2D) subunits after 3 days in culture. The functional expression of AMPA/kainate and NMDA receptors was investigated using Ca(2+) imaging and whole-cell patch-clamp recording techniques in cells pulse-labeled with bromodeoxyuridine (BrdU) for 1-4 hr. The recorded cells were then double-immunostained for BrdU incorporation and neuron-specific beta-tubulin (TuJ1). The results show that AMPA/kainate and NMDA induced increases in cytosolic Ca(2+) and inward currents only in differentiating neurons. In contrast, proliferating (BrdU(+)TuJ1(-)) cells failed to respond to any ionotropic glutamate receptor agonists. Interestingly, Ca(2+) imaging revealed that a subpopulation of BrdU(+)TuJ1(+) cells also responded to AMPA, indicating the emergence of functional ionotropic AMPA/kainate receptors during terminal cell division and the earliest commitment to neuronal cell lineage. These in vitro results were supported by flow cytometric sorting of AMPA-responsive cells pulse-labeled with BrdU for 1 hr in vivo, which revealed that functional AMPA receptors appear in BrdU(+)TuJ1(+) cells under physiological conditions and may play a role in terminal cell division.

Animals↗

PubMed2000

Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription/translation reactions.

We have developed a novel biochemical method to simultaneously amplify and immobilize a target gene onto insoluble particles using PCR. This method employs the covalent attachment of one of two PCR primers to a particle surface either directly during DNA synthesis of the primer or post-DNA synthesis, through the use of chemical crosslinkers. Immobilization of the target gene can be achieved directly during PCR amplification, with one bead-bound primer and one soluble primer. Alternatively, this can be achieved post-PCR, through covalent attachment of a chemically modified primer incorporated into the amplicon to an activated particle. All of the immobilized DNA templates containing appropriate regulatory regions were fully competent for transcription and translation reactions and several could be re-used in serial reactions. The most successful strategy utilized amino-silanized controlled pore glass beads, which were coupled to phosphorylated primers using carbodiimide chemistry. These bead-bound primers were used during PCR to generate attached DNA templates that could be collected and re-used for at least seven sequential transcription reactions without significant loss in efficiency. This method has also been successfully applied to the amplification, transcription and translation of multiple DNA templates using a single, immobilized primer. The combined PCR-based amplification/immobilization method was shown to be more durable than post-PCR chemical immobilization and affords the convenience of performing sequential PCR amplification, transcription and translation reactions in a single tube.

Animals↗

PubMed2000

Immobilization of neural cells in three-dimensional matrices for biosensor applications.

To overcome logistical difficulties with current designs of cell- or tissue-based biosensors which have individual cells or tissue slices immobilized on membranes or microelectrode arrays, we have proposed a system that uses three-dimensional cultures of neural cells immobilized in hydrogel matrices. In this design, immobilized cells would be maintained in a reservoir and then transferred to a detector platform when needed for analysis. The development of such a system relies upon a renewable supply of cells and the ability to culture cells for long periods of time in three-dimensions while maintaining their physiological function. To investigate the ability to culture neural cells in 3D matrices, embryonic rat cortical neurons and astrocytes were immobilized by matrix entrapment in a novel sugar poly(acrylate) hydrogel and collagen gels. The sugar poly(acrylate) hydrogel does not appear to support neural cell growth as a result of a lack of cell adherence, small pore size and, possibly, harshness of synthesis conditions. In contrast, collagen gels support the growth of cortical neurons, astrocytes, as well as neural progenitor cells. Evidence is also presented from immunocytochemistry and patch-clamp measurements which shows that neural progenitor cells proliferate in culture and can be induced to differentiate into neural cell types. Thus, they potentially represent a renewable cell source.

Animals↗

PubMed2000

Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

Acetylcholine↗

PubMed1998

Substrate mutations that bypass a specific Cpn10 chaperonin requirement for protein folding.

The bacteriophage T4 GroES homologue, gp31, in conjunction with the Escherichia coli chaperonin GroEL, is both necessary and sufficient to fold the T4 major capsid protein, gp23, to a state competent for capsid assembly as shown by in vivo expression studies. GroES is unable to function in this role as a productive co-chaperonin. The sequencing and characterization of mutations within gp23 that confer GroEL and gp31 chaperonin-independent folding of the mutant protein suggest that the chaperonin requirements are due to specific sequence determinants or structures in critical regions of gp23 that behave in an additive fashion to confer a chaperonin bypass phenotype. Conservative amino acid substitutions in these critical regions enable gp23 to fold in a GroEL-gp31 chaperonin-independent mode, albeit less efficiently than wild type, both in vivo and in vitro. Although the presence of functional GroEL-gp31 enhances folding of the mutated gp23 in vivo, GroEL-GroES has no such effect. Site-directed mutagenesis experiments suggest that a translational pausing mechanism is not responsible for the bypass mutant phenotype. Polyhead reassembly experiments are also consistent with direct, post-translational effects of the bypass mutations on polypeptide folding. Given our finding that gp31 is not required for the binding of the major capsid protein to GroEL and that active GroES is incapable of folding the gp23 polypeptide chain to native conformation, our results suggest co-chaperonin specificity in the folding of certain substrates.

Amino Acid Substitution↗